CN101824440A - Method for separating microorganisms from grease - Google Patents
Method for separating microorganisms from grease Download PDFInfo
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- CN101824440A CN101824440A CN200910010566A CN200910010566A CN101824440A CN 101824440 A CN101824440 A CN 101824440A CN 200910010566 A CN200910010566 A CN 200910010566A CN 200910010566 A CN200910010566 A CN 200910010566A CN 101824440 A CN101824440 A CN 101824440A
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Abstract
The invention discloses a method for separating microorganisms from grease, which relates to a method for directly extracting grease from microorganism fermenting liquor for producing oil and co-producing microbial dreg and belongs to the technical field of downstream post treatment of biological engineering. The invention adopts the following technical scheme: mixing the microorganism fermenting liquor for producing oil and organic solvent according to certain ratio; stirring and leaching for a certain time; performing the solid-liquid separating operation; removing solvent from an organic solvent phase to obtain grease; and drying a solid to obtain the microbial dreg. The invention has the advantages of simple process, mild operating conditions, high extraction rate and yield of grease, good quality of microbial dreg, low energy consumption and favorability for scale industrial production.
Description
Technical field
The present invention relates to the greasy method of separation and Extraction, be a kind of from the oleaginous microorganism fermented liquid method of separation and Extraction grease and coproduction bacterium slag, belong to post-processing technology field, biotechnology downstream.
Background technology
Certain micro-organisms is converted into organism glycerin fatty acid ester under certain condition and is stored in the cell as yeast, mould, algae etc., and wherein fat content can surpass the bacterial strain of biological total amount more than 20% and be called oleaginous microorganism in the born of the same parents.Some oleaginous microorganism Pseudomonas is the substratum fermentation of carbon source with the saccharine material, can accumulate and account for grease (Li, the Y. of its dry cell weight more than 70%; Zhao, Z.; Bai, F.Enzyme Microb.Technol., 2007,41,312-317).The grease that utilizes fermentation method to obtain is called microbial oil, is called Unicell Oils and Fats again.The lipid acid of part microbial oil is formed similar with common Vegetable oil lipoprotein such as rapeseed oil, plam oil, soybean wet goods, mainly contains palmitinic acid, Zoomeric acid, stearic acid, oleic acid and other long chain polyunsaturated fatty acids etc.Advantage such as utilize the microorganisms producing grease to have not to be subjected to season and climatic influences, raw material sources is extensive, with short production cycle, product is high-valued.
The microbial oil large-scale industrial production still is unrealized in the world at present, and its major cause is that cost is than higher.The cost of microbial oil mainly comprises raw materials cost and separation and Extraction cost.The height of raw materials cost depends on the production efficiency of bacterial classification and the zymotechnique that is adopted, and the separation and Extraction cost then depends primarily on the technology of employing.Because microbial oil is present in the somatic cells mostly, conventional art all needs to isolate earlier thalline with methods such as centrifugal or filtrations from fermented liquid, extract according to the similar method of the extraction of Vegetable oil lipoprotein again, main extracting method comprises milling process (Matthews R F, Braddock R J.Food Technology, 1987,41:57-61), super critical extraction (Sovova H, Zarevucka M, Vacek M, et al.J.Supereritical Fluids, 2001, (20): 15-28) and organic solvent extractionprocess (Bligh E G, Dyer W J.J.Biochem.Physiol., 1959,37:911-917) etc.The shortcoming of these methods will be carried out solid-liquid separation exactly before extraction, but because the oleaginous microorganism fermentation has the cell concentration height in a later stage, viscosity height, characteristic such as cell density is low, thalline difficult separation and recycling, cost height.The shortcoming of these methods also has in addition: the milling process loss is bigger, and extraction yield is not high, just can carry out after will drying the thalline that obtains before extracting, and required equipment is more; The super critical extraction facility investment is big, the cost height; Conventional organic solvent method needs the microbial oil in the wet thallus after separating is carried out purifying after the cytoclasis, broken method has freeze-thaw method (WO00/13660), enzyme hydrolysis method (WO02/10423) and acid heat method (CN1923960A) etc., it is bigger that some pair cell of these methods carries out the energy consumption that break process need use, the complex steps that needs; Wherein acidic treatment carries out broken wall treatment to thalline, causes biomolecules hydrolysis or degraded such as albumen in the thalline, has reduced the using value of bacterium slag, and is less economical.
