CN110257265A - A kind of cultural method of oleaginous yeast, microbial oil production method and its application - Google Patents
A kind of cultural method of oleaginous yeast, microbial oil production method and its application Download PDFInfo
- Publication number
- CN110257265A CN110257265A CN201910362911.1A CN201910362911A CN110257265A CN 110257265 A CN110257265 A CN 110257265A CN 201910362911 A CN201910362911 A CN 201910362911A CN 110257265 A CN110257265 A CN 110257265A
- Authority
- CN
- China
- Prior art keywords
- oleaginous yeast
- culture
- microbial oil
- cells
- production method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 69
- 230000000813 microbial effect Effects 0.000 title claims abstract description 31
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 22
- 239000002351 wastewater Substances 0.000 claims abstract description 75
- 241000894006 Bacteria Species 0.000 claims abstract description 55
- 239000001963 growth medium Substances 0.000 claims description 56
- 239000004519 grease Substances 0.000 claims description 34
- 238000009423 ventilation Methods 0.000 claims description 28
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 15
- 229910052760 oxygen Inorganic materials 0.000 claims description 15
- 239000001301 oxygen Substances 0.000 claims description 15
- 239000003225 biodiesel Substances 0.000 claims description 14
- 238000000855 fermentation Methods 0.000 claims description 14
- 230000004151 fermentation Effects 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 12
- 238000000605 extraction Methods 0.000 claims description 4
- 244000005700 microbiome Species 0.000 abstract description 16
- 230000005526 G1 to G0 transition Effects 0.000 abstract description 8
- 238000012545 processing Methods 0.000 abstract description 3
- 231100000033 toxigenic Toxicity 0.000 abstract description 2
- 230000001551 toxigenic effect Effects 0.000 abstract description 2
- 241000221523 Rhodotorula toruloides Species 0.000 description 61
- 239000003921 oil Substances 0.000 description 31
- 238000005119 centrifugation Methods 0.000 description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 230000001954 sterilising effect Effects 0.000 description 14
- 238000011218 seed culture Methods 0.000 description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 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 description 8
- 239000000356 contaminant Substances 0.000 description 8
- 239000001888 Peptone Substances 0.000 description 7
- 108010080698 Peptones Proteins 0.000 description 7
- 239000002253 acid Substances 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 235000019319 peptone Nutrition 0.000 description 7
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 6
- 229940041514 candida albicans extract Drugs 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 238000004659 sterilization and disinfection Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 239000012138 yeast extract Substances 0.000 description 6
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 5
- 239000008121 dextrose Substances 0.000 description 5
- 238000004090 dissolution Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 239000005416 organic matter Substances 0.000 description 5
- 229910052698 phosphorus Inorganic materials 0.000 description 5
- 239000011574 phosphorus Substances 0.000 description 5
- 241000235342 Saccharomycetes Species 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000005265 energy consumption Methods 0.000 description 4
- 239000003337 fertilizer Substances 0.000 description 4
- 241000276457 Gadidae Species 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- YUWBVKYVJWNVLE-UHFFFAOYSA-N [N].[P] Chemical compound [N].[P] YUWBVKYVJWNVLE-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000005276 aerator Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 239000002283 diesel fuel Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013505 freshwater Substances 0.000 description 2
- 239000010842 industrial wastewater Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 239000001965 potato dextrose agar Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000013535 sea water Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 235000019737 Animal fat Nutrition 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 240000002791 Brassica napus Species 0.000 description 1
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 240000001973 Ficus microcarpa Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 239000002803 fossil fuel Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000010773 plant oil Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000009333 weeding Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- 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
- C12P7/6445—Glycerides
- C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/32—Nature of the water, waste water, sewage or sludge to be treated from the food or foodstuff industry, e.g. brewery waste waters
- C02F2103/325—Nature of the water, waste water, sewage or sludge to be treated from the food or foodstuff industry, e.g. brewery waste waters from processes relating to the production of wine products
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Mycology (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Botany (AREA)
- Biodiversity & Conservation Biology (AREA)
- Water Supply & Treatment (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to technical field of waste water processing more particularly to a kind of cultural methods of oleaginous yeast, microbial oil production method and its application.The present invention provides a kind of cultural methods of oleaginous yeast, it is cultivated the following steps are included: oleaginous yeast is seeded in the culture solution that pH value is 3.3~4, wherein, initial concentration of the oleaginous yeast in the culture solution is (1~2) × 108cells/mL.In the cultural method of oleaginous yeast of the present invention, initial concentration of the oleaginous yeast in culture solution is (1~2) × 108Cells/mL can greatly shorten the time that oleaginous yeast grows into stationary phase, dominant bacteria can be rapidly become before culture solution indigenous microorganisms grow, avoid the influence of culture solution indigenous microorganisms bring, it is the culture that aseptic condition can carry out oleaginous yeast without controlling condition of culture, reduces the toxigenic capacity of oleaginous yeast.
Description
Technical field
The invention belongs to technical field of waste water processing more particularly to a kind of cultural methods of oleaginous yeast, microbial oil
Rouge production method and its application.
Background technique
Biodiesel is a kind of green renewable resource, is had good burning performance, sulfur content is low, being capable of substitute fossil fuels.It is raw
The production technology of object diesel oil is divided into three generations, and first generation biodiesel technology is raw from consumption oil crops such as rapeseed and soybean
Generation diesel oil, the technology maturation have simultaneously realized industrialization, but plant oil crops and need large amount of land resources, it may appear that with grain
The problem of food production competition soil, while its yield is not able to satisfy the demand of rapid growth much yet；Second generation biodiesel skill
Art produces biodiesel from non-consumption oil crops such as manioca, and the technology is not yet mature, and has and first generation biology bavin
The similar problem of oil tech, or biodiesel is produced from waste home appliance such as gutter oil or animal fat, it can come because of raw material
Source is limited and is restricted；Third generation biodiesel technology produces biodiesel using oleaginous microorganism, becomes biodiesel
The new hope of industry.
