CN101037639A - Method for producing biologic grease and diesel oil - Google Patents
Method for producing biologic grease and diesel oil Download PDFInfo
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- CN101037639A CN101037639A CNA2006100460918A CN200610046091A CN101037639A CN 101037639 A CN101037639 A CN 101037639A CN A2006100460918 A CNA2006100460918 A CN A2006100460918A CN 200610046091 A CN200610046091 A CN 200610046091A CN 101037639 A CN101037639 A CN 101037639A
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- rice grass
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- thalline
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- 238000004519 manufacturing process Methods 0.000 title claims abstract description 11
- 239000004519 grease Substances 0.000 title claims description 17
- 239000002283 diesel fuel Substances 0.000 title abstract description 7
- 239000003921 oil Substances 0.000 claims abstract description 39
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 26
- 239000012075 bio-oil Substances 0.000 claims abstract description 20
- 235000000346 sugar Nutrition 0.000 claims abstract description 20
- 241000894006 Bacteria Species 0.000 claims abstract description 10
- 239000003225 biodiesel Substances 0.000 claims abstract description 10
- 239000002994 raw material Substances 0.000 claims abstract description 8
- 235000003675 Paspalum scrobiculatum Nutrition 0.000 claims description 50
- 238000000855 fermentation Methods 0.000 claims description 35
- 230000004151 fermentation Effects 0.000 claims description 35
- 239000007788 liquid Substances 0.000 claims description 29
- 239000000243 solution Substances 0.000 claims description 29
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- 239000002551 biofuel Substances 0.000 claims description 14
- 230000007062 hydrolysis Effects 0.000 claims description 12
- 238000006460 hydrolysis reaction Methods 0.000 claims description 12
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- 108010046845 tryptones Proteins 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
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- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
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- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
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- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 1
- -1 iginates Species 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
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- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
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- 229960004232 linoleic acid Drugs 0.000 description 1
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- 230000001473 noxious effect Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
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Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P30/00—Technologies relating to oil refining and petrochemical industry
- Y02P30/20—Technologies relating to oil refining and petrochemical industry using bio-feedstock
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明涉及生物能源领域，具体地说是一种生产生物油脂和生物柴油的方法，以海洋滩涂植物大米草为原料，采用酵母工程菌将大米草分步糖化发酵生产生物油脂和生物柴油。本发明是一种由海洋滩涂植物大米草经酸水解产糖后利用微生物中的酵母工程菌发酵转化为油脂或柴油的新工艺，该工艺产油率高、转化快，在变废为宝和环境保护等方面具有广阔的应用前景。The invention relates to the field of bioenergy, in particular to a method for producing bio-oil and bio-diesel. The marine beach plant Spartina is used as a raw material, and yeast engineering bacteria are used to saccharify and ferment Spartina in steps to produce bio-oil and bio-diesel. The present invention is a new process of producing sugar from marine tidal flat plant Spartina through acid hydrolysis, and then using yeast engineering bacteria in microorganisms to ferment and convert it into oil or diesel oil. Environmental protection and other aspects have broad application prospects.
Description
Technical field
The present invention relates to the bioenergy field; specifically a kind of production bio-oil and method of bio-diesel oil; with ocean beach plant rice grass as raw material; adopt the method for Yeast engineering bacteria with rice grass substep diastatic fermentation; the noxious plant material at seabeach is become bioenergy-diesel oil and grease, is a kind ofly to turn waste into wealth and environment protection combines and has the novel technique of great economic and social benefit.
Background technology
Rice grass (S.anglica), English name common cordgrass is the cross-fertilize seed of spartina alterniflora loisel (S.alterniflora) and fox rice grass (S.patens), originates in Britain's south coast, rice grass plant height 20～50cm, the highest reached at 100cm.Well developed root system, the base portion axillalry bud can grow new tiller and terrestrial stem, spreads growth, and salt tolerant, anti-flooding property are strong, can tide band growth in the seabeach that other plant can not be grown.In early days, the rice grass for can resist than high sea, is had created land with silt by communications and reports, protection sea wall, the pioneer plant of the effect of raising seabeach soil fertility and good forage feed.So all parts of the world all begins to introduce a fine variety on a large scale; cause rice grass rapid spread in the world; [Liu builds now to have become a kind of global crop smothering; Huang Jianhua. careful alien crop is watched out for negative impact. plant protection, 2002,28 (4): 51～53. Qin Wei China; Wang Zhi; Jiang Mingkang. the spartina alterniflora loisel is to the invasion of two wetland wilderness areas, entrance of Changjiang River. weeds science, 2004,4:15～16.]
