CN108374026A - Utilize the method for saccharomycetes to make fermentation Synthetic Oil - Google Patents
Utilize the method for saccharomycetes to make fermentation Synthetic Oil Download PDFInfo
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- CN108374026A CN108374026A CN201810050458.6A CN201810050458A CN108374026A CN 108374026 A CN108374026 A CN 108374026A CN 201810050458 A CN201810050458 A CN 201810050458A CN 108374026 A CN108374026 A CN 108374026A
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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
- C12P7/6409—Fatty acids
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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
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
The present invention provides a kind of methods using saccharomycetes to make fermentation Synthetic Oil comprising following steps：The seed liquor of oleaginous yeast is subjected to fermented and cultured in limit phosphorus culture medium, until exponential phase latter stage obtains zymotic fluid；Separation and fermentation liquid collects sediment and therefrom extracts grease.The saccharomycete of the present invention carries out fermented and cultured in limit phosphorus culture medium, compared with saccharomycete carries out fermented and cultured in unlimited phosphorus culture medium, it realizes the optimization of grease composition, so that the content of monounsaturated fatty acids is improved, the content of polyunsaturated fatty acid is reduced, to achieve the purpose that the performance after enhancement microbiological grease is converted to biodiesel；In addition, its fat content is improved, promote the significant effect of oil synthesis.Therefore, the present invention for solve the problems, such as the biomass fermentations such as sludge zymotic fluid in practical applications due to contain a large amount of ammonia nitrogens inhibit oleaginous yeast accumulate grease provide a new approaches.
Description
Technical field
The invention belongs to microbial grease synthesis technical fields, and in particular to a kind of using saccharomycetes to make fermentation Synthetic Oil
Method.
Background technology
As the mankind are to the growing day by day of demand for fuel, the conventional fossil fuel from energy such as oil, natural gases exists
The problem of environmental pollution, non-renewable etc., is increasingly prominent.For response environment problem and energy crisis, various novel energies
Exploitation be emphasis of people's attention all the time.Biodiesel refers to open countries such as oil crops such as soybean, rape, cotton, palm
The water plants such as oil generation tannin plant and engineering microalgae grease and animal fat, microbial grease, food garbage oil etc. are raw material
Oil recyclability diesel fuel made of ester exchange process is a kind of long-chain that the technologies such as biomass economy thermal cracking obtain
The monoalkyl ester of aliphatic acid, element composition is simple, fuel is pollution-free.Wherein had by microbial grease preparation biodiesel and is not accounted for
With arable land, not by season environmental restrictions, it is with short production cycle, convenient for collect and management, production efficiency is high the advantages that, it is considered to be most
The biodiesel raw material of tool foreground.
Oleaginous microorganism refers to by certain culture, and microbial cell fat content can be more than the micro- of the 20% of dry cell weight
Biology.The main Types of oleaginous microorganism include microalgae, bacterium, saccharomycete and fungi, wherein saccharomycete has growth fast
The advantages that speed, fat content are high, substrate utilization scope is wide is interested to researchers extensively, the microbial oil generated by saccharomycete
Fat has 90% or more can be converted into biodiesel, however its culture medium raw material cost it is higher be current Limit exploitation it is main because
Element.At present in the developmental research of oleaginous yeast cheap carbon source, being generated by organic waste anaerobic fermentations such as sludge, kitchen garbages
Volatile fatty acid (Volatile fatty acids, VFAs) synthesized micro-organism grease, on the one hand realize debirs to
On the other hand the conversion of high value added product reduces microbial grease production cost and promotes its popularization and application.
Oleaginous microorganism should be in the base containing excessive carbon source and one or several specific limiting factors (being usually nitrogen)
It is cultivated in matter, carries out oil and fat accumulation, however since protein hydrolysis discharges a large amount of ammonia nitrogen limits in bio-waste fermentation and acid liquid
Its practical application is made.
Invention content
The present invention is directed to deficiency in the prior art, it is therefore an objective to provide a kind of side using saccharomycetes to make fermentation Synthetic Oil
Method.
In order to achieve the above objectives, solution of the invention is：
A method of utilizing saccharomycetes to make fermentation Synthetic Oil comprising following steps：
(1), the seed liquor of oleaginous yeast is subjected to fermented and cultured in limit phosphorus culture medium, until exponential phase latter stage obtains
To zymotic fluid；
(2), separation and fermentation liquid collects sediment and therefrom extracts grease.
Preferably, oleaginous yeast is bending Cryptococcus.