Summary of the invention
The purpose of this invention is to provide a kind of method of from the oleaginous microorganism fermented liquid, extracting microbial oil and coproduction bacterium slag effectively, simplify technological process, shorten the operational cycle, reduce production costs, promote comprehensive utilization of resources.Method of the present invention can widespread use in the microbial oil large-scale industrial production.
The present invention is achieved by following technical proposals:
(1) directly with oleaginous microorganism fermented liquid and 1: 0.25 by volume～1: 5 mixed of organic solvent,, obtains the lixiviate mixture 20 ℃～90 ℃ following lixiviates 10 minutes～24 hours;
(2) the lixiviate mixture is carried out the solid-liquid separation operation, obtain organic solvent phase, water and solid-phase material;
(3) organic solvent is removed organic solvent mutually according to a conventional method, obtains microbial oil;
(4) the solid-phase material drying obtains the bacterium slag.
The organic solvent that the present invention uses includes but not limited to chloroform, methylene dichloride, toluene, dimethylbenzene, sherwood oil, contain the Fatty Alcohol(C12-C14 and C12-C18) of 4～12 carbon atoms, contain the ester of the Fatty Alcohol(C12-C14 and C12-C18) of 1～12 carbon atom, No. four solvents, No. six solvents or the mixture between them.And can also contain volumn concentration in these organic solvents and be no more than 60% methyl alcohol, ethanol or propyl alcohol.
That the solid-liquid separation that the present invention uses is operating as is centrifugal, filtration, sedimentation or the combination of their necessity.
The present invention removes the method that organic solvent obtains microbial oil mutually from organic solvent and is distillation or evaporation.
The oleaginous microorganism that the present invention uses can surpass fungi, bacterium or little algae of dry cell weight 20% (w/w) for thalline fat content after fermentation culture.They include but not limited to, the produce oil fungi is as Rhodosporidium toruloides, Cryptococcus curvatus, Yarrowia lipolytica, Rhodotorula glutinis, Rhizopus arrhizus, Lipomyces starkeyi, Mortierellaisabellina, Mucor circinelloides and Cunninghamella; The produce oil bacterium is as Croynebacterium, Nocardia and Mycobacterium; The little algae of produce oil is as Botryococcusbraunii, Crypthecodinium cohnii, Chlorellaprotothecoides, Nannochloropsis sp. and Schizochytrium limacinum.
The oleaginous microorganism fermented liquid that the present invention uses is cultivated the mash that contains thalline that obtains as produce oil fungi, bacterium or little algae at liquid culture condition bottom fermentation.
The present invention uses actual fat content reference literature (Li, the Y. of thalline material; Zhao, Z.; Bai, F.Enzyme Microb.Technol., 2007,41, method 312-317) is measured, and calculates the grease extraction yield of other method as benchmark.
The invention has the beneficial effects as follows: 1) compare with traditional microbial oil extracting method, the present invention uses whole beer directly to extract, saved from fermented liquid and reclaimed preprocessing process such as the solid-liquid separation operation of thalline and thalline drying, simplified processing step, shortened the operational cycle; 2) the present invention can effectively reduce fermentation broth viscosity by extracting grease in the cell, changes cell density, helps solid-liquid separation operation and bacterium slag to reclaim; 3) the present invention need not thalline is carried out broken wall treatment, has reduced energy consumption, has reduced the use of corrosive chemical; 4) bacterium slag pollutent of the present invention is few, and rate of recovery height has improved the Technological Economy of oil fermentation process.In a word, the technology of the present invention is simply effective, and facility investment is few, but operate continuously, and energy consumption is low, helps large-scale industrial production.
