CN103045491A - Mixed culture method of microorganism containing oil - Google Patents
Mixed culture method of microorganism containing oil Download PDFInfo
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- CN103045491A CN103045491A CN2011103138490A CN201110313849A CN103045491A CN 103045491 A CN103045491 A CN 103045491A CN 2011103138490 A CN2011103138490 A CN 2011103138490A CN 201110313849 A CN201110313849 A CN 201110313849A CN 103045491 A CN103045491 A CN 103045491A
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Abstract
The invention discloses a mixed culture method of a microorganism containing oil, comprising the following steps: inoculating a bioreactor containing mixed culture medium with a yeast seed solution containing oil and an autotrophic microalgae seed solution for secondary enlarging culture, wherein the yeast seed solution containing oil is obtained by primary culture, and the main component of the mixed culture medium is the basic culture medium needed by the yeast cells; adding inorganic salts and trace elements needed for the growth of the microalgae; adding the yeast fermentation solution containing oil and the microalgae fermentation solution into the same bioreactor for mixed culture when the OD value of the oil-containing yeast fermentation solution subjected to enlarging culture is 5-15 and the OD value of the microalgae fermentation solution is 2.0-10. The mixed culture method has the advantages that the harvest yield of microbial cells and the content of oil in the microorganism are improved, the cost is reduced and the culture device is simplified.
Description
Technical field
The invention belongs to biological technical field, relate in particular to and a kind ofly utilize saccharomyces olei and autotrophy oil-producing microalgae to carry out mixed culture to obtain the method that high cell harvesting amount is gathered in the crops microbial oil simultaneously.
Background technology
Along with day by day highlighting and problem of environmental pollution more outstanding of the rising steadily of day by day in short supply, the oil price of petroleum resources, oil product imbalance between supply and demand, developing by all kinds of means renewable grease resource becomes inevitable.From the diesel oil of microbial oil have that energy density height, sulphur content are low, the performance such as sufficient combustion, oilness are good, also have the characteristics such as renewable, readily biodegradable, storing and transporting security, the capability of antidetonance be good, can be used as the substitute of fossil energy.
Document shows that under suitable condition, the grease of certain micro-organisms production and storage accounts for more than 20% of its biological total amount, and this quasi-microorganism is called as the oil-containing microorganism.The bacterial strain of producing grease is arranged in known bacterium, yeast, mould, the algae.Microbial oil claims again Unicell Oils and Fats, and its lipid acid forms similar to general Vegetable oil lipoprotein, is lipid acid with C16, C18, is main such as saturated and undersaturated lipid acid such as palmitinic acid, stearic acid, oleic acid and linolic acid.Microbial fermentation utilizes the substrate scope wider, can directly utilize glucose, fructose, sucrose, molasses, starch and cellulosic hydrolysate etc.New grease production method not only can be provided, and can utilize cheap abandoned biomass, reduce the grease production cost, protection of the environment.Therefore, microbial oil is potential animal-plant oil alternate resources.Have broad application prospects.
Except saccharomyces olei, little algae is an other class oil-containing microorganism, and the nutritive ingredients (such as spirulina) such as little algae rich in proteins, polysaccharide, unsaturated fatty acids can be used for food, medicine and energy aspect; Can accumulate in a large number lipid acid, slightly algae such as chlorella are arranged, its body fat acid content can account for 30%～60% of dry weight.Utilize the little algae of cultivation to accumulate oil resource, become and utilized at present the most popular research field of solar energy development renewable resources.Not only have powerful market potential, and have outstanding social value.
It is fermented bacterium (CN200610113582.X) that the cultivation of microbial fermentation grease and preparation method generally adopt yeast, carries out biomass accumulation through conventional microorganism culturing, obtains the oil-containing microbial cells, and then processes this thalline, obtains bio-oil.This technical process adopts single microorganism fermentation culture process, and its bio-oil is accumulated in the cell, and the cell harvesting amount is the key factor of restriction grease harvest yield.
