CN101294171A - Method for preparing microorganism with xylem filber raw material - Google Patents
Method for preparing microorganism with xylem filber raw material Download PDFInfo
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- CN101294171A CN101294171A CNA2008101146166A CN200810114616A CN101294171A CN 101294171 A CN101294171 A CN 101294171A CN A2008101146166 A CNA2008101146166 A CN A2008101146166A CN 200810114616 A CN200810114616 A CN 200810114616A CN 101294171 A CN101294171 A CN 101294171A
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- hydrolyzed solution
- wood fibre
- microbial oil
- fibre raw
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Abstract
A method for preparing microbial oils from wood fiber materials, which belongs to the field of biochemical technology, comprises the following steps: preparing hydrolysate from wood fiber materials by two hydrolysis steps, and preparing the microbial oils from the hydrolysate by two material feeding and fermenting processes. The method can prepare microbial oils with high output and content from the wood fiber materials. The fermentation substrate and the feeding substrate can be selected from the hydrolysate of the wood fiber materials, thus sufficiently utilizing the wood fiber resources and lowering the production cost.
Description
Technical field
The invention belongs to technical field of biochemical industry, particularly a kind of method for preparing microbial oil by the wood fibre raw material.
Background technology
Biofuel is the renewable new forms of energy of a kind of alternative fossil oil of growing up based on environmental degradation and energy dilemma in recent years, and biofuel is exactly a kind of renewable and clean energy resource with the potentiality that develop on a large scale very much.The domestic and international at present research for biodiesel manufacture mainly concentrates on vegetables oil or food and drink waste grease aspect, but utilize vegetables oil to be the raw materials cost height, its cost accounts for 70%～85% of total cost of production, with the food and drink abendoned oil is raw material, also exist raw material to be difficult to the difficulty of collecting and transporting, these have all seriously restricted the industrialization process of biofuel, and the raw material of therefore seeking a kind of cheapness becomes the key of biofuel industrialization.
Microbial oil (microbial oil) claims Unicell Oils and Fats (single cell oil) again, be under certain conditions by microorganisms such as yeast, mould, bacterium and algae, utilize carbohydrate, hydrocarbon polymer and common grease as carbon source, a large amount of greases that in thalline, produce.Biofuel by various animal and plant greases through esterification or transesterification metallization processes and get, and the lipid acid of most of microbial oil is formed and generally vegetables oil is close, so the alternative Vegetable oil lipoprotein production of microbial oil biofuel [Li Xiaosong, surplus setting sail. microbial oil. food science and technology, 1997, (5): 8-9.Das UN.Essential fatty acid metabolism in patients withessential hypertension, diabetes mellitus and coronary heart disease.Prostaglandins, Leukotrienes ﹠amp; Essential Fatty Acids, 1995,52 (6): 387-391.]
Lignocellulose is renewable resource abundant, the most cheap on the earth.Have data to show, the plant materials growing amount in whole world every year is up to 1.55 * 1011 tons of dry-matteies.The main component of wooden Biological resources is Mierocrystalline cellulose, hemicellulose and xylogen.Wherein, Mierocrystalline cellulose, hemicellulose are the sources of fermentable sugars, and content accounts for 66%-79% (the over-dry weight amount of cellulose raw material).China is a large agricultural country, if can be hydrolyzed into saccharification liquid to a large amount of agricultural fibre raw materials and the Mierocrystalline cellulose in the industrial fiber waste material economically, effectively, and be used, this will help improving present resource growing tension, the situation that environment is abominable day by day has important economy and social effect.
At present, the research of microbial oil mainly is that raw material adopted batch fermentation 5-7 days with glucose, and fat content can reach the 20-50% of dry cell weight.Therefore, still need to develop new technology, improve fat content, biomass, and to adopt cheap raw material be substrate, thereby reduce production costs.
Summary of the invention
The object of the present invention is to provide a kind ofly to prepare the method for microbial oil, improve fat content, biomass, and to adopt cheap raw material be substrate, thereby reduce production costs by the wood fibre raw material.
