CN103224884A - Method for culturing oleaginous microorganism - Google Patents
Method for culturing oleaginous microorganism Download PDFInfo
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Abstract
The invention discloses a novel method for culturing oleaginous microorganism, which is characterized in that a carbohydrate polymer is taken as a carbon source for microbial cultivation, polymer hydrolase and oleaginous microorganism seeds are added, air is introduced under condition of proper temperature and pH, a fluid suspension is cultured, thereby microbial oil containing long chain aliphatic acid and its derivative can be accumulated. The microbial oil can be a raw material for preparing biodiesel and oil chemical product. The method provided by the invention develops the carbon source for culturing oleaginous microorganism from a traditional water-soluble organic compound to the carbohydrate polymer, the processes of biomass hydrolysis and oil fermentation are integrated, cost of raw material is reduced, technology is simplified, the efficiency is increased, and the method for culturing oleaginous microorganism has obvious economic benefit.
Description
Technical field
The present invention relates to a kind of cultural method of oleaginous microorganism, belong to biological technical field.More particularly, be directly to utilize the carbon source of carbohydrate polymer for substratum, in the presence of lytic enzyme, cultivate oleaginous microorganism, realization is converted into carbon source and is stored in intracellular grease.This grease contains one or more longer chain fatty acids and derivative thereof, can be used for preparing biofuel, functional grease or other high value added products.
Background technology
Some yeast, mould, bacterium and algae etc. are under certain condition, can be carbon source with carbohydrate, hydrocarbon polymer etc., synthetic in vivo and store a large amount of greases, every can be in born of the same parents the oil and fat accumulation microorganism that surpasses dry cell weight 20% be called oleaginous microorganism (Ratledge C, Wynn JP Adv Appl Microbiol, 2002,51:1-52).Some oleaginous microorganism in addition can accumulate 70% microbial oil above its dry cell weight.The grease that microbial oil, especially yeast produce, its major ingredient is a triglyceride level, lipid acid is formed similar to commercial animal-plant oil, with C
14-C
22Longer chain fatty acid is main.With respect to animal-plant oil, microbial oil is with short production cycle, not limited by season and weather, and raw material sources are wide, and the outer cultivated land resource of occupying volume not is easy to realize scale production substantially, is considered to very potential new oil resource.Therefore, microbial oil not only may also may in the industry development of regeneratable liquors fuel biofuel, play a significant role as the substitute of edible oil or other functional greases (Zhao Zongbao. Chinese biological engineering magazine, 2005,25 (2): 8-11).
The carbon source that oleaginous microorganism is cultivated is water-soluble organic compounds normally.Utilize sucrose, glucose, fructose, wood sugar, lactose, cellobiose, glycerine, water-soluble oligofructose (inulin), ethanol etc. to cultivate oleaginous microorganism, existing bibliographical information (Ageitos JM, et al.Appl Microbiol Biotechnol.2011,90:1219-1227).Common biomass as timber, crop material, crop seeds, corps nutrient stem tuber etc., contain a large amount of carbohydrate polymers.Because the polymerization degree is higher, they are generally water insoluble.Therefore, adopt various technique means usually, at first carbohydrate polymer is degraded to water-soluble cpds, utilize the microbial transformation preparation to comprise the various meta-bolitess of grease again.Especially, be fermentable sugar with ligocellulose degradation, utilize the carbohydrate admixture of these solubilities to be seen in report then for carbon source accumulation grease.Fermentable trichosporon Trichosporon fermentans utilizes the straw olefin(e) acid hydrolyzed solution fermentation of detoxification, and after 8 days, the thalline fat content reaches 40.1% (Huang C, et al.Bioresour Technol, 2009,100 (19): 4535-4538); Utilize oleaginous yeast Cryptococcus curvatus to the fermentation of straw olefin(e) acid hydrolyzed solution, the thalline fat content is 35.5%.Utilize saccharomyces oleaginosus Lipomyces starkeyi to straw olefin(e) acid hydrolyzed solution fermentation, the thalline fat content reach 31.2% (Yu XC, et al.Bioresour Technol, 2011,102:6134-6140).
Adopt the solid state fermentation pattern, biomass material can be converted into grease.As, with being raw material through the pretreated straw of gas explosion, but cultivate the plant endogenesis epiphyte of the plain enzyme of a kind of eccrine fiber, under the cellulase condition of extra interpolation 10FPU/g substrate, by fermentation in 10 days, the grease yield be the 80mg/g dry-matter (Peng XW, Chen HZ.Bioresour Technol.2008,99:3885-3889).Under optimal conditions, aspergillus oryzae Aspergillus oryzae A-4 transforms straw and produces microbial oil, cultivate after 6 days the grease yield and be the 62mg/g dry-matter (Lin H, et al.Bioresour Technol.2010,101:7556-7562).In the above-mentioned example, though microorganism possesses production of cellulose enzyme and the greasy function of accumulation simultaneously, yet its enzymatic productivity is low, the enzymic hydrolysis weak effect, and adopt the solid state fermentation pattern, and fermentation period is long, and transformation efficiency is low, and grease yield is low.
Because it is main carbon source that existing oleaginous microorganism culture technique is utilized water-soluble organic compounds, the raw materials cost height, whole efficiency is low, is difficult to large-scale production.Therefore, the researchdevelopment new technology is expanded the carbon source that oleaginous microorganism is cultivated, and is significant.