The microalgae cell growth pattern divides and is generally two kinds of light autotrophy and heterotrophism carbon sources, and light autotrophy process will consume CO
2, CO
2Effective utilization absorb, be the key of culture effect of realizing ideal, exist simultaneously and replenish CO
2Produce O with photosynthesis
2Desorb, the problem of discharge.Liu Jianguo etc. " microalgae mass cultivate pipeline bioreactor " (CN200410020978.0) and the Miao JianRen wait " a kind of little algae industrial production photosynthetic organism reactor assembly " (CN03128138.9) all to adopt a kind of installation method of adding in photo-bioreactor system, realize CO
2Supply, can realize also that simultaneously certain oxygen resolves effect.The heterotrophic growth process is to utilize organic carbon source to be the CO in the alternative autotrophy process of substrate
2Carry out the accumulation of little algal biomass, the Growth of Cells speed, but the oil and fat accumulation level lacks comparatively lower in the cell.
Carry out CO for yeast microorganism cultivation production grease and the little algae of autotrophy in the prior art
2Supply and oxygen resolving are produced the oil-containing micro-algae biomass to be had related to obtain the aspects such as grease, but all be that the oil-containing microorganism is cultivated separately, realize that for the collaborative training method of the mixing between the dissimilar oil-containing microorganisms research of the aspects such as high-efficiency grease results then has no report.In the oil-containing microbial cultivation process ubiquity that cost is high, slow, the deficiency such as fat content is low of microorganism cells accumulative total.
Summary of the invention
For the deficiencies in the prior art, the invention provides the method that a kind of saccharomyces olei and little algae carry out mixed culture, the method has the microorganism cells harvest yield of raising and microbial oil content, reduces cost, simplifies the advantages such as culture apparatus.
A kind of method of oil-containing microorganism mixed culture, comprise following content: will cultivate the saccharomyces olei seed liquor that obtains and the little algae seed liquor of autotrophy through one-level and be linked into respectively and carry out the secondary enlarged culturing in the bio-reactor that contains mixed culture medium, described mixed culture medium composition is take the required basic medium of yeast cell as main, add simultaneously the required inorganic salt of micro algae growth and trace element, when the saccharomyces olei fermented liquid OD of enlarged culturing value is 5-15, when little algae fermented liquid OD value is 2.0-10, saccharomyces olei fermented liquid and little algae fermented liquid is linked into carries out mixed culture in the same bio-reactor.
Enlarged culturing described in the inventive method and mixed culture condition are as follows: 25 ℃ ~ 30 ℃ of leavening temperatures; Air flow 0.1 vvm ~ 1.0 vvm, mixing speed 100 rpm ~ 400rpm, pH 6.5 ~ 7.5, and the organic carbon source mass concentration is 1.0% ~ 2.0%.
In the inventive method, the saccharomyces olei fermented liquid that adds during mixed culture and little algae fermented liquid can any volume mixture, and preferred volume ratio is 2:1～1:2.
In the inventive method, yeast comprises
S.cerevisiaeYeast saccharomyces cerevisiae,
R.glutinisRhodotorula glutinis,
Trichosporon cutaneumTrichosporon cutaneum etc.Little algae comprises chlorella, grape algae, little ring algae, diatom etc.
In the inventive method, bioreactor can be the closed reactors such as board-like, tubular type, airlift agitation formula.Mainly take the air that passes into as back-mixing power, when the air that passes into enters bioreactor, a plurality of auxiliary points preferably evenly are set and baffle plate is set in reactor, realize uniform gas distribution.This reactor also is provided with the determinator of culture system carbonic acid gas and dissolved oxygen content, adjusts as required air flow, to obtain good effect.
PH control acid neutralizing agent is HCL, H in present method
2SO
4, HNO
3Etc. mineral acid commonly used, the alkali neutralizing agent is NaOH, NaHCO
3Deng mineral alkali.
In the inventive method, described organic carbon source can be glucose, fructose, starch, cellulosic hydrolysate etc., the adding employing stream of this organic carbon source adds arbitrary way to carry out, according to nutrient solution sample analysis of components, the organic carbon source concentration level of detection system carries out stream with the organic carbon source mother liquor that configures by spending rate and adds at any time.