The present invention is equipped with hydrolyzed solution by the wood fibre raw material by two one-step hydrolysis legal systems, and hydrolyzed solution is through the method for twice fed-batch fermentation prepared microbial oil.Its concrete processing step is as follows:
1, with pulverizer wood fibre raw materials such as bagasse, corn cob is pulverized, then with the NaOH of 1-2% by liquid-solid ratio 15-25: 1 handles raw material 15-20 hour on vibrator, with deionized water raw material is cleaned the back oven dry again, with the 110-130 ℃ of hydrolysis of 1-2% sulfuric acid of the raw material after the oven dry, liquid-solid mass ratio: 8-10 in the hydrolyzation system: 1 (being mass percent) obtains hydrolyzed solution behind the hydrolysis 2-4h.
2, furfural is removed in the underpressure distillation of gained hydrolyzed solution, and hydrolyzed solution is concentrated into total sugar concentration at 30-50g/L, and regulating hydrolyzed solution pH with KOH is 6-7,
3, centrifuging and taking supernatant liquor adds ammonium sulfate or saltpetre and trace element solution according to C/N=40-50 (mass ratio), after dissolving fully, and 121 ℃ of sterilization 15min.
4, with rhodotorula glutinis, Si Dakaiyi yeast, mortierella, mortierella ramanniana etc. can produce the yeast or the mould of microbial oil and insert seed culture medium cultivation 24-30h, with (percent by volume) inoculum size access fermention medium of 5%-10%.25-35 ℃ of shake-flask culture temperature, shaking speed 150-250r/min.Fermentor cultivation pH=6.0-7.0, air flow 0.5-1.1vvm, mixing speed 300-600r/min.
5, fermentation culture to thalline logarithmic phase 24-48h and the fermentation later stage 60-96h carry out feed supplement, 1) or carbon source such as wood fibre matter hydrolyzed solution in substratum, adds the mixing solutions (wood sugar: glucose=2～2.5:, and carry out adding of nitrogenous sources such as sulfate of ammoniac or saltpetre simultaneously of 10g/L-20g/L glucose and wood sugar with C/N=40.
6, fermentation 72-144h finishes, and 4 ℃, the centrifugal thalline of 4000-5000r/min extracts microbial oil and measures.
Beneficial effect of the present invention
Adopt two one-step hydrolysis legal systems to be equipped with wood fibre raw material hydrolyzed solution and can effectively remove the murder by poisoning to thalli growth of acetate and furfural, wood fibre raw material hydrolyzed solution can prepare the microbial oil of high level and output through twice fed-batch fermentation technology.Fermentation substrate and current adding substrate all can adopt wood fibre raw material hydrolyzed solution, have made full use of wood fibre matter resource, have reduced production cost.
Embodiment
The invention provides and a kind ofly prepare the method for microbial oil by the wood fibre raw material, technology comprises: be equipped with hydrolyzed solution by the wood fibre raw material by two one-step hydrolysis legal systems, and hydrolyzed solution is through the method for twice fed-batch fermentation prepared microbial oil.
Lifting specific embodiment is below again further specified the present invention.
Example one:
1. bagasse pre-treatment
The bagasse of getting certainweight 20～40 orders oven dry constant weight is in Erlenmeyer flask, and the NaOH with 1% (massfraction) on vibrator handles bagasse 20 hour by liquid-solid ratio at 25: 1 earlier, and temperature is fixed as 30 ℃, and the vibrator rotating speed is 140r/min.After pre-treatment finishes, with deionized water wash and suction filtration, will wash to neutral bagasse 105 ℃ of baking 4h in air dry oven, and be cooled to room temperature in the moisture eliminator, it is standby to place sample sack to preserve.Bagasse after the oven dry is carried out dilute acid hydrolysis.Sulfuric acid concentration=1.24%, time=2.64h, temperature=123 ℃, liquid-solid ratio stuck-at-0: 1.Furfural is removed in the underpressure distillation of gained hydrolyzed solution, and hydrolyzed solution is concentrated into total sugar concentration about 30g/L, and regulating hydrolyzed solution pH with KOH is 6, and 3000r/min is centrifugal.
