WO2011050578A1 - 一种微藻培养方法及其光生物反应器系统 - Google Patents

一种微藻培养方法及其光生物反应器系统 Download PDF

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WO2011050578A1
WO2011050578A1 PCT/CN2010/001715 CN2010001715W WO2011050578A1 WO 2011050578 A1 WO2011050578 A1 WO 2011050578A1 CN 2010001715 W CN2010001715 W CN 2010001715W WO 2011050578 A1 WO2011050578 A1 WO 2011050578A1
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reactor
primary
microalgae
light source
algae
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French (fr)
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韩春梅
李静
张蕊
刘敏胜
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ENN Science and Technology Development Co Ltd
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ENN Science and Technology Development Co Ltd
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G33/00—Cultivation of seaweed or algae
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001—Culture apparatus for tissue culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/02—Photobioreactors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/34—Internal compartments or partitions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/58—Reaction vessels connected in series or in parallel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M31/00—Means for providing, directing, scattering or concentrating light
    • C12M31/10—Means for providing, directing, scattering or concentrating light by light emitting elements located inside the reactor, e.g. LED or OLED
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture
    • 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; 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

Definitions

  • This invention relates to the field of microalgae cultivation and, in particular, to a method of culturing microalgae using a novel photobioreactor system and a novel photobioreactor system for use in the method. Background technique
  • Microalgae is one of the most efficient photosynthetic plants, and the yield per unit area can be several tens of times higher than that of crops.
  • Microalgae is rich in active substances such as proteins, polysaccharides, and pigments, and can be widely used in the fields of vocabulary, food, medicine and health care products, cosmetics, and renewable fuels.
  • active substances such as proteins, polysaccharides, and pigments
  • Microalgae biodiesel technology has also made great progress. In the United States, Australia, Japan, Western Europe, India and South Africa, both the government and enterprises have invested heavily in the development of oil-producing microalgae.
  • the traditional method has artificial control of illumination, temperature, relative aseptic conditions, etc., but the cost is high. More of these methods are used to control illumination.
  • Existing lighting control technology some use the built-in artificial light source to fill light, but require the use of waterproof materials, the cost is higher; some use external single or double row light, but the area is large, and the light utilization rate Low.
  • High-efficiency microalgae culture must first have a highly efficient photobioreactor. Secondly, algae species should have strong resistance to stress, high growth rate and high total lipid content. Microalgae species that meet the above conditions are mainly chlorella, diatom, and Scenedesmus.
  • the two-step culture method is generally used: in the first step, the optimal growth conditions and nutrient conditions are ensured during the exponential culture period, and the biomass is rapidly accumulated; the second step is to start the transformation culture conditions at the end of the index and the early stage of the stabilization period, thereby Accumulate substances such as oils and pigments in the body.
  • the most similar implementation with the present invention is CN200410017249.X.
  • the invention selects a multi-stage airlift reactor, adds a culture liquid in the first-stage reactor, transfers the algae liquid by gravity, and adds an artificial The light source, creating better conditions, can produce a large number of highly active algae species in the logarithmic growth phase.
  • the technical solution of the invention does not have a supplemental light system, so it is difficult to ensure rapid growth of microalgae in the first-stage reactor, and there are many reactors, and the liquid-liquid transportation between the reactors is cumbersome and consumes a large amount of energy. Summary of the invention
  • the present invention provides a novel microalgae photobioreactor system and a culture process using the same in the large-scale cultivation of microalgae, high-density culture and rapid accumulation of products, which directly restricts large-scale cultivation. .
  • the invention provides a new process and device for the large-scale cultivation of microalgae.
  • the present invention provides a method of producing a cultured algae of oil and fat, the method comprising the following steps -
  • step (b) continuing the algae liquid harvested in step (a) in the secondary reactor for the log phase and stationary phase growth of the microalgae; (c) concentrating the algae solution obtained in step (b), and then Nutrient and/or environmental stress induction is carried out in the tertiary reactor to promote oil accumulation in the microalgae, preferably the nutrient stress is carried out by nitrogen stress culture in a nitrogen-deficient medium.
