WO2013013433A1 - 小球藻突变株及其应用 - Google Patents

小球藻突变株及其应用 Download PDF

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WO2013013433A1
WO2013013433A1 PCT/CN2011/078739 CN2011078739W WO2013013433A1 WO 2013013433 A1 WO2013013433 A1 WO 2013013433A1 CN 2011078739 W CN2011078739 W CN 2011078739W WO 2013013433 A1 WO2013013433 A1 WO 2013013433A1
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chlorella
mutant
fat
algae
mutant strain
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PCT/CN2011/078739
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English (en)
French (fr)
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王艳
秦松
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中国科学院烟台海岸带研究所
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Priority to LU92145A priority Critical patent/LU92145B1/de
Publication of WO2013013433A1 publication Critical patent/WO2013013433A1/zh

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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
    • 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
    • C12N1/125—Unicellular algae isolates
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
    • 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
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/89—Algae ; Processes using algae
    • 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

Definitions

  • Chlorella mutant strain and application thereof
  • the present invention relates to a selected chlorella mutant for producing single cell oil and biodiesel and an application thereof. Background technique
  • Biodiesel is mainly obtained from bio-fat as a raw material and converted into fatty acid methyl ester by esterification reaction. Biodiesel produces only 16% to 40% of the carbon dioxide from conventional diesel, and the resulting emissions of exhaust particles are also reduced by about 30%. It does not require any modifications to existing diesel engines to be mixed or used alone. There is a need to change the way energy is distributed and the market for energy resources, which can be directly used as fuel for civil fuels and internal combustion engines.
  • Biodiesel bio-fat raw materials are mainly derived from vegetable oils, waste oils and animal fats.
  • the production of biodiesel from the production of microalgae is the advantage that other fat-producing organisms can't match: (1) Microalgae are easy to breed and culture for a short time. Generally, higher plants need to grow for several months or even years to complete a generation. The time of microalgae breeding is only 2-5 days; (2) Microalgae are not affected by climate and seasonal changes like high plants, can maintain pure culture, and can be continuously produced on a large scale throughout the year, which can guarantee raw materials.
  • microalgae Although the production of microalgae is currently the best source of industrial biodiesel production, its oil content is not high, which leads to the high cost of large-scale cultivation of microalgae to produce oil. Ultimately, it is difficult to obtain economic benefits by using microalgae to prepare biodiesel. The process slowed down. Increasing the oil content of microalgae cells is the key to reducing costs at present, and it is expected to fundamentally solve the bottleneck problem of high cost of biodiesel industry. Therefore, it has been necessary to make unremitting efforts to develop excellent algae strains with higher fat yield and biomass, and to provide competitive raw materials for the industrial production of single-cell fats and biodiesel.
  • the object of the present invention is to provide a mutant strain of Chlorella and its use.
  • a chlorella mutant a chlorella mutant was deposited with the China Microbial Culture Collection Management Committee General Microbiology Center (CGMCC) on May 27, 2011, under the number CGMCC No. 4917, and the taxonomic name Chlorella Kessleri.
  • CGMCC China Microbial Culture Collection Management Committee General Microbiology Center
  • the chlorella mutant is a single-cell lipid high-fat mutant, which can be applied to In the diesel production process.
  • Chlorella ChreUa kessleri cultured in Kuhl medium to logarithmic growth phase is treated by EMS chemical mutagenesis; Chlorella is inoculated into Kuhl medium at a temperature of 18-35 °C, rotation speed For 100-200 rpm, light intensity of 20-150 ⁇ ⁇ m- 2 s- 1 continuous light in a shaker shake culture to logarithmic growth phase, add 10-100 per ml of algae cultured to logarithmic growth phase ⁇ 1 EMS solution, treated at 18-35 ° C for 15 - 180 min in the dark, and then added to the EMS solution with an equal volume of sodium thiosulfate solution to terminate the mutagenesis reaction;
  • the Kuhl medium is: 10 g / L glucose , lg/L potassium nitrate, 89 mg/L disodium hydrogen phosphate dihydrate, 621 mg/L sodium dihydrogen phosphate dihydrate, 246 mg/L magnesium sulfate hept
  • step 1) the algae body is transferred to a high-fat directional screening plate, and the expanded single-cell algae body is screened for subculture and subculture; that is, after the mutagen treatment, the algae body is washed and observed.