There are many advantages compared to using oil crops using oleaginous microorganism production biodiesel, grease yield compares oil plant
Crop is high, and water consumption is less, can grow in a variety of environment, does not need to fight for land resource with traditional agriculture, does not need weeding
Agent and insecticide.But the microdisk electrode of early stage production biodiesel needs to ensure yield, the use of chemical fertilizer using a large amount of chemical fertilizer
It will increase cost, and have the risk for causing secondary pollution.So using organic matter and nutrients or agricultural residue in waste water
Fertilizer as culture oleaginous microorganism is better choice；On the other hand, food industrial wastewater high organic content, it is very suitable
Close the growth of oleaginous microorganism.
However, carrying out the culture of oleaginous microorganism using waste water, living contaminants problem is highlighted, organic matter and battalion in waste water
It is more to support object, suitable for the breeding of many types microorganism, and the speed of growth faster primary bacterium and fungi than additional oil-producing
Microorganism has more growth vigor, adversely affects to the growth and oil-producing of oleaginous microorganism.Such as in animal husbandry waste water, nothing
The grease yield of microalgae under the conditions of bacterium is 1.3-1.7 times under non-sterile conditions, i.e. the growth of miscellaneous bacteria can make the grease of microalgae
Yield reduces 20%-40%.
The third generation biodiesel technology of early stage, uses seawater or fresh water to cultivate microalgae.Due to seawater or fresh water
The nutrition contents such as organic matter and nitrogen phosphorus are low, solid suspension is few and viscosity is relatively low is easy to the features such as filtering, mainly pass through
The method of filtration sterilization controls living contaminants.At the same time, the low grease yield that also results in of nutrition content is low, needs to add
Chemical fertilizer is added to ensure yield.But the solid suspension in waste water is relatively more, the nutriments content of material such as organic matter and nitrogen phosphorus
Height, effluent part have it is slightly viscous, be easy blocking strainer, filtration sterilization is not convenient to use, so that using waste water culture oil-producing
Yeast production microbial oil predominantly stays in the laboratory research stage, and control living contaminants mainly use high-temp steam sterilizing.
But high-temp steam sterilizing needs to consume a large amount of energy.From the angle of production, for the huge water of waste water, using high temperature
Requirement of the steam sterilizing to equipment is high.It can be likely to generate secondary pollution using bacteriostatic agent or antibiotic etc., influence at subsequent water
Manage the efficiency of technical process.
Summary of the invention
In view of this, the present invention provides a kind of cultural method of oleaginous yeast, microbial oil production method and its
Using for solving the problems, such as that preparing microbial oil living contaminants using waste water highlights, and controls miscellaneous bacteria using autoclave sterilization
The problem of energy consumption is high for pollution, equipment requirement height etc..
The specific technical solution of the present invention is as follows:
The present invention provides a kind of cultural methods of oleaginous yeast, comprising the following steps:
Oleaginous yeast is seeded in the culture solution that pH value is 3.3~4 and is cultivated, wherein the oleaginous yeast
Initial concentration in the culture solution is (1~2) × 108cells/mL。
In the cultural method of oleaginous yeast of the present invention, initial concentration of the oleaginous yeast in culture solution be (1~2) ×
108Cells/mL can greatly shorten the time that oleaginous yeast grows into stationary phase, can be raw in culture solution indigenous microorganisms
It is long to get up to rapidly become dominant bacteria before, the influence of culture solution indigenous microorganisms bring is avoided, is without controlling condition of culture
Aseptic condition can carry out the culture of oleaginous yeast, reduce the toxigenic capacity of oleaginous yeast.
The present invention also provides a kind of preparation methods of microbial oil, comprising the following steps:
A) oleaginous yeast is seeded in the culture solution that pH value is 3.3~4 and is fermented, obtain tunning, wherein
Initial concentration of the oleaginous yeast in the culture solution is (1~2) × 108cells/mL；
B) tunning is collected, then carries out grease extraction, obtain microbial oil.
In the present invention, fermented in the culture solution of acid condition using oleaginous yeast, oleaginous yeast is in waste water
In initial concentration be (1~2) × 108Cells/mL substantially reduces the time that oleaginous yeast grows into stationary phase, so that
Oleaginous yeast rapidly becomes the dominant bacteria in culture solution before indigenous microorganisms growth is got up, and avoids the original in culture solution
Raw miscellaneous bacteria bring influences, and is that aseptic condition can be fermented to obtain tunning, tunning without controlling fermentation condition
With rich grease-contained thallus, then grease extraction is carried out, obtains microbial oil, microbial oil can be carried out in culture solution
Effective production, should preparation method is simple, without autoclave sterilization create aseptic condition, sterilizing can be substantially reduced and caused
Energy consumption and equipment requirement, be easy to industrially apply.
Preferably, the temperature of the step a) fermentation is 20 DEG C~35 DEG C；
The time of the step a) fermentation is 12h~for 24 hours.
The ventilation ratio of the step a) fermentation is 3vvm~4.5vvm；
The dissolved oxygen concentration of the step a) fermentation is 30%~100% air saturation.
Preferably, the culture solution is brewing wastewater；
The COD (COD) of the brewing wastewater be 17,120mg/L~65,075mg/L, more preferably 20,
315mg/L~22,736mg/L.
