WO2009105927A1 - 一种利用异养小球藻高密度发酵产生生物柴油的方法 - Google Patents

一种利用异养小球藻高密度发酵产生生物柴油的方法 Download PDF

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WO2009105927A1
WO2009105927A1 PCT/CN2008/001318 CN2008001318W WO2009105927A1 WO 2009105927 A1 WO2009105927 A1 WO 2009105927A1 CN 2008001318 W CN2008001318 W CN 2008001318W WO 2009105927 A1 WO2009105927 A1 WO 2009105927A1
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biodiesel
culture
chlorella
density fermentation
algae
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PCT/CN2008/001318
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English (en)
French (fr)
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吴庆余
周文广
熊伟
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清华大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/003—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fatty acids with alcohols
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
    • 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

Definitions

  • the present invention relates to the field of renewable bioenergy, and more particularly to a method for producing biodiesel by high-density fermentation of heterotrophic chlorella.
  • a chlorella species with high oil content and fast growth is used as an oil-producing algae, and is cultured in a high-density fermentation in a bioreactor, and the obtained algal oil is further subjected to transesterification to prepare biodiesel.
  • BACKGROUND OF THE INVENTION The increasingly severe environmental pollution and energy shortages in today's world have made human beings face unprecedented challenges. To this end, people have begun to develop renewable carbon-neutral fuels to replace traditional ones from the perspective of environmental and economic sustainability. Fossil fuel.
  • Biodiesel is such an environmentally friendly renewable clean energy source.
  • Biodiesel refers to long-chain fatty acid alkyl monoesters obtained by esterification of animal, plant or bio-oil, and is widely used in various parts of the world, especially in Europe and America.
  • the high raw material price causes the production cost of biodiesel to be higher than that of traditional petrochemical diesel. This has become a bottleneck restricting the industrialization of biodiesel. Therefore, trying to reduce the raw material cost of biodiesel is the main direction for the breakthrough of biodiesel industry.
  • economic crops such as soybean, rapeseed and sunflower seeds are the main raw materials for the production of biodiesel.
  • Microalgae is a microorganism that can perform photosynthesis.
  • the concept of "microalgae biofuels" has been proposed long ago, but so far, the cost of producing biodiesel using microalgae is still high and cannot meet the requirements of commercialization.
  • the main reason is that algae with high oil content grow slowly under autotrophic conditions, while algae species with rapid growth tend to have relatively low oil content.
  • the invention is characterized in that the chlorella algae forest with high oil content and rapid growth is screened, the conditions of heterotrophic fermentation of the algae strain are further optimized, the strategy and method for adding nutrients to the bioreactor are established, and the stirring rate and the aeration rate are controlled.
  • the resulting microalgal cell density (unit yield) is 108g (dry weight) / L (the following cell density or concentration is dry weight), cell fat content is 61%, both are much higher than our earlier published research Data (highest cell density of 15.5 g/L, maximum oil content of 55.2%) [1, 2, 3].
  • biodiesel raw materials such as vegetable oil, animal fat, and food waste oil
  • the cell density of up to 108g/L and the oil content of 61% makes this technology an economical preparation of biodiesel raw material oil. An efficient way.
  • the method for producing biodiesel by high-density fermentation of Heterotrophic Chlorella is to use high-density fermentation of heterotrophic chlorella in a bioreactor as a raw material for preparing biodiesel, and the following steps are carried out: (1) screening one oily oil a high-volume, fast-growing chlorella strain; (2) directly inoculate the selected algae forest into a shake flask for primary seed culture; (3) transfer the seed culture to the fermenter for secondary high Density fermentation; (4) adding nutrient solution, condition optimization and process control to the optimal cell density in the first-stage seed culture and the second-stage high-density fermentation; (5) collecting the chlorella cells by centrifugation and drying; (6) Extracting algae oil from dry algal flour; (7) preparing biodiesel by transesterification using the obtained fat as raw material.
  • the C. protothecoides strain for heterotrophic high-density fermentation has high oil content, preferably
  • High oil content algae strains were screened by centrifugation. Algae cells in the supernatant The density is small and the oil content is high. These cells are seeded on agar plates and streaked to form a single algal. In order to screen for algal strains that grow fast, screening involves the step of picking large, monoclonal colonies from the plate in one step.
  • the cell density in the fermenter is preferably between 15 and 108 g/L.
  • a strain of C. protothecoides 0710 having a fast growth rate and a high oil content is screened.
  • the culture solution cultured in the primary seed culture and the secondary fermentation tank is supplemented with carbohydrates.
  • the additional carbohydrates in the primary seed culture and the secondary fermentation broth include, but are not limited to, glucose and other monosaccharides, disaccharides and polysaccharides, and the concentration is preferably between 0.01 and 100 g/L.
  • These carbohydrates include glucose, fructose, corn starch hydrolysate, tapioca hydrolysate, wheat starch hydrolysate, and sorghum juice.
  • the inoculum amount in the first-stage seed culture and the second-stage fermentation culture is preferably between 0.01% and 50%.
  • the shaker or agitation speed of the first-stage seed culture and the second-stage fermentation culture is preferably between 5 and 1000 rpm.
  • the dissolved oxygen in the first-stage seed culture and the second-stage fermentation culture is generally maintained at 5% or more, preferably at least 20%.
  • Organic nitrogen sources that are added during primary seed culture and secondary fermentation include, but are not limited to, glycine, yeast powder, commercially available yeast extract, and corn steep liquor.
  • the P H value in the first-stage seed culture and the second-stage fermentation culture is preferably between 6.0 and 8.0, and the acidity of the medium is lowered by adding an alkaline solution, preferably potassium hydroxide is an alkaline solution.
  • the temperature control during the first-stage seed culture and the second-stage fermentation culture is preferably 20-45. Between C.
  • the dry weight of the cells in the first-stage seed culture and the second-stage fermentation culture is preferably between 20 and 108 g/liter, and the oil content is preferably between 40% and 61%.
  • the technical flow, method and steps involved in the present invention are shown in FIG. 1 and are described in detail as follows: (1) screening the growth rate of the algae strain with high growth rate and high oil content, preferably 8000 ⁇ 12000 g, 30 min, 4 °C The heterotrophic cultured Chlorella cells were centrifuged twice. The algae cells in the supernatant have a lower density and a higher oil content.
  • the cells were inoculated on agar plates, streaked, and cultured for 240 to 360 hours to form single algal blooms.
  • the relatively large single colonies in the plates were inoculated into liquid cultures in shake flasks, respectively, and each flask was compared.
  • Cell growth rate and oil content From a group of cultures with the highest growth rate and oil content, a forest with high oil content and fast growth rate was obtained.