  • the cell concentration is diluted 10 3 -10 6 times according to the number of cells, and respectively coated on a high-fat directional screening plate, placed in a programmable constant temperature incubator at 18-35 ° C, without light static culture, screening list
  • the increased volume of algae in the Kuhl medium is oscillated in a shaker at a temperature of 18-35 ° C, 100-200 rpm, and a light intensity of 20-150 ⁇ mol m 2 s" 1 continuous illumination. Subculture to logarithmic growth phase.
  • step 3) The algae body is determined by gas chromatography (GC) to determine the lipid content of the mutant strain and the wild strain.
  • GC gas chromatography
  • the ratio of the lipid content of the mutant strain to the fat content of the wild strain is greater than 110%, that is, the mutant strain is a single cell fat and fat.
  • GC gas chromatography
  • the gas chromatographic conditions are: using a split mode, with nitrogen as the carrier gas; the inlet temperature is 250 V FID detector temperature is 260 ° C; the temperature of the column oven is from 140 ° C / min heating rate from 140 °C rose to 240. C.
  • the high-fat mutant Chlorella vulgaris (Oz/ore//a kessleri) A ⁇ obtained by the above technical scheme has been deposited with the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee on May 27, 2011. , numbered CGMCC No. 4917, the classification name Chlorellala kessleri.
  • CGMCC General Microbiology Center
  • the chlorella Ch reUa kessleri) Al CGMCC No. 4917 has a characteristic structure of a typical green algae, green algae, chlorella, chlorella, and chlorella.
  • the vegetative cells are about 5-10 ⁇ m in size, spherical, green and fast.
  • the physiological and biochemical characteristics of the chlorella Chrella kessleri) Al CGMCC No. 4917 are as follows: 1. Autotrophic medium and heterotrophic medium such as BG-11, BBM, Basak CZ-M1, Kuhk KM1 It can grow in medium or in polyculture. It grows best when mixed. The most suitable carbon source for polyculture is glucose. The best source of nitrogen is nitrate. Lower dissolved oxygen favors autotrophic growth, while saturated dissolved oxygen favors heterotrophic growth. The right amount of magnesium can improve its growth.
  • the cell proliferation is carried out by spore propagation.
  • the specific growth rate is 0.025-0.065 h- 1 , and the generation time is 27.72-10.66 h; the optimum pH range for growth: 4-10; the optimum temperature for growth: 18-
  • the optimum light intensity for growth was 20-150 ⁇ mol m 2 s" 1 ; the optimum speed for shaking culture was 100-200 rpm.
  • Chlorella Al CGMCC No. 4917 cells rapidly become larger, lipid synthesis speed is accelerated, lipid production is significantly improved, and fat production rate can be increased. Up to 2.93 mg g 1 h" 1 , up to 2.14 times the lipid production rate of ChhreUa kessleri wild strain. The carbohydrate content also increased, but the total protein content decreased.
  • the main pigments in the cells are chlorophyll a, b, neoxanthin, violax, epoxy zeaxanthin, zeaxanthin, lutein and beta-carotene.
  • the pigment composition of the cells is unchanged, and the proportion of each pigment varies with induction and culture conditions, but the total amount of pigments decreases.
  • the main components of fat in cells C16, C18 series saturated and unsaturated fatty acids, one or several of myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acidkind.
  • the invention adopts the wild strain of Chlorella as the material, adopts the mutation breeding method of chemical mutagenesis and lipid synthesis inhibitor screening, and obtains a lipid production rate of 2.14 times that of the starting strain, stable physiological and biochemical characteristics, and stable inheritance. Performance of chlorella excellent mutant strains.
  • the difference between the present invention and the existing high-fat microalgae mutant strain breeding method is that the microalgae mutant strain resistant to the lipid synthesis inhibitor is directly selected from the strains after the mutagen treatment. Therefore, the interference of low-fat mutants and mutants with insignificant changes in lipid content is avoided, and the targeted screening of high-fat mutants is achieved, thereby avoiding blindness, thereby greatly improving the breeding efficiency, reducing the useless workload, and saving. R&D costs and development time. detailed description
  • the original wild strain Chlorella kessleri which grows well on the solid medium, is picked and inoculated into a 50 ml flask containing 10 ml of sterile Kuhl medium. , shake culture in a shaker with a temperature of 25 V, a speed of 150 rpm, and a light intensity of 100 ⁇ mol m 2 s" 1 continuous illumination.