Wherein, limit phosphorus culture medium includes：16.6 ± 0.1g/L potassium acetates, 0.2 ± 0.1g/L yeast extracts, 3.0176 ±
0.0002g/L ammonium chlorides, 1.5 ± 0.2g/L magnesium chloride hexahydrates and 1 ± 0.3g/L sodium sulphate.
It limits in phosphorus culture medium：Initial pH value is 5.5-6.5；Carbon-phosphorus ratio is (1400-1500):1, carbon-nitrogen ratio is (4.5-
5.5):1。
Preferably, it is 5.9-6.1 to limit initial pH value in phosphorus culture medium.
In fact, the mass concentration for limiting acetic acid in the potassium acetate in phosphorus culture medium is 10g/L；Limit the phosphorus member in phosphorus culture medium
Usually from yeast extract, content is 1.372% (i.e. the mass concentration of P elements is 1.372% (g/g))；Nitrogen comes from
Ammonium chloride and yeast extract, the content in yeast extract are that 11.7% (i.e. the mass concentration of nitrogen is 11.7%
(g/g))。
Preferably, the fermented and cultured in step (1) includes：The seed liquor of oleaginous yeast is inoculated with volume ratio 9-11%
Fermented and cultured is carried out to limiting in phosphorus culture medium.
Preferably, in step (1), the time of fermented and cultured is 46-50h, and the temperature of fermented and cultured is 29-31 DEG C；Shaking table
Rotating speed be 190-210rpm.
Preferably, the generation process of the seed liquor of the oleaginous yeast in step (1) includes the following steps：
(1-1), the oleaginous yeast being stored in inclined-plane pipe is seeded on tablet and is cultivated, obtain oleaginous yeast
Single bacterium colony；
(1-2), the single bacterium colony of oleaginous yeast is cultivated in the first culture medium, until exponential phase latter stage obtains
The seed liquor of oleaginous yeast.
Preferably, in step (1-1), inclined-plane pipe is wort agar inclined-plane pipe；Tablet is wort agar tablet.
Preferably, in step (1-2), the first culture medium is yeast extract powder peptone dextrose culture-medium (i.e. YPD Liquid Cultures
Base), including：20 ± 5g/L glucose, 10 ± 2g/L fish meal proteins peptone and 10 ± 2g/L yeast extracts；Initial pH value is 5.9-
6.1。
Preferably, in step (1-2), in the single bacterium colony incubation of oleaginous yeast, the time of culture is 34-38h, training
Foster temperature is 29-31 DEG C；The rotating speed of shaking table is 190-210rpm.
Preferably, in step (2), the process of extraction is：Sediment is cleaned with physiological saline；In broken sediment
Yeast cell, in conjunction with organic solvent extract to extract grease.
Wherein, organic solvent is methanol, chloroform with volume ratio 1:0.9‐1:1.1 mixed liquor.
In fact, specific extraction process is：Zymotic fluid is centrifuged into 5min in the case where centrifugal force is 4000 × g, discards supernatant liquid,
The thalline of saccharomycete in lower sediment thing is primary with physiological saline eccentric cleaning, and the thalline of obtained saccharomycete is cold to be done to perseverance
Weight adds the bead of 0.5mm diameters to grind 5-10s with the rotating speed of 3500rpm, is extracted with organic solvent (mixed liquor of methanol chloroform)
It takes twice, the lower layer's solution extracted twice is extracted and merged after centrifugation, then primary with 0.15wt% sodium chloride solution eccentric cleanings,
And lower layer's solution after cleaning is extracted, blowing method volatile organic solvent using nitrogen at 50 ± 5 DEG C obtains grease, adds after weighing weight
Enter 1mL chloroforms to re-dissolve, be stored in -20 DEG C of (or 4 DEG C) environment.
Then the measurement of aliphatic acid composition is carried out：Grease chloroformic solution is blown into 20-30min using nitrogen evaporator nitrogen, by solvent
0.5mol/L sodium hydrate methanol solutions are added after chloroform drying and carry out saponification, it is molten to add 0.7mol/L hydrochloric acid (HCl) methanol
Liquid and 14wt% boron trifluorides (BF3) methanol solution progress esterification, saturated nacl aqueous solution then is added and chromatographic grade is different pungent
Alkane centrifuges 5min after fully shaking mixing under the rotating speed of 3600 × g, and upper layer isooctane is taken mutually GC-FID 6890N to be used to measure
Aliphatic acid forms.
Wherein, in step (2), grease includes：Palmitic acid, stearic acid, oleic acid and linoleic acid.