Summary of the invention
Directly utilize carbohydrate polymer to be carbon source, cultivate oleaginous microorganism, might reduce raw materials cost significantly, the technology that simplifies the operation is raised the efficiency, and improves the Technological Economy of microbial oil.Therefore, the contriver studies the culture condition of oleaginous microorganism, finds in the environment of cellulose suspension and cellulase coexistence, after adding oleaginous microorganism viable bacteria body, hatching certain hour, can accumulate a large amount of greases in the cell, and Mierocrystalline cellulose is consumed simultaneously.And then the contriver optimizes culture condition.Based on above-mentioned discovery, the present invention proposes a kind of cultivation novel method of oleaginous microorganism, the carbon source that it is characterized in that substratum is a carbohydrate polymer, and the enzyme that has the hydrolysis carbon source in the substratum, the oleaginous microorganism inoculum size is 0.05～0.50 gram dry mycelium/gram carbohydrate polymer, 20～45 ℃ of culture temperature, pH 3～7, the blowing air fluid suspension culture.It directly is raw material with biomass that this method is cultivated oleaginous microorganism, improves the Technological Economy that microbial oil is produced, and has broad prospect of application.The microbial oil that this method obtains contains one or more lipid acid and derivative thereof, can be used for preparing biofuel or other high value added products.
The present invention is achieved through the following technical solutions:
1, preparation produce oil substratum.Carbohydrate polymer or the material, carbohydrate polymer lytic enzyme and the water that contain carbohydrate polymer are mixed, make suspension, be the produce oil substratum.
2, preparation oleaginous microorganism seed.Adopt the abundant relatively substratum of nutrition, at 20～37 ℃, pH 3～8, and the aerated culture oleaginous microorganism after cultivation finishes, adopts centrifugation or filtering method to collect wet thallus, obtains the oleaginous microorganism viable cell, is the oleaginous microorganism seed.
3, cultivate oleaginous microorganism accumulation grease.The oleaginous microorganism seed is added in the produce oil substratum by the inoculum size that is equivalent to 0.05～0.5 gram dry mycelium/gram carbohydrate polymer, and at 20～45 ℃, pH 3.0～7.5, and aerated culture when carbon source content is lower than 5g/L in the substratum, stops cultivating.
4, the mensuration of biomass and oil quantity.Reference literature (Li YH, et al.Enzyme Microb Technol, 2007, method 41:312-317), fermented liquid is through centrifugal, washing, collecting precipitation.Be deposited in 105 ℃ and be dried to constant weight, and reference literature (Updegraff DM.Anal Biochem, 1969, method 32:420-424) is measured residual Mierocrystalline cellulose in the fermented liquid, calculates biomass; Reference literature (Li Zhifeng, etc. microbiology circular, 2001,28 (6): 72-75) use acid heat-organic solvent method extracting to obtain grease, calculate the thalline oil quantity.
The present invention prepares the oleaginous microorganism seed with reference to microbial culture method commonly used, uses substratum nutritious, that help the thalline fast breeding.In the later stage in growing microorganism stage, the photoabsorption in that wavelength 600 nanometers are measured fermentation liquid promptly obtains the OD600nm value, adopts method separated and collected oleaginous microorganism cell from fermentation liquid of centrifugal or membrane filtration.Fermentation liquid is centrifugal under the centrifugal action more than the 800g, obtains wet thallus; Fermentation liquid also usage aperture obtains wet thallus less than the membrane filtration of 5um, is used to inoculate the produce oil substratum.According to the volume of produce oil substratum and the OD600nm value in liquid seeds later stage, can calculate inoculum density during inoculation.
The carbohydrate polymer that the present invention was suitable for is that Mierocrystalline cellulose, hemicellulose, xylan, starch, major ingredient are that biological material, the major ingredient of Mierocrystalline cellulose and hemicellulose is the biological material of starch or several mixture in them.Wherein major ingredient is that the biological material of Mierocrystalline cellulose and hemicellulose is one or two or more kinds combination in maize straw, straw, straw, bagasse, corn cob, production of forestry tankage, pine, the dragon spruce; Major ingredient is that the biological material of starch is one or two or more kinds combination in corn, paddy, Chinese sorghum, wheat, barley, cassava, the sweet potato.
The enzyme of hydrolysis carbon source of the present invention is the enzyme with the polymerization degree that reduces carbohydrate polymer, comprises cellulase, hemicellulase, zytase, glucuroide, gives birth to diastatic enzyme, amylase, saccharifying enzyme a kind of or their necessity combination.Wherein, cellulase, hemicellulase, zytase, dextran glycosides enzyme, amylase and saccharifying enzyme are buied from Ningxia jade of the He family Bioisystech Co., Ltd, are respectively 50,000 U/g, 1,200,000 U/g, 150,000 U/g, 1,200,000 U/g, 30,000 U/g and 30,000 U/g than vigor.Living diastatic enzyme reference literature method (Lee or Na, etc. use and the environmental organism journal 2010,16 (5): 714-718) preparation is 80,000 U/g than vigor.Under the vigor test condition, 1g zymin hydrolysis carbohydrate polymer discharges out the reducing sugar amount that is equivalent to 1mg glucose, is defined as 1 enzyme activity unit (U).
It is the lytic enzyme of the carbohydrate polymer of 5～100U that the every gram carbohydrate polymer of the present invention adds enzyme activity, makes polymkeric substance be hydrolyzed into the product that can be utilized by oleaginous microorganism in culturing process.According to the physico-chemical property of carbohydrate polymer material and the properties and characteristics of oleaginous microorganism, the present invention prepares the combination that the produce oil substratum can use the plurality of enzymes preparation.For example, when being carbon source,, need use cellulase, hemicellulase, zytase and glucuroide simultaneously in order to utilize carbon source better with pretreated stalk.