2. fermentation
(1) bacterial classification: glutinous rhodotorula (Rhodotorula gutini As2.107), available from Chinese science research institute institute of microbiology.
(2) substratum:
1. solid slant culture base
Yeast powder: 10g/L; Peptone: 20g/L; Glucose: 20g/L; Agar: 20g/L; PH=5.
2. seed culture medium
Glucose: 15g/L; (NH
4)
2SO
4: 2g/L; Yeast powder: 1g/L; KH
2PO
4: 7g/L; Na
2HPO
4: 2g/L; MgSO
4: 1.5g/L; PH=5.5.
3. fermention medium: add KNO in the centrifugal gained supernatant liquor in the step 1
3: 3.8g/L; Yeast powder: 4g/L; KH
2PO
4: 6g/L; Na
2HPO
4: 2g/L; MgSO
4: 2g/L; PH=6,115 ℃ of sterilization 20min.
(3) fermentation mode: the bacterial classification of solid slant culture is connected to cultivates 24h in the seed culture medium, be forwarded to fermention medium by 5% inoculum size then, thalline is carried out fermentation culture.500ml shakes bottled amount 100ml, 30 ℃ of culture temperature, shaking speed 150r/min.Fermentation 24h and 60h feed supplement twice respectively adds 10g/l wood sugar and glucose mixing solutions (2.5: 1), adds saltpetre with C/N=40 simultaneously.
(4) fermentation result,
After 72h had fermented, its biomass reached 15.1g/L, and fat content reaches 32.5%.
Embodiment two
1. bagasse pre-treatment
The bagasse of getting certainweight 20～40 orders oven dry constant weight is in Erlenmeyer flask, and the NaOH with 1% (massfraction) on vibrator handles bagasse 20 hour by liquid-solid ratio at 25: 1 earlier, and temperature is fixed as 30 ℃, and the vibrator rotating speed is 140r/min.After pre-treatment finishes, with deionized water wash and suction filtration, will wash to neutral bagasse 105 ℃ of baking 4h in air dry oven, and be cooled to room temperature in the moisture eliminator, it is standby to place sample sack to preserve.Bagasse after the oven dry is carried out dilute acid hydrolysis.Sulfuric acid concentration=1.24%, time=2.64h, temperature=123 ℃, liquid-solid ratio stuck-at-0: 1.Furfural is removed in the underpressure distillation of gained hydrolyzed solution, and hydrolyzed solution is concentrated into total sugar concentration about 50g/L, and regulating hydrolyzed solution pH with KOH is 6, and 3000r/min is centrifugal.
3. fermentation
(5) bacterial classification: glutinous rhodotorula (Rhodotorula gutini As2.107), available from Chinese science research institute institute of microbiology.
(6) substratum:
1. solid slant culture base
Yeast powder: 10g/L; Peptone: 20g/L; Glucose: 20g/L; Agar: 20g/L; PH=5.
2. seed culture medium
Glucose: 15g/L; (NH
4)
2SO
4: 2g/L; Yeast powder: 1g/L; KH
2PO
4: 7g/L; Na
2HPO
4: 2g/L; MgSO
4: 1.5g/L; PH=5.5.
3. fermention medium: add KNO in the centrifugal gained supernatant liquor in the step 1
3: 3.8g/L; Yeast powder: 4g/L; KH
2PO
4: 6g/L; Na
2HPO
4: 2g/L; MgSO
4: 2g/L; PH=6,115 ℃ of sterilization 20min.
(7) fermentation mode: the bacterial classification of solid slant culture is connected to cultivates 24h in the seed culture medium, be forwarded to fermention medium by 5% inoculum size then, thalline is carried out fermentation culture.1L fermentor tank loading amount 750ml, 30 ℃ of culture temperature, shaking speed 550r/min.Air flow 1.0vvm.Fermentation 36h and 96h feed supplement twice respectively, adding total sugar concentration is 15g/l bagasse hydrolyzed solution, adds saltpetre with C/N=40 simultaneously.
(8) fermentation result,
After 132h had fermented, its biomass reached 17.5g/L, and fat content reaches 52.1%.