The rice grass of China is to introduce from Britain in 1963, so far the history in year surplus in the of existing 40.At first plant experimentally, be used for coastal bank protection and improve the soil, produce feed and paper making raw material simultaneously at the beach, Jiangsu.To 1981 the end of the year China have 3.6 ten thousand hm south gets north, Hepu of Guangxi county to Jinxi, Liaoning county
2The rice grass.In recent years, the rice grass in Fujian, the mad diffusion in coastlands such as Guangdong, Jiangsu, Shandong, its area coverage is increasing, has arrived unmanageable situation, has a strong impact on the development of coastal aquaculture.Brought serious problems therefore for the coastal economy and the ecology of China.Rice grass serious threat China seashore ecological safety has now belonged to first 16 kinds of exotic invasive species that country announces.
Along with the surge of development of modern industry and world population, energy dilemma is aggravated day by day.At present, countries in the world are launched new forms of energy one after another, particularly the research and development of the recyclable organism energy.Biofuel is one of most important renewable energy product-derived, and its chemical ingredients mainly is the low-carbon ester of longer chain fatty acid.The relevant both at home and abroad lignocellulosic material based on straw, stalk is hydrolyzed into fermentable sugars, utilizes microbial fermentation to produce grease and biofuel, bio-ethanol etc.And be not appearing in the newspapers as yet of raw material with the rice grass; the main component Mierocrystalline cellulose hemicellulose of rice grass accounts for 60%; can be used as a kind of affluent resources of production biofuel, can play the effect of turning waste into wealth and protecting environment to this utilization simultaneously with disastrous invasive species.
Summary of the invention
The purpose of this invention is to provide a kind of rice grass substep diastatic fermentation that utilizes effective, that hydrolysis time is short, diastatic fermentation efficient is high and produce bio-oil and method of bio-diesel oil.
The method principle: the rice grass mainly is made up of Mierocrystalline cellulose, hemicellulose, accounts for 60%～70% of biomass dry weight.The structure of lignocellulose is complicated, and hemicellulose and xylogen in the cell walls are formed network structure by covalent bond, and Mierocrystalline cellulose is inlayed wherein.Hemicellulose is very easy to be hydrolyzed into five-carbon sugar under the acid effect, and the destroyed further glycosidic link fracture of cellulosic crystalline structure is hydrolyzed into hexose and other fermentable sugars.And sufficient and some nutritive ingredient (particularly nitrogenous source) of oleaginous microorganism carbon source in fermention medium (rice grass hydrolyzed solution) is when lacking, and the somatic cells split speed falls sharply, and Metabolic activity transfers to consume carbon source and based on synthetic and accumulate grease.In the oil and fat accumulation phase, microorganism no longer carries out cell proliferation basically, but excessive carbohydrate is converted into grease.
For achieving the above object, the technical solution used in the present invention is:
A kind of production bio-oil and method of bio-diesel oil are raw material with ocean beach plant rice grass, adopt Yeast engineering bacteria that rice grass substep diastatic fermentation is produced bio-oil and biofuel.
Detailed process is as follows:
1) saccharifying: the Mierocrystalline cellulose under the concentrated acid low-temperature atmosphere-pressure in the rice grass, hydrolysis of hemicellulose become five, hexose and other fermentable sugars.