By adopting the above scheme, the beneficial effects of the invention are as follows：
The first, saccharomycete of the invention limit phosphorus culture medium (take P elements limit and combines initial pH value regulation and control) it is interior into
Row fermented and cultured realizes the optimization of grease composition, makes compared with saccharomycete carries out fermented and cultured in unlimited phosphorus culture medium
The content of monounsaturated fatty acids (such as oleic acid) is improved, polyunsaturated fatty acid (such as linoleic acid, octatecatrienoic acid and
Eicosapentaenoic acid) content be reduced, to reach the usability after enhancement microbiological grease is converted to biodiesel
The purpose of energy.
The second, saccharomycete of the invention limit phosphorus culture medium (take P elements limit and combines initial pH value regulation and control) it is interior into
Row fermented and cultured, compared with saccharomycete carries out fermented and cultured in unlimited phosphorus culture medium, fat content is improved, to promote
Into the synthesis of grease.
Third, the present invention saccharomycete limit phosphorus culture medium (P elements is taken limit and combines initial pH value regulation and control) it is interior into
Row fermented and cultured applies in general to the high (NH containing 1400-1500mg in every liter of solution of ammonia-nitrogen content4 +- N) sludge fermentation production
Acid solution provides the new approaches for meeting practical application for the recycling of sludge, and can reduce sludge production acid solution
Pretreated cost, while can also realize the recycling of phosphor resource, mitigate water body eutrophication degree and solve sludge treatment
Secondary pollution problem.
In short, the saccharomycete of the present invention limit phosphorus culture medium (P elements is taken limit and combines initial pH value regulation and control) it is interior into
The method of row fermented and cultured Synthetic Oil, not only optimizes the composition of grease, and also achieve volatile fatty acid acetic acid to
The Efficient Conversion of microbial grease presses down due to containing a large amount of ammonia nitrogens in practical applications to solve the biomass fermentations zymotic fluids such as sludge
The problem of oleaginous yeast accumulation grease processed, provides a new approaches.
Description of the drawings
Fig. 1 is growing state and fat content schematic diagram of the saccharomycete of the present invention in embodiment and comparative example.
Fig. 2 is that of the invention utilizing limits the reaction that P elements are restricted in phosphorus culture medium in saccharomycetes to make fermentation Synthetic Oil
Principle schematic.
Specific implementation mode
The present invention provides a kind of methods using saccharomycetes to make fermentation Synthetic Oil.
Saccharomycete in the embodiment of the present invention and comparative example is bending Cryptococcus (Cryptococcus
Curvatus MUCL 29819), it buys in Japanese national technology and evaluation studies institute Biological Resource Center (Biological
Resource Center,National Institute of Technology and Evaluation,Tokyo,Japan)。
<Utilize the method for saccharomycetes to make fermentation Synthetic Oil>
A kind of method using saccharomycetes to make fermentation Synthetic Oil includes the following steps：
(1), the oleaginous yeast that will be inoculated in inclined-plane pipe (wort agar inclined-plane pipe) is cultivated 2-3 days at 30 DEG C,
Then it is preserved at 4 DEG C, carries out within 2-3 months primary switching passage；Then by its streak inoculation to wort agar tablet,
Activation 3-5 days in 30 DEG C of incubator, up to growing single bacterium colony to get to the single bacterium colony of oleaginous yeast；
(2), picking one is expired the single bacterium colony of the oleaginous yeast of ring and is seeded to containing 50mL the first culture medium (YPD Liquid Cultures
Base) 250mL conical flasks in cultivated, until exponential phase latter stage obtains the seed liquor of oleaginous yeast；
(3), the seed liquor of treated oleaginous yeast is seeded to the limit phosphorus culture medium containing 45mL (takes P elements to limit
And combine initial pH value regulate and control) 250mL conical flasks in carry out fermented and cultured, until exponential phase latter stage obtain zymotic fluid；
(4), separation and fermentation liquid collects sediment (i.e. third sediment) and therefrom extracts grease.
Wherein, in step (1), the preparation process of wort agar inclined-plane pipe is：Weigh the training of 145.1g wort agars
It supports base and is dissolved in 100mL distilled water, agitating and heating is boiled to being completely dissolved, and test tube, the high pressure sterilization at 115 DEG C are dispensed
20min obtains strain tube, and the saccharomycete in strain tube is seeded in inclined-plane pipe after cooled and solidified and is placed in incubator 3d progress
Dry and steriling test.