Embodiment three
1. corn cob pre-treatment
The corn cob raw material of getting certainweight 20～40 orders oven dry constant weight is in Erlenmeyer flask, and the NaOH with 1% (massfraction) on vibrator handles bagasse 20 hour by liquid-solid ratio at 25: 1 earlier, and temperature is fixed as 30 ℃, and the vibrator rotating speed is 140r/min.After pre-treatment finishes, with deionized water wash and suction filtration, will wash to neutral corn cob raw material 105 ℃ of baking 4h in air dry oven, and be cooled to room temperature in the moisture eliminator, it is standby to place sample sack to preserve.Bagasse after the oven dry is carried out dilute acid hydrolysis.Sulfuric acid concentration=1.15%, time=1h, temperature=115 ℃, liquid-solid ratio stuck-at-0: 1.Furfural is removed in the underpressure distillation of gained hydrolyzed solution, and hydrolyzed solution is concentrated into total sugar concentration about 50g/L, and regulating hydrolyzed solution pH with KOH is 6, and 3000r/min is centrifugal.
2. fermentation
(1) bacterial classification: glutinous rhodotorula (Rhodotorula gutiniAs2.107), available from Chinese science research institute institute of microbiology.
(2) substratum:
1. solid slant culture base
With embodiment 1
2. seed culture medium
With embodiment 1
3. fermention medium: add KNO in the centrifugal gained supernatant liquor in the step 1
3: 3.8g/L; Yeast powder: 4g/L; KH
2PO
4: 6g/L; Na
2HPO
4: 2g/L; MgSO
4: 2g/L; PH=6,115 ℃ of sterilization 20min.
3. fermentation mode: the bacterial classification of solid slant culture is connected to cultivates 24h in the seed culture medium, be forwarded to fermention medium by 5% inoculum size then, thalline is carried out fermentation culture.1L fermentor tank loading amount 750ml, 30 ℃ of culture temperature, shaking speed 550r/min.Air flow 1.0vvm.Fermentation 36h and 96h feed supplement twice respectively, adding total sugar concentration is 15g/l corn cob hydrolyzed solution, adds saltpetre with C/N=40 simultaneously.
4. fermentation result,
After 132h had fermented, its biomass reached 14.5g/L, and fat content reaches 42.5%.
Claims (6)
1, a kind ofly prepares the method for microbial oil, it is characterized in that technology comprises: be equipped with hydrolyzed solution by the wood fibre raw material by two one-step hydrolysis legal systems, and hydrolyzed solution is through twice fed-batch fermentation prepared microbial oil by the wood fibre raw material.
2, in accordance with the method for claim 1, it is characterized in that: describedly comprise by the processing step that two one-step hydrolysis legal systems are equipped with hydrolyzed solution by the wood fibre raw material:
(1) with pulverizer wood fibre raw materials such as bagasse, corn cob is pulverized before the hydrolysis, NaOH with 1-2% presses liquid-solid ratio 15-25 then: 1 handles the wood fibre raw material 15-20 hour on vibrator, with deionized water raw material is cleaned the back oven dry again, with the 110-130 ℃ of hydrolysis of 1-2% sulfuric acid of the raw material after the oven dry, liquid-solid mass ratio: 8-10 in the hydrolyzation system: 1, obtain hydrolyzed solution behind the hydrolysis 2-4h.
(2) furfural is removed in the underpressure distillation of gained hydrolyzed solution, and hydrolyzed solution is concentrated into total sugar concentration at 30-50g/L, and regulating hydrolyzed solution pH with KOH is 6-7.