  • the culture in which the algae cells were streaked on the agar plates used a basal medium supplemented with 30 g/L of glucose and 4 g/L of the commercially available yeast extract, and the formulation of the basal medium was the same as that of the next part of the seed culture and the high-density fermentation culture.
  • the optimized A5 drum element liquid contains: H 3 B0 3 2.86gL-', Na 2 Mo0 4 -2H 2 0 0.039 gL" 1 , ZnS0 4 -7H 2 0 0.222 MnCl 2 -4H 2 0 1.81 g ⁇ -1 , CuSO 4 '5H 2 O 0.074 gL _1 . 0
  • the effect of culture conditions in a constant temperature shaking culture shake flask on the growth of Chlorella cells including but not limited to, for example, glucose concentration, Nitrogen source, temperature, speed, light intensity, etc.
  • the growth conditions were as follows: 5 to 50 g/L of glucose and 1 to 10 g/L of commercially available yeast extract were separately added to the base medium.
  • the seed cell culture is transferred to the fermenter for secondary high-density fermentation, and the seed culture at the end of the logarithmic growth period is transferred to the secondary bioreactor for process control and optimization after 168 hours of cultivation.
  • This step reaction is preferably carried out in a 5-11,000 L fermentor.
  • 30 g/L of glucose and 4 g/L of commercially available yeast extract were added to the basal medium.
  • the fermentation process of Heterotrophic Chlorella was repeatedly optimized by adjusting and controlling the inoculum amount, bottom stream addition, dissolved oxygen, stirring rate, temperature and pH. The optimization results show that the organic carbon source flow addition strategy based on dissolved oxygen feedback is essential for the accumulation of biomass and fat.
  • the high-density fermentation conditions of Heterotrophic Chlorella were set as follows: Inoculum size 25%; Temperature 28 ⁇ 0.5 °C; Ventilation rate 180 IJh; Passing force ⁇ 10g/L KOH, the H value is controlled within the range of 6.3 soil 0.1; the glucose concentration in the fermentation broth is maintained at 5-18 by adding 500g/L glucose Fluctuating in the range of g/L; continuously adding organic nitrogen source (commercial yeast extract) at a rate of 0.75 g/1/h, and after 88 hours of cultivation, the dissolved oxygen in the fermentation broth is saturated by gradually increasing the stirring rate.
  • organic nitrogen source commercial yeast extract
  • the degree was maintained above 20%; nitrogen source and glucose were added simultaneously after 128 hours; at 213 hours, the cell density reached 108 g/L (Fig. 2), and the oil content reached 61% of the dry weight of the cell, and the fermentation was terminated.
  • the concentration of the cells of the collected algae cells is preferably 20-108 g/L, and after the cell concentration is measured by sampling, the algal cells are separated from the fermentation broth. This process includes, but is not limited to, filtration and centrifugation processes.
  • Extraction of oil from dried algal cells Methods for extracting oil from dried algal cells include, but are not limited to, a sock extraction method.
  • the oil is extracted from the dry algae powder by a cable extraction method using n-hexane as a standard extraction solvent. The n-hexane was repeatedly rinsed until no more fat remained in the fineness, and then the solvent was removed under reduced pressure.
  • Biodiesel can be prepared by esterification.
  • the conversion of fatty acids to fatty acid esters can be catalyzed by a strong acid, such as concentrated sulphuric acid, or enzymatically.
  • Lipase Triacylglycerol Acylhydrolase, EC 3.1.1.3 catalyzes the hydrolysis of long-chain fatty acid glycerides at the oil/water interface, effectively catalyzing esterification and transesterification.
  • the lipase was extracted from Candida sp. 99-125 and the catalytic activity was up to 12000 U g.
  • esterification conditions pH 7.0, adding 2.5 volumes of n-hexane solvent, 60% immobilized lipase ( Relative to the quality of the oil, the enzyme activity was 12000 U), 10% by mass of water, and the temperature was raised in a constant temperature shaker at 38 ° C and 160 rpm.
  • the molar ratio of alcohol to oil was 3:1, and the reaction system was added in three portions.
  • the fatty acid methyl ester catalyzed by lipase is the main component of biodiesel.
  • the intention of the present invention is to screen the chlorella strain with high oil content and rapid growth, further optimize the conditions of heterotrophic fermentation of the algae forest, adopt the method of adding nutrients to the bioreactor, control the stirring rate, Aeration, etc., the resulting microalgae cell density (unit yield) is 108g (dry weight) / L, cell fat content of 61% of high-density cultured heterotrophic chlorella cells, reducing the cost of biodiesel raw materials, to meet
  • the industrial application requirements for the production of biodiesel using heterotrophic chlorella cells make this technology an economical and efficient way to prepare biodiesel raw materials.
  • Biodiesel Long-chain fatty acid mercapto monoesters obtained from renewable biological lipid resources such as vegetable oils or animal fats. "Biological” means that it is a renewable biological resource relative to petroleum-based diesel; “diesel” means that it can be used in diesel engines or as a fuel like diesel.
  • Alkyl monoester/monoester generally refers to biodiesel having a chemical composition of a fatty acid ester. For example, fatty acid alkyl monoester biodiesel is composed of oleic acid, linoleic acid, stearic acid, palmitoleic acid, and other fatty acids.
  • Algae Refers to a class of aquatic photosynthesis organisms, including unicellular algae and multicellular large algae such as kelp, seaweed, etc. Algae was once classified as a plant, but because it lacks plant characteristics such as roots, stems, leaves, and embryos, it is currently classified separately.
  • Microalgae Individually very small algae, generally referred to as a single fine-packed aquatic algae that can be observed and identified by means of a microscope.
  • Heterotrophy The way in which organisms use organic compounds as a source of carbon and nitrogen.
  • Transesterification A reaction in which an alkoxy group is exchanged by introducing a compound having a hydroxyl group.
  • Long-chain fatty acid alkyl monoester/fatty acid methyl ester A class of organic compounds formed by the addition of sterols and fatty acids and the removal of one molecule of water.
  • Bioreactor A container or device (including culture flasks, thermostated shake flasks, fermenters, etc.) used for biological cell culture or involving biochemical reaction processes with appropriate environmental conditions.
  • Heterotrophic culture/fermentation The process by which microbial cells are grown under the aerobic conditions using glucose or other organic carbon sources.
  • Yeast fermentation The process of using yeast to convert sugar into alcohol under anaerobic conditions.
  • Fuel ethanol A biofuel prepared from the alcohol obtained by microbial fermentation.
  • Oil/Fat A class of natural hydrocarbons with a long chain structure.
  • Nile Red is a lipophilic fluorescent dye that emits golden yellow fluorescence in a fat-rich environment.
  • Pyrolysis The process of high temperature chemical degradation of organic matter under anoxic conditions.
  • Single colony/algal bloom Colonies or algal blooms formed by the growth and division of individual cells on agar plates.