  • Kuhl medium formula 10 g/L glucose, lg/L potassium nitrate, 89 Mg/L disodium hydrogen phosphate dihydrate, 621 mg/L sodium dihydrogen phosphate dihydrate, 246 mg/L magnesium sulfate heptahydrate, 9.3 mg/L EDTA, 6.9 mg/L ferrous sulfate heptahydrate, 14.7 mg/ L calcium chloride dihydrate, 0.29 mg/L zinc sulfate heptahydrate, 0.17 mg/L manganese sulfate monohydrate, 0.06 mg/L boric acid, 0.002 mg/L copper sulfate pentahydrate, 0.012 mg/L ammonium molybdate tetrahydrate, Add water to a total volume of 1000 ml, pH 6.5.
  • the algae liquid grown to the exponential phase in the first step is subjected to EMS chemical mutagenesis treatment.
  • EMS chemical mutagenesis treatment takes 3 ml of algae solution into a covered sterile glass test tube, add 300 W 1 EMS solution (1 M), and mix well. Treated at 25 °C for 30 min, with 0 min as a blank control.
  • the mutagenesis reaction was terminated by adding an equal volume of sterile sodium thiosulfate solution (10%, w/v) to the EMS solution.
  • the mutagenized cells in the second step were washed twice with fresh sterile Kuhl medium (3000 rpm, 10 min), and the precipitate was collected and suspended in 3 ml of fresh sterile Kuhl medium. in, Store at 4°C in the dark.
  • the fourth step observe the cell concentration of the suspension in the third step, and then dilute the suspension in the third step to the cell density of the blank control group.
  • the cell density of the blank control group is about 1 ⁇ 10 3 cells/ml, and take 100 ⁇ .
  • the diluted algal liquid was coated with a high-fat oriented screening plate, and 500 plates coated with the algal liquid were placed in a 25 ° C programmable constant temperature incubator without light static culture.
  • the prepared high-fat oriented screening plate was prepared by preparing a Kuhl medium having a high initial C/N ratio (30 g/L glucose, 1 g/L potassium nitrate) containing 15 g/L agar at 120 ° C. After autoclaving for 20 minutes, it was cooled to 50 °C, and the filter synthesis sterilized lipid synthesis inhibitor, Cerulenin, was added to a final concentration of 200 ⁇ M, and the mixture was gently shaken to form a high fat orientation. Screen the plate.
  • the strain which can be grown in the fourth step is picked out from the control (primitive chlorella) by a monoclonal algae which is significantly enlarged by the monoclonal algae, and suspended in a liquid culture containing 3 ml of fresh sterile Kuhl liquid.
  • shaking culture was carried out in a shaker at a temperature of 25 ° C, a rotation speed of 150 rpm, and a light intensity of 100 w mol m - 2 s - 1 for continuous propagation, and subculture was carried out;
  • the sterilized 50% glucose mother liquor 250 g glucose dissolved in water to a total volume of 500 ml is added to the algae liquid grown in the fifth step to the exponential phase to a final concentration of 50 g/L, induced.
  • Mass synthesis of algal cell lactam Mass synthesis of algal cell lactam.
  • the seventh step when the culture is induced for 6 days in the sixth step, 50 ml of algae solution is taken, and the mixture is washed twice with deionized water at 3000 rpm, the supernatant is removed, and the algal sludge is subjected to vacuum freeze-drying treatment for 24 hours to obtain dry algae. powder.
  • the extract described in the eighth step was determined by gas chromatography (GC).
  • the chromatographic conditions were: DB-23 capillary gas chromatography column (30 m X 0.25 mm X 0.25 rn); nitrogen as carrier gas; split mode, injection volume 1 ⁇ 1; inlet temperature 250 ° C; FID detection The temperature of the column was 260 ° C; the temperature of the column oven was increased from 140 ° C to 240 ° C at a heating rate of 2.5 ° C / min.
  • Fatty acid methyl esters were identified by comparing the residence time of fatty acid methyl esters with a trusted standard, and fatty acid methyl esters were quantified by comparing the peak areas of fatty acid methyl esters with internal standards (see Table D o).
  • the chlorella CMorella kessler was used as the original strain, treated with chemical mutagen EMS, and the high-fat directional screening was used to screen the plate.
  • 80 new high-fat mutant strains were obtained and screened by gas chromatography. The results showed that These 80 new lines are indeed all high-fat mutants, in which the ratio of the lipid content of the mutant to the fat content of the wild strain is 110-150%, 9%, and 150-200% is 43%, more than 200%. 48%.
  • Chlorella ChhreUa kessleri Chlorella ChhreUa kessleri
  • the new high-fat mutant strain was submitted to the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee on May 27, 2011, and was numbered CGMCC No. 4917.
  • the algae strain was named Chlorella kessleri. ⁇ CGMCC No. 4917.