Oleaginous microorganism liquid nutrient medium of the present invention is except that containing carbohydrate polymer and lytic enzyme preparation, also can contain other microorganism culturing nutritive substance commonly used, other microorganism culturing nutritive substance commonly used comprises that quality is the nitrogenous source of carbon source total mass 0.1%～20%, phosphorus source that quality is carbon source total mass 0.01%～15% or the quality sulphur source that is carbon source total mass 0.01%～5% or two kinds or three kinds combination in them.
Nitrogenous source of the present invention is that ammonium salt and warp can discharge the material that forms ammonium ion in the aqueous solution.
Phosphorus of the present invention source is phosphoric acid salt and the material that can discharge phosphate radical through hydrolysis.
Oleaginous microorganism of the present invention is the eukaryotic microorganisms that the thalline fat content can surpass dry cell weight 20% after fermentation culture, and they include but not limited to, saccharomyces oleaginosus reaches saccharomyces oleaginosus Lipomyces starkeyi like that; Rhodotorula is as circle red winter spore Rhodosporidium toruloides, rhodotorula glutinis Rhodotorula glutinis and shadow yeast Sporobolomyces roseus; Trichosporon is as skin shape trichosporon Trichosporon cutaneum and fermentable trichosporon Trichosporon fermentans; Cryptococcus is as white Cryptococcus Cryptococcus albidus and crooked Cryptococcus Cryptococcus curvatus; Mould is intended endomyces Endomycopsis burtonii, strong mould doughtily Geotrichum robustum, Mortierella isabellina Mortierella isabellina and volume branch Mucor Mucor circinelloides as Christian Breton; Excellent bacillus Croynebacterium, Nocardia Nocardia and rhodococcus Rhodococcus in the produce oil bacterium.These bacterial strains can directly separate from Chinese common micro-organisms culture presevation administrative center (CGMCC), American type culture collection (ATCC) or the purchase of Chinese industrial microbial strains preservation administrative center culture presevation mechanisms such as (CICC) or from occurring in nature, also can use the artificial or spontaneous mutation bacterial strain different with former bacterial strain proterties.
Microbial oil of the present invention is mainly the glyceryl ester of longer chain fatty acid and derivative thereof.Reference literature (Liu B, Zhao Z B.J Chem Technol Biotechnol, 2007,82 (8): method 775-780), directly utilize the oleaginous microorganism thalline to be raw material, can under the acid catalysis condition, transesterificationization prepare biofuel; Or reference literature (lining is big, etc. the process engineering journal, 2007,7 (1): method 137-140), utilize the microbial oil that extracts, can under the enzyme catalysis condition, transesterificationization prepare biofuel.
Microbial oil of the present invention also can be used for preparing other high value added products or oil-fat chemical products.
The invention has the beneficial effects as follows:
1) the present invention directly utilizes carbohydrate polymer to carry out oil fermentation, provides a kind of novel method for cultivating oleaginous microorganism and preparing microbial oil;
2) Yu with carbohydrate polymer at first be degraded to fermentable sugar, cultivating the conventional art of oleaginous microorganism again compares, the present invention has simplified technology with the enzymatic saccharification and the oil fermentation PROCESS COUPLING of carbohydrate polymer, has improved enzymolysis and fermentation efficiency;
3) hydrolysis of lignocellulose resource obtains the mixing sugar hydrolysate based on glucose and wood sugar, and oleaginous microorganism utilizes mixing sugar when fermentation, often has glucose effect, and fermentation period is long, and the xylose utilization rate is low.The present invention directly utilizes lignocellulose to carry out oil fermentation, and hydrolysis and fermentation are carried out simultaneously, no water-soluble sugar accumulation in the substratum, avoided glucose effect, Mierocrystalline cellulose and hemicellulose all are utilized effectively, and have improved utilization of resources degree, and substrate is to greasy transformation efficiency height.
4) because the product that carbohydrate polymer enzymatic saccharification of the present invention discharges is utilized by oleaginous microorganism rapidly, avoided the inhibition of hydrolysate to lytic enzyme, the enzyme activity length of holding time, therefore, enzyme dosage is few; And because the culturing process mild condition, the fermentation supernatant that contains enzyme can reuse.Therefore, the present invention not only can reduce the enzyme cost, also can reduce discharge of wastewater.
5) the lignocellulose resource inevitably produces many inhibitor in preprocessing process, as furfural, hydroxymethylfurfural, acetate, levulinic acid, p-Hydroxybenzaldehyde, syringic aldehyde and vanillin food grade,1000.000000ine mesh etc., they have significant inhibition effect to the growth and breeding of oleaginous microorganism, thereby influence ferment effect.Acetate, formic acid, furfural and vanillin food grade,1000.000000ine mesh to the fermentation of oleaginous yeast have stronger restraining effect (Chen X, et al.Appl Biochem Biotechnol, 2009,159:591-604); The furfural of 1mM and derivative thereof to the growth-inhibiting of oleaginous yeast Rhodosporidium toruloides Y4 45% (Hu CM, et al.Bioresour Technol, 2009,100:4843-4847); To maize straw through cultivating oleaginous yeast Trichosporon cutaneum CX1 behind the biological detoxication, fat content significantly improve (Huang X, et al.Bioresour Technol, 2011,102:9705-9709).Traditional oil fermentation must carry out detoxification treatment to the inhibition compound that produces in the preprocessing process.Oleaginous microorganism seed inoculum size is bigger among the present invention, and thalline is not bred substantially, mainly carries out oil and fat accumulation, and in the oil and fat accumulation stage, bacterium is strong to the inhibitor tolerance, can pretreated lignocellulosic material not carried out detoxification treatment.