  • the primary reactor is a column reactor, a plate reactor or a bag reactor, wherein the core is hollow and the hollow is provided with an artificial light source.
  • the secondary reactor is a hollow column reactor, a plate reactor or a bag reactor.
  • the primary reactor and the secondary reactor are concentrically distributed, the secondary reactor inner diameter being greater than the primary reactor inner diameter, preferably in the gap between the primary reactor and the secondary reactor An additional artificial light source is provided.
  • the volume ratio of the secondary reactor to the primary reactor is 8-6:1.
  • the tertiary reactor is an open reactor. In one embodiment, the tertiary reactor is a racetrack shaped reactor (runway reactor), a plate reactor or a bag reactor.
  • the semi-continuous culture is carried out in a first-stage reactor, 20-80% by volume of fresh culture solution is added daily, and 20-80% by volume of algae solution is collected, wherein 20 by volume The amount of -80% is achieved by the difference in height between the initial algae solution and the remaining algae in the first stage reactor.
  • C0 2 is simultaneously introduced to adjust the pH to a range suitable for the growth of the microalgae and to supplement the carbon source required for growth.
  • the algal fluid is cultured in a secondary reactor for 3-5 days.
  • the transfer of the algae solution from the primary reactor to the secondary reactor is carried out by gravity through a tee.
  • the amount of the transferred algal fluid is achieved by the difference in height between the initial algal fluid and the remaining algal fluid.
  • the plurality of algae such as the diatoms, the green algae, the diatoms, and the red algae are preferably chlorella, diatoms or Scenedesmus.
  • the alga is selected from Chaetoceros sinensis, Phaeodactylum tricornutum, Chlorella, Haematococcus pluvialis, Chlorella vulgaris, Algae, and other algae.
  • the artificial light source is continuously turned on during the cultivation, or the artificial light source is turned off partially or completely depending on the difference in the concentration of the inoculated algae and the algae.
  • the artificial light source in the initial stage of inoculation of the primary reactor, the artificial light source is completely turned off, relying solely on natural light growth; after 3-6 hours of inoculation, the artificial light source is partially turned on; after 7-10 hours of inoculation, all open Artificial light source.
  • the present invention provides a photobioreactor system, as shown in Figures 1 and 3, the photobioreactor system comprising a stage reactor and a secondary reactor, wherein the first stage reactor is a column reaction , plate reactor or bag reactor, with a hollow central position, an artificial light source in the hollow, a secondary column reactor, a hollow column reactor, a plate reactor or a bag reactor, a primary reactor And the secondary reactor is concentrically distributed, the primary reactor and the secondary reactor are connected by a three-way connection, wherein the secondary reactor inner diameter is greater than the primary reactor outer diameter, preferably in the primary reactor and the secondary reaction Additional artificial light sources are provided in the gap between the devices to supplement the light source required for microalgae growth.
  • the first stage reactor is a column reaction , plate reactor or bag reactor, with a hollow central position, an artificial light source in the hollow, a secondary column reactor, a hollow column reactor, a plate reactor or a bag reactor, a primary reactor
  • the secondary reactor is concentrically distributed, the primary reactor and the secondary
  • the photobioreactor system further comprises a three-stage reactor that is independently disposed.
  • the tertiary reactor is an open reactor.
  • the tertiary reactor is a racetrack shaped reactor (runway pool reactor) or a plate reactor.
  • the primary reactor has an inner diameter of 50-70 mm, an outer diameter of an inner diameter of + (10-20 mm), and a secondary reactor having an inner diameter of a first-stage reactor having an outer diameter of +10 mm and an outer diameter of The inner diameter of the secondary reactor + (50-100mm).
  • the volume ratio of the secondary reactor to the primary reactor is 8-6:1.