  • the fatty acid composition of the Chlorella Chhrella kessler Al CGMCC No. 4917 strain, the ratio of each fatty acid to total fatty acids, and the fat production rate are shown in Table 1:
  • the lipid production rate of the mutant strain is higher than that of the wild plant.
  • the growth rate is compared with the wild strain compared with the wild strain (h- 1 ).

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Abstract

本发明涉及一种经选育的用以生产单细胞油脂及生物柴油的小球藻突变株及其应用。所述小球藻突变株已于2011年5月27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（CGMCC），编号为CGMCC No.4917。所述小球藻突变株为单细胞油脂高脂突变株，可应用于生物柴油生产工艺中。

Description

小球藻突变株及其应用 技术领域
本发明涉及一种经选育的用以生产单细胞油脂及生物柴油的小球藻突变株及其 应用。 背景技术
生物柴油突出的环保性和可再生性引起了世界发达国家尤其是资源贫乏国家的 高度重视。 生物柴油主要以生物脂为原料， 经酯化反应转化为脂肪酸甲酯而得。 生物 柴油产生的二氧化碳仅为传统柴油的 16%到 40%， 所产生的尾气微粒排放量也降低 了 30%左右， 同时不需要对现有柴油发动机作任何改装即可混合或单独使用，也不需 要改变能源的分配方式以及能源资源的市场， 可直接作为民用燃料和内燃机燃料。
生物柴油的生物脂原料主要来源于植物油脂、废油和动物脂肪。其中以产脂微藻 作为原料制备生物柴油， 具有其他产脂生物无法比拟的优势： （1 )微藻很容易繁殖并 且培养的时间短，一般高等植物需要生长好几个月甚至几年才能完成一代，微藻繁殖 一代的时间仅为 2-5天； （2 )微藻不像高等植物那样受气候、 季节变化的影响， 可保 持纯培养， 一年四季都可连续大规模生产， 可保证原料供应充足； （3 )微藻的生长繁 殖是在水域中，不依靠土壤，可以在占地有限的设备上进行而得到高产，不与粮争地; ( 4 )藻类所需酯化反应的条件相对较低， 使生产成本降低， 炼制工艺相对较为简单。
虽然产脂微藻是目前最好的生物柴油工业生产来源，但它的含油量不高， 导致大 规模培养微藻生产油脂成本昂贵， 最终使得利用微藻制备生物柴油难以获得经济效 益， 产业化进程减缓。提高微藻细胞的油脂含量是目前降低成本的关键， 有望从根本 上解决生物柴油产业成本过高的瓶颈问题。 因此， 一直需要努力不懈的研究， 以开发 出产脂量及生物量更高的优良藻株，为单细胞油脂及生物柴油的工业生产提供具有竞 争力的原材料。
为了提高微藻细胞的产脂量， 过去的研究主要集中在以下几个方面： （1 )培养基 优化； （2 ) 培养过程控制； （3 ) 代谢工程方法改造藻株； （4 ) 诱变选育高脂突变株。 前两种方法虽然可在一定程度上有效促进藻细胞的产脂量， 但提高的百分比数有限 (通常提高 20%以下）， 仍无法满足产业化规模开发的要求。 代谢工程方法改造微藻 是解决藻细胞脂含量低的根本方法，需要全面考虑藻株的整体代谢网络的结构和调控 特点， 有待进一步进行研究。 诱变育种方法研发成本低， 选育过程简单、 耗时少， 是 提高微藻细胞产脂量的有效途径。 发明内容
本发明目的在于提供一种小球藻突变株及其应用。
为实现上述目的， 本发明采用的技术方案为：
一种小球藻突变株：小球藻突变株已于 2011年 5月 27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（CGMCC ) , 编号为 CGMCC No. 4917, 分类名称小 球藻 Chlorella kessleri。
小球藻突变株的应用： 所述小球藻突变株为单细胞油脂高脂突变株， 可应用于生 物柴油生产工艺中。