In the present invention, brewing wastewater is the yellow water waste liquid that ferments obtained in soybean-flavor liquor production process.The present invention adopts
Microbial oil is prepared with brewing wastewater, brewing wastewater can be recycled, is carried out using the organic matter in brewing wastewater
The culture of oleaginous yeast is realized that oleaginous yeast synchronizes purification and oil-producing to brewing wastewater, is created without high temperature and pressure
Aseptic condition has the advantages that equipment requirement is low, easy to operate, the fermentation reaction time is short, is easy to industrially apply.Also,
The preparation method purifies brewing wastewater, solves brewing wastewater while carrying out resource utilization to brewing wastewater
The problem that discharge amount is huge and growing, processing is difficult.Brewing wastewater high organic content, is very suitable to oleaginous yeast
Growth, and discharge amount is huge and growing, be it is a kind of it is good can recovery energy resource.
It should be noted that similar food industrial wastewater also can be used in culture solution, it is not specifically limited herein.
When the COD of brewing wastewater is higher, sanitary wastewater, tap water or distilled water can be used to brewing wastewater
It is diluted.The pH value of brewing wastewater is not required to adjust when being higher than 3.3, and pH value is adjusted pH value to 4 when being lower than 3.3.
In the present invention, initial concentration of the oleaginous yeast in brewing wastewater is preferably 2 × 108Cells/mL, experiment knot
Fruit shows that under the initial concentration, the grease yield of oleaginous yeast is big, and fat content is stablized, and it is fast that grease produces speed, also, makes to produce
Initial concentration of the oily saccharomycete in brewing wastewater reaches 2 × 108Cost is relatively low needed for cells/mL.
Preferably, the oleaginous yeast is selected from circle rhodosporidium toruloides.
Step a) of the present invention is preferred are as follows: and it is 20,315mg/L~22 that oleaginous yeast, which is seeded to COD,
In 736mg/L, the brewing wastewater that pH value is 3.3, initial concentration of the oleaginous yeast in brewing wastewater is 2 × 108cells/
ML, then under conditions of temperature is 30 DEG C, ventilation ratio 4vvm, dissolved oxygen concentration are 60% air saturation carry out 12h hair
Ferment obtains rich grease-contained yeast thallus/bacterium mud/biomass.
Preferably, the oleaginous yeast preparation method the following steps are included:
The oleaginous yeast strain is seeded in the expansion culture medium that pH value is 4~5, expands culture, obtain institute
State oleaginous yeast.
Preferably, the temperature for expanding culture is 20 DEG C~35 DEG C, more preferably 30 DEG C；
The time for expanding culture is 8h~for 24 hours, more preferably 8~16h, further preferably 8~12h.
The ventilation ratio for expanding culture is 3vvm~4.5vvm, more preferably 4vvm；
The dissolved oxygen concentration for expanding culture is 40%~100% air saturation, and more preferably 60%~90% is empty
Gas saturation.
It in the present invention, expands culture without controlling condition of culture for aseptic condition, has using oleaginous yeast strain
Good acid resistance, the pH value that will be enlarged by culture medium are adjusted to 4~5, and the pollution of environmental microorganism can be effectively prevented, and exempt high
Warm sterilization steps.
In the present invention, oleaginous yeast strain is 1%~5% (v/v) in the bacterium amount that initially connects for expanding culture medium, preferably
5% (v/v), initially connects that bacterium amount is bigger, and the time for expanding culture is shorter.
In the present invention, expand culture expansion culture medium used be selected from yeast extract powder peptone glucose (YPD) culture medium and/
Or potato glucose (PDA) agar medium, preferably yeast extract powder peptone dextrose culture-medium, expansion culture medium have been placed in
The open container of aerator.
In the present invention, the concentration of the oleaginous yeast is (4~10) × 109Cells/mL, more preferably 5 ×
109cells/mL。
In the present invention, by the way that oleaginous yeast is collected by centrifugation, 4000rpm is centrifuged 10min, goes supernatant collection bacterium
Body counts thallus using blood counting chamber, and according to count value adjust oleaginous yeast concentration to (4~10) ×
109cells/mL。
Preferably, expand before cultivating, further includes:
Seed culture is carried out to the oleaginous yeast strain in the seed culture medium of sterilizing.
Final concentration of (1.19 ± 0.18) × 10 of thallus in the present invention, in seed culture medium8cells/mL。
Seed culture medium is selected from yeast extract powder peptone dextrose culture-medium or potato dextrose agar, preferably
Yeast extract powder peptone dextrose culture-medium.Seed culture medium, which is placed in conical flask, to seal and sterilizes.
It carries out seed culture to the oleaginous yeast strain in the seed culture medium of sterilizing to specifically include: by oleaginous yeast
Strain is seeded in seed culture medium in super-clean bench with oese, in 30 DEG C of constant-temperature tables, is shaken and is planted for 24 hours under fast 200rpm
Son culture.
In the present invention, oleaginous yeast strain the preparation method is as follows:
Initial productive barms are activated, oleaginous yeast strain is obtained.
Initial productive barms are carried out activation to specifically include: the oleaginous yeast bacterium colony on solid medium, inoculation
To fresh solid seed culture medium, 30 degree are cultivated 2-3 days, until it is observed that apparent yeast colony.
In the present invention, when the environment for expanding culture is the corrupt acid-producing bacteria such as neighbouring refuse depot more place, expand culture
The pH of base is preferably 4.5~5, and oleaginous yeast strain is 5% (v/v) in the bacterium amount that connects for expanding culture medium, expands the time of culture
For 8h, the risk for producing sour acid fast bacteria pollution is reduced；When the environment for expanding culture is surrounding enviroment corruption acid-producing bacteria less place,
The pH for expanding culture medium is preferably 4, also, can directly be inhaled after the activation of initial productive barms without carrying out seed culture
Expansion culture medium is taken, is expanded in culture medium from thallus is eluted on solid plate, carries out expanding culture for 24 hours.