  • Constant temperature oscillation culture shake flask It is a device that can control the environmental conditions such as temperature and humidity for microbial growth or other biochemical reactions.
  • Organic carbon A family of carbon-containing compounds (such as sucrose, fat, etc.) derived from organisms, which are distinguished from inorganic carbon sources such as co 2 .
  • Organic nitrogen refers to nitrogen-containing compounds derived from organisms such as proteins, urea, and the like.
  • Sorghum Gramineae are annual high herbaceous plants.
  • Sweet sorghum Sorghum varieties with a sucrose content generally above 10%.
  • Cassava A shrubby perennial crop rich in starch at the roots.
  • Cable extractor A semi-automatic solvent extraction device used in the present invention to separate oils and fats from algal cells.
  • Calorific value The amount of heat released by the complete combustion of a unit mass of fuel.
  • Monosaccharide Can not be further hydrolyzed, the molecular formula is generally CnH2nOn carbohydrates, such as glucose, fructose, xylose and the like.
  • Disaccharide A carbohydrate consisting of two monosaccharide units.
  • Polysaccharide A carbohydrate consisting of two or more monosaccharide units.
  • Hydrolyzate A compound obtained by a hydrolysis reaction, in the present invention, a mixture of monosaccharides or short-chain oligosaccharides obtained by hydrolysis of cellulose, starch or other polysaccharide substances.
  • FIG. 1 is a method and a step for producing biodiesel by high density fermentation using heterotrophic chlorella.
  • Figure 2 is a graph showing cell growth and glucose consumption of high density fermentation of heterotrophic microalgae in a 5 L bioreactor.
  • Figure 3 shows the relative oil content of the fluorescence intensity of Nile red staining cells under fluorescence microscope.
  • Figure 4 is a graph showing the composition change of the product in the transesterification reaction with the reaction time.
  • DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for producing biodiesel using high density fermentation of heterotrophic Chlorella. Based on the existing research results and patented technology, it further increases the yield and oil content of C. ratot/zeco ifes in bioreactors, and uses high-density culture techniques to screen chlorella algae with high oil content and rapid growth.
  • A5 elemental element liquid comprises H 3 BO 3 , Na 2 MoO 4 -2H 2 O , ZnSO 4 '7H 2 O, and
  • the optimized A5 elemental solution contains: H 3 BO 3 2.86gL _1 , Na 2 MoO 4 -2H 2 O 0.039 gL" 1 , ZnSO 4 -7H 2 0 0.222 gL -1 , MnCl 2 -4H 2 0 1.81 gL" 1 , CuSO 4 -5H 2 0 0.074 gL -1 .
  • the registration number is 2578.
  • the proposed classification is named Chlorella protothecoides.
  • the state of survival is survival.
  • Algal strain Compared with C. protothecoides sp 0710 H, the color is golden, the maximum biomass is up to 108 g / liter, and the oil content is as high as 61%.
  • the highest cell density of the starting strain was 15.5 g/l and the highest oil content was 55.2%.
  • Table 1 shows the difference in composition and content of biodiesel prepared by high-density fermentation of C. protothecoides sp 0710 and biodiesel prepared by fermentation of the original starting strain.
  • the selected strains were inoculated into a 500 ml shake flask containing basal medium for primary seed culture at a culture temperature of 28 ° C and a shake flask speed of 200 rpm.
  • a basal medium of a constant temperature shaking culture shake flask 30 g/L of glucose and 4 g/L of a commercially available yeast extract were added.
  • the cells were cultured for 168 hours (cell density was about 15 g/L) and the cells entered the end of logarithmic growth. Low light irradiation of 5 mol.m-2s-l was also used in the culture process.
  • the effects of different culture conditions such as glucose concentration, nitrogen source, temperature and rotation speed on the growth of Chlorella cells were investigated in a constant temperature shaking culture shake flask.
  • the growth conditions are optimized as follows: Concentrations of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 g/L of glucose and concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g/L of commercially available yeast extract was added to the above basic medium at a culture temperature of 20, 25, 28, 30, 33, 35, 40 ° C, and the shake flask speed was 200 rpm.
  • the culture was carried out under the conditions of the fractions, and preferably, 30 g/L glucose and 4 g/L of the commercially available yeast extract were added to the basal medium at a culture temperature of 28 ° C and a shake flask speed of 200 rpm. The fine monthly bag grows best. Cells in the logarithmic growth phase are inoculated into the fermentor to continue the secondary high-density culture.
  • the fermentation culture was carried out in a 5 L stirred bioreactor (MINIFORS, Switzerland), and the original chlorella cells in the logarithmic growth phase were inoculated into a basal medium supplemented with 30 g/L glucose and 4 g/L of commercially available yeast extract.
  • the inoculum size is 25% (VV).
  • High-density culture conditions were as follows: 28 ⁇ 0.5. C, aeration rate of 180L / h, pH 6.3 ⁇ 0.1, online monitoring of dissolved oxygen changes in the fermenter, once dissolved oxygen is below 20% of saturation, by gradually increasing the stirring rate, while continuously adding glucose, glucose concentration Controlled in the range of 5-18 g / L, the dissolved oxygen saturation in the fermentation broth is maintained at 20% or more.
  • the commercially available yeast extract and glucose are added simultaneously, and the nitrogen source rate is 0.75 g/ Lh, establish a high-density culture strategy by using fed-batch and process control in a 5L fermentor, and monitor the changes in glucose, glycine, phosphorus concentration, pH and dissolved oxygen in the fermenter.
  • the fermentation broth was centrifuged at 10,000 rpm for 2 minutes at 4 ° C, and the precipitate was collected, vacuum dried, and weighed to obtain a total of 108 g / liter of dry algae.
  • the oil is extracted from the dry algae powder by a cable extraction method using a semi-automatic solvent extraction device-sand extractor using n-hexane as a standard extraction solvent.
  • a cable extractor the cells are repeatedly rinsed with n-hexane until no more fat remains in the cells.
  • the acid value of the oil and fat, the calculated molecular weight of the microalgae oil is a dimensionless value.
  • the oil obtained by the cable extraction method was dried, and then weighed, and the dry weight of the cells was divided by the dry weight of the cells to obtain a dry weight of 61% (w/w).
  • the esterification reaction is catalyzed by a lipase derived from Candida sp. 99-125.
  • the reaction was carried out in a constant temperature shaker at 180 rpm. After the reaction, the mixed reaction system is divided into upper and lower layers, and the biodiesel in the upper layer is separated and rinsed in warm water at 50 ° C, and the solvent is removed by rotary evaporation to obtain pure biodiesel.
  • the reaction products meet the requirements of triglyceride, diester, monoester, methanol and glycerol in the national biodiesel national standard ( ASTM 6751).