With the rice grass hydrolysis under water bath with thermostatic control, mechanical stirring condition in the inorganic concentrated acid aqueous solution after drying, the pulverizing; Be neutralized to neutrality with alkali lye after reacting the thin up that finishes, carry out solid-liquid separation with circulation ability of swimming vacuum pump suction filtration suction filtration again, or use dense H
3PO
4Readjustment pH to 6.0, suction filtration removes Ca
3(PO
4)
2Precipitation; Measure solubility sugar contents in the hydrolyzed solution with anthrone method then, standby with the Rotary Evaporators concentrating hydrolysate;
Be specially,
A. the rice grass is obtained 20～60 order particles through cleaning, prescind, dry, pulverize and sieving;
B. be 5～12.5% to add the inorganic concentrated acid (as: sulfuric acid) of weight concentrations 10～80% according to the weight content of solid substance, 40～120 ℃ of hydrolysis 1～4 hour; Make every effort under optimum hydrolysising condition, get maximum sugar yield;
C. hydrolyzed solution transfers to neutrality with alkali, carries out solid-liquid separation, and filtrate concentrates standby in 4 ℃ of refrigerations as microbial fermentation solution.
2) fermenting process: the weight inoculum size with 10% is inoculated in the liquid seeds nutrient solution of oleaginous yeast in the rice grass hydrolyzed solution after 110～121 ℃ of sterilizations, and under 28 ℃～30 ℃, the condition of 180～200r/min, shaking table was cultivated 48～96 hours; Measure biomass, oil quantity and the residual sugar of thalline after the fermentation ends;
Be specially,
A. will be on the fresh inclined-plane long good produce oil inoculation was cultivated 20～48 hours in 28 ℃～30 ℃, 180～200r/min shaking table in liquid seed culture medium;
B. be inoculated in the fermention medium (the rice grass hydrolyzed solution after 110～121 ℃ of sterilizations) with 10% inoculum size, shaking table was cultivated 48～96 hours under identical condition; (this fermentation condition plays a key effect to greasy formation of later stage)
C. measure biomass, oil quantity and the residual sugar of thalline after the fermentation ends; Or measure the oleaginousness of the corresponding dry weight in every strain bacterium fermentation back with Soxhlet fat test instrument, calculate oil yield rate then.
3) thalline separates with grease, gets bio-oil; Bio-oil carries out transesterificationization just can obtain biofuel.
The present invention has following advantage:
1. technology is simple.The present invention is hydrolyzed to the Mierocrystalline cellulose in the rice grass, hemicellulose with mineral acid (as: sulfuric acid) from the angle of rice grass substep diastatic fermentation, utilizes the oleaginous microorganism fermentation that carbohydrate is converted into grease again; Realized the process of rice grass substep diastatic fermentation production bio-oil and biofuel.
2. conversion coefficient height, the rate of recovery and the utilization ratio height of sugar.The present invention selects for use spore yeast produce oil bacterial strain of red winter of the circle of purchase to carry out the shake flask fermentation experiment, can utilize the main monose (glucose, wood sugar and pectinose) in the rice grass hydrolyzed solution to be converted into the grease storage in vivo preferably, and the thalline fat content is up to 47.9%; Fermentation ends obtains grease, and grease just can obtain the main component of diesel oil through transesterificationization; Be inoculated in the fermention medium with 10% inoculum size, shaking table was cultivated 96 hours, and fermentation ends obtains 7.75% biofuel (pressing rice grassland material butt calculates).
3. production cost is low, and is good in economic efficiency.The present invention a kind ofly utilizes Yeast engineering bacteria fermentation in the microorganism to be converted into the novel process of grease or diesel oil after sugar is produced in acid hydrolysis by ocean beach plant rice grass; this technology oil yield rate height, transform soon, turning waste into wealth and aspect such as environment protection has broad application prospects.
4. obvious social benefit.The inventive method to rice grass concentrated acid hydrolysis extract fermentable sugars, and carbohydrate is converted into grease by the oleaginous microorganism fermentation, carry out the transesterification change again and can get biofuel, can alleviate the energy dilemma that the fossil oil shortage is brought.
Description of drawings
Fig. 1 produces the process flow sheet of bio-oil and biofuel for the present invention with rice grass substep diastatic fermentation.
Embodiment
Embodiment 1
Raw material rice grass is collected in jiangsu coast in by the end of September, 2005 in, its main component content is: hemicellulose: 23.28%, and Mierocrystalline cellulose: 35.18%.