Compared with prior art, the method for a kind of oil-containing microorganism of the present invention mixed culture has following advantage:
1, in general, different microorganisms is difficult to carry out mixed culture in same reactor, this is not only because its culture condition of different microorganisms is different, and also can mutually restrict between the microorganism, the inventive method is mixed the first order seed nutrient solution of saccharomyces olei and the little algae of autotrophy respectively and is carried out the secondary enlarged culturing with mixed culture medium, and enlarged culturing saccharomyces olei fermented liquid and little algae fermented liquid that will be in the certain growth stage carry out mixed culture, make the enhancing of two kinds of compatible property of oleaginous microorganism of the little algae of saccharomyces olei and autotrophy, therefore can in same reactor, carry out better mixed culture;
2, in the inventive method, the carbonic acid gas that is in the saccharomyces olei release of proper growth phase in the mixed cultivation process can be absorbed fast by the little algae of autotrophy, two kinds of oil-containing microorganism advantage iuntercellulars produce the dependence of symbiosis, set up stable culture system, realized the efficient process of growth of two kinds of oil-containing microorganisms, improved the accumulation content of grease, test-results shows that unit stem cell grease harvest yield is brought up to 5.12g/L from 2.58g/L respectively under the same terms;
3, the inventive method obviously improved oil-containing micro-algae culture efficiency and quality, greatly reduce production cost, can for the preparation biofuel high-quality oil-containing microorganism raw material is provided, be suitable for industrial application.
Description of drawings
Fig. 1 is a kind of concrete technology schematic flow sheet of the present invention.
Wherein: the little algae seed of 1-, the 2-yeast starter, the little algae of 3-is cultivated A reactor, 4-yeast culture A reactor, the little algae of 5-is cultivated second reactor, 6-yeast culture second reactor, 7-mixed culture reactor, the agent of 8-acid-base neutralisation, 9-inlet mouth, the outlet of 10-mixed culture tail gas.
Embodiment
Further specify process and the effect of the inventive method below in conjunction with embodiment, but be not limited to following examples.
In present method, used medium composed as follows:
Micro-algae culture medium (in every liter):
| NaNO 3 | 0.20g |
| K 2HPO 4 . 3H 2O | 0.07g |
| MgSO 4 . 7H 2O | 0.07g |
| CaCl 2 . 2H 2O | 0.03g |
| KH 2PO 4 | 0.18g |
| NaCl | 0.03 |
| Soil extract (soil extract) | 40mL |
| FeCl 3·6H 2O | 0.01 |
| Fe—EDTA | 1mL |
Secondary enlarged culturing and mixed culture used medium: (in every liter):
| Glucose | 20.00g |
| Potato liquid | 1000mL |
| NaNO 3 | 0.20g |
| K 2HPO 4 . 3H 2O | 0.07g |
| MgSO 4 . 7H 2O | 0.07g |
| CaCl 2 . 2H 2O | 0.03g |
| KH 2PO 4 | 0.18g |
| NaCl | 0.03 |
| Soil extract (soil extract) | 40mL |
| FeCl 3·6H 2O | 0.01 |
| Fe—EDTA | 1mL |
Yeast culture base: (in every liter):
Glucose 20.0g, potato liquid 1000mL, pH nature.The wherein making of potato nutritional liquid: boiled 20 ~ 40 minutes after the 200g potato is cut into small pieces, the multilayer filtered through gauze is settled to 1000.0mL.
Embodiment 1
Use in shaking flask rhodotorula glutinis and chlorella separately that substratum carries out single culture, obtain the OD value and be respectively 10 and 6 seed liquor.With cultured rhodotorula glutinis seed liquor, chlorella seed liquid is 10% to be linked into respectively in the 10L bio-reactor by inoculum size (V/V), all add mixed culture medium and carry out enlarged culturing, concrete culture condition is as follows: 30 ℃ of leavening temperatures, air flow (air) 0.4 vvm, mixing speed 200 rpm, pH7.0, the organic carbon source mass concentration is 1.5%, when the rhodotorula glutinis fermented liquid OD value that detects enlarged culturing is 5.0, chlorella fermented liquid OD value is 2.0 o'clock, two kinds of fermented liquids are changed over to carry out mixed culture in the 20L bioreactor, culture condition is identical with the enlarged culturing condition, continues to cultivate 5 days end fermenting processs.Collect microorganism cells, survey dry weight and fat content, obtain grease harvest yield; Measure the organic carbon source Expenditure Levels, the amount that consumes organic carbon source according to culturing process is calculated carbon oil transformation efficiency index.