The present invention also provides the microbial oils that above-mentioned technical proposal the method is prepared.
The present invention also provides application of the above-mentioned technical proposal microbial oil in preparation biodiesel.
The microbial oil that the present invention is prepared can be further converted to biodiesel, realize and be not necessarily to high temperature and pressure
Sterilising conditions carry out the resource utilization of brewing wastewater, and synchronous water purification oil-producing greatly reduces process energy consumption and wants to equipment
It asks, is easy in practical application.
In conclusion the present invention provides a kind of preparation methods of microbial oil, comprising the following steps: a) by oil-producing ferment
Female bacterium, which is seeded in the culture solution that pH value is 3.3~4, ferments, and obtains tunning, wherein the oleaginous yeast is in institute
Stating the initial concentration in culture solution is (1~2) × 108cells/mL；B) by the grease-contained thallus of richness in the tunning
It is collected, then carries out grease extraction, obtain microbial oil.The method that the present invention prepares microbial oil using culture solution,
It is that aseptic condition can be fermented to obtain tunning without controlling fermentation condition, tunning has rich grease-contained bacterium
Body can be realized oleaginous yeast oil-producing, and equipment requirement is low, easy to operate, the fermentation reaction time is short, is easy to industrially answer
With.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 is that blank YPD culture medium and Rhodosporidium toruloides kind are trained in the expansion of different pH value in the embodiment of the present invention 1
It supports and expands the result figure of culture for 24 hours in base, wherein front-seat 3 reactors are the blank without being inoculated with Rhodosporidium toruloides kind
YPD culture medium, heel row are successively from left to right that Rhodosporidium toruloides kind expands in the expansion culture medium that pH value is 4,4.5 and 5
The result figure of big culture for 24 hours；
Fig. 2 is blank YPD culture medium in the embodiment of the present invention 1 (pH value is 4~5) and Rhodosporidium toruloides kind in pH value
To expand the result figure after culture 40h centrifugation in 4~5 YPD culture medium, wherein left figure is without being inoculated with circle rhodosporidium toruloides
Result figure after the blank YPD culture medium open culturing 40h centrifugation of strain, it in pH value is 4 that right figure, which is Rhodosporidium toruloides kind,
Result figure after expanding culture 40h centrifugation in~5 YPD culture medium；
Fig. 3 is that Rhodosporidium toruloides kind expands culture 40h in the YPD culture medium that pH value is 4 in the embodiment of the present invention 1
Result figure after centrifugation；
Fig. 4 is that Rhodosporidium toruloides are sent out in the brewing wastewater of different CODs in the embodiment of the present invention 2
The result figure of ferment, the COD of brewing wastewater is followed successively by 17,120mg/L, 22,736mg/L, 25,457mg/L from left to right
And 28,080mg/L；
Fig. 5 is that difference initially connects bacteria concentration (0.5 × 10 in the embodiment of the present invention 38cells/mL、1×108cells/mL、
1.5×108Cells/mL and 2 × 108Cells/mL) corresponding different ventilation ratios (0.9vvm, 1.8vvm, 2.7vvm and 3.6vvm)
Under the figure that changes over time of dissolved oxygen concentration；
Fig. 6 is that initial concentration of the Rhodosporidium toruloides in brewing wastewater is not sent out simultaneously in the embodiment of the present invention 3
The result figure of ferment, wherein from left to right initial concentration of the Rhodosporidium toruloides in brewing wastewater be followed successively by 0.5 respectively ×
108cells/mL、1×108cells/mL、1.5×108Cells/mL and 2 × 108cells/mL；
Fig. 7 is that initial concentration of the Rhodosporidium toruloides in brewing wastewater is not sent out simultaneously in the embodiment of the present invention 3
Total dry cell weight and miscellaneous bacteria dry weight figure after ferment, wherein solid line is total dry cell weight, and dotted line is miscellaneous bacteria dry weight figure；
Fig. 8 is that initial concentration of the Rhodosporidium toruloides in brewing wastewater is not sent out simultaneously in the embodiment of the present invention 3
Grease yield figure and fat content figure after ferment, wherein (a) grease yield, (b) fat content in dry cell weight；
Fig. 9 is that Rhodosporidium toruloides ferment in brewing wastewater under different ventilation ratios in the embodiment of the present invention 4
Dry cell weight and grease yield figure afterwards, (a) real diagram are dry cell weight, and dashed line view is the primary miscellaneous bacteria dry weight of white, (b) grease
Yield；
Figure 10 is that Rhodosporidium toruloides ferment in brewing wastewater under different ventilation ratios in the embodiment of the present invention 4
Every water quality parameter variation diagram of brewing wastewater afterwards, COD (COD) (a), total phosphorus (b), total nitrogen (c), ammonia nitrogen (d),
PH (e) and dissolved oxygen (f).
Specific embodiment
The present invention provides a kind of cultural method of oleaginous yeast, microbial oil production method and its applications, are used for
It solves the problems, such as to highlight using waste water preparation microbial oil living contaminants, living contaminants energy consumption is controlled using autoclave sterilization
Greatly, the problem of equipment requirement height etc..
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
The preparation of the present embodiment progress Rhodosporidium toruloides, comprising the following steps:
(1) prepare seed culture medium: seed culture medium is yeast extract powder peptone dextrose culture-medium (Yeast Extract
Peptone Dextrose Medium, YPD culture medium), according to YPD culture medium prescription (yeast powder 10g/L, peptone 20g/L,
Glucose 20g/L) 40mL YPD culture medium is prepared, it is placed in conical flask and seals and sterilize.