  • the manipulated variables were analyzed. These variables include the amount of lipase in the reaction system, the amount of methanol, the acceleration of methanol flow, the content of fat, the amount of organic solvent, the content of 7J, and the pH and reaction time.
  • gas chromatography-mass spectrometry whereby the conversion rate from fat to fatty acid oxime ester can be calculated. Analysis was performed by gas chromatography-mass spectrometer using a DSQ GC ( Thermo, USA, VARIAN VF-5ms capillary column 30M*0.25MM). The flow rate is set to 10 ml min - 1 - Temperature program: first increase to 70 °C for 2 min; then increase the temperature to 300 °C at 10 °C min for 20 min. The injection port temperature is 250 °C and the split ratio is 30:1.
  • This depository registration number is CGMCC No. 2578
  • Chlorella protothecoides The microorganism (strain) was 2 ⁇ 8 in July 0S date of receipt by the Depositary. And were registered.

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一种利用异养小球藻高密度发酵生产
生物柴油的方法 技术领域 本发明属于可再生生物能源领域，特别涉及一种利用异养小球藻高密度 发酵生产生物柴油的方法。 具体说是以一株含油量高生长快的原始小球藻为 产油藻种， 在生物反应器中高密度发酵培养， 所得藻油再经过转酯反应制备 生物柴油。 背景技术 当今世界日益严峻的环境污染和能源匮乏问题使人类正面临前所未有 的巨大挑战， 为此， 人们开始从环境和经济可持续性发展的角度考虑， 开发 可再生的碳中性燃料来取代传统的化石燃料。 生物柴油就是这样一种环境友 好的可再生清洁能源。 生物柴油指的是动、 植物或 ：生物油脂经酯化反应后 得到的长链脂肪酸烷基单酯， 在世界各地特别是欧美地区有着广泛的应用。 但是， 高昂的原料价格， 造成生物柴油的生产成本高于传统的石化柴油， 这 成为制约生物柴油产业化发展的瓶颈， 因此，设法降低生物柴油的原料成本， 是生物柴油产业突破的主要方向。 目前， 大豆、 油菜、 葵花籽等经济作物是生产生物柴油的主要原料。 这 种以传统农业为基础的生物能源生产方式不仅产量氏而且与粮食作物争夺耕 地、 淡水、 肥料等资源， 因此不能满足生物柴油产业对原料油持续增长的需 求。 微藻是一种能进行光合作用的微生物。 "微藻生物燃料" 的概念在很早 以前就已提出， 但是迄今为止， 利用微藻生产生物柴油成本依然高昂， 无法 满足商业化的要求。 主要原因在于自养状态下， 含油量高的藻生长緩慢， 而 生长迅速的藻种往往含油量相对较低。 为此， 吴庆余课题组以一株淡水绿藻 Chlorella protothecoides为材料， 率先开展了利用异养 4鼓藻来生产生物柴油的研究。 在该课题组已经发表的论 文： ( 1 ) Miao XL, Wu QY. 2006. Biodiesel production from heterotrophic microalgal oil. Bioresour Technol 97:841-846; (2) Xu H, Miao XL., Wu QY. 2006. High quality biodiesel production from microalga Chlorella protothecoides by heterotrophic growth in fermenters. J Biotechnol 126: 499-507; (3) Xiufeng Li, Han Xu, Qingyu Wu, 2007, Large-scale biodiesel production from microalga Chlorella protothecoids through heterotrophic cultivation in bioreactors. Biotechnology and Bioengineering. 98(4): 764-771.和 已经 申请专 利 CN1699516A CN1837352A的内容显示， C. protothecoides细胞的牟位产量 低于 16 g (干重） /L, 含油量低于 56 % , 如此低的微藻细胞产量和含油量还 不能满足工业化应用的要求。 发明内容 本发明的目的在于提供一种利用异养小球藻高密度发酵生产生物柴油 的方法。 它是在已有研究成果和专利技术的 上， 进一步提高生物反应器 中 C. pratot/ze w'cfey的产量和含油量， 在保证高含油量的同时， 实现高密度 培养来满足利用异养小球藻细胞生产生物柴油的工业化应用要求。 本发明的 特征在于筛选含油量高、 生长迅速的小球藻藻林， 进一步优化该藻株异养发 酵的条件， 建立向生物反应器中流加营养物的策略和方法、 控制搅拌速率、 通气量等等， 最终得到的微藻细胞密度（单位产量）为 108g (干重） /L (以 下细胞密度或浓度均是干重）， 细胞油脂含量为 61% , 均大大高于我们早先 发表的研究数据 （最高细胞密度为 15.5 g/L, 最高含油量 55.2% ) [1 , 2, 3]。 相对于植物油、 动物脂肪、 餐饮废油等生物柴油原料， 高达 108g/L的细胞密 度和 61%的含油量， 降氏生物柴油的原料成本， 使本技术成为一条生物柴油 原料油脂制备的经济、 高效途径。 所述利用异养小球藻高密度发酵生产生物柴油的方法是以在生物反应 器中高密度发酵培养异养小球藻作为制备生物柴油的原料， 按照如下步骤进 行： （1 ) 筛选一株含油量高、 生长迅速的小球藻藻株； （2 ) 将筛选到的藻 林直接接种至摇瓶中作为一级种子培养； （3 ) 将种子培养物转接至发酵罐 中进行二级高密度发酵； （4 )在一级种子培养和二级高密度发酵过程中流加 营养液、 条件优化和过程控制至细胞密度达到最佳； （5 ) 离心收集小球藻细 胞并干燥； （6 ) 从干藻粉中抽提藻油； （7 ) 以所得油脂为原料， 经过转酯 化反应制备生物柴油。 所述用于异养高密度发酵的 C. protothecoides藻株的含油量高， 优选于