In step (1), the preparation process of wort agar tablet is：Weigh 14.51g wort agars culture medium and molten
In 100mL distilled water, agitating and heating is boiled to being completely dissolved, and the high pressure sterilization 20min at 115 DEG C is sub-packed in flat at room temperature
It is placed on plate in superclean bench and carries out cooled and solidified, obtain wort agar tablet, then set wort agar tablet
It is dried and steriling test in incubator 3d.
In step (2), YPD fluid nutrient mediums include following ingredient：Glucose, fish meal protein peptone and yeast extract.
The preparation process of YPD fluid nutrient mediums is：It weighs 10g fish meal proteins peptone and 10g yeast extracts is dissolved in 900mL
In distilled water, it is 6.0 to be used in combination 4mol/L hydrochloric acid (HCl) to adjust its pH value, obtains the first solution A 1；Weigh the dissolving of 20g glucose
The second solution B 1 is obtained in 100mL distilled water.The first solution As of packing 45mL 1, mix 5mL second in each shaking flask after sterilizing
Solution B 1 can be obtained YPD fluid nutrient mediums.
In step (2), in the single bacterium colony incubation of oleaginous yeast：The time of culture can be 34-38h, preferably
For 36h；The temperature of culture can be 29-31 DEG C, preferably 30 DEG C；The rotating speed of shaking table can be 190-210rpm, preferably
200rpm。
In step (2), the last handling process of the seed liquor of oleaginous yeast is：The seed liquor of oleaginous yeast is being turned
After centrifuging 5min under fast 3000rpm, liquid is discarded supernatant, obtains the first sediment, and add 50mL physiology in the first sediment
Brine is stirred to the bacterium colony in the seed liquor of oleaginous yeast with glass bar and is uniformly cleaned, and 5min is centrifuged at rotating speed 3000rpm
Afterwards, liquid is discarded supernatant, the second sediment is obtained, and adds 50mL distilled water in the second sediment and obtains clean oleaginous yeast
The seed liquor of bacterium, it is this to wash the inoculation of bacterium inoculation method progress, it is therefore an objective to nutriment in YPD fluid nutrient mediums can be excluded
Influence.
In step (3), the seed liquor of treated oleaginous yeast is seeded to limit phosphorus culture with volume ratio 9-11%
Fermented and cultured is carried out in base, volume ratio is preferably 10%.
Wherein, the volume ratio of the seed liquor of oleaginous yeast is the seed of oleaginous yeast during fermented and cultured
Liquid volume shared in limiting phosphorus culture medium.
In step (3), limit phosphorus culture medium includes following ingredient：Potassium acetate (CH3COOK), yeast extract (YE), chlorine
Change ammonium (NH4Cl), magnesium chloride hexahydrate (MgCl2·6H2) and sodium sulphate (Na O2SO4)。
In fact, the preparation process of limit phosphorus culture medium is：Weigh 16.6gCH3COOK、0.2gYE、3.0176gNH4Cl、
1.5gMgCl2·6H2O and 1gNa2SO4Be dissolved in 1000mL distilled water, be used in combination 4mol/L hydrochloric acid (HCl) adjust its pH value to
5.5-6.5, pH value are preferably 5.9-6.1, and more preferably 6.0, the high pressure sterilization 20min at 115 DEG C is placed in room temperature asepsis ring
Carry out cooling in border in case follow-up use, wherein carbon-phosphorus ratio (C/P) can be (1400-1500):1, preferably 1482:1；Carbon
Nitrogen ratio (C/N) can be 4.5-5.5:1, preferably 5:1.
The principle of fermented and cultured in limit phosphorus culture medium is：As shown in Fig. 2, when P elements are restricted in culture medium,
Cell adenylate (Adenosine Monophosphate, AMP) will decompose, and discharge inorganic phosphorous sources for other intramicellar reactions, i.e.,
The inorganic phosphorous sources of release have been transferred to from cell adenylate (AMP) in other substances by biochemical reaction, further by other objects
The cell of matter is utilized, so that the content of cell adenylate (AMP) significantly reduces, and cell adenylate (AMP) is as different
The activator of citric dehydrogenase (Isocitrate Dehydrogenase, IDH), due to the content of cell adenylate (AMP)
It reduces, therefore, the activity of isocitric dehydrogenase (IDH) can be caused to reduce, so that the content of isocitric acid increases, lemon
The content of acid also increases, and citric acid passes through mitochondrial membrane to enter in cytoplasmic matrix the accumulation for participating in grease.