6) the present invention is because oleaginous microorganism seed inoculum size is bigger, and dissolved organic matter concentration is low in the fermention medium, and no water-soluble sugar accumulates in the culturing process, and assorted bacterium is difficult to growth.Therefore, fermention medium does not need sterilising treatment, can simplify technology, reduces production costs.
In a word, the present invention is converted into microbial oil with carbohydrate polymer, have generally that technical process is brief, prepared using efficient height, grease yield height, technical superiority that cost is low, can significantly improve the Technological Economy of microbial oil, significant to bioenergy industry and biorefinery.
Specific embodiment
Following examples have been chosen and used typical carbohydrate polymer is the example that material is cultivated typical oleaginous microorganism, helps to understand this patent, does not apply the present invention to other materials or produce oil bacterial strain but do not limit in any form.
Comparative Examples 1
(1) fermention medium: the suspension of Mierocrystalline cellulose 40g/L, cellulase 320U/L and glucuroide 600U/L at 4.8,50 ℃ of enzymic hydrolysis 72h of pH, is adjusted pH 5.5.
(2) seed preparation: substratum is formed (g/L): glucose 20, yeast powder 10, peptone 10, pH 6.0, behind 121 ℃ of sterilization 15min, insert Cryptococcus curvatus ATCC 20509 (available from U.S. representative microbial culture presevation administrative centers), cultivate 24h for 30 ℃, nutrient solution in the centrifugal 5min of 8000rpm, is obtained wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=15 (being equivalent to dry mycelium 0.27g/L) in the described fermention medium of 50mL step (1), cultivate 52h at 30 ℃ of following blowing airs.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, determines that dry mycelium content is 13.5g/L, and residual sugar is 3.1g/L in the fermented liquid.Reference literature (Li Zhifeng etc. the microbiology circular, 2001,28 (6): 72-75) use acid heat-organic solvent method to extract grease, obtain grease 6.5g/L, the thalline fat content is 48.1%.
Embodiment 1
(1) fermention medium: Yu Shuizhong, Mierocrystalline cellulose 40g/L, cellulase 320U/L, glucuroide 600U/L, pH 5.2.
(2) seed preparation: substratum is formed (g/L): glucose 20, yeast powder 10, peptone 10, pH 6.0, behind 121 ℃ of sterilization 15min, insert Cryptococcus curvatus ATCC 20509 (available from U.S. representative microbial culture presevation administrative centers), cultivate 24h, centrifugal collection wet thallus for 30 ℃.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=15 (being equivalent to dry mycelium 0.27g/L) in the described fermention medium of 50mL step (1), cultivate 52h at 37 ℃ of following blowing airs.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, and other gets a certain amount of precipitation and surveys content of cellulose, determines that dry mycelium content is 13.2g/L, residual Mierocrystalline cellulose 1.9g/L.Reference literature (Li Zhifeng etc. the microbiology circular, 2001,28 (6): 72-75) use acid heat-organic solvent method to extract grease, obtain grease 7.6g/L, the thalline fat content is 57.6%.
Compare found that of Comparative Examples 1 and embodiment 1, the carbohydrate polymer zymin identical with vigor that functional quality equates improved 19% by technical scheme grease yield of the present invention.The more important thing is that Comparative Examples 1 is in order to obtain glucose, with Mierocrystalline cellulose at 4.8,50 ℃ of following enzymic hydrolysis 72h of pH; Embodiment 1 has then exempted this hydrolytic process fully.Therefore, with being the prior art scheme ratio of representative with Comparative Examples 1, the present invention has the significant advantage that improves grease yield and production efficiency, reduces cost, reduces facility investment.
Embodiment 2
(1) fermention medium: Yu Shuizhong, Mierocrystalline cellulose 50g/L, potassium primary phosphate 7g/L, Sodium phosphate dibasic 0.5g/L, cellulase 750FPU/L, pH 4.8.
(2) seed preparation: substratum is formed (g/L): glucose 20, yeast powder 10, peptone 10, pH 6.0, behind 121 ℃ of sterilization 15min, insert Rhodosporidium toruloides AS 2.1389 (available from Chinese common micro-organisms culture presevation administrative center), 30 ℃ of aerated culture 24h, in the centrifugal 5min of 8000rpm, obtain wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=10 (dry mycelium 0.18g/L) in the described fermention medium of 50mL step (1), cultivate 72h at 34 ℃ of following blowing airs.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, and other gets a certain amount of precipitation and surveys content of cellulose, determines that dry mycelium content is 12.9g/L, residual Mierocrystalline cellulose 9.2g/L.Press acid heat-organic solvent method and extract grease, obtain grease 8.3g/L, fat content is 64.3%.
Embodiment 3
(1) fermention medium: Yu Shuizhong, sweet potato starch 30g/L, ammonium sulfate 1.0g/L gives birth to diastatic enzyme 900U/L, and pH 3.0.
(2) seed preparation: in the PDA liquid nutrient medium, insert a volume branch Mucor Mucor circinelloides AS 3.2208 (available from Chinese common micro-organisms culture presevation administrative center), 28 ℃ of aerated culture 24h, centrifugal collection wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=10 (dry mycelium 0.18g/L) in the described fermention medium of 50mL step (1), cultivate 60h at 40 ℃ of following blowing airs.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, determines that dry mycelium content is 8.9g/L.Press acid heat-organic solvent method and extract grease, obtain grease 5.0g/L, fat content is 56.1%.
Embodiment 4
(1) fermention medium: Yu Shuizhong, Mierocrystalline cellulose 30g/L, yeast powder 0.01g/L, cellulase 3000FPU, pH 5.5.