(2) it prepares Rhodosporidium toruloides kind: original circle rhodosporidium toruloides strain is activated in solid medium
Afterwards, it is seeded in seed culture medium in super-clean bench with oese, in 30 DEG C of constant-temperature tables, shakes and planted for 24 hours under fast 200rpm
Son culture, obtains Rhodosporidium toruloides kind.
(3) preparation expands culture medium: expansion culture medium is YPD culture medium, prepares 500mL according to YPD culture medium prescription
YPD culture medium, the pH value that will be enlarged by culture medium are adjusted to 4, are placed directly in the open container of aerator without sterilizing.
(4) the expansion culture of Rhodosporidium toruloides kind is carried out: under open condition, by step (2) circle rhodosporidium toruloides
Strain is seeded in the expansion culture medium that pH value is 4~5, then in temperature is 20 DEG C~30 DEG C, ventilation ratio 4vvm, initial dissolution
Oxygen concentration is to expand culture under 80%~90% air saturation, obtains Rhodosporidium toruloides.Wherein, the red winter spore ferment of circle
Female strain is (1% and 5%, v/v) in the inoculum concentration for expanding culture medium.
Referring to Fig. 1, Fig. 1 is blank YPD culture medium in the embodiment of the present invention 1 and Rhodosporidium toruloides kind in different pH
Expanding for value expands the result figure of culture for 24 hours in culture medium, wherein front-seat 3 reactors are without being inoculated with circle rhodosporidium toruloides
The blank YPD culture medium of strain, from left to right pH value is followed successively by 4,4.5 and 5；Heel row is successively from left to right round rhodosporidium toruloides
Strain expands the result figure of culture for 24 hours in the expansion culture medium that pH value is 4,4.5 and 5.
Referring to Fig. 2, Fig. 2 is that blank YPD culture medium and Rhodosporidium toruloides kind are in the embodiment of the present invention 1 in pH value
4~5 result figure expanded after expanding culture 40h centrifugation in culture medium, wherein left figure is without inoculation Rhodosporidium toruloides
Result figure after the blank YPD culture medium centrifugation of kind, right figure is the expansion culture that Rhodosporidium toruloides kind is 4~5 in pH value
Result figure after expanding culture 40h centrifugation in base.
In the present embodiment, Fig. 2 left figure is centrifuged to obtain by collecting the front row Fig. 1 blank YPD culture medium, and Fig. 2 right figure passes through receipts
Collection Fig. 2 heel row Rhodosporidium toruloides are centrifuged to obtain, and the revolving speed of centrifugation is 10000rpm, and the time of centrifugation is 10min.Fig. 1 and
Fig. 2 shows that Rhodosporidium toruloides kind expands culture in the YPD culture medium that pH value is 4,4.5 and 5 and stationary phase is presented for 24 hours
Red, and the YPD culture that the blank YPD culture medium and Rhodosporidium toruloides kind that pH value is 4,4.5 and 5 are 4~5 in pH value
There is no miscellaneous bacteria mud (white) appearance after expanding culture 40h centrifugation in base, when showing that expanding Medium's PH Value is 4~5, it is not easy to quilt
Pollution；Expanding culture simultaneously need to only be shorter than Rhodosporidium toruloides culture to growth period, incubation time for 24 hours, living contaminants wind
Danger is lower to stationary phase than cultivating.Seed liquor of the Rhodosporidium toruloides that the present embodiment is prepared as subsequent embodiment,
In traditional yeast bacterium shaking flask culture, saccharomycete 48h reaches stationary phase, and culture solution for 24 hours can be used as seed liquor, so the condition
The Rhodosporidium toruloides that lower culture 12h is prepared meet the needs of seed liquor.
Referring to Fig. 3, Fig. 3 be in the embodiment of the present invention 1 Rhodosporidium toruloides kind in the YPD culture medium that pH value is 4
Result figure after expanding culture 40h centrifugation.The revolving speed of centrifugation is 10000rpm, and the time of centrifugation is 10min, and centrifugation goes supernatant rich
The number for collecting thallus is twice.Fig. 3 shows to expand the pH value of culture medium when being 4, without control condition of culture be aseptic condition into
Row expand culture, Rhodosporidium toruloides kind expand culture after bacterium mud do not have other living contaminants (if there is pollution, through twice from
Among two layers of bacterium mud of heart enrichment and top will appear white miscellaneous bacteria band), Rhodosporidium toruloides are dominant bacteria, and bacterium mud is all in
It is red.
Embodiment 2
Rhodosporidium toruloides are collected by centrifugation in the present embodiment, and 4000rpm is centrifuged 10min, go supernatant collection bacterium
Body is concentrated into 4 50mL centrifuge tubes, is counted using blood counting chamber to the thallus in centrifuge tube, according to count value tune
The cell concentration in centrifuge tube is saved to 5 × 109Cells/mL, then by cell concentration be 5 × 109The red winter spore ferment of the circle of cells/mL
Female bacterium is seeded in the brewing wastewater of 500mL difference COD, and Rhodosporidium toruloides are initial dense in brewing wastewater
Degree is 1 × 108Cells/mL, then pH value is 3.3, temperature is 23 DEG C~30 DEG C, ventilation ratio 1.8vvm, initial dissolution oxygen
Concentration is that the fermentation of 48h is carried out under 35~55% air saturations, obtains tunning.
The brewing wastewater of different CODs is diluted to obtain by using distilled water, and the present embodiment brewing wastewater is set
Four groups have been set, every group of extension rate is followed successively by 4,3,2.5 and 2, and corresponding COD is followed successively by 17,120mg/L, 22,
736mg/L, 25,457mg/L and 28,080mg/L, every group sets 2 parallel tests.