Description of drawings
Fig. 1 extracts and coproduction bacterium slag process flow diagram for microbial oil of the present invention.
Embodiment
Following examples have been chosen some typical oleaginous microorganism bacterial classifications, help to understand this patent, do not use the present invention but be not limited in any form on other bacterial strain material.
Embodiment 1
According to document (Li, Y.; Zhao, Z.; Bai, F.Enzyme Microb.Technol., 2007,41:312-317) described method, be linked in the fermention medium growing in the inoculum size of oleaginous yeast Rhodosporidiumtoruloides AS 2.1389 in the YEPD substratum (bacterium source in Chinese common micro-organisms culture presevation administrative center) by 10%, 30 ℃, air flow 0.8vvm, the glucose 500ml of feed supplement 1000g/L five times cultivates the fermented liquid that obtains dry cell weight 92g/L (fat content 60.1%) behind the 120h.Get above-mentioned fermented liquid 50ml, add the 50ml chloroform, under 20 ℃, stirring and leaching 1h, a layer organic solvent phase taken off in the sedimentation layering, and the distillation after drying obtains grease 2.06g, grease extraction yield 74.6%.The residuum centrifugal treating obtains the solid phase, and drying gets bacterium slag 2.31g.
Embodiment 2
According to document (Huang Jianzhong, Shi Qiaoqin, Xiaolan ZHOU, etc.The microbiology circular, 1998, (4): 187-191) described method, to cultivate 28h produce oil mould Mortierella isabellina AS 3.3410 (bacterium source in Chinese common micro-organisms culture presevation administrative center) seed liquor is linked in the fermention medium with 10% inoculum size, 280rpm, cultivate 70h down for 25 ℃, obtain the fermented liquid of dry cell weight 34g/L (fat content 47.2%).Get above-mentioned fermented liquid 25ml, add the 75ml chloroform: methyl alcohol 2: 1 (V/V), under 70 ℃, stirring and leaching 1h takes off a layer organic solvent phase after centrifugal, and the evaporation after drying obtains grease 0.37g, grease extraction yield 92%.The upper strata is filtered and is obtained wet bacteria slag, dry back residue quality 0.25g.
Embodiment 3
With reference to embodiment 1 described method, twice of feed supplement, fermentation obtains oleaginous yeast Lipomyces starkeyi AS 2.1560 (bacterium source in Chinese common micro-organisms culture presevation administrative center) the fermented liquid 100ml of dry cell weight 48g/L (fat content 55.2%) behind the 62h, add No. six solvents of 25ml, under 30 ℃, the upper strata solvent phase is got in sedimentation layering behind the stirring and leaching 10min, the evaporation of dry back obtains grease 0.72g, grease extraction yield 27%.Lower floor's phase centrifugal treating obtains the solid phase, dry back bacterium slag 2.80g.
Embodiment 4
According to document (Wang Jufang, Liang Shizhong, Wu Zhenqiang, etc.South China Science ﹠ Engineering University's journal (natural science edition) 2008,28 (10): 28-31) described method, the seed liquor that to cultivate 24h Crypthecodinium cohnii Crypthecodinium cohnii ATCC 30556 (bacterium source is in U.S. common micro-organisms culture presevation administrative centers) is linked in the fermention medium with 5% inoculum size, at 25 ℃, obtain the fermented liquid of dry cell weight 15g/L (fat content 30.1%) under the 170rpm condition behind the cultivation 65h.Get above-mentioned fermented liquid 50ml, add the 50ml n-Octanol, under 80 ℃, stirring and leaching 2h, centrifuging and taking upper strata organic solvent phase, the distillation of dry back obtains grease 0.158g, grease extraction yield 70.1%.Residuum continues centrifugal treating, obtains the solid phase, and drying obtains bacterium slag 0.49g.