Take the limitations of P elements with removing in sludge fermentation liquid compared with ammonia nitrogen with two in the limit phosphorus culture medium of the present invention
A advantage：First, since P elements have the characteristics that one-way flow in sewage disposal process, cannot directly be decomposed consumption,
Most phosphorus can only be stored in essential elements in organism, finally be discharged in the form of excess sludge, by sludge fermentation
P elements in liquid are recycled by ad hoc approach, can be mitigated the secondary pollution problem of sludge treatment while be realized holding for phosphorus
It is continuous to utilize.Second, the phosphorus recovery technology of many new high-efficiencies has been developed at present, and such as ion-exchange is adsorbed analytic method and received
Filter method etc. so that phosphorus recycling can reach relatively higher organic efficiency and recycling efficiency.In summary, acid is produced to sludge fermentation
Liquid is used to cultivate oleaginous yeast progress oil-producing after carrying out phosphorus recycling, can be realized simultaneously phosphor resource and carbon energy, and
With environmental benefit and economic benefit.
In step (3), the seed liquor of oleaginous yeast is when limiting progress fermented and cultured in phosphorus culture medium：The time of culture
Can be 46-50h, preferably 48h；The temperature of culture can be 29-31 DEG C, preferably 30 DEG C；The rotating speed of shaking table can be
190-210rpm, preferably 200rpm.
In step (4), zymotic fluid is centrifuged into 5min at 4000 × g of centrifugal force, discards supernatant liquid, obtains third precipitation
Object.The third sediment sodium chloride solution of 0.9wt% is mixed, eccentric cleaning 1 time, freeze-drying is contained afterwards for 24 hours
The thalline of the saccharomycete of grease.
Grease is extracted from the thalline of the saccharomycete containing grease combines organic solvent to extract using mechanical lapping is broken
The method being combined：1. preparing homogeneous tubule：Be added into 2mL homogeneous tubules the bead of a diameter of 0.5mm to about with lower end
Line is flat；2. in the thalline to homogeneous tubule (2mL) for weighing the grease-contained saccharomycete of about 0.1g, recording weight；3. being added thereto
1mL methanol, after simply shaking up at 4 DEG C overnight；4. with homogenizer homogeneous 10s, rotating speed is selected most high-grade；5. will with 4mL chloroforms
Sample in homogeneous tubule is carefully transferred in the first centrifuge tubes of 100mL and (rinses homogeneous tubule 4 times)；6. into the first centrifuge tube
10mL, 4mol/L hydrochloric acid (HCl) solution and 20mL methanol chloroform solution is added, with the rotating speed of 2500rpm on multitube oscillator
Shake 30min；7. sample removes leveling (the 4mol/L hydrochloric acid for using water phase), 10min is centrifuged with the rotating speed of 5500rpm；8. using band
Needle injection takes out in the first lower liquid to the second centrifuge tube, and records volume (about 10-14mL)；Add again in first centrifuge tube
Enter 20mL chloroform methanol solution, 10min is mixed in multitube oscillator, 10min is centrifuged with the rotating speed of 5500rpm, is taken under second
Layer liquid (about 5mL) merges with the first lower liquid；9. being added in equal volume (about in the second lower liquid into the second centrifuge tube
0.15wt% sodium chloride solutions 5mL), after mixing 1min, leveling (uses sodium chloride solution), is centrifuged with the rotating speed of 5500rpm
10min；10. carefully taking out in lower layer's organic phase to pre-weighed ml headspace bottle, nitrogen is born at 45 DEG C and blows evaporation 15-20min, cooling
It weighs again after to room temperature, difference is to extract grease obtained quality.Extract it is grease obtained re-dissolved with 1mL chloroforms after,
Grease chloroformic solution is obtained, is stored in -20 DEG C of (or 4 DEG C) environment.