20%-61 %之间。 高含油量的藻株采用离心的方式筛选。 在上清液中的藻细胞 密度较小， 含油量较高， 将这些细胞接种于琼脂平板中， 划线分离， 形成单 个藻落。 为了筛选到生长速度快的藻株， 筛选包括一步从平板中挑取大的、 单克隆菌落的步骤。 发酵罐中细胞密度优选于 15-108g/L之间。 本发明中筛 选到一株生长速度快、 含油量高的藻株 C. protothecoides 0710。 本发明中，一级种子培养和二级发酵罐培养的培养液都补加含碳水化合 物。 一级种子培养和二级发酵培养液中补加的碳水化合物包括但不限于葡萄 糖以及其他单糖， 二糖和多糖等， 浓度优选于 0.01-100 g/L之间。 这些碳水 化合物包括葡萄糖、 果糖、 玉米淀粉水解物、 木薯淀粉水解物、 小麦淀粉水 解物和高粱汁。 本发明中， 一级种子培养和二级发酵培养时接种量优选于 0.01%-50%之间。 一级种子培养和二级发酵培养时摇床转动或搅拌速度优选 于 5-1000转 /分之间。一级种子培养和二级发酵培养时溶氧一般保持在 5%以 上， 优选 20%以上。 一级种子培养和二级发酵培养时流加的有机氮源包括但 不限于甘氨酸、 酵母粉、 市售酵母提取物和玉米浆。 本发明中， 一级种子培 养和二级发酵培养时 PH值优选 6.0-8.0之间， 通过流加碱性溶液来降低培养 基的酸性， 优选氢氧化钾为碱性溶液。 一级种子培养和二级发酵培养时温度 控制优选 20-45。C之间。 本发明中， 一级种子培养和二级发酵培养时细胞干 重优选 20-108克 /升之间， 油脂含量优选 40%-61 %之间。 本发明中涉及的其 他对象、 技术流程、 优势和特点将会出现在下文的详细描述中。 本发明内容涉及的技术流程及方法与步骤如图 1所示， 详细描述如下： ( 1 ) 筛选生长速率快， 含油量高的藻株 优选离心率为 8000〜12000 g, 30 min, 4 °C对异养培养的小球藻细胞进 行两次离心。 上清中的藻细胞密度较小， 含油量较高。 接种该细胞于琼脂平 板中， 划线分离， 经过 240至 360小时的培养形成单个藻落， 平板中体积相 对较大的单菌落被分别接种至摇瓶中液体培养， 分别考察比较各摇瓶中细胞 的生长速度及含油量。 从细胞的生长速度及含油量最高的一组培养中得到一 林含油量高、 生长速度快的藻林。 藻细胞在琼脂平板上划线分离后的培养使 用了添加 30g/L葡萄糖和 4g/L市售酵母提取物的基础培养基，基础培养基的 配方与下一部分种子培养和高密度发酵培养相同。倒制平板时， 添加 1.5%的 琼脂粉形成固体培养基。 平板培养时培养箱的温度控制在 28 ± 0.5。C。 ( 2 )在一级反应器中进行藻细胞种子培养 将筛选得到的藻林接种至含有基础培养基的摇瓶中作种子培养，基础培 养基配方:^下：
KH2PO4 0.7g丄-1 , K2HP04 0.3g.L_1 , MgSO4-7H2O 0.3g丄-1 , FeS04.7H20 3mg.L-1 , 甘氨酸 O.lg丄 维生素 Bl O.Olmg丄— 1 , Α5微量元 素液 lml.L^ 其中 A5 微量元素液包含 H3B03, Na2Mo04-2H2O , ZnS04.7H20,和 MnCl2'4H20, CuS04'5H20 等组分。 优化的 A5 鼓量元素液包含： H3B03 2.86g.L-', Na2Mo04-2H20 0.039 g.L"1, ZnS04-7H20 0.222
Figure imgf000006_0001
MnCl2-4H20 1.81 g丄 -1, CuSO4'5H2O 0.074 g.L_1.0 在恒温振荡培养摇瓶中的培养条件对小球藻细胞生长的影响，包括但不 限于如葡萄糖浓度、 氮源、 温度、 转速、 光强等。 生长条件如下： 将 5 至 50 g/L 的葡萄糖和 1至 10 g/L市售的酵母提取物分别加入到基 培养基中。 其中，采用 30g/L的葡萄糖和 4g/L市售的酵母提取物时，细胞生长达到最优， 在 20-45 °C的范围内， 28 。C为最佳培养温度。 摇瓶速度， 200转 /分对细胞 生长最佳。 培养过程中还采用了 5 mol.m-2s-l的弱光照射
( 3 )种子细胞培养物转接至发酵罐中进行二级高密度发酵， 将经过 168小时培养，处于对数生长末期的种子培养物转接至二级生物 反应器中进行过程控制与优化，该步反应优选在 5-11,000 L的发酵罐中进行。 发酵开始时，在基础培养基中添加了 30g/L的葡萄糖和 4g/L市售的酵母提取 物。 通过调整和控制接种量、 底物流加、 溶氧、 搅拌速率、 和温度、 pH值等 参数对异养小球藻发酵工艺进行了反复优化。 优化结果显示， 基于溶氧反馈 的有机碳源流加策略对生物量和脂肪的积累至关重要。 在培养基中分别尝试 流加葡萄糖、 果糖、 蔗糖， 玉米淀粉、 木薯淀粉、 小麦淀粉水解物以及高粱 汁、 含糖废水等碳水化合物溶液， 然后通过在线监测耗氧量的变化考察细胞 生长的状况。 发酵工艺优化表明由搅拌速度和通气量变化引起发酵液溶氧 ( DO )波动对细胞的高密度培养极为重要。通过反复的发酵条件优化和过程 控制实验， 异养小球藻的高密度发酵条件设定如下： 接种量为 25%; 温度 28 士 0.5 °C; 通气速率 180IJh; 通过补力 σ 10g/L的 KOH， 将 H值控制在 6.3 土 0.1的范围之内； 通过补加 500g/L的葡萄糖维持发酵液中葡萄糖浓度在 5-18 g/L的范围内波动；以 0.75 g/1/h的速度连续补加有机氮源（市售酵母提取物）， 培养 88 小时以后， 通过逐步提高搅拌速率， 使发酵液中的溶氧饱和度维持 在 20%以上； 在 128小时以后同时补加氮源和葡萄糖； 213 小时时， 细胞密 度达到 108g/L (图 2 ) , 油含量达到细胞干重的 61%, 终止发酵。 ( 4 ) 收集藻细胞 细胞浓度优选 20-108g/L, 通过取样测定细胞浓度以后， 将藻细胞从发 酵液中分离出来。 该过程包括但不限于过滤和离心工艺。 分离得到的藻细胞 干粉或者其他固体形态。
( 5 )从干燥的藻细胞中提取油脂 从干燥的藻细胞中提取油脂的方法包括但不限于索式抽提法。采用索式 抽提法， 以正己烷为标准萃取溶剂， 从干藻粉中提取油脂。 正己烷反复淋洗， 直至细^ <中不再残留脂肪， 然后减压去除溶剂。