(2) seed preparation: substratum is formed (g/L): glucose 20, yeast powder 10, peptone 10, pH 5.5, behind 121 ℃ of sterilization 20min, rhodotorula glutinis Rhodotorula glutinis AS 2.703 (available from Chinese common micro-organisms culture presevation administrative center), 26 ℃, 200rpm shaking culture 28h, membrane filtration obtains wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=42 (dry mycelium 0.75g/L) in the described fermention medium of 50mL step (1), cultivate 24h at 40 ℃ of following blowing airs.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, determines that dry mycelium content is 20.9g/L.Press acid heat-organic solvent method and extract grease, obtain grease 5.2g/L, fat content is 24.9%.
Embodiment 5
(1) fermention medium: Yu Shuizhong, xylan 36g/L, yeast powder 1.0g/L, potassium primary phosphate 0.8g/L, zytase 180U/L, pH 7.5.
(2) seed preparation: substratum is formed (g/L): glucose 20, yeast powder 10, peptone 10, pH 6.0, behind 121 ℃ of sterilization 15min, insert Trichosporon cutaneum AS 2.571 (available from Chinese common micro-organisms culture presevation administrative center), 30 ℃ of aerated culture 20h, nutrient solution in the centrifugal 5min of 8000rpm, is obtained wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=5 (dry mycelium 0.09g/L) in the described fermention medium of 50mL step (1), cultivate 36h at 30 ℃ of following blowing airs.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, and other gets a certain amount of precipitation and surveys content of cellulose, determines that dry mycelium content is 8.4g/L, residual xylan 4.2g/L.Press acid heat-organic solvent method and extract grease, obtain grease 4.1g/L, fat content is 48.8%.
Embodiment 6
(1) fermention medium: Yu Shuizhong, Mierocrystalline cellulose 120g/L, yeast powder 0.5g/L, potassium primary phosphate 7.0g/L, Sodium phosphate dibasic 2.0g/L, cellulase 7200U/L, glucuroide 1200U/L, pH 5.0.
(2) seed preparation: in malt juice liquid medium, insert shadow yeast Sporobolomyces roseus IAM 13481 (available from Japanese JCM bacterial strain preservation centers), 25 ℃ of aerated culture 24h, centrifugal collection wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=40 (dry mycelium 15.6g/L) in the described fermention medium of 1.0L step (1), at 28 ℃, blowing air amount 0.8vvm cultivates 84h down.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, and other gets a certain amount of precipitation and surveys content of cellulose, determines that dry mycelium content is 36.7g/L, residual Mierocrystalline cellulose 2.9g/L.Press acid heat-organic solvent method and extract grease, obtain grease 19.7g/L, fat content is 53.7%.
Embodiment 7
(1) fermention medium: Yu Shuizhong, Mierocrystalline cellulose 40g/L, pH 5.2 is transferred in containing in the enzymic fermentation supernatant after being resuspended in embodiment 1 step (4) thalline and separating.
(2) seed preparation: substratum is formed (g/L): glucose 20, yeast powder 10, peptone 10, pH 6.0, behind 121 ℃ of sterilization 15min, insert Cryptococcus curvatus ATCC 20509 (available from U.S. representative microbial culture presevation administrative centers), 30 ℃ of aerated culture 24h, centrifugal collection wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=20 (dry mycelium 0.36g/L) in the described fermention medium of 50mL step (1), cultivate 66h at 37 ℃ of following blowing airs.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, and other gets a certain amount of precipitation and surveys content of cellulose, determines that dry mycelium content is 15.1g/L, residual Mierocrystalline cellulose 6.2g/L.Press acid heat-organic solvent method and extract grease, obtain grease 7.2g/L, fat content is 47.7%.
The result of present embodiment shows that the lytic enzyme of carbohydrate polymer can recycle and reuse.This is because oil and fat accumulation cultivation stage mild condition helps enzyme activity and keeps.
Embodiment 8
(1) fermention medium preparation: reference literature (Zhang J, et a1.Bioresour Technol, 2011,102:4480-4488), be raw material with the straw, clean, drying was pulverized 60 mesh sieves, and flooded 18h with 2.5% sulfuric acid, behind 190 ℃ of steam treatment 3min, when being cooled to 50 ℃, adding water to solid concentration is 80g/L, transfer pH 5.2, add cellulase 1000U/L, zytase 800U/L obtains fermention medium.
(2) seed preparation: substratum is formed (g/L): glucose 20, yeast powder 10, peptone 10, pH 6.0, behind 121 ℃ of sterilization 15min, insert Lipomyces starkeyi AS 2.1560 (available from Chinese common micro-organisms culture presevation administrative center), 25 ℃ of aerated culture 40h, nutrient solution in the centrifugal 5min of 8000rpm, is obtained wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=15 (dry mycelium 0.27g/L) in the described fermention medium of 50mL step (1), cultivate 80h at 30 ℃ of following blowing airs.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, presses acid heat-organic solvent method and extracts grease, obtains grease 8.3g/L.
Embodiment 9
(1) fermention medium preparation: reference literature (Krishnan C, et al.Biotechnol Bioeng, 2010,107:441-450), be raw material with the dragon spruce, clean, drying was pulverized 60 mesh sieves, the ammoniacal liquor of 2: 1 (w/w) and dragon spruce, 140 ℃ of ammonia expansion process 30min, when being cooled to 50 ℃, adding water to solid concentration is 50g/L, transfer pH 4.8, add cellulase 600U/L, zytase 500U/L, glucuroide 300U/L.