Aliphatic acid composition measuring：Grease chloroformic solution is blown into 20-30min using nitrogen evaporator nitrogen, solvent chloroform is dried up, is added
Enter 0.5mol/L sodium hydrate methanol solution 8mL, imports tens of seconds drying nitrogens into ml headspace bottle rapidly and exhaust air, tighten
Lid, in shaking 5min on multitube oscillator, then the water-bath 30min in 80 DEG C of thermostatic water baths, during which every up to oil droplet disappearance
30-60s slowly shakes ml headspace bottle, is attached in bottle wall with preventing sodium hydroxide from forming solid-state, and water-bath finishes, and room temperature water-bath will push up
Empty bottle is cooled to room temperature.Add 8mL, 0.7mol/L hydrochloric acid (HCl) methanol solution and 8mL, 14wt% boron trifluoride (BF3) first
Alcoholic solution imports rapidly tens of seconds drying nitrogens into ml headspace bottle and exhausts air, tightens lid, shaken on multitube oscillator
5min, then the water-bath 30min in 80 DEG C of thermostatic water baths, is cooled to room temperature.2mL saturated nacl aqueous solutions and 10mL chromatographies is added
Grade isooctane, be placed on multitube oscillator shake 10min after, hold ml headspace bottle up and down rock so that mixed liquor is mixed well after from
Mental and physical efforts are to centrifuge 15min under 3600 × g, take upper layer isooctane that GC-FID 6890N is mutually used to measure aliphatic acid composition.In fact,
Chromatographic column：TR-FAME quartz capillary chromatographic columns (column length 60m, internal diameter 0.25mm, film thickness 0.25mm), detector is
Hydrogen flame ionization detector (FID).Injection port：Shunt mode, carrier gas are nitrogen, split ratio 20:1, bypass flow is
30.0mL/min, heater settings are 250 DEG C, pressure value 4561.7MPa, and total flow value is 33.7mL/min.Injector：
1. 2.0 μ L of sample size；2. it is isooctane to wash needle liquid solvent, respectively cleaned 5 times before and after sample introduction, it is 8 that needle core number is pushed and pulled before sample introduction.Column
Incubator temperature program：Initial temperature is 120 DEG C, retention time 0min；First stage is warming up to 140 DEG C with 4 DEG C/min, keeps
Time is 5min；Second stage is warming up to 175 DEG C with 5 DEG C/min, retention time 0min；Phase III is heated up with 1 DEG C/min
To 178 DEG C, retention time 3min；Fourth stage is warming up to 216 DEG C with 4 DEG C/min, retention time 3min；5th stage with
1 DEG C/min is warming up to 220 DEG C, retention time 0min；6th stage was warming up to 240 DEG C with 4 DEG C/min, and the retention time is
3min, operation total time are 47.50min；Afterwards running temperature be 50 DEG C, retention time 0min, operation total time be
47.50min.Detector：Fid detector, heter temperature setting value are 260 DEG C；Compensation settings of lighting a fire are 2.0；Hydrogen stream
Amount setting value is 40.0mL/min；Air mass flow setting value is 450mL/min；Make-up gas is nitrogen, flow setting value 45mL/
min。
In fact, organic solvent can be methanol, chloroform is with volume ratio 1:0.9‐1:1.1 mixed liquor, preferably methanol,
Chloroform is with volume ratio 1:1 mixed liquor.
Wherein, in step (4), grease may include：Palmitic acid, stearic acid, oleic acid and linoleic acid.
The present invention will be further described with reference to the accompanying drawings.
Embodiment 1：
The method using saccharomycetes to make fermentation Synthetic Oil of the present embodiment includes the following steps：
(1), the oleaginous yeast in wort agar inclined-plane pipe will be inoculated in cultivate 3 days at 30 DEG C, then at 4 DEG C
It preserves, carries out within 2 months primary switching passage；Then by its streak inoculation to wort agar tablet, in 30 DEG C of incubators
Activation 3 days, until growing single bacterium colony to get to the single bacterium colony of oleaginous yeast；
(2), the single bacterium colony of the oleaginous yeast of a full ring is taken to be seeded to the 250mL cones of the YPD fluid nutrient mediums containing 50mL
It is cultivated in shape bottle, is 30 DEG C in temperature, enrichment culture 36h, i.e. exponential phase latter stage in the shaking table that rotating speed is 200rpm
Obtain the seed liquor of oleaginous yeast；
(3), by the seed liquor of treated oleaginous yeast be seeded to containing 45mL limit phosphorus culture medium (initial pH value 6.0,
C/P is 1482:1, C/N 5:1) in 250mL conical flasks, it is 30 DEG C in temperature, training is enriched in the shaking table that rotating speed is 200rpm
48h is supported, i.e. exponential phase latter stage obtains zymotic fluid；
(4), separation and fermentation liquid discards supernatant liquid and collects sediment；
(5), grease is extracted from sediment, and carries out aliphatic acid composition measuring, and test result is as shown in table 1.
Wherein, in step (2), in the single bacterium colony incubation of oleaginous yeast：The time of culture within 34-38h,
The temperature of culture is within 29-31 DEG C, the rotating speed of shaking table is all possible within 190-210rpm.
In step (3), limit in phosphorus culture medium：Initial pH value within 5.5-6.5, carbon-phosphorus ratio (C/P) is in (1400-
1500):Within 1, carbon-nitrogen ratio (C/N) is at (4.5-5.5):It is all possible within 1.