1) rice grass is cleaned, prescinds, dries, pulverizes and sieves gets less than 20 purpose parts; (quality: volume) add the sulfuric acid of weight concentration 60%, 50 ℃ of water-baths were also stirred 1 hour according to 1: 10 ratio of solid-to-liquid ratio;
2) with its dilute with water, and add proper C a (OH) after reaction finishes
2Transfer to neutrality, carry out solid-liquid separation, adopt the anthrone colorimetry to survey the solubility total sugar content;
3) resulting filtrate is concentrated as microbial fermentation solution;
4) will be on the fresh inclined-plane long good produce oil inoculation was cultivated 20～24 hours in 28 ℃, 180r/min shaking table in liquid seed culture medium; Be inoculated in the microbial fermentation solution (the rice grass hydrolyzed solution after 121 ℃ of sterilizations) with (solid is that weight, liquid are the solid-to-liquid ratio of volume) 10% inoculum size, shaking table was cultivated 96 hours under identical condition;
Described produce oil bacterial strain is for the red winter spore yeast As2.1389 of circle, available from Chinese common micro-organisms DSMZ; (wherein As2.1389 was the numbering in the chief editor's of China Committee for Culture Collection of Microorganisms in 1992 the microbial strains data, and this data has gone out supplementary issue again and has been renamed as Chinese common micro-organisms DSMZ in 2003)
Substratum: a) culture presevation substratum (1L): yeast powder 2～14g, Tryptones 10～20g, agar powder 16～30g;
B) YEPD substratum (g/L) is formed: glucose 10～30, yeast powder 5～16, peptone 8～15, pH5.8～6.0; Solid medium adds the 15g agar powder on the YEPD basis;
Liquid seed culture medium (g/L) is formed: glucose 16～28, (NH
4)
2SO
42～8, yeast powder 0.1～0.6, KH
2PO
40.5～3, MgSO
47H
2O0.1～0.6, pH5.8～6.0;
Limit nitrogen fermention medium (g/L) is formed: glucose 50～90, (NH
4)
2SO
41.0～4.0, yeast powder 0.1～0.6, KH
2PO
40.5～3.0, MgSO
47H
2O0.2～.08, pH5.8～6.0;
Above substratum is all at 110～121 ℃ of following saturation steam sterilization 15min;
5) biomass, oil quantity and the residual sugar of mensuration thalline after the fermentation ends;
6) utilizing extracting process to carry out thalline separates with greasy; Transesterificationization just can obtain biofuel, and per 100 gram dried rice protocols produce thing diesel oil 7.75 grams.
Embodiment 2
Raw material rice grass is that to be collected in Zhejiang in May, 2005 coastal, and its main component content is: hemicellulose: 27.99%, and Mierocrystalline cellulose: 25.30%.
1) rice grass is cleaned, prescinds, dries, pulverizes and sieves gets 20～40 purpose parts; According to 1: 10 ratio of solid-to-liquid ratio (quality: volume)
) adding the sulfuric acid of weight concentration 50%, 80 ℃ of water-baths were also stirred 4 hours;
2) with its dilute with water, and add an amount of Ba (OH) after reaction finishes
2Transfer to neutrality, carry out solid-liquid separation, adopt the anthrone colorimetry to survey the solubility total sugar content;
3) resulting filtrate is concentrated as microbial fermentation solution;
4) will be on the fresh inclined-plane long good produce oil inoculation was cultivated 40～48 hours in 30 ℃, 200r/min shaking table in liquid seed culture medium; Be inoculated in the microbial fermentation solution (the rice grass hydrolyzed solution after 110 ℃ of sterilizations) with weightmeasurement ratio 10% inoculum size, shaking table was cultivated 48 hours under identical condition;
Described produce oil bacterial strain is identical with embodiment 1;
Substratum: a) culture presevation substratum (1L): yeast powder 2～14g, Tryptones 10～20g, agar powder 16～30g;
B) YEPD substratum (g/L) is formed: glucose 10～30, yeast powder 5～16, peptone 8～15, pH5.8～6.0; Solid medium adds the 15g agar powder on the YEPD basis;
Liquid seed culture medium (g/L) is formed: glucose 16～28, (NH
4)
2SO
42～8, yeast powder 0.1～0.6, KH
2PO
40.5～3, MgSO
47H
2O0.1～0.6, pH5.8～6.0;
Limit nitrogen fermention medium (g/L) is formed: glucose 50～90, (NH
4)
2SO
41.0～4.0, yeast powder 0.1～0.6, KH
2PO
40.5～3.0, MgSO
47H
2O0.2～.08, pH5.8～6.0;
Above substratum is all at 110～121 ℃ of following saturation steam sterilization 15min;
5) biomass, oil quantity and the residual sugar of mensuration thalline after the fermentation ends;
6) utilizing extracting process to carry out thalline separates with greasy; Transesterificationization just can obtain biofuel, and per 100 gram dried rice protocols produce thing diesel oil 5.65 grams.