( 6 ) 酯化反專制备生物柴油 从藻细胞中提取的油脂制备生物柴油可采用酯化反应。脂肪酸到脂肪酸 酯的转化可通过强酸， 如浓石克酸催化， 或酶催化。 脂肪酶 （Triacylglycerol Acylhydrolase, EC 3.1.1.3)在油 /水界面催化水解长链脂肪酸甘油酯， 有效催 化酯化和酯交换反应。脂肪酶从 Candida sp. 99-125中提取的，催化活力可达 12000 U g 最佳曱酯化条件的组合为： pH值 7.0, 加入 2.5倍体积的正己 烷溶剂， 60 %固定化脂肪酶（相对于油的质量， 酶活 12000 U )、 10 %质 量分数的水， 38 °C、 160 rpm恒温摇床中温育， 醇油摩尔比 3: 1 , 分三次加 入反应体系。 用脂肪酶催化生成的脂肪酸甲酯为生物柴油的主要成分。 本发明的有意效果是本方法以筛选含油量高、 生长迅速的小球藻藻株， 进一步优化该藻林异养发酵的条件，采用向生物反应器中流加营养物的方式、 控制搅拌速率、 通气量等等， 最终得到的微藻细胞密度（单位产量）为 108g (干重） /L, 细胞油脂含量为 61%的高密度培养异养小球藻细胞， 降低生物 柴油的原料成本，满足利用异养小球藻细胞生产生物柴油的工业化应用要求， 使本技术成为一条生物柴油原料油脂制备的经济、 高效途径。 定义 除非特别说明， 本文中涉及的名词定义如下: 生物柴油： 从可再生生物脂质资源，如植物油或动物脂中得到的长链脂 肪酸皖基单酯。 "生物"表示相对于石油基柴油来言， 它是一种可再生的生物 资源； "柴油" 指的是它可用于柴油发动机或像柴油一样作为燃料使用。 烷基单酯 /单酯：一般是指化学组成为脂肪酸酯的生物柴油。例如，脂肪 酸烷基单酯生物柴油是由油酸， 亚油酸， 硬脂酸， 棕榈油酸以及其他脂肪酸 等成份组成的。 藻类： 指一类水生的光合作用生物， 包括单细胞藻和多细胞大型藻类如 海带、 紫菜等。 藻类曾一度被划归为植物， 但由于它缺乏根、 茎、 叶和胚胎 等植物特征， 因此目前被单独分为一类。 微藻： 个体非常微小的藻类， 一般指需借助于显微镜才能观察和识别的 单细包水生藻。 异养： 生物体以有机化合物作为碳源和氮源的营养方式。 转酯化反应： 通过引入含有羟基的化合物， 使烷氧基发生交换的反应。 长链脂肪酸烷基单酯 /脂肪酸甲酯： 曱醇和脂肪酸加成并脱去一分子水 生成的一类有机化合物。 生物反应器：是一种用于生物细胞培养或涉及生物化学反应过程并具有 适当环境条件的容器或设备（包括培养瓶、 恒温振荡培养摇瓶和发酵罐等）。 异养培养 /发酵： 微生物细胞在耗氧条件下利用葡萄糖或其他有机碳源 生长的过程。 酵母发酵： 在厌氧条件下利用酵母菌将糖转化为酒精的过程。 燃料乙醇： 以微生物发酵得到的酒精为原料制备的一种生物燃料。 油 /脂肪： 一类具有长链结构的天然碳氢化合物。 在本发明中， 指的是 脂肪酸及其衍生物， 包括甘油单酯、 二酯、 三酯、 磷脂以及其他的脂溶性甾 醇类物质 （如胆固醇等） 尼罗红： 是一种亲脂的荧光染料，在富含脂肪的环境中能发出金黄色荧 光。 热解： 在缺氧条件下， 有机质高温化学降解的过程。 单菌落 /藻落： 在琼脂平板上由单个细胞生长分裂堆积形成的菌落或藻 落。 恒温振荡培养摇瓶： 是一种温度、 湿度等环境条件可控， 供微生物生长 或进行其他生化反应的设备。 有机碳： 是一族来自于有机体的含碳化合物 （如蔗糖、 脂肪等）， 它区 别于 co2等无机碳源。 有机氮： 是指来自于有机体的含氮化合物， 如蛋白质、 尿素等。 高梁： 禾本科萄黍属一年生高大草本植物。 甜高梁： 蔗糖含量一般在 10%以上的高粱品种。 木薯： 一种根部富含淀粉的灌木状多年生作物。 索式抽提器： 是一种半自动的溶剂萃取装置， 在本发明中用于从藻细胞 中分离油脂。 热值： 单位质量的燃料完全燃烧所释放的热量。 单糖： 不能被进一步水解， 分子式一般为 CnH2nOn的碳水化合物， 如 葡萄糖、 果糖、 木糖等。 二糖： 由两个单糖单元构成的碳水化合物。 多糖： 由两个以上的单糖单元构成的碳水化合物。 水解物： 经水解反应得到的化合物， 在本发明中， 一 ^指纤维素、 淀粉 或其他多糖物质经水解后得到的单糖或短链寡糖混合物。 附图说明 图 1为利用异养小球藻进行高密度发酵生产生物柴油的方法及步骤。 图 2为在 5L生物反应器中异养微藻高密度发酵的细胞生长和葡萄糖消 耗曲线。 图 3为荧光显微镜下尼罗红染色细胞荧光强度测定相对油脂含量。 图 4为转酯化反应中随反应时间的产物组成变化。 具体实施方式 本发明提供一种利用异养小球藻高密度发酵生产生物柴油的方法。它是 在已有研究成果和专利技术的基础上， 进一步提高生物反应器中 C. ratot/zeco ifes的产量和含油量， 利用高密度培养技术， 筛选含油量高、 生长 迅速的小球藻藻株 C. protothecoides sp 0710。 在保证高含油量的同时， 实现 高密度培养来满足利用异养小球藻细胞生产生物柴油的工业化应用要求。 下面列举实施例于本发明予以进一步说明。 实施例 原始小球藻（C/w/ore〃a rato ecozWe 出发藻林购自美国 Texas 大学藻 种中心， I) 1990 年起， 清华大学生物科学与技术系微藻生物能源实验室长 期培养该藻种用于生物能源的研究， 其^出培养基配方如下：
KH2PO4 0.7 g/L, K2HPO4 0.3 g/L, MgSO4 ·7Η20 0.3 g/L, FeS04 -7H2O 3m g L , 甘氨酸 0.1 g/L , 维生素 0.01m g/L, A5 ^啟量元素液 lml/L。 其中 A5 史量元素液包含 H3BO3, Na2MoO4-2H2O , ZnSO4'7H2O,和
MnCl2-4H2O, CuS04-5H20 等组分。 优化的 A5 量元素液包含： H3BO3 2.86g.L_1, Na2MoO4-2H2O 0.039 g.L"1, ZnSO4-7H20 0.222 g.L-1, MnCl2-4H20 1.81 g.L"1, CuSO4-5H20 0.074 g.L-1.。 通过步骤 1中所描述的定向筛选技术，得到一株生长迅速、含油量高的 藻株 C. protothecoides sp 0710。该藻株于 2008年 7月 8日保藏于中国微生物 菌种保藏管理委员会普通微生物中心， 登记入册编号为 2578 , 建议的分类命 名为原始小球藻 Chlorella protothecoides„保藏时状态为存活。 与出发藻株相 比， C. protothecoides sp 0710 H, 色泽金黄， 最大生物量可达 108克 /升， 含油量高达 61%。 相比之下， 在我们过去已经发表的文献中， 出发藻株的最 高细胞密度为 15.5克 /升，最高含油量为 55.2%。