(2) seed preparation: substratum is formed (g/L): glucose 80g/L, urea 3g/L, yeast powder 1g/L, dipotassium hydrogen phosphate 1g/L, behind 121 ℃ of sterilization 15min, insert Mortierella isabellina AS 3.3410 (available from Chinese common micro-organisms culture presevation administrative center), 25 ℃, 200rpm shaking culture 36h, centrifugal collection wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=10 (dry mycelium 0.2g/L) in the described fermention medium of 50mL step (1), cultivate 52h at 30 ℃ of following blowing airs.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, presses acid heat-organic solvent method and extracts grease, obtains grease 6.1g/L.
Comparative Examples 2
(1) fermention medium preparation: reference literature (Kim S, et al.Bioresour Technol, 2005,96:1994-2006), be raw material with the maize straw, clean, drying is crushed to 2.5mm, presses the 0.073g/g dry straw and adds mixing behind the calcium hydroxide, in 55 ℃ of 4 weeks of ventilation treatment, after removing the xylogen more than 80%, adding water to solid concentration is 70g/L, transfers pH 4.8, add cellulase 1050U/L, zytase 700U/L, glucuroide 300U/L, enzymic hydrolysis 72h under 50 ℃ of conditions, obtain that glucose is 32g/L in the hydrolyzed solution, wood sugar is 14g/L.
(2) seed preparation: substratum is formed (g/L): glucose 20, yeast powder 10, peptone 10, pH 6.0, behind 121 ℃ of sterilization 15min, insert Cryptococcus curvatus ATCC 20509 (available from U.S. representative microbial culture presevation administrative centers), 28 ℃ of aerated culture 24h, in the centrifugal 5min of 8000rpm, obtain wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=15 (dry mycelium 0.29g/L) in the described hydrolyzed solution of the described step of 50mL step (1) (1), cultivate 65h at 30 ℃ of following blowing airs, glucose is 1.5g/L during fermentation ends, and wood sugar is 8g/L.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, presses acid heat-organic solvent method and extracts grease, obtains grease 6.9g/L.
Embodiment 10
(1) fermention medium preparation: reference literature (Kim S, et al.Bioresour Technol, 2005,96:1994-2006), be raw material with the maize straw, clean, drying is crushed to 2.5mm, presses the 0.073g/g dry straw and adds mixing behind the calcium hydroxide, in 55 ℃ of 4 weeks of ventilation treatment, remove the xylogen more than 80% after, adding water to solid concentration is 70g/L, transfer pH 4.8, add cellulase 1050U/L, zytase 700U/L, glucuroide 300U/L.
(2) seed preparation: substratum is formed (g/L): glucose 20, yeast powder 10, peptone 10, pH 6.0, behind 121 ℃ of sterilization 15min, insert Cryptococcus curvatus ATCC 20509 (available from U.S. representative microbial culture presevation administrative centers), 28 ℃ of aerated culture 24h, in the centrifugal 5min of 8000rpm, obtain wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=15 (dry mycelium 0.29g/L) in the described fermention medium of 50mL step (1), cultivate 65h at 37 ℃ of following blowing airs.Detect less than glucose in the substratum behind the fermentation 65h, wood sugar is 0.8g/L.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, presses acid heat-organic solvent method and extracts grease, obtains grease 8.1g/L.
Compare found that of Comparative Examples 2 and embodiment 10, the maize straw zymin identical with vigor that functional quality equates improved 17% by technical scheme grease yield of the present invention.The maize straw that Comparative Examples 2 will be handled is at 4.8,50 ℃ of following enzymic hydrolysis 72h of pH, and glucose and xylose concentration are respectively 32g/L and 14g/L in the hydrolyzed solution, and at the produce oil cultivation stage, xylose utilization speed is slow, and residual wood sugar is 8g/L behind the fermentation 65h.Embodiment 9 is owing to hydrolysis and fermenting process coupling, exempted independent hydrolysis process; The more important thing is that do not detect glucose in the substratum behind the fermentation 65h, wood sugar is 0.8g/L, illustrates that technical scheme of the present invention has significantly improved the utilization of carbon source rate.Therefore, the present invention has the significant advantage that improves utilization of carbon source rate, grease yield and production efficiency.
Embodiment 11
(1) fermention medium preparation: reference literature (Tucker MP, et al.Appl Biochem Biotechnol, 2003,105:165-178), be raw material with the sawdust, clean, drying was pulverized 80 mesh sieves, the sulfuric acid dipping 4h with 0.98%, natural air drying is to water content 50% (w/w), after sulfuric acid concentration reaches 1.0%, 190 ℃ of processing 90s then, when being cooled to 50 ℃, adding water to solid concentration is 100g/L, transfers pH 4.5, add cellulase 1500U/L, zytase 500U/L.
(2) seed preparation: substratum is formed (g/L): glucose 20, yeast powder 20, peptone 10, behind 5.5,121 ℃ of the pH sterilization 20min, rhodotorula glutinis Rhodotorula glutinis AS 2.703 (available from Chinese common micro-organisms culture presevation administrative center), 30 ℃, 200rpm shaking culture 28h in the centrifugal 5min of 8000rpm, obtains wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=24 (dry mycelium 0.42g/L) in the described fermention medium of 50mL step (1), cultivate 72h at 35 ℃ of following blowing airs.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, presses acid heat-organic solvent method and extracts grease, obtains grease 9.1g/L.
Embodiment 12
(1) fermention medium preparation: Yu Shuizhong, ground corn 30g/L, high-temperature sterilization when treating that temperature is cooled to 30 ℃, adds amylase 450U/L, and saccharifying enzyme 300U/L transfers pH 6.5.