Embodiment 5
With reference to embodiment 1 described method, feed supplement four times, fermentation obtain oleaginous yeast Rhodotorula glutinis AS 2.703 (bacterium source in the Chinese common micro-organisms culture presevation administrative center) fermented liquid of dry cell weight 79g/L (fat content 49%) behind the 90h.Get above-mentioned fermented liquid 50ml, add 200ml dimethylbenzene, under 90 ℃, the upper strata solvent phase is got in sedimentation layering behind the stirring and leaching 1h, and the evaporation after drying obtains grease 1.01g, grease extraction yield 52%.With lower floor's phase centrifugal treating, collect the solid phase, drying obtains bacterium slag 2.63g.
Embodiment 6
With reference to embodiment 4 described methods, obtain the little algae Botryococcus of produce oil braunii LB572 (bacterium source is in the Texas ,Usa university) fermented liquid of dry cell weight 20g/L (fat content 37.4%) behind the fermentation 54h.Get above-mentioned fermented liquid 30ml, add the 100ml sherwood oil, under 50 ℃, centrifuging and taking upper strata solvent phase behind the stirring and leaching 20min, the evaporation after drying obtains grease 0.1g, grease extraction yield 42.6%.Continue to remove the middle level water, collect the solid phase, drying obtains bacterium slag 0.39g.
Embodiment 7
With reference to embodiment 1 described method, feed supplement three times, fermentation obtain oleaginous yeast Lipomyces starkeyi AS 2.1560 (bacterium source in the Chinese common micro-organisms culture presevation administrative center) fermented liquid of dry cell weight 70g/L (fat content 50.3%) behind the 72h.Get above-mentioned fermented liquid 50ml, add the 125ml chloroform: ethyl acetate 3: 1 (V/V), under 35 ℃, a layer organic solvent phase taken off in sedimentation layering behind the stirring 24h, and the evaporation after drying obtains grease 1.43g, grease extraction yield 81%.With the residuum centrifugal treating, obtain the solid phase, obtain bacterium slag 1.35g after the drying.
Embodiment 8
With reference to embodiment 4 described methods, obtain produce oil chlorella Chlorella protothecoides CS-41 (bacterium source is in the little algae of the Australian CSIRO research centre) fermented liquid of dry cell weight 21.7g/L (fat content 24.3%) behind the fermentation 64h.Get above-mentioned fermented liquid 50ml, add No. four solvents of 100ml, under 80 ℃, stir 2h, the upper strata solvent phase is got in the sedimentation layering, and the evaporation after drying obtains grease 0.14g, grease extraction yield 53.2%.Lower floor's water filtration treatment obtains bacterium slag 0.68g behind the filtration cakes torrefaction.
Embodiment 9
With reference to embodiment 1 described method, feed supplement three times, fermentation obtain oleaginous yeast Yarrowia lipolytica AS 2.1398 (bacterium source in the Chinese common micro-organisms culture presevation administrative center) fermented liquid of dry cell weight 65g/L (fat content 62.7%) behind the 130h.Get above-mentioned fermented liquid 50ml, add the 150ml chloroform, under 30 ℃, a layer solvent phase taken off in sedimentation layering behind the stirring 1.5h, and the evaporation after drying obtains grease 1.73g, grease extraction yield 85%.Centrifugal treating is removed the residuum of organic solvent phase, collects the solid phase, and drying obtains bacterium slag 0.95g.
Claims (9)
1. the separation method of a microbial oil, it is characterized in that: the method that is separation and Extraction grease and coproduction bacterium slag from oleaginous microorganism fermented liquid raw material, with oleaginous microorganism fermented liquid and organic solvent mixing lixiviate certain hour, carry out the solid-liquid separation operation, organic solvent is removed solvent mutually, obtain grease; Solid is carried out drying mutually, obtain the bacterium slag.
2. it is characterized in that in accordance with the method for claim 1: described organic solvent is chloroform, methylene dichloride, toluene, dimethylbenzene, sherwood oil, contain the Fatty Alcohol(C12-C14 and C12-C18) of 4～12 carbon atoms, contain the ester of the Fatty Alcohol(C12-C14 and C12-C18) of 1～12 carbon atom, a kind of in No. four solvents or No. six solvents or more than one mixture.