表 1显示了以 C. protothecoides sp 0710 高密度发酵制备的生物柴油与原初出发藻株一般发酵制备的生物柴 油在成分与含量上的差别。 将筛选得到的藻株接种至含有基础培养基的 500ml 摇瓶中作一级种子 培养， 培养温度为 28°C, 摇瓶速度为 200转 /分。 在恒温振荡培养摇瓶的基 础培养基中， 加入 30g/L的葡萄糖和 4g/L市售的酵母提取物。 培养至 168小 时（细胞密度约为 15 g/L), 细胞进入对数生长末期。 培养过程中还采用了 5 mol.m-2s-l的弱光照射。 在一级种子培养和二级高密度发酵过程中，在恒温振荡培养摇瓶中考察 了不同培养条件如葡萄糖浓度、 氮源、 温度、 转速等对小球藻细胞生长的影 响。 生长条件优化过程如下： 将浓度为 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 g/L 的葡萄糖和浓度为 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g/L 的市售 酵母提取物分别加入到上述基础培养基中， 在培养温度为 20、 25、 28、 30、 33、 35、 40 °C, 摇瓶速度为 200转 /分的条件下进行考察培养， 其中优选后， 采用在基础培养基中加入 30g/L葡萄糖和 4g/L市售酵母提取物，培养温度为 28 °C, 摇瓶速度为 200转 /分时， 细月包生长最佳。 处于对数生长期的细胞被接种至发酵罐中继续进行二级高密度培养。发 酵培养在 5L搅拌式生物反应器（MINIFORS, 瑞士） 中进行， 处于对数生长 期的原始小球藻细胞接种至加入 30g/L葡萄糖和 4g/L市售酵母提取物的基础 培养基中， 接种量为 25% (VV)。 高密度培养条件如下： 28±0.5。C， 通气 速率 180L/h, pH值 6.3 ±0.1, 在线监测发酵罐中的溶氧变化， 一旦溶氧低于 饱和度的 20%以下时， 通过逐步提高搅拌速率， 同时连续添加葡萄糖， 葡萄 糖浓度控制在 5-18 g/L范围内， 使发酵液中的溶氧饱和度维持在 20%以上， 在 128 小时以后同时补加市售酵母提取物和葡萄糖， 补加氮源速度为 0.75g/lh,通过在 5L发酵罐中采用补料流加和过程控制的方式建立高密度培 养策略， 同时监控发酵罐中葡萄糖、 甘氨酸、 磷浓度以及 pH值和溶氧的变 化。 发酵完成后， 从发酵液离心收集细胞， 并真空干燥。 高密度发酵过程中 细胞密度通过定时测定光密度值 ( OD540nm ) 来估算。 OD54Qnm与细胞干重的 线性关系可以用以下公式表示： y = 0.4155x, (R2 = 0.9933, P<0.05), 其中 y 表示细胞密度（克 /升）， X表示 540 nm处光密度值。 发酵完成后，发酵液于 4°C以 10000转 /分的速度离心 2分钟，收集沉淀， 真空干燥， 称重， 共得到 108克 /升的干藻分。 采用索式抽提法， 利用半自动的溶剂萃取装置 -索式抽提器， 以正己烷 为标准萃取溶剂， 从干藻粉中提取油脂。 在索式抽提器中， 用正己烷反复淋 洗细胞， 直至细胞中不再残留脂肪。 异养藻油脂的分子量计算公式如下： 分子量（ M ) = 56.1 X 1000 X 3/(SV-AV) , 式中 SV ( Saponification Value ) 代表微藻油脂的皂化值， AV ( Acid Value )代表微藻油脂的酸值， 计算得到 的微藻油脂的分子量是无量纲数值。 经过溶剂减压旋转蒸馏后对索式抽提法获得的油脂干燥后进行称中，以 油脂净中除以细胞干重， 得细胞干重的油脂含量为 61 % ( w/w )。 酯化反应由来源于 Candida sp. 99-125的脂肪酶催化完成。 反应在 180 转 /分的恒温摇床中进行。 反应结束后， 混合反应体系分成上下两层， 分离位 于上层的生物柴油， 并置于 50 °C的温水中淋洗， 旋转蒸发， 去除溶剂， 得 到纯净的生物柴油。 为了考察不同操作条件对生物柴油产率的影响，使反应产物满足美国生 物柴油国家标准（ ASTM 6751 ) 中对甘油三酯、 二酯、 一酯、 甲醇和甘油等 含量的要求， 我们对各种操作变量进行了分析。 这些变量包括反应体系中脂 肪酶的用量、 甲醇用量、 甲醇流加速度、 脂肪含量、 有机溶剂用量、 7J含量 等参数以及 pH值和反应时间等。 通过利用气相色谱-质谱连用技术可分析体 系中甘油三酯、 二酯、 一酯、 甲醇和甘油等的含量， 据此可计算从油脂到脂 肪酸曱酯的转化率。 采用 DSQ GC ( Thermo, USA , VARIAN VF-5ms 毛细 管柱 30M*0.25MM ) 气相色谱-质谱串联仪进行分析。 流速设定为 10 ml min-1 - 升温程序： 先升温到 70 °C , 保持 2 min; 然后以 10 °C min 的速度将 温度上升到 300 °C , 保持 20 min。 注射口温度 250 °C , 分流比为 30: 1。 在 上述条件下， 经过 213小时的高密度培养， 细胞密度达到 108克 /升， 油含量 为细胞干重的 61%。 在 12小时的酯化反应过程中， 98.15%的微藻油脂转化 为脂肪酸曱酯 （生物柴油）， 生物柴油的产率为 64.66克 /升。 共检测到 9种 脂肪酸曱酯， 其中含量最高的是油酸甲酯、 亚油酸甲酯和棕榈酸甲酯， 其含 量占全部生物柴油的 80 %以上。 主要结果如表 1所示。 表 1 利用异养小球藻制备的生物柴油的组成成分与含量
Figure imgf000013_0001
参考文献
[1] Miao XL, Wu QY. 2006. Biodiesel production from heterotrophic microalgal oil. Bioresour Technol 97:841-846.
[2] Xu H, Miao XL., Wu QY. 2006. High quality biodiesel production from microalga Chlorella protothecoides by heterotrophic growth in fermenters. J Biotechnol 126: 499-507.
[3] Xiufeng Li, Han Xu, Qingyu Wu, 2007 , Large-scale biodiesel production from microalga Chlorella protothecoids through heterotrophic cultivation in bioreactors. Biotechnology and Bioengineering. 98(4): 764-771.