(2) seed preparation: substratum is formed (g/L): glucose 20, yeast powder 20, peptone 10, pH 5.5, behind 121 ℃ of sterilization 20min, white Cryptococcus Cryptococcus albidus ATCC56298 (available from U.S. representative microbial culture presevation administrative center), 22 ℃, 200rpm shaking culture 28h, centrifugal collection wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=10 (dry mycelium 0.22g/L) in the described fermention medium of 50mL step (1), cultivate 48h at 20 ℃ of following blowing airs.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, presses acid heat-organic solvent method and extracts grease, obtains grease 5.4g/L.
Embodiment 13
(1) fermention medium preparation: Yu Shuizhong, the cassava that 40g/L pulverizes, high-temperature sterilization when treating that temperature is cooled to 40 ℃, adds amylase 8 00U/L, and saccharifying enzyme 400U/L transfers pH 7.0.
(2) seed preparation: the growing microorganism stage cultivates and somatic cells is collected: the MSM minimum medium, with molasses as carbon source, behind 121 ℃ of sterilization 15min, insert opaque rhodococcus Rhodococcus opacus PD630 (German microbial strains preservation center), 30 ℃ of aerated culture 20h, nutrient solution in the centrifugal 15min of 10000rpm, is obtained wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=13 (dry mycelium 0.25g/L) in the described fermention medium of 50mL step (1), cultivate 40h at 45 ℃ of following blowing airs.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, presses acid heat-organic solvent method and extracts grease, obtains grease 5.1g/L.
Embodiment 14
(1) fermention medium preparation: reference literature (Krishnan C, et al.Biotechnol.Bioeng.2010,107:441-450), with bagasse is raw material, pulverizes 20 mesh sieves, adds water and becomes 150% (w/w) bagasse, mix ammoniacal liquor and bagasse by 2: 1 (w/w), 140 ℃ of ammonia expansion process 30min, when being cooled to 50 ℃, being adjusted to solid concentration is 70g/L, transfer pH 5.3, add cellulase 3500U/L, zytase 300U/L, glucuroide 2100U/L.
(2) seed preparation: substratum is formed (g/L): glucose 80, urea 3, yeast powder 1, dipotassium hydrogen phosphate 1, behind 121 ℃ of sterilization 15min, insert strong mould doughtily Geotrichum robustum CICC 1256 (available from Chinese industrial microbial strains preservation administrative center), 28 ℃ of shaking culture 24h, centrifugal collection wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=45 (dry mycelium 16.2g/L) in the described fermention medium of 1.0L step (1), under 34 ℃, blowing air is cultivated, air flow 1.0vvm, treat that the amount of bagasse reduces to below the 2g/L, divide and add the pretreated bagasse of 120g 3 times, to the 142h fermentation ends;
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, presses acid heat-organic solvent method and extracts grease, obtains grease 15.7g/L.
Embodiment 15
(1) fermention medium preparation: reference literature (Zhang J, et al.Bioresour Technol, 2011,102:4480-4488), be raw material with the straw, clean, drying was pulverized 60 mesh sieves, and flooded 18h with 2.5% sulfuric acid, behind 190 ℃ of steam treatment 3min, adding water to solid concentration is 80g/L, and reference literature (Tang Qi. Chongqing Jiaotong University's journal, 2008,27 (1): after method 148-151) is carried out dephosphorization treatment to pretreated straw, make phosphorus source content reduce to below 0.1% of carbon source total mass, when being cooled to 50 ℃, transfer pH 5.2, add cellulase 1000U/L, zytase 2400U/L.
(2) seed preparation: substratum is formed (g/L): glucose 20, yeast powder 10, peptone 10, pH 6.0, behind 121 ℃ of sterilization 15min, insert Lipomyces starkeyiAS 2.1560 (available from Chinese common micro-organisms culture presevation administrative center), 30 ℃ of aerated culture 40h, centrifugal collection wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=15 (dry mycelium 0.27g/L) in the described fermention medium of 50mL step (1), cultivate 80h at 30 ℃ of following blowing airs.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, presses acid heat-organic solvent method and extracts grease, obtains grease 8.7g/L.
Embodiment 16
(1) fermention medium preparation: reference literature (Kim S, et al.Bioresour Technol, 2005,96:1994-2006), with the maize straw is raw material, cleaning, drying are crushed to 2.5mm, press the 0.073g/g dry straw and add mixing behind the calcium hydroxide, in 55 ℃ of 4 weeks of ventilation treatment, remove the xylogen more than 80% after, adding water to solid concentration is 70g/L, reference literature (Li Zhu, Deng. gas chemical industry .2009, (34): after 24-26) the employing chemical precipitation method is carried out denitrogenation processing to raw material, make nitrogenous source content reduce to below 0.1% of carbon source total mass, when being cooled to 50 ℃, transfer pH 4.8, add cellulase 1400U/L, zytase 1400U/L.
(2) seed preparation: substratum is formed (g/L): glucose 20, yeast powder 10, peptone 10, pH 6.0, behind 121 ℃ of sterilization 15min, insert Cryptococcus curvatus ATCC 20509 (available from U.S. representative microbial culture presevation administrative centers), 28 ℃ of aerated culture 24h, centrifugal collection wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=15 (dry mycelium 0.29g/L) in the described fermention medium of 50mL step (1), cultivate 72h at 37 ℃ of following blowing airs.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, presses acid heat-organic solvent method and extracts grease, obtains grease 8.3g/L.
Embodiment 17
(1) fermention medium preparation: reference literature (Tucker MP, et al.Appl Biochem Biotechnol, 2003,105:165-178), (comprise corn with corn plants, corn cob and maize straw etc.) be raw material, clean, drying was pulverized 40 mesh sieves, the sulfuric acid dipping 4h with 0.98%, natural air drying is to water content 50% (w/w) then, after sulfuric acid concentration reaches 1.0%, 190 ℃ of processing 90s, when being cooled to 50 ℃, adding water to solid concentration is 80g/L, transfer pH 4.8, add cellulase 1600U/L, zytase 800U/L, amylase 8 00U/L, saccharifying enzyme 400U/L.