Embodiment 2
When the rhodotorula glutinis fermented liquid OD value that detects enlarged culturing is 5.0, chlorella fermented liquid OD value is 10.0 o'clock, two kinds of fermented liquids is changed over to carry out mixed culture in the 20L bioreactor, and all the other conditions are with embodiment 1.Collect microorganism cells, survey dry weight and fat content, obtain grease harvest yield; Measure the organic carbon source Expenditure Levels, the amount that consumes organic carbon source according to culturing process is calculated carbon oil transformation efficiency index.
Embodiment 3
When the rhodotorula glutinis fermented liquid OD value that detects enlarged culturing is 10.0, chlorella fermented liquid OD value is 2.0 o'clock, two kinds of fermented liquids is changed over to carry out mixed culture in the 20L bioreactor, and all the other conditions are with embodiment 1.Collect microorganism cells, survey dry weight and fat content, obtain grease harvest yield; Measure the organic carbon source Expenditure Levels, the amount that consumes organic carbon source according to culturing process is calculated carbon oil transformation efficiency index.
Embodiment 4
When the rhodotorula glutinis fermented liquid OD value that detects enlarged culturing is 10.0, chlorella fermented liquid OD value is 10.0 o'clock, two kinds of fermented liquids is changed over to carry out mixed culture in the 20L bioreactor, and all the other conditions are with embodiment 1.Collect microorganism cells, survey dry weight and fat content, obtain grease harvest yield; Measure the organic carbon source Expenditure Levels, the amount that consumes organic carbon source according to culturing process is calculated carbon oil transformation efficiency index.
Embodiment 5
When the rhodotorula glutinis fermented liquid OD value that detects enlarged culturing is 15.0, chlorella fermented liquid OD value is 2.0 o'clock, two kinds of fermented liquids is changed over to carry out mixed culture in the 20L bioreactor, and all the other conditions are with embodiment 1.Collect microorganism cells, survey dry weight and fat content, obtain grease harvest yield; Measure the organic carbon source Expenditure Levels, the amount that consumes organic carbon source according to culturing process is calculated carbon oil transformation efficiency index.
Embodiment 6
When the rhodotorula glutinis fermented liquid OD value that detects enlarged culturing is 15.0, chlorella fermented liquid OD value is 10.0 o'clock, two kinds of fermented liquids is changed over to carry out mixed culture in the 20L bioreactor, and all the other conditions are with embodiment 1.Collect microorganism cells, survey dry weight and fat content, obtain grease harvest yield; Measure the organic carbon source Expenditure Levels, the amount that consumes organic carbon source according to culturing process is calculated carbon oil transformation efficiency index.
Comparative example 1
When the rhodotorula glutinis fermented liquid OD value that detects enlarged culturing is 5.0, do not carry out mixed culture among the embodiment 1, but continue fermentation 5 days separately.Collect microorganism cells, survey dry weight and fat content, obtain grease harvest yield; Measure the organic carbon source Expenditure Levels, the amount that consumes organic carbon source according to culturing process is calculated carbon oil transformation efficiency index.
Comparative example 2
When the chlorella fermented liquid OD value that detects enlarged culturing is 2.0, do not carry out mixed culture among the embodiment 1, but continue fermentation 5 days separately.Collect microorganism cells, survey dry weight and fat content, obtain grease harvest yield; Measure the organic carbon source Expenditure Levels, the amount that consumes organic carbon source according to culturing process is calculated carbon oil transformation efficiency index.
Above-described embodiment experimental result such as following table:
| Scheme | The microorganism dry weight | Oil quality content | Grease harvest yield | The carbon oil transformation efficiency, % |
| Embodiment 1 | 14.70g/L | 34.8% | 5.12g/L | 5.70 |
| Embodiment 2 | 15.20g/L | 34.4% | 5.23g/L | 5.82 |
| Embodiment 3 | 16.00g/L | 35.0% | 5.60g/L | 6.00 |
| Embodiment 4 | 16.60g/L | 34.8% | 5.78g/L | 6.19 |
| Embodiment 5 | 14.36g/L | 34.0% | 4.88g/L | 5.23 |
| Embodiment 6 | 16.80g/L | 34.6% | 5.81g/L | 6.23 |
| Comparative example 1 | 7.02g/L | 31.2% | 2.19g/L | 2.31 |
| Comparative example 2 | 5.21g/L | 7.4% | 0.39g/L | 0.43 |
Can find out that from above-mentioned data the inventive method (scheme 1 ~ 6) has greatly improved the harvest yield of oil-containing microorganism cells, fat content also is improved.Therefore adopt the inventive method, under identical culture condition, compare with oil-containing microorganism single culture method, through mixed culture, the grease harvest yield that obtains has obtained larger raising with two kinds of oil-containing microorganisms.The carbon oil transformation efficiency of mixed culture is improved significantly.