  • the height order between the artificial light source, the primary reactor, and the secondary reactor is: artificial light source > primary reactor > secondary reactor, height difference between primary reactor and secondary reactor , can avoid light occlusion, make maximum use of natural light and artificial light source.
  • the central location of the primary reactor is provided with an artificial light source, and the artificial light source is a plurality of light tubes arranged in a circular bundle shape.
  • the artificial light source can adjust the light intensity according to the cell density of the microalgae.
  • the artificial light source is continuously turned on during the cultivation process or some or all of the artificial light source is turned off depending on the difference in concentration of the inoculated strain of algae and algae.
  • the artificial light source in the initial stage of inoculation of the primary reactor, is completely turned off, relying solely on natural light growth; after 3-6 hours of inoculation, the artificial light source is partially turned on; after 7-10 hours of inoculation, all open Artificial light source, which can achieve the most efficient use of artificial light source, while also saving costs.
  • the first-stage reactor is made of a light-transmissive glass material, which can be easily cleaned and sufficiently transparent, and the secondary reactor can also receive part of the artificial light source to promote cell growth.
  • the main advantages of this reactor are: (1) Adding a separate artificial light source to the hollow of the first-stage reactor, the artificial light source can achieve the effect of the built-in light source of the reactor, preventing the light intensity from being insufficient due to the attenuation of the light source at the center, and at the same time, because it is independently placed, the artificial light source
  • the material can be selected from general materials and the cost is much lower than the built-in light source of the reactor using waterproof material.
  • the artificial light source can adjust the light intensity according to the concentration of the algae cells. When the concentration of algae cells is very low, the artificial light source can be turned off in whole or in part at the beginning of the seeding.
  • the difference in height between the primary reactor and the secondary reactor allows maximum use of light.
  • the primary reactor can receive part of the natural light, and the artificial light source of the primary reactor can also be irradiated into the secondary reactor through the reactor, thereby promoting the growth of the algae cells in the secondary reactor.
  • the entire invention is as shown in FIG. 4, in which a semi-continuous culture is carried out in a first-stage reactor, 20-80% by volume of fresh culture solution is added every day, and 20-80% by volume of algae liquid is collected. , wherein 20-80% by volume is achieved by the difference in height between the initial algae solution and the remaining algae in the first-stage reactor, so that the microalgae is always in the logarithmic growth phase, and the growth rate is 1-3. Times / day.
  • C0 2 is introduced to adjust the pH to maintain an optimal growth pH environment and to supplement the carbon source required for growth.
  • the harvested algae solution is added to the secondary reactor to achieve the log phase and stable growth of the algae, cultured for 3-5 days, the algae solution is concentrated, and then the nitrogen-deficient culture solution is added and placed.
  • the three-stage reactor can select an open reactor such as a plate type or a runway pool.
  • the invention selects an open-type runway pool reactor, so that the light receiving area is large, the natural light can be utilized to the maximum, and the oil is promoted. Rapid accumulation.
  • the present invention adopts different photobioreactors in different stages of the three-step cultivation of microalgae, and the design of each stage of the reactor rationally considers the optimal culture conditions of the microalgae at this stage to achieve high density culture and The product has accumulated rapidly.
  • the present invention employs a three-step culture method in which cell growth and product accumulation are combined. Make full use of the advantages of microalgae growing faster in logarithmic period, and create a continuous logarithmic growth period by intermittently supplementing nutrient solution, which can be quickly and effectively Achieve biomass accumulation. Thirdly, nitrogen stress is added at the end of the logarithm to ensure rapid conversion of the product, reduce the probability of pollution, and rely entirely on natural light, which is economically feasible.
  • the invention adopts a novel photobioreactor system, which can realize the combination of algae cultivation and microalgae expansion, and has simple operation. '
  • the fill light system is arranged in a circular bundle shape, similar to the structure of the reactor, and the artificial light source can be utilized to the utmost extent, and the effect is similar to that of the ordinary built-in artificial light source, but the waterproof material is not required, and the cost is greatly reduced.