In step (3), the seed liquor of oleaginous yeast is when limiting progress fermented and cultured in phosphorus culture medium：The time of culture
Within 46-50h, the temperature of culture within 29-31 DEG C, the rotating speed of shaking table is all possible within 190-210rpm.
Comparative example 1：
The method using saccharomycetes to make fermentation Synthetic Oil of this comparative example includes the following steps：
(1), the oleaginous yeast in wort agar inclined-plane pipe will be inoculated in cultivate 3 days at 30 DEG C, then at 4 DEG C
It preserves, carries out within 2 months primary switching passage；Then by its streak inoculation to wort agar tablet, in 30 DEG C of incubators
Activation 3 days, until growing single bacterium colony to get to the single bacterium colony of oleaginous yeast；
(2), the single bacterium colony of the oleaginous yeast of a full ring is taken to be seeded to the 250mL cones of the YPD fluid nutrient mediums containing 50mL
It is cultivated in shape bottle, is 30 DEG C in temperature, enrichment culture 36h, i.e. exponential phase latter stage in the shaking table that rotating speed is 200rpm
Obtain the seed liquor of oleaginous yeast；
(3), the seed liquor of treated oleaginous yeast is seeded to containing the unlimited phosphorus culture medium (C/P 18 of 45mL:1, C/
N is 5:1) it is 30 DEG C in temperature, enrichment culture 48h in the shaking table that rotating speed is 200rpm, i.e. logarithm give birth in 250mL conical flasks
Long-term latter stage obtains zymotic fluid；
(4), separation and fermentation liquid discards supernatant liquid and collects sediment；
(5), grease is extracted from sediment, and carries out aliphatic acid composition measuring, and test result is as shown in table 1.
Wherein, the preparation process of unlimited phosphorus culture medium is：Weigh 16.6gCH3COOK、0.2gYE、3.0176gNH4Cl、
1.5gMgCl2·6H2O、1gNa2SO4And 1gKH2PO4It is dissolved in 1000mL distilled water, 4mol/L hydrochloric acid (HCl) is used in combination to adjust
Its pH value is 6.0, the high pressure sterilization 20min at 115 DEG C.
In fact, P elements come from potassium dihydrogen phosphate and yeast extract in unlimited phosphorus culture medium, nitrogen comes from chlorination
Ammonium and yeast extract.
Aliphatic acid forms situation in the grease of 1 embodiment 1 of table and comparative example 1
As shown in Table 1, with palmitic acid (C16 in grease:0), stearic acid (C18:0), oleic acid (C18:And linoleic acid 1)
(C18:2) four kinds of aliphatic acid are main component, account for 90% or more of total ingredient, wherein oleic acid (C18:1) content highest, with plant
The aliphatic acid composition of grease is suitable, is the quality raw materials of biodiesel synthesis.Therefore, single in embodiment 1 compared with comparative example 1
Unsaturated fatty acid content increases 8.47%, and content of polyunsaturated fatty acid reduces 4.51%, so that the group of grease
At being optimized.Monounsaturated fatty acids is considered as the best fat for keeping resistance to low temperature and combustibility to reach balance
Acid, wherein oleic acid (C18:1) content higher can make the low temperature flow of grease more preferable.
Wherein, saturated fatty acid is not double bond containing aliphatic acid, and monounsaturated fatty acids is the fat containing 1 double bond
Acid, polyunsaturated fatty acid are containing 2 or 2 or more double bonds and straight chain fatty acid that carbon chain lengths are 18-22 carbon atom.
In fact, saturated fatty acid includes：Stearic acid (C18:And palmitic acid (C16 0):0)；Monounsaturated fatty acids packet
It includes：Oleic acid (C18:1)；Polyunsaturated fatty acid includes：Linoleic acid (C18:2), octatecatrienoic acid (C18:And 20 light dydrocarbons 3)
Olefin(e) acid (C20:5)
As shown in Figure 1, in embodiment 1：Biomass is 3.03g/L, grease yield 0.92g/L, and fat content is
30.35%；In comparative example 1：Biomass is 3.5g/L, grease yield 0.64g/L, fat content 18.29%, therefore, with
Comparative example 1 is compared, and fat content increases 12.06% in embodiment 1, increases 1.7 times, and grease yield increases 0.28g/L, increases
1.4 times, so that the synthetic effect of grease is notable.
In short, the saccharomycete of the present invention limit phosphorus culture medium (P elements is taken limit and combines initial pH value regulation and control) it is interior into
The method of row fermented and cultured Synthetic Oil not only optimizes compared with saccharomycete carries out fermented and cultured in unlimited phosphorus culture medium
The composition of grease, and Efficient Conversion of the volatile fatty acid acetic acid to microbial grease is also achieved, it is raw to solve sludge etc.