中国微生物菌种保藏管理委员会
普通效生物中心
China General Microbiological Culture Collection Center(CGMCC) 地址： 北京市朝阳区大屯路， 中国科学院微生物研究所， 邮政编码： 100101， http://www.cgmcc.net 电话： 010-64807355 传真： 010-64807288 电子邮件： cgmcc@sun.im.ac.cn 受理通知书 （收据）
存活性 艮告书 用于专利程序的微生物保存 发出日期 2008年 07 月 08 日
(请求保藏人或代理人的姓名、 地址）
吴庆余
清华大学
北京市海淀区清华大学生物系微藻生物技术实验室
本保藏中心登记入册编号 CGMCC No. 2578
你（们）提供的请求保藏并注明以下鉴定
参椐的微生物 （株） ： sp 0710 上述请求保藏的微生物 （株） 附有
□ 科学描述
0 建议的分类命名： 原、 /J、雜
Chlorella protothecoides
Figure imgf000015_0001
该微生物（株） 已于 2∞8 年 07 月 0S日由本保藏中心收到， .并登记入册。
根据你（们） 的请求， 由该日起保存三十年， 在期满前收到提供微生物样品的请求后再延 续保存五年。
该微生物（株） 的存活性经本保藏中心于 2∞8年 Q7 月 D8日检测， 结果是
( 1 )存活 (2) 失活 _/
Figure imgf000015_0002

Claims

权 利 要 求 书
1. 一种利用异养小球藻高密度发酵生产生物柴油的方法， 其特征在于， 该方法是以在生物反应器中高密度发酵培养的异养小球藻作为制备生 物柴油的原料， 具体步骤包括： （1) 筛选一株生长速率快， 含油量高的 小球藻藻株； (2) 将筛选得到的藻株接种至含有基础培养基的摇瓶或小 型生物反应器中进行种子培养， 还添加碳水化合物和有机氮源为细胞 生长提供营养； （3 )将种子培养物转接至含有营养液的发酵罐或其他 生物反应器中进行藻细胞高密度发酵即二级培养， 发酵罐或生物反应 器中细胞密度介于 15-120g/L之间； （4 )在一级种子培养和二级高密 度发酵过程中， 向发酵罐或生物反应器中流加营养液并通过其他条件 控制至细胞密度达到最佳； （5 ) 离心收集小球藻细胞并干燥； （6 )从 干藻粉中抽提藻油； （7 ) 以所得油脂为原料， 经过转酯反应制备生物 柴油。
2. 根据权利要求 1 所述利用异养小球藻高密度发酵生产生物柴油的方 法， 其特征在于， 所述筛选一株生长速率快， 含油量高的小球藻藻林， 还包括使 C. protothecoides油脂含量达到细胞干重 61%的异养培养技 术； 由于高含油量的藻株密度小， 离心后仍悬浮于上清液中， 而平板 中， 体积大的单菌落具有较快的生长速率， 因此， 筛选得到的藻林是 由离心取上清和筛选大的单菌落的方式得到的， 所得藻株被命名为 C. protothecoides sp 0710。
3. 根据权利要求 1 所述利用异养小球藻高密度发酵生产生物柴油的方 法， 其特征在于， 将筛选得到的藻株 C. ?rotot/zecoz'cfey (?770接种至含 有碳水化合物浓度为 0.01-100 g/L的基础培养基的摇瓶或小型生物反 应器中作种子培养， 按接种量为 0.01%-50%, 摇瓶转动或搅拌速度为 5-1000转 /分， 溶氧保持在 5% - 20%或 20%以上； 在恒温、 搅拌培养 摇瓶或小型生物反应器中间歇或连续补加入浓度为 5 ~ 50 g/L 的碳水 合物和 1 ~ 10 g/L的有机氮源，通过流加碱性溶液来降 4氏培养基的酸 性， 使 pH值为 6-8, 温度控制在 20-45。C之间。 根据权利要求 1或 3所述利用异养小球藻高密度发酵生产生物柴油的 方法， 其特征在于， 所述种子细包培养物转接至二级发酵罐或生物反 应器中进行高密度发酵与过程控制， 其接种量介于总体积的 0.01% 50%之间， 向发酵罐或生物反应器中流加营养液的方法还包括 间歇或连续流加操作。 发酵罐或生物反应器中原有营养液或添加营养 液含有的碳水化合物包括葡萄糖或其他单糖、 二糖或多糖， 碳水化合 物来源包括葡萄糖、 果糖、 玉米淀粉水解物、 木薯淀粉水解物、 小麦 淀粉水解物和高粱汁； 碳水化合物浓度控制在 0.01-100克 /升之间； 发 酵罐或生物反应器中原有的营养液或添加的营养液含有的有机氮源包 括甘氨酸、 酵母粉、 市售酵母提取物或玉米浆； 高密度发酵方法还包 括搅拌操作和通气，搅拌速率控制在 5-1000转 /分之间， 通气操作保持 培养基中溶氧（ DO )值保持在 5% ~ 20%或 20%以上； 所述高密度发 酵方法还包括向发酵罐中流加碱液用以维持体系 pH值在 6.0-8.0之间， 碱液包括氢氧化钾及其他强碱性化合物； 发酵罐内发酵的温度控制在 20-45。C之间。 根据权利要求 1 所述利用异养小球藻高密度发酵生产生物柴油的方 法， 其特征在于， 所述酯化反应制备生物柴油， 首先采用索式抽提法， 以正己烷或氯仿为标准萃取溶剂， 从发酵液中分离收集的小球藻细胞 干粉中提取藻粉油脂， 然后以酯交换反应： 在藻粉油脂中加入醇油摩 尔比 2 ~ 6:1 的曱醇， 分三次加入反应体系， 在脂肪酶 Triacylglycerol Acylhydrolase, EC 3.1.1.3的有效催化酯化作用下，加热至 30。C ~ 90°C, 160 rpm恒温摇床中温育， 反应生成的产物， 经石油醚及水洗涤' 离心 取有机相， 将有机溶剂蒸发、 干燥后获得生物柴油， 并分离出副产品 甘油。 根据权利要求 1 所述利用异养小球藻高密度发酵生产生物柴油的方 法， 其特征在于， 所述的种子培养和高密度发酵营养液含有的基础培 养基配方如下： KH2PO4 OJg.L"1 , K2HPO4 0.3g.L_1 , MgSO4'7H20 O gX"1 , FeS04'7H20 3mg丄 ，甘氨酸 O.lg丄 -1 , 维生素 Bl O.Olmg.L"1 , A54啟量元素液 lmLL^ 其中 A5 数量元素液的组成为： H3B03 2.86g.L"1, Na2MoO4-2H20 0.039
Figure imgf000017_0001
ZnSO4-7H2O 0.222 g.L"1, MnCl2'4H2O 1.81 g.L"1, CuSO4-5H20 0.074 g丄 。
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