所述小球藻突变株的筛选过程：
1 )将在 Kuhl培养基中培养至对数生长期的小球藻 Ch reUa kessleri)采用 EMS 化学诱变处理； 将小球藻接种到 Kuhl培养基中， 在温度为 18-35 °C， 转速为 100-200 rpm, 光照强度为 20-150 μ ηιοΐ m- 2 s-1连续光照的摇床中振荡培养至对数生长期， 在 培养至对数生长期的每毫升藻体内加入 10-100 μ 1 EMS溶液，在黑暗条件下以 18-35 °C处理 15-180 min, 而后加入 EMS溶液等体积的硫代硫酸钠溶液终止诱变反应； 所述 Kuhl培养基为： 10 g/L葡萄糖、 l g/L硝酸钾、 89 mg/L十二水磷酸氢二钠、 621 mg/L二水磷酸二氢钠、 246 mg/L七水硫酸镁、 9.3 mg/L EDTA、 6.9 mg/L七水 硫酸亚铁、 14.7 mg/L二水氯化钙、 0.29 mg/L七水硫酸锌、 0.17 mg/L—水硫酸锰、 0.06 mg/L硼酸、 0.002 mg/L五水硫酸铜、 0.012 mg/L 四水钼酸铵， pH 6.5。
2 ) 将步骤 1 ) 诱变处理后藻体转接培养在高脂定向筛选平板上， 筛选出体积增 大的单细胞藻体进行扩种继代培养；即将诱变处理后藻体洗涤后观测其细胞浓度， 根 据细胞数将藻体稀释 103-106倍， 分别涂布到高脂定向筛选平板上， 置于 18-35 °C的程 控恒温培养箱中无光静置培养， 筛选单细胞体积增大的藻体于 Kuhl培养基中， 在温 度为 18-35 °C， 转速为 100-200 rpm， 光照强度为 20-150 μ mol m 2 s"1 连续光照的摇 床中振荡扩种继代培养至对数生长期。
3 )将上述扩种继代培养至对数生长期的藻体进行诱导培养 6-20天， 待用； 将扩 种继代培养至对数生长期的藻体中加入 50%葡萄糖母液（取 250 g葡萄糖溶于水中至 总体积 500 ml)至终浓度为 30-60 g/L， 诱导培养 6-20天， 使藻细胞内脂的大量合成， 而后用去离子水 3000 g低温离心洗涤， 去除上清， 藻泥沉淀进行真空冷冻干燥处理 24 h, 得到干藻粉。
4 ) 取步骤 3 ) 藻体利用气相色谱 （GC ) 测定突变株与野生株的脂含量， 突变株 的脂含量与野生株的脂含量的比值大于 110%， 即突变株为单细胞油脂高脂突变株； 将诱导培养后藻体中加入甲苯、 1%硫酸一甲醇 （体积比硫酸： 甲醇 =1 :99 ) 和十九烷 酸（C19:0 ) 内标液混合均匀后， 置于 50-60 振荡水浴过夜； 取出， 冷却后， 加入 5% NaCl水溶液和正己烷， 混合均匀后， 离心收集上层液体， 向收集的上层液中加入 2% KHC03水溶液混合均匀， 漩涡仪上混匀离心收集上层液体， 用氮气吹干溶剂后， 用正己烷定容， 利用气相色谱 （GC ) 测定突变株中脂含量。 所述气相色谱条件为： 采用分流模式， 以氮气为载气； 进样口温度为 250 V FID检测器温度为 260°C ; 柱 温箱的温度为以 2.5 °C/min的升温速率从 140 °C升至 240 。C。
经上述技术方案选育获得的高脂突变株小球藻（Oz/ore//a kessleri) A\ , 已于 2011 年 5月 27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 （CGMCC ) , 编 号为 CGMCC No. 4917，分类名称小球藻 Chlordla kessleri。
所述的小球藻 Ch reUa kessleri) Al CGMCC No. 4917具有典型的绿藻门、 绿 藻纲、 绿球藻目、 小球藻科、 小球藻属的特征性结构。 在无菌 BG-11、 BBM、 Basak CZ-M1、 Kuhk KM1等培养液中、适宜条件下培养时， 其营养细胞大小约 5-10 μ m， 球形， 绿色， 生长快速。
所述的小球藻 Ch rella kessleri) Al CGMCC No. 4917生理生化特征如下： 1 . 在 BG-11、 BBM、 Basak CZ-M1、 Kuhk KM1 等自养培养基和异养培养基 中均能生长， 也可以混养培养。 混养时生长最好。 混养时最适的碳源是葡萄糖， 最好 的氮源是硝酸盐。 较低的溶解氧有利于自养生长， 而饱和的溶解氧有利于异养生长。 适量的镁能改善其生长。