3. it is characterized in that in accordance with the method for claim 1: described wood fibre raw material hydrolyzed solution through the processing step of twice fed-batch fermentation prepared microbial oil is:
(1) centrifuging and taking supernatant liquor adds ammonium sulfate or saltpetre and trace element solution according to the C/N=40-50 mass ratio, after dissolving fully, and 121 ℃ of sterilization 15min
(2) with rhodotorula glutinis, Si Dakaiyi yeast, mortierella, mortierella ramanniana can produce the yeast or the mould of microbial oil and insert seed culture medium cultivation 24-30h, with the percent by volume inoculum size access fermention medium of 5%-10%; 25-35 ℃ of shake-flask culture temperature, shaking speed 150-250r/min; Fermentor cultivation pH=6.0-7.0, air flow 0.5-1.1vvm, mixing speed 300-600r/min;
(3) fermentation culture to thalline logarithmic growth later stage 24-48h and the fermentation later stage 60-96h carry out feed supplement, in substratum, add the mixing solutions or the wood fibre raw material hydrolyzed solution carbon source of 10g/L-20g/L glucose and wood sugar, and carry out adding of sulfate of ammoniac or saltpetre nitrogenous source simultaneously with the C/N=40-50 mass ratio;
(4) fermentation 72-144h finishes, and 4 ℃, the centrifugal thalline of 4000-5000r/min extracts microbial oil and measures.
4. in accordance with the method for claim 1, it is characterized in that: the described bacterial strain that is used to produce microbial oil comprises: the Si Dakaiyi yeast, mortierella, mortierella ramanniana can produce the yeast or the mould of microbial oil.
5. it is characterized in that in accordance with the method for claim 1: described wood fibre raw material comprises bagasse, corn cob, sorghum rod or stalk.
6. it is characterized in that in accordance with the method for claim 1: described wood sugar: glucose=2-2.5: 1.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102135509A (en) * | 2011-01-13 | 2011-07-27 | 浙江大学 | Method for measuring characteristic molecule group in bio-oil |
| CN103627513A (en) * | 2013-12-02 | 2014-03-12 | 东北农业大学 | Fed-batch pretreatment solid-state fermentation auxiliary cold-pressing oil extraction process for oil crops |
| CN104263771A (en) * | 2014-09-16 | 2015-01-07 | 清华大学 | Method for producing microbial oils by using non-detoxified cellulosic hydrolysate |
| CN104388484A (en) * | 2014-11-18 | 2015-03-04 | 清华大学 | Method for fermenting and producing microbial grease by adopting volatile fatty acid as raw material |
| CN107828829A (en) * | 2017-12-09 | 2018-03-23 | 四川金英科技有限责任公司 | A kind of method using low value biomass ferment microbial grease |
| CN112522126A (en) * | 2020-12-29 | 2021-03-19 | 南京理工大学 | High-yield microbial oil-and-fat rhodotorula genetic engineering bacteria and construction and culture method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101153299B (en) * | 2007-08-31 | 2010-06-02 | 清华大学 | Method for high production of microorganism grease by stuffing batch culture |
-
2008
- 2008-06-05 CN CN2008101146166A patent/CN101294171B/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102135509A (en) * | 2011-01-13 | 2011-07-27 | 浙江大学 | Method for measuring characteristic molecule group in bio-oil |
| CN103627513A (en) * | 2013-12-02 | 2014-03-12 | 东北农业大学 | Fed-batch pretreatment solid-state fermentation auxiliary cold-pressing oil extraction process for oil crops |
| CN104263771A (en) * | 2014-09-16 | 2015-01-07 | 清华大学 | Method for producing microbial oils by using non-detoxified cellulosic hydrolysate |
| CN104388484A (en) * | 2014-11-18 | 2015-03-04 | 清华大学 | Method for fermenting and producing microbial grease by adopting volatile fatty acid as raw material |
| CN104388484B (en) * | 2014-11-18 | 2018-04-10 | 清华大学 | A kind of method that microbial grease is produced using volatile fatty acid as fermenting raw materials |
| CN107828829A (en) * | 2017-12-09 | 2018-03-23 | 四川金英科技有限责任公司 | A kind of method using low value biomass ferment microbial grease |
| CN112522126A (en) * | 2020-12-29 | 2021-03-19 | 南京理工大学 | High-yield microbial oil-and-fat rhodotorula genetic engineering bacteria and construction and culture method thereof |
| CN112522126B (en) * | 2020-12-29 | 2022-07-22 | 南京理工大学 | High-yield microbial oil-and-fat rhodotorula genetic engineering bacteria and construction and culture method thereof |
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