Embodiment 3
The bacterial classification that adopts is: 1, justify red winter spore yeast As2.1515; 2, rhodotorula glutinis As2.703; 3, rhodotorula glutinis As2.704; Available from Chinese common micro-organisms DSMZ.
Substratum: a) culture presevation substratum (1L): yeast powder 2～14g, Tryptones 10～20g, agar powder 16～30g;
B) YEPD substratum (g/L) is formed: glucose 10～30, yeast powder 5～16, peptone 8～15, pH5.8～6.0; Solid medium adds the 15g agar powder on the YEPD basis;
Liquid seed culture medium (g/L) is formed: glucose 16～28, (NH
4)
2SO
42～8, yeast powder 0.1～0.6, KH
2PO
40.5～3, MgSO
47H
2O0.1～0.6, pH5.8～6.0;
Limit nitrogen fermention medium (g/L) is formed: glucose 50～90, (NH
4)
2SO
41.0～4.0, yeast powder 0.1～0.6, KH
2PO
40.5～3.0, MgSO
47H
2O0.2～.08, pH5.8～6.0;
Above substratum is all at 110～121 ℃ of following saturation steam sterilization 15min.The shake flask fermentation of oleaginous microorganism is cultivated: will be on the fresh inclined-plane long good produce oil inoculation in liquid seed culture medium, cultivated 20～48 hours in 28 ℃～30 ℃, 180～200r/min shaking table.
The rice grass that picks up from Lianyun Harbour is suitably cleaned, baking oven is preheated to 110 ℃～115 ℃, then rice grass sample is inserted baking oven, pulverize rice grass sample to particle diameter 30 orders with pulverizer, H in 105 ℃ of constant temperature 8 hours
2SO
4Concentration was by 1: 10 (quality: volume) add, at 110 ℃ of following hydrolysis 3h.After suction filtration was removed rice grass residue, hydrolyzed solution was crossed with milk of lime and is neutralized to pH10.0, and suction filtration removes CaSO
4The dense H of post precipitation
3PO
4Readjustment pH to 6.0, suction filtration removes Ca
3(PO4)
2Precipitation, the gained hydrolyzed solution is standby in 4 ℃ of refrigerations.