2. 营养生长时以孢子繁殖的方式进行细胞增殖， 比生长速率为 0.025-0.065 h-1 , 代时为 27.72-10.66 h; 生长最适 pH范围： 4-10; 生长最适宜温度： 18-35 °C ; 生长 最适光照强度为 20-150 μ mol m 2 s"1 ; 摇床振荡培养时的最适转速 100-200 rpm。
3.在氮限制、磷或硫缺乏、盐胁迫等不利环境条件下，补给足够的碳源， Chlorella Al CGMCC No. 4917细胞迅速变大， 脂合成速度加快， 脂产量显著提高， 产 脂率可达 2.93 mg g 1 h"1 , 高达 ChhreUa kessleri野生株产脂率的 2.14倍。碳水化合物 含量也相应增加， 但总蛋白含量下降。
4. 细胞中主要色素为叶绿素 a、 b， 新黄质， 紫黄质， 环氧玉米黄质， 玉米黄质， 叶黄素以及 β -胡萝卜素。 在高脂诱导、 合成过程中， 细胞的色素组成不变， 各色素 的比例随诱导及培养条件的不同而有所变化， 但色素总量呈下降趋势。
5. 在高脂诱导、 合成过程中， 细胞的呼吸速率上升， 光合速率下降。
6. 细胞中脂的主要成分： C16、 C18系饱和和不饱和脂肪酸， 为肉豆蔻酸、 棕榈 酸、 棕榈油酸、 硬脂酸、 油酸、 亚油酸、 亚麻酸中的一种或几种。
本发明以小球藻的野生株系为材料，采用化学诱变，脂合成抑制剂筛选的诱变育 种方法， 结果得到了一个产脂率高达出发株 2.14倍、 生理生化特性稳定、 具有稳定 遗传性能的小球藻优良突变株。
本发明所具有的优点： 本发明与现有的高脂微藻突变株选育方法区别在于， 从诱 变处理后的藻株中， 直接选育耐受脂合成抑制剂的微藻突变株， 因而避免了低脂突变 株和脂含量变化不显著的突变株的干扰， 实现了高脂突变株的定向筛选，避免了盲目 性， 因此大大提高了选育效率， 减少了无用工作量， 节约了研发成本和研发时间。 具体实施方式
以下结合实施例为本发明作进一步描述。
第一步、在无菌操作台上，挑取固体培养基上生长状态良好的原始野生株小球藻 ( Chlorella kessleri) 藻落， 接种到含 10 ml无菌 Kuhl培养基的 50 ml三角瓶中， 在 温度为 25 V , 转速为 150 rpm， 光照强度为 100 μ mol m 2 s"1 连续光照的摇床中振 荡培养。 Kuhl培养基配方： 10 g/L葡萄糖、 l g/L硝酸钾、 89 mg/L十二水磷酸氢二 钠、 621 mg/L二水磷酸二氢钠、 246 mg/L七水硫酸镁、 9.3 mg/L EDTA、 6.9 mg/L七 水硫酸亚铁、 14.7 mg/L二水氯化钙、 0.29 mg/L七水硫酸锌、 0.17 mg/L一水硫酸锰、 0.06 mg/L硼酸、 0.002 mg/L五水硫酸铜、 0.012 mg/L 四水钼酸铵， 加水至总体积 1000ml, pH 6.5。
第二步、 取第一步中生长至指数期的藻液， 进行 EMS化学诱变处理。 在黑暗无 菌条件下， 取 3 ml藻液到有盖的无菌玻璃试管中， 加入 300 W 1 EMS溶液 （1M)， 充分混匀。 25 °C处理 30 min， 以处理 0 min作为空白对照。 加入 EMS溶液等体积无 菌现配的硫代硫酸钠溶液 （10%， w/v) 终止诱变反应。
第三步、 将第二步中诱变处理完毕的藻细胞分别用新鲜无菌 Kuhl 培养基离心 ( 3000 rpm, 10 min)清洗两次， 收集沉淀将其悬浮在 3 ml新鲜无菌 Kuhl培养基中， 4°C避光存放。
第四步、观测第三步中悬浮液的细胞浓度， 而后将第三步中悬浮液稀释至空白对 照组细胞密度， 空白对照组细胞密度约为 1 X 103 cells/ml， 取 100 μ ΐ稀释的藻液涂 布高脂定向筛选平板， 将涂布了藻液的 500个平板置于 25 °C的程控恒温培养箱中无 光静置培养。
所述配制的高脂定向筛选平板为， 配制含有 15 g/L琼脂的高起始 C/N比（30 g/L 葡萄糖、 1 g/L硝酸钾） 的 Kuhl培养基， 于 120°C 下高压蒸汽灭菌 20分钟后冷却至 50°C， 加入过滤灭菌后的脂合成抑制剂浅蓝菌素 （Cerulenin) 至终浓度为 200 μ M， 轻轻摇动混匀， 倒制成高脂定向筛选平板。