Referring to Fig. 4, being Rhodosporidium toruloides in the embodiment of the present invention 2 in the brewing wastewater of different CODs
The result figure fermented, the COD of brewing wastewater is followed successively by 17,120mg/L, 22,736mg/L, 25 from left to right,
457mg/L and 28,080mg/L.Fig. 4 shows that the extension rate of brewing wastewater is 3~4, and corresponding COD is 22,
When 736mg/L~17,120mg/L, the red of normal growth is presented in Rhodosporidium toruloides, and the extension rate of brewing wastewater is
2~2.5, corresponding COD is 25,457mg/L~28, and when 080mg/L, Rhodosporidium toruloides present suppressed black
Color, it is known that, the extension rate of brewing wastewater is preferably 3, and COD is preferably 22,736mg/L.
Embodiment 3
Rhodosporidium toruloides are collected by centrifugation in the present embodiment, and 4000rpm is centrifuged 10min, go supernatant collection bacterium
Body is concentrated into 4 50mL centrifuge tubes, is counted using blood counting chamber to the thallus in centrifuge tube, according to count value tune
The cell concentration in centrifuge tube is saved to 5 × 109Cells/mL, then by cell concentration be 5 × 109The red winter spore ferment of the circle of cells/mL
Female bacterium is seeded in the brewing wastewater that 500mL COD is 20,163mg/L, and brewing wastewater is provided with four groups, and every group sets 2
A parallel test, initial concentration of the Rhodosporidium toruloides in brewing wastewater are followed successively by 0.5 × 10 respectively8cells/mL、1×
108cells/mL、1.5×108Cells/mL and 2 × 108Cells/mL, then pH value is 3.3, temperature is 23 DEG C~30 DEG C,
Initial dissolution oxygen concentration be 35%-55% air saturation under ferment, every group of ventilation ratio be followed successively by respectively 0.9vvm,
1.8vvm, 2.7vvm and 3.6vvm, per a sample is taken for 24 hours, sample is centrifuged 10min in 4000rpm, obtains thallus, thallus is used again
Deionized water is cleaned 1 time, and 105 DEG C drying to constant weight, measures dry cell weight and grease yield, grease yield measuring method is using acid
Thermal method.
Referring to Fig. 5, initially connecing bacteria concentration (0.5 × 10 to be different in the embodiment of the present invention 38cells/mL、1×
108cells/mL、1.5×108Cells/mL and 2 × 108Cells/mL) corresponding different ventilation ratios (0.9vvm, 1.8vvm,
2.7vvm and 3.6vvm) under the figure that changes over time of dissolved oxygen concentration.Fig. 5 shows Rhodosporidium toruloides in brewing wastewater
In initial concentration difference when, different ventilation ratios need to be correspondingly arranged, identical initial dissolution oxygen concentration could be obtained.
Referring to Fig. 6, be Rhodosporidium toruloides in the embodiment of the present invention 3 in brewing wastewater initial concentration difference when
The result figure fermented, wherein initial concentration of the Rhodosporidium toruloides in brewing wastewater is followed successively by respectively from left to right
0.5×108cells/mL、1×108cells/mL、1.5×108Cells/mL and 2 × 108cells/mL.Fig. 6 shows that circle is red
Initial concentration of the winter spore saccharomycete in brewing wastewater is 1 × 108When cells/mL, Rhodosporidium toruloides are possible to cannot be at
For the dominant bacteria in brewing wastewater, and replaced by the primary miscellaneous bacteria of white, when Rhodosporidium toruloides are initial in brewing wastewater
Concentration is more than or equal to 1.5 × 108When cells/mL, Rhodosporidium toruloides become dominant bacteria in brewing wastewater, and bacterium solution is in red
Color.
Referring to Fig. 7, be Rhodosporidium toruloides in the embodiment of the present invention 3 in brewing wastewater initial concentration difference when
Total dry cell weight and miscellaneous bacteria dry weight figure after being fermented, wherein solid line is total dry cell weight, and dotted line is miscellaneous bacteria dry weight figure.Fig. 7
Show that the initial concentration relative to Rhodosporidium toruloides in brewing wastewater is 0.5 × 108Cells/mL, initial concentration are
(1~2) × 108Cells/mL can greatly shorten the time that oleaginous yeast grows into stationary phase, can be in brewing wastewater original
Raw microorganism growth rapidly becomes dominant bacteria before getting up, and brewing wastewater indigenous microorganisms bring is avoided to influence.Also, originally
Beginning concentration is 2 × 108When cells/mL, thallus gross weight reaches maximum value in two days, and numerical value is noticeably greater than other experiments
Group (p < 0.05).
Referring to Fig. 8, be Rhodosporidium toruloides in the embodiment of the present invention 3 in brewing wastewater initial concentration difference when
Fat content figure in grease yield figure and dry cell weight after being fermented, wherein (a) grease yield, it is (b) oily in dry cell weight
Rouge content.Fig. 8 a shows that initial concentration of the Rhodosporidium toruloides in brewing wastewater is 2 × 108When cells/mL, the circle red winter
The grease yield of spore saccharomycete reaches maximum value in 1 day, and grease yield is noticeably greater than other experimental groups (p < 0.05), figure
8b shows that fat content reaches maximum value when cultivating 1 day.When cultivating 1 day, Rhodosporidium toruloides are in brewing wastewater
Initial concentration is 2 × 108Cells/mL, it is 1 × 10 that fat content, which is significantly higher than initial concentration,8Cells/mL and 1.5 ×
108cells/mL(p<0.05).In terms of fat content, initial concentration is 2 × 108Cells/mL and initial concentration be 0.5 ×
108Cells/mL is compared and is not significantly different (p > 0.05), but initial concentration is 0.5 × 108The standard error of cells/mL is bright
Showing greater than bacteria concentration is initially connect is 2 × 108The experimental group of cells/mL, and grease yield be also significantly less than the experimental group (p <
0.05).Fig. 8 shows that initial concentration is 2 × 108When cells/mL, grease yield highest, fat content is opposite in dry cell weight
Height, and it is more stable, and the grease yield arrival highest time can foreshorten to for 24 hours.