  • the semi-continuous culture method for the primary culture of the present invention supplements light at night to maintain the cells in the logarithmic growth phase, and the algae species are excellent.
  • the invention combines the concentrated algae liquid and replenishes the nitrogen-deficient culture liquid phase to ensure the micro-growth of the cells during the product conversion period, and at the same time consume the residual nitrogen source, which can greatly shorten the conversion time and also reduce the conversion time. The probability of pollution.
  • Figure 1 is a first stage reactor (column reactor) and a secondary reactor (column reactor) in the photobioreactor system of the present invention:
  • 1 is the area where the artificial light source is placed, 2 is the first-stage reactor zone, and 3 is the secondary reactor zone.
  • Figure 2 is a distribution diagram of the artificial light source used in the primary reactor.
  • the selected lamp is T8, with a length of 120cm and a power of 36 watts. It is a special lamp for cultivating microalgae. By arranging a plurality of them in a circular bundle shape, the reactor can be utilized to the maximum extent possible.
  • Figure 3 is the head view of Figure 1.
  • 1 is a first-stage reactor
  • 2 is a secondary reactor
  • 3 is an artificial light source zone
  • 4 is a three-way transfer zone.
  • the transfer of the algae liquid is carried out into the secondary reactor by gravity through the tee, and the transfer volume is the height of the initial algae liquid and the remaining algae liquid through the primary reactor.
  • the difference is shown in Fig. 3.
  • the culture system is first inoculated to the height A, and when the culture algae liquid is discharged to the mark B, it is 30%. Similarly, When the cultured algae liquid is discharged to the scale line C, it is 50%.
  • Fig. 4 is a flow chart showing the cultivation of the algae liquid by the culture method of the present invention.
  • Fig. 5 is a graph showing the growth curve of the semi-continuous culture of the first-stage reactor and the conventionally cultured Chaetoceros sinensis.
  • Figure 6 A comparison of the total lipid content of C. faecalis obtained by the method of the present invention and the conventional culture method.
  • Figure 7 Comparison of the growth curves of the semi-continuous culture of the first-stage reactor and the traditional culture of the Phytophthora.
  • Figure 8 Top view of the runway shape reactor.
  • the racetrack shape reactor achieves algae fluid flow by agitating the rotor.
  • Figure 9 Top view of a primary reactor (inside, plate reactor) and a secondary reactor (outer, column reactor) in a photobioreactor system according to another embodiment of the invention:
  • 1 is the algae culture zone
  • 2 is the fill zone
  • 3 is the void fill zone.
  • Figure 10 Top view of a primary reactor (inside, plate reactor) and a secondary reactor (outer, plate reactor) in a photobioreactor system according to another embodiment of the invention:
  • 1 is the algae culture zone
  • 2 is the fill zone
  • 3 is the void fill zone.
  • Figure 11 is a top plan view of a primary reactor (inside, column reactor) and a secondary reactor (outer, column reactor) in a photobioreactor system in accordance with another embodiment of the present invention:
  • 1 is the algae culture zone
  • 2 is the fill zone
  • 3 is the void fill zone.
  • Figure 12 A modification of a primary reactor in a photobioreactor system in accordance with another embodiment of the present invention.
  • a mirror On the first-stage reactor, a mirror is installed, which can be rotated according to the position and intensity of the sunlight, thereby concentrating and adding light to the dead zone.
  • the example arrangement is as shown in Fig. 12.
  • the angle is input to the computer, and then the angle of rotation is set to ensure optimal illumination. This maximizes the use of natural light sources, where 1 is the support column, 2 is the mirror, and 3 is the axis of rotation.
  • 1 is the support column
  • 2 is the mirror
  • 3 is the axis of rotation.
  • the artificial light source In the first 3 hours, the artificial light source is completely turned off. In 4-7 hours, the artificial light source is turned on halfway, and the remaining time is continuously irradiated under the illumination.
  • the illumination is the lamp bundle designed by the invention, and the continuous illumination is 126 ⁇ 10umol/( M2.s), culture temperature