Fermentation of materials liquid in practical applications due to contain a large amount of ammonia nitrogens and inhibit oleaginous yeast accumulate grease the problem of provide one
A new approaches.
The above-mentioned description to embodiment is that this hair can be understood and used for the ease of those skilled in the art
It is bright.Those skilled in the art obviously readily can make various modifications to these embodiments, and described herein one
As principle be applied in other embodiment, without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments.
Those skilled in the art's principle according to the present invention, not departing from improvement that scope of the invention is made and modification all should be at this
Within the protection domain of invention.
Claims (10)
1. a kind of method using saccharomycetes to make fermentation Synthetic Oil, it is characterised in that：It includes the following steps：
(1), the seed liquor of oleaginous yeast is subjected to fermented and cultured in limit phosphorus culture medium, until exponential phase latter stage is sent out
Zymotic fluid；
(2), the zymotic fluid is detached, sediment is collected and therefrom extracts grease.
2. according to the method described in claim 1, it is characterized in that：The oleaginous yeast is bending Cryptococcus.
3. according to the method described in claim 1, it is characterized in that：The limit phosphorus culture medium includes：16.6 ± 0.1g/L acetic acid
Potassium, 0.2 ± 0.1g/L yeast extracts, 3.0176 ± 0.0002g/L ammonium chlorides, 1.5 ± 0.2g/L magnesium chloride hexahydrates and 1 ±
0.3g/L sodium sulphate；And/or
In the limit phosphorus culture medium：Initial pH value is 5.5-6.5；Carbon-phosphorus ratio is (1400-1500):1, carbon-nitrogen ratio is (4.5-
5.5):1；
Preferably, initial pH value is 5.9-6.1 in the limit phosphorus culture medium.
4. according to the method described in claim 1, it is characterized in that：Fermented and cultured in step (1) includes：By the oil-producing ferment
The seed liquor of female bacterium is seeded in limit phosphorus culture medium with volume ratio 9-11% and carries out fermented and cultured.
5. according to the method described in claim 1, it is characterized in that：In step (1), the time of the fermented and cultured is 46-
The temperature of 50h, the fermented and cultured are 29-31 DEG C；And/or
The rotating speed of shaking table is 190-210rpm during the fermented and cultured.
6. according to the method described in claim 1, it is characterized in that：The generation of the seed liquor of oleaginous yeast in step (1)
Process includes the following steps：
(1-1), the oleaginous yeast being stored in inclined-plane pipe is seeded on tablet and is cultivated, obtain the list of oleaginous yeast
Bacterium colony；
(1-2), the single bacterium colony of the oleaginous yeast is cultivated in the first culture medium, until exponential phase latter stage obtains
The seed liquor of oleaginous yeast.
7. according to the method described in claim 6, it is characterized in that：In step (1-1), the inclined-plane pipe is that wort agar is oblique
Facial canal：And/or
The tablet is wort agar tablet.
8. according to the method described in claim 6, it is characterized in that：In step (1-2), first culture medium leaches for yeast
Powder peptone dextrose culture-medium, including：20 ± 5g/L glucose, 10 ± 2g/L fish meal proteins peptone and 10 ± 2g/L yeast extracts；
Initial pH value is 5.9-6.1；And/or
In step (1-2), in the incubation of the single bacterium colony of the oleaginous yeast：The time of culture is 34-38h, culture
Temperature is 29-31 DEG C；And/or
The rotating speed of shaking table is 190-210rpm in the incubation of the single bacterium colony of the oleaginous yeast.
9. according to the method described in claim 1, it is characterized in that：In step (2), the process of the extraction is：It will be described heavy
Starch is cleaned with physiological saline；The yeast cell being crushed in the sediment is extracted in conjunction with organic solvent to extract
Go out grease；
Preferably, the organic solvent is methanol, chloroform with volume ratio 1:0.9‐1:1.1 mixed liquor.
10. according to the method described in claim 1, it is characterized in that：In step (2), the grease includes：Palmitic acid, tristearin
Acid, oleic acid and linoleic acid.
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| CN112391421A (en) * | 2020-11-19 | 2021-02-23 | 中国科学院青岛生物能源与过程研究所 | Culture method for improving oil content of saccharomyces cerevisiae by adjusting total salt content of saccharomyces cerevisiae fermentation liquor and high-oil-yield saccharomyces cerevisiae |
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