Embodiment 4
Rhodosporidium toruloides are collected by centrifugation in the present embodiment, and 4000rpm is centrifuged 10min, go supernatant collection bacterium
Body is concentrated into 4 50mL centrifuge tubes, is counted using blood counting chamber to the thallus in centrifuge tube, according to count value tune
The cell concentration in centrifuge tube is saved to 5 × 109Cells/mL, then by cell concentration be 5 × 109The red winter spore ferment of the circle of cells/mL
Female bacterium is seeded in the brewing wastewater that 500mL COD is 20,315mg/L, and Rhodosporidium toruloides are in brewing wastewater
Initial concentration be 2 × 108Cells/mL, brewing wastewater are provided with four groups, and every group sets 2 parallel tests, every group of ventilation score
Be not followed successively by 3vvm, 3.5vvm, 4vvm and 4.5vvm, then pH value be 3.4, temperature be 23 DEG C~30 DEG C, dissolved oxygen concentration
To ferment under 30%~100% air saturation, every 12h takes a sample, and sample is collected in 4000rpm centrifugation 10min, obtained
To bacterium mud (thallus), the top layer of bacterium mud is white miscellaneous bacteria, and bacterium mud top layer white miscellaneous bacteria is resuspended by slightly concussion, then will contain
The supernatant 10000rpm centrifugation 10min of miscellaneous bacteria collects miscellaneous bacteria thallus, 4 DEG C of preservations of filtrate, for every water quality parameter such as COD
Measurement, water quality parameter are measured according to National Standard Method.Thallus is cleaned 1 time with deionized water again, and 105 DEG C drying to constant weight, measures thallus
Dry weight and grease yield, grease yield measuring method use acid heat method.
Referring to Fig. 9, for Rhodosporidium toruloides in the embodiment of the present invention 4 under different ventilation ratios in brewing wastewater into
Dry cell weight and grease yield figure after row fermentation, (a) real diagram are dry cell weight, and dashed line view is the primary miscellaneous bacteria dry weight of white,
(b) grease yield.Referring to Fig. 10, useless in wine brewing under different ventilation ratios for Rhodosporidium toruloides in the embodiment of the present invention 4
Every water quality parameter variation diagram of brewing wastewater after being fermented in water, COD COD (a), total phosphorus (b), total nitrogen
(c), ammonia nitrogen (d), pH (e) and dissolved oxygen (f).
Fig. 9 shows that grease yield reaches maximum value in 12h.When ventilation ratio is 4 and 3.5vvm, grease yield and thallus
Gross weight difference is not significant (p > 0.05).But when 12h, the average value of grease yield when ventilation ratio is 4vvm is than ventilation ratio
The sample of 3.5vvm is big, and standard error is smaller；When ventilation ratio is 4vvm, grease yield is larger and more stable, preferably ventilates
Than for 4vvm.When ventilation ratio is 4vvm, Rhodosporidium toruloides can grow into stationary phase in 12h, and grease yield is maximum, and
Rhodosporidium toruloides accounting is big under this condition, and the primary miscellaneous bacteria of white just occurs in 48h, and accounting very little, grease yield are reachable
1.92 ± 0.24g/L, lipid-producing is up to 160mg/ (Lh).
Figure 10 show ventilation ratio be 4 and 3.5vvm when, can be completed in preceding 12h major part COD, total nitrogen,
The removal of total phosphorus and ammonia nitrogen, every water quality parameter variation is unobvious after 12h and ammonia nitrogen concentration can become larger, and the reaction time can shorten
To 12h.In terms of COD, total nitrogen, total phosphorus and ammonia nitrogen removal, ventilation ratio is that the experimental group of 4 and 3.5vvm is not significant
Difference (p > 0.05), but in 12h, ventilation ratio be 4 and the experimental group of 3.5vvm to be significantly higher than ventilation ratio be 3 and 4.5vvm
Experimental group.Because when cultivating 12h, grease yield average value is bigger when ventilation ratio is 4vvm and standard error is smaller, more stable,
It is preferred that ventilation ratio is 4vvm.(the initial dissolution oxygen concentration corresponding with 4.5vvm of ventilation ratio 3,3.5,4 respectively is 2%~
60%, 40%~60%, 50%~70% and 83%~103% air saturation).Figure 10 shows the COD, total of brewing wastewater
Phosphorus, total nitrogen and ammonia nitrogen removal frank are respectively successively up to 71.82 ± 0.52%, 79.53 ± 1.77%, 54.39 ± 6.45% Hes
83.42 ± 3.07%.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of cultural method of oleaginous yeast, which comprises the following steps:
Oleaginous yeast is seeded in the culture solution that pH value is 3.3~4 and is cultivated, wherein the oleaginous yeast is in institute
Stating the initial concentration in culture solution is (1~2) × 108cells/mL。
2. a kind of microbial oil production method, which comprises the following steps:
A) oleaginous yeast is seeded in the culture solution that pH value is 3.3~4 and is fermented, obtain tunning, wherein described
Initial concentration of the oleaginous yeast in the culture solution is (1~2) × 108cells/mL；
B) tunning is collected, then carries out grease extraction, obtain microbial oil.
3. microbial oil production method according to claim 2, which is characterized in that the temperature of the step a) fermentation is
20 DEG C~35 DEG C；
The time of the step a) fermentation is 12h~for 24 hours；
The ventilation ratio of the step a) fermentation is 3vvm~4.5vvm；
The dissolved oxygen concentration of the step a) fermentation is 30%~100% air saturation.