Will be available from the above-mentioned three strain bacterium of Chinese common micro-organisms DSMZ preservation inclined-plane kind and glycerine kind respectively.Slant strains one ring inserts respectively in the liquid seed culture medium, 500mL triangular flask liquid amount 100mL, and totally three bottles, under 30 ℃, shaking speed 200rPmin cultivates 48h down.Shake adding 95mL rice grass hydrolyzed solution in the bottle in 500mL then, insert the 5mL liquid seeds, at 180-200rPmin, 30 ℃ of bottom fermentations.The centrifugal repeatedly supernatant that goes of gained fermented liquid, centrifugal gained thalline is beaten centrifugal 3 times with the distilled water suction, put into 9 centimetres of culture dish of diameter then less than the thick packing of 2mm, in-20 ℃ refrigerator freezing 60 minutes, treat sample freeze good after, insert low-temperature freeze-drying machine lyophilized overnight at (first day in afternoon put into 8 taking-ups in second day) at 5.The sample that drying is good divides bacterial strain directly to measure the grease quantum of output in Soxhlet fat test instrument, and the gained result is as follows:
Table 1. takes by weighing the result that the rice grass 20g (hydrolysis in the 1L acid solution) after the oven dry operates:
| Numbering | Dry cell weight (g) | Thalline oil offtake (g) | Oil yield rate |
| 1 | 0.45 | 0.038 | 8.44 |
| 2 | 1.45 | 0.150 | 10.3 |
| 3 | 1.52 | 0.561 | 37.3 |
Embodiment 4
Be crushed to particle diameter 30 orders with picking up from the rice grass sample that dries by the fire well in Lianyun Harbour, by 1: 10 (quality: the H of adding 10% volume) of solid-to-liquid ratio
2SO
4, at 110 ℃ of following hydrolysis 3h.After suction filtration was removed rice grass residue, hydrolyzed solution was crossed with milk of lime and is neutralized to pH10.0, and suction filtration goes the CaSO4 post precipitation to adjust back pH to 6.0 with dense H3PO4, and suction filtration goes Ca3 (PO4) 2 precipitations, and the gained hydrolyzed solution is standby in 4 ℃ of refrigerations.
The bacterial strain that adopts is identical with embodiment 3, inserts respectively in the liquid seed culture medium protecting good slant strains one ring, and 500mL triangular flask liquid amount 100mL, totally three bottles, under 30 ℃, shaking speed 200rPmin cultivation 48h down.Shake adding 95mL rice grass hydrolyzed solution in the bottle in 500mL then, insert the 5mL liquid seeds, at 180-200rPmin, 30 ℃ of bottom fermentations.The centrifugal repeatedly supernatant that goes of gained fermented liquid, centrifugal gained thalline is beaten centrifugal 3 times with the distilled water suction, put into 9 centimetres of culture dish of diameter then less than the thick packing of 2mm, put into baking oven, 105 ℃ of constant temperature toasted 8 hours, the sample that drying is good divides bacterial strain to measure fat content with the Soxhlet extraction apparatus, measuring method is to dry the thalline filter paper packaging, then in baking oven 105 degree bakings 5 hours, the weight of weighing filter paper packet is then put into the apparatus,Soxhlet's lixiviate more than 7 hours with paper bag, paper bag is taken out put into baking oven 105 degree constant temperature more than 30 minutes then, the weight of weighing paper bag again, twice weighing data subtracted each other the weight that is the contained bio-oil of thalline:
Table 2. takes by weighing the result that the rice grass 18g (hydrolysis in the 1L acid solution) after the oven dry operates:
| Numbering | Dry cell weight | Packing back weight | Weight after the lixiviate | Oil yield rate |
| 1 | 0.6380 | 1.5338 | 1.5971 | 9.92 |
| 2 | 1.3271 | 2.3273 | 2.4349 | 8.11 |
| 3 | 1.2372 | 2.0527 | 2.5060 | 36.64 |
Table 3. takes by weighing the result that the rice grass 25g (hydrolysis in the 1L acid solution) after the oven dry operates:
| Numbering | Dry cell weight | Packing back weight | Weight after the lixiviate | Oil yield rate |
| 1 | 0.5620 | 1.6994 | 1.7765 | 13.7 |
| 2 | 0.4124 | 1.4891 | 1.5280 | 9.43 |
| 3 | 0.7026 | 2.0935 | 2.3902 | 42.23 |
This shows that when rice grass sample dry weight was 25g, the thalline oil offtake was the highest.