(2) seed preparation: substratum is formed (g/L): glucose 20, yeast powder 10, peptone 10, pH 6.0, behind 121 ℃ of sterilization 15min, insert Cryptococcus curvatus ATCC 20509 (available from U.S. representative microbial culture presevation administrative centers), 28 ℃ of aerated culture 24h, in the centrifugal 5min of 8000rpm, obtain wet thallus.
(3) oil and fat accumulation is cultivated: add step (2) gained thalline to inoculum density and reach OD600nm=15 (dry mycelium 0.29g/L) in the described fermention medium of 50mL step (1), cultivate 80h at 35 ℃ of following blowing airs.Detect less than glucose and wood sugar in the substratum behind the fermentation 80h.
(4) grease extracts: step (3) gained fermented liquid is centrifugal, and collecting precipitation is dried to constant weight under 105 ℃, presses acid heat-organic solvent method and extracts grease, obtains grease 9.0g/L.
Embodiment 18
Reference literature (Li YH, et al.Enzyme Microb Technol, 2007, method 41:312-317) obtains to extract microbial oil the thalline from embodiment 1.(lining is big for reference literature, Deng. the process engineering journal, 2007,7 (1), method 137-140) is got 0.3g grease transesterification preparation and purifying biological diesel oil under the enzyme catalysis condition, obtain 0.28g, biofuel yield 93%, the octane value of gained biofuel is 58, is suitable as the substitute of diesel oil.
Embodiment 19
Reference literature (Liu B, Zhao Z B.J Chem Technol Biotechnol, 2007,82 (8): method 775-780), the dry-matter 2.0g that obtains with embodiment 6 is a material, the preparation biofuel, get biofuel 1.0g, yield is 94%, and the octane value of gained biofuel is 57, is suitable as the substitute of diesel oil.
Claims (8)
1. the cultural method of an oleaginous microorganism, it is characterized in that: be the carbon source of liquid nutrient medium with the carbohydrate polymer, adding enzyme activity by every gram carbohydrate polymer is that the lytic enzyme and the dry mycelium quality of the carbohydrate polymer of 5～100U is the oleaginous microorganism viable bacteria body of 0.05～0.50 gram, 20～45 ℃ of temperature, pH 3.0～7.5, under the blowing air condition, fluid suspension culture.
2. in accordance with the method for claim 1, its feature also is: described carbohydrate polymer is that Mierocrystalline cellulose, hemicellulose, xylan, starch, major ingredient are that the biological material of Mierocrystalline cellulose and hemicellulose or major ingredient are the biological material of starch or the mixture more than two kinds in them.
3. according to claim 1 or 2 described methods, its feature also is: the lytic enzyme of described carbohydrate polymer is the enzyme with the polymerization degree that reduces carbohydrate polymer, comprises a kind of or combination more than two kinds in them of cellulase, hemicellulase, zytase, glucuroide, living diastatic enzyme, amylase, saccharifying enzyme.
4. in accordance with the method for claim 1, its feature also is: also can contain other microorganism culturing nutritive substance commonly used in the described oleaginous microorganism liquid nutrient medium, other microorganism culturing nutritive substance commonly used comprises that quality is the nitrogenous source of carbohydrate polymer total mass 0.1%～20%, phosphorus source that quality is carbohydrate polymer total mass 0.01%～15% or the quality sulphur source that is carbohydrate polymer total mass 0.01%～5% or two kinds or three kinds combination in them.
5. in accordance with the method for claim 2, its feature also is: described major ingredient is that the biological material of Mierocrystalline cellulose and hemicellulose is one or two or more kinds combination in maize straw, straw, straw, bagasse, corn cob, production of forestry tankage, pine, the dragon spruce.
6. in accordance with the method for claim 2, its feature also is: described major ingredient is that the biological material of starch is one or two or more kinds combination in corn, paddy, Chinese sorghum, wheat, barley, cassava, the sweet potato.
7. in accordance with the method for claim 1, it is characterized in that: described oleaginous microorganism can surpass the microorganism of dry cell weight 20% for thalline fat content after fermentation culture, comprise one or two or more kinds in the following microorganism, this in the saccharomyces oleaginosus reaches saccharomyces oleaginosus Lipomyces starkeyi; Spore Rhodosporidium toruloides of red winter of circle in the rhodotorula, rhodotorula glutinis Rhodotorula glutinis and shadow yeast Sporobolomyces roseus; Skin shape trichosporon Trichosporon cutaneum in the trichosporon and fermentable trichosporon Trichosporon fermentans; White Cryptococcus Cryptococcus albidus in the Cryptococcus and crooked Cryptococcus Cryptococcus curvatus; Christian Breton in the mould is intended endomyces Endomycopsis burtonii, strong mould doughtily Geotrichum robustum, Mortierella isabellina Mortierella isabellina and volume branch Mucor Mucor circinelloides; Excellent bacillus Croynebacterium, Nocardia bacteria Nocardia and rhodococcus Rhodococcus in the produce oil bacterium.
8. in accordance with the method for claim 1, its feature also is: the thalline accumulation that described oleaginous microorganism obtains after cultivating contains the grease of one or more longer chain fatty acids and derivative thereof, and fat content accounts for more than 20% of dry cell weight; The grease of accumulation in the described oleaginous microorganism thalline can be used as the raw material for preparing biofuel.
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