3. according to claim 1 or 2 described methods, it is characterized in that: can add one or more formation mixed solvents in methyl alcohol, ethanol or the propyl alcohol in the described organic solvent, add their volumn concentration≤60% in mixed solvent of back.
4. in accordance with the method for claim 1, it is characterized in that: described lixiviate operational condition is service temperature: 20 ℃～90 ℃, 10 minutes～24 hours operating time.
5. it is characterized in that in accordance with the method for claim 1: described solid-liquid separation is operating as a kind of in centrifugal, filtration, the sedimentation or more than one necessity combination.
6. it is characterized in that in accordance with the method for claim 1: described fermented liquid is 1: 0.25～1: 5 with the volume of organic solvent ratio.
7. it is characterized in that in accordance with the method for claim 1: the removal method of described organic solvent is distillation or evaporation.
8. it is characterized in that in accordance with the method for claim 1: described oleaginous microorganism fermented liquid is that produce oil fungi, bacterium or little algae are cultivated the mash that contains thalline that obtains at liquid culture condition bottom fermentation.
9. it is characterized in that in accordance with the method for claim 1: described oleaginous microorganism is for surpassing dry cell weight 20% greasy fungi, bacterium or little algae at intracellular accumulation content under certain culture condition.
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| CN101967452A (en) * | 2010-09-13 | 2011-02-09 | 华南理工大学 | Fermentable silk spore yeast strains and application for preparing microbial oil thereof |
| CN101985637A (en) * | 2010-11-02 | 2011-03-16 | 嘉吉烯王生物工程（武汉）有限公司 | Method for extracting microbial oil |
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| CN101967452B (en) * | 2010-09-13 | 2012-08-22 | 华南理工大学 | Fermentable silk spore yeast strains and application for preparing microbial oil thereof |
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| CN102061317B (en) * | 2010-11-09 | 2013-04-17 | 佛山市正合生物能源有限公司 | Method for producing biolipid by cassava fermentation |
| CN102533430B (en) * | 2010-12-28 | 2013-09-18 | 中国科学院大连化学物理研究所 | Extraction method of micro-algae oil |
| CN102533430A (en) * | 2010-12-28 | 2012-07-04 | 中国科学院大连化学物理研究所 | Extraction method of micro-algae oil |
| US10100341B2 (en) | 2011-02-02 | 2018-10-16 | Corbion Biotech, Inc. | Tailored oils produced from recombinant oleaginous microorganisms |
| US11401538B2 (en) | 2012-04-18 | 2022-08-02 | Corbion Biotech, Inc. | Structuring fats and methods of producing structuring fats |
| US9909155B2 (en) | 2012-04-18 | 2018-03-06 | Corbion Biotech, Inc. | Structuring fats and methods of producing structuring fats |
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| CN103937596A (en) * | 2013-01-22 | 2014-07-23 | 富士电机株式会社 | Grease manufacturing method |
| CN103184097A (en) * | 2013-02-28 | 2013-07-03 | 黄俊超 | Method for extracting microalgae by utilizing dual-solvent system and obtaining extract |
| US10053715B2 (en) | 2013-10-04 | 2018-08-21 | Corbion Biotech, Inc. | Tailored oils |
| US10316299B2 (en) | 2014-07-10 | 2019-06-11 | Corbion Biotech, Inc. | Ketoacyl ACP synthase genes and uses thereof |
| US9969990B2 (en) | 2014-07-10 | 2018-05-15 | Corbion Biotech, Inc. | Ketoacyl ACP synthase genes and uses thereof |
| CN105669258A (en) * | 2016-01-06 | 2016-06-15 | 济南开发区星火科学技术研究院 | Resource utilization method for oil-producing microbial fermentation culture liquid |
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Application publication date: 20100908 |