All oil-producing yeast strain of the prior art all can be used the inventive method, utilize rice grass substep diastatic fermentation to produce the operation of bio-oil and biofuel, for example:
Common oleaginous yeast has: shallow white Cryptococcus (Cryptococcus albidus), curved Cryptococcus (Cryptococcus albi2dun), this Da Shi saccharomyces oleaginosus (Lipomyces), Trichosporon pullulans (Trichospiron pullulans), produce oil saccharomyces oleaginosus (Lipomysli2pofer), gluing rhodotorula (Rhodotorula giutinis), red winter of class yeast full (Rhodosporidium toru2 loides);
At Chinese biological engineering magazine 2005.25 (12): in 39～44, Li Yonghong etc. have carried out preliminary investigation to the different monose of 10 saccharomycete utilizations for the greasy ability of accumulation in the carbon source condition hypothallus.Test-results shows, all experimental strains can both assimilate multiple monose, the broad spectrum that shows utilization of carbon source of L.starkeyi AS2.1390, R.toruloides AS 2.1389 and L.sta rkey i AS 2.1608 wherein, can transform five-carbon sugar wood sugar and pectinose and accumulate grease in thalline, fat content is up to 26%.Fatty acid compositional analysis is the result show, bacterium oil is rich in saturated and low unsaturated longer chain fatty acid, and wherein palmitinic acid, oleic acid and linolic acid three sum account for more than 90% of total fatty acids composition, and lipid acid is formed distributional class and is similar to common vegetables oil.To obtain the research of oil resource significant to utilizing oleaginous microorganism to transform lignocellulose hydrolysis mixing sugar for these results.
Executed peace brightness, cycle in 2003 by rhodotorula glutinis GRL513 being produced the discussion discovery of the best small-sized technology fermentation condition of grease, final grease yield can reach 67.12% of dry cell weight;
Biological processing (magazine name), the progress of microbial oil and prospect (title of article) vol.3 in 2005, No.1 Xue Fei swallow, Zhang Xu, Tan Tianwei (author);
(magazine name) the yeast mutagenesis in 2000 27 (2) of microbiology circular obtains superior strain LipomycesStarkeyi HL.
Claims (4)
1. produce bio-oil and method of bio-diesel oil for one kind, it is characterized in that: with ocean beach plant rice grass is raw material, adopts Yeast engineering bacteria that rice grass substep diastatic fermentation is produced bio-oil and biofuel.
2. according to described production bio-oil of claim 1 and method of bio-diesel oil, it is characterized in that: detailed process is as follows,
1) saccharifying: the rice grass after drying, the pulverizing is stirred hydrolysis in the inorganic concentrated acid aqueous solution; Be neutralized to neutrality with alkali lye after reacting the thin up that finishes, suction filtration carries out solid-liquid separation, or uses dense H
3PO
4Readjustment pH to 6.0, suction filtration removes Ca
3(PO
4)
2Precipitation; Concentrating hydrolysate is standby;
2) fermenting process: the weight inoculum size with 10% is inoculated in the liquid seeds nutrient solution of oleaginous yeast in the rice grass hydrolyzed solution after 110～121 ℃ of sterilizations, and under 28 ℃～30 ℃, the condition of 180～200r/min, shaking table was cultivated 48～96 hours; Measure biomass, oil quantity and the residual sugar of thalline after the fermentation ends;
3) thalline separates with grease, gets bio-oil; Bio-oil carries out transesterificationization just can obtain biofuel.
3. according to described production bio-oil of claim 2 and method of bio-diesel oil, it is characterized in that:
Described step 1) saccharifying is,
A. the rice grass is obtained 20～60 order particles through cleaning, prescind, dry, pulverize and sieving;
B. be 5～12.5% to add the inorganic concentrated acid of weight concentrations 10～80% according to the weight content of solid substance, 40～120 ℃ of hydrolysis 1～4 hour;
C. hydrolyzed solution transfers to neutrality with alkali, carries out solid-liquid separation, and filtrate concentrates standby in 4 ℃ of refrigerations as microbial fermentation solution.
4. according to described production bio-oil of claim 2 and method of bio-diesel oil, it is characterized in that:
Described step 2) fermenting process is,
A. will be on the fresh inclined-plane long good produce oil inoculation was cultivated 20～48 hours in 28 ℃～30 ℃, 180～200r/min shaking table in liquid seed culture medium;
B. be inoculated in the rice grass hydrolyzed solution with 10% inoculum size, shaking table was cultivated 48～96 hours under identical condition;
C. measure biomass, oil quantity and the residual sugar of thalline after the fermentation ends; Or measure the oleaginousness of the corresponding dry weight in every strain bacterium fermentation back with Soxhlet fat test instrument, calculate oil yield rate then.
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