第五步、将能够在第四步中生长的菌株与对照（原始小球藻）相比单克隆藻明显 增大的单克隆藻落挑出， 悬浮到装有 3 ml新鲜无菌 Kuhl液体培养基的玻璃试管中， 在温度为 25 °C， 转速为 150 rpm， 光照强度为 100 w mol m- 2 s- 1 连续光照的摇床中振 荡培养， 进行扩种继代培养；
第六步、 将第五步中生长至指数期的藻液中加入已灭菌的 50%葡萄糖母液 （取 250 g葡萄糖溶于水中至总体积 500 ml) 至终浓度为 50 g/L, 诱导藻细胞内脂的大量 合成。
第七步、 在第六步中诱导培养 6天时， 取 50 ml藻液， 用去离子水 3000 rpm低 温离心洗涤 2次， 去除上清， 藻泥沉淀进行真空冷冻干燥处理 24 h， 得到干藻粉。
第八步、 称取第七步中所述干藻粉 20 mg， 加入 l ml甲苯、 2 ml 1%硫酸一甲醇 (体积比为硫酸： 甲醇 =1 :99)、 0.8 ml十九烷酸 （C19:0 ) 内标液， 漩涡仪上混匀后， 置于 50 。C 振荡水浴过夜。取出， 冷却后， 加入 5 ml 5% NaCl水溶液、 3 ml正己烷， 漩涡仪上混匀， 离心收集上层液体， 重复多次直至提取完全， 向收集的上层液中加入 6 ml 2% KHC03水溶液， 漩涡仪上混匀， 离心收集上层液体， 用氮吹仪吹干溶剂后， 用正己烷定容至 l ml， 去除杂质后转移入色谱进样瓶中， 4 °C保存。将样品中的有效 脂肪酸提取并甲酯化以便利用气相色谱 （GC ) 测定。
利用气相色谱 （GC ) 法测定第八步所述提取液。 色谱条件为： DB-23 毛细管气 相色谱柱（30 m X 0.25 mm X 0.25 rn) ; 氮气为载气； 采用分流模式， 进样体积 为 1 μ 1;进样口温度为 250°C ; FID检测器温度为 260°C ;柱温箱的温度为以 2.5 °C/min 的升温速率从 140 °C 升至 240 °C。通过比较脂肪酸甲酯与可信标准的停留时间来识 别脂肪酸甲酯， 并且通过比较脂肪酸甲酯与内标的峰面积对脂肪酸甲酯进行量化（参 见表 D o
本实施例以小球藻 CMorella kessler 为原始株， 经化学诱变剂 EMS处理， 高 脂定向筛选平板初筛， 获得 80个高脂突变株新品系， 经气相色谱法复筛， 分析结果 显示， 这 80个新品系确实全部为高脂突变株， 其中突变株的脂含量与野生株的脂含 量的比值为 110-150%的占 9%， 为 150-200%的占 43%， 200%以上的占 48%。
其中的一个新品系小球藻 ChhreUa kessleri) Al， 其目的产物脂的产脂率高达 野生株的 2.14倍， 而生长状况与出发株一致， 是一个理想的优良突变株， 可应用于 单细胞油脂及生物柴油的工业生产有一定的优势。 该高脂突变株新品系于 2011年 5 月 27日提交中国微生物菌种保藏管理委员会普通微生物中心（CGMCC )保藏， 编号 为 CGMCC No. 4917,该藻株命名为：小球藻（ Chlorella kessleri) \ CGMCC No. 4917。 小球藻 Chhrella kessler Al CGMCC No. 4917藻株的脂肪酸组成、 各脂肪酸占总 脂肪酸的比例、 以及产脂率如下表 1 :
表 1 突变株 A1诱导脂合成六天时脂肪酸组成及含量与野生株的比较。
Figure imgf000006_0001
另夕卜， 将高脂突变株小球藻 Chlorella kessler Al CGMCC No. 4917藻株在扩 种继代培养不同代数时其产脂率和比生长速率与原始野生株的比较如下表 2。
比生长速率的测定方法： 在藻细胞的指数生长期初期和后期， 各取 5 ml藻液称 细胞干重， 以公式 μ = ( Ln t-Ln o ) I (t-t0) 计算生长速率， μ为比生长速率， Nt为 t时间的细胞干重值， NQ为起始细胞干重值。
表 2 突变株 A1继代培养时产脂率和比生长速率与野生株的比较。
突变株 产脂率比野生株提高 比生长速率 生长状况与野生株相比 倍数 ( h-1 )
第一代 2.18 0.050 相似 第二代 2.14 0.048 相似 第三代 2.20 0.049 相似 第四代 2.15 0.051 相似 继代培养 6个月 2.18 0.050 相似 继代培养 1年后 2.14 0.049 相似