4. microbial oil production method according to claim 2, which is characterized in that the culture solution is brewing wastewater；
The COD of the brewing wastewater is 17,120mg/L~65,075mg/L.
5. microbial oil production method according to claim 2, which is characterized in that the oleaginous yeast is the circle red winter
Spore yeast.
6. microbial oil production method according to claim 2, which is characterized in that the preparation side of the oleaginous yeast
Method are as follows:
Oleaginous yeast strain is seeded in the expansion culture medium that pH value is 4~5, expands culture, obtain the oil-producing ferment
Female bacterium.
7. microbial oil production method according to claim 6, which is characterized in that the temperature for expanding culture is 20
DEG C~35 DEG C；
The time for expanding culture is 8h~for 24 hours；
The ventilation ratio for expanding culture is 3vvm~4.5vvm；
The dissolved oxygen concentration for expanding culture is 40%~100% air saturation.
8. microbial oil production method according to claim 2, which is characterized in that the concentration of the oleaginous yeast is
(4~10) × 109cells/mL。
9. the microbial oil that microbial oil production method produces described in claim 2 to claim 8 any one.
10. application of the microbial oil described in claim 9 in preparation biodiesel.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910362911.1A CN110257265A (en) | 2019-04-30 | 2019-04-30 | A kind of cultural method of oleaginous yeast, microbial oil production method and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910362911.1A CN110257265A (en) | 2019-04-30 | 2019-04-30 | A kind of cultural method of oleaginous yeast, microbial oil production method and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110257265A true CN110257265A (en) | 2019-09-20 |
Family
ID=67914053
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910362911.1A Pending CN110257265A (en) | 2019-04-30 | 2019-04-30 | A kind of cultural method of oleaginous yeast, microbial oil production method and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110257265A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101108997A (en) * | 2006-07-19 | 2008-01-23 | 中国科学院大连化学物理研究所 | Process for preparing microbe oil |
| CN102080119A (en) * | 2009-11-26 | 2011-06-01 | 北京化工大学 | Method for producing oil by mixed culture of yeast and alga |
-
2019
- 2019-04-30 CN CN201910362911.1A patent/CN110257265A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101108997A (en) * | 2006-07-19 | 2008-01-23 | 中国科学院大连化学物理研究所 | Process for preparing microbe oil |
| CN102080119A (en) * | 2009-11-26 | 2011-06-01 | 北京化工大学 | Method for producing oil by mixed culture of yeast and alga |
Non-Patent Citations (4)
| Title |
|---|
| JIAYIN LING ET AL.: "Enhancement of lipid productivity of Rhodosporidium toruloides in distillery wastewater by increasing cell density", 《BIORESOURCE TECHNOLOGY》 * |
| 林金涛 等: "圆红冬孢酵母两阶段培养法生产微生物油脂", 《生物工程学报》 * |
| 沈宏伟 等: "恒化培养稀释率和碳氮比对圆红冬孢酵母油脂积累的影响", 《生物工程学报》 * |
| 王琦: "圆红冬孢酵母（Rhodosporidium toruloides）21167菌株产油脂的研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101705190B (en) | Chlorella sorokiniana CS-01 and culture method thereof for producing grease | |
| CN102037849B (en) | Tremella fuciformis strain culture method | |
| CN106467896B (en) | A kind of kelvin being resistant to high PH intends chlorella and its culture application | |
| CN103598010A (en) | Original ecological imitative wild cultivation method for inonotus sanghuang | |
| CN103484396B (en) | New strain of streptomyces thermocarboxydus and application thereof | |
| CN104611227B (en) | Scenedesmus obliquus with tolerance to high pH and breeding method thereof | |
| CN1232632C (en) | New strain APC-20 of Paecilomyces cicadae and fementation process for artificial culture | |
| CN106801017A (en) | The cordyceps militaris link bacterial strain screening of a kind of high yield thermophilic protease and cordycepin and method of mutagenesis | |
| CN107903092A (en) | A kind of method and application using mushroom bran discarded object production bio-feritlizer synergist | |
| CN105948831B (en) | A method of biological and ecological methods to prevent plant disease, pests, and erosion fertilizer is produced using brewex's grains | |
| CN107460216A (en) | A kind of method that microalgae grease is produced using flue gas | |
| CN105494715A (en) | Process for producing dark tea through inoculation method | |
| CN105648023A (en) | Method used for preparing grease via microalgae mixed cultivation | |
| CN103704069A (en) | Method of three-dimensionally preventing and controlling tomato bacterial wilt | |
| CN109479622A (en) | A kind of tea tree mushroom strains industrial production method | |
| CN107460215A (en) | A kind of method of microalgae mixed culture production grease | |
| CN106520630B (en) | A kind of bacillus megaterium QBJP-F6 and its application | |
| CN103571756A (en) | Test tube screening method of isaria cicadae strain and culture medium | |
| CN104651267A (en) | Microorganism strain having fermenting alkali producing function and application of microorganism strain in organic fertilizer | |
| CN105713934B (en) | A method of producing microalgae grease | |
| CN109182151A (en) | The separating screening method of gingko endogenous fungus | |
| CN103981103A (en) | DSE (Dark Septate Endophyte) strain J-N3 and applications thereof in dendrobium candidum production | |
| CN110229757A (en) | One plant effectively facilitates the tangerine green trichoderma JS84 of plant growth and its biological organic fertilizer of development | |
| CN107460217A (en) | A kind of method that mixed culture prepares microalgae grease | |
| CN108148768B (en) | Tortomium globosum strain and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190920 |