Claims (7)
1. the method for an oil-containing microorganism mixed culture, it is characterized in that: comprise following content: will cultivate the saccharomyces olei seed liquor that obtains and the little algae seed liquor of autotrophy through one-level and be linked into respectively and carry out the secondary enlarged culturing in the bio-reactor that contains mixed culture medium, described mixed culture medium composition is take the required basic medium of yeast cell as main, add simultaneously the required inorganic salt of micro algae growth and trace element, when the saccharomyces olei fermented liquid OD of enlarged culturing value is 5-15, when little algae fermented liquid OD value is 2.0-10, saccharomyces olei fermented liquid and little algae fermented liquid is joined carry out mixed culture in the same bio-reactor.
2. method according to claim 1, it is characterized in that: described enlarged culturing and mixed culture condition are as follows: 25 ℃ ~ 30 ℃ of leavening temperatures; Air flow 0.1 vvm ~ 1.0 vvm, mixing speed 100 rpm ~ 400rpm, pH 6.5 ~ 7.5, and the organic carbon source mass concentration is 1.0% ~ 2.0%.
3. method according to claim 1 is characterized in that: the saccharomyces olei fermented liquid that adds during described mixed culture and little algae fermentating liquid volume are than being 2:1～1:2.
4. method according to claim 1, it is characterized in that: described yeast comprises
S.cerevisiaeYeast saccharomyces cerevisiae,
R.glutinisRhodotorula glutinis,
Trichosporon cutaneumTrichosporon cutaneum.
5. method according to claim 1, it is characterized in that: described little algae comprises chlorella, grape algae, little ring algae, diatom.
6. method according to claim 1 is characterized in that: described bioreactor is board-like, tubular type or airlift agitation formula.
7. method according to claim 1, it is characterized in that: described organic carbon source is glucose, fructose, starch, cellulosic hydrolysate, this organic carbon source adopts stream to add arbitrary way to carry out.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560749A (en) * | 2013-10-29 | 2015-04-29 | 中国石油化工股份有限公司 | Method for open-type fast cultivation of oil-bearing microbes |
| CN106754383A (en) * | 2016-11-14 | 2017-05-31 | 华南理工大学 | A kind of method for improving microbes biomass and grease yield |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102080119A (en) * | 2009-11-26 | 2011-06-01 | 北京化工大学 | Method for producing oil by mixed culture of yeast and alga |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102080119A (en) * | 2009-11-26 | 2011-06-01 | 北京化工大学 | Method for producing oil by mixed culture of yeast and alga |
Non-Patent Citations (3)
| Title |
|---|
| CHEIRSILP B ET AL: "Mixed culture of oleaginous yeast Rhodotorula glutinis and microalga chlorella vulgaris for lipid production from industrial wastes and its use as biodiesel feedstock", 《NEW BIOTECHNOLOGY》, vol. 28, no. 4, 19 January 2011 (2011-01-19) * |
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| 苗金鑫 等: "粘红酵母和钝顶螺旋藻混合培养生产微生物油脂培养基优化", 《生物加工工程》, vol. 5, no. 3, 15 August 2007 (2007-08-15) * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560749A (en) * | 2013-10-29 | 2015-04-29 | 中国石油化工股份有限公司 | Method for open-type fast cultivation of oil-bearing microbes |
| CN104560749B (en) * | 2013-10-29 | 2017-08-22 | 中国石油化工股份有限公司 | A kind of method of open type fast culture oil-containing microorganism |
| CN106754383A (en) * | 2016-11-14 | 2017-05-31 | 华南理工大学 | A kind of method for improving microbes biomass and grease yield |
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Application publication date: 20130417 |