Claims

权 利 要 求 书
1 . 一种小球藻突变株， 其特征在于： 小球藻突变株已于 2011年 5月 27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 （CGMCC), 编号为 CGMCC No. 4917 ο
2. 一种权利要求 1所述的小球藻突变株的应用， 其特征在于： 所述小球藻突变 株为单细胞油脂高脂突变株， 可应用于生物柴油生产工艺中。
3. 按权利要求 2所述的小球藻突变株的应用， 其特征在于： 所述小球藻突变株 的筛选过程：
1 )将在 Kuhl培养基中培养至对数生长期的小球藻 Ch rdla kesslerD采用 EMS 化学诱变处理；
2) 将步骤 1 ) 诱变处理后藻体转接培养在高脂定向筛选平板上， 筛选出体积增 大的单细胞藻体进行扩种继代培养；
3 ) 将上述扩种继代培养至对数生长期的藻体进行诱导培养 6-20天， 待用；
4)取步骤 3 )藻体利用气相色谱（GC)测定其脂含量以及测定野生株的脂含量， 突变株的脂含量与野生株的脂含量的比值大于 110%， 即突变株为单细胞油脂高脂突 变株；其高脂突变株已于 2011年 5月 27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 （CGMCC), 编号为 CGMCC No. 4917。
4. 按权利要求 3所述的小球藻突变株的应用， 其特征在于： 所述步骤 1 ) 将小 球藻接种到 Kuhl培养基中， 在温度为 18-35 V， 转速为 100-200 rpm, 光照强度为
20-150μηιο1 ηι-2 s"1连续光照的摇床中振荡培养至对数生长期，在培养至对数生长期的 每毫升藻体内加入 10-100 μΐ EMS溶液， 在黑暗条件下以 18-35 °C处理 15-180 min, 而后加入 EMS溶液等体积的硫代硫酸钠溶液终止诱变反应；
所述 Kuhl培养基为： 10 g/L葡萄糖、 l g/L硝酸钾、 89 mg/L十二水磷酸氢二钠、 621 mg/L二水磷酸二氢钠、 246 mg/L七水硫酸镁、 9.3 mg/L EDTA、 6.9 mg/L七水 硫酸亚铁、 14.7 mg/L二水氯化钙、 0.29 mg/L七水硫酸锌、 0.17 mg/L—水硫酸锰、 0.06 mg/L硼酸、 0.002 mg/L五水硫酸铜、 0.012 mg/L 四水钼酸铵， pH 6.5。
5. 按权利要求 3所述的小球藻突变株的应用， 其特征在于： 将诱变处理后藻体 洗涤后涂布到高脂定向筛选平板上， 置于 18-35 °C的恒温培养箱中无光静置培养， 筛 选单细胞体积增大的藻体于 Kuhl培养基中，在温度为 18-35 V ,转速为 100-200 rpm, 光照强度为 20-150 μηιοΐ ηι-2 s"1连续光照的摇床中振荡扩种继代培养至对数生长期。
6. 按权利要求 3所述的小球藻突变株的应用， 其特征在于： 将扩种继代培养至 对数生长期的藻体中加入 50%葡萄糖母液至终浓度为 30-60 g/L， 诱导培养 6-20天， 使藻细胞内脂的大量合成， 而后用去离子水 3000 g低温离心洗涤， 去除上清， 藻泥 沉淀进行真空冷冻干燥处理 24 h， 得到干藻粉。
7. 按权利要求 3或 6所述的小球藻突变株的应用， 其特征在于： 将诱导培养后 藻体中加入甲苯、 1%硫酸一甲醇和十九烷酸（C19:0)内标液混合均匀后，置于 50-60°C 振荡水浴过夜； 取出， 冷却后， 加入 5% NaCl水溶液和正己烷， 混合均匀后， 离心 收集上层液体， 向收集的上层液中加入 2% KHC03水溶液混合均匀， 漩涡仪上混匀 离心收集上层液体， 用氮气吹干溶剂后， 用正己烷定容， 利用气相色谱 （GC ) 测定 突变株中脂含量。
8. 按权利要求 3所述的小球藻突变株的应用， 其特征在于： 所述气相色谱条件 为： 采用分流模式， 以氮气为载气； 进样口温度为 250 °C ; FID检测器温度为 260°C ; 柱温箱的温度为以 2.5 °C/min的升温速率从 140 °C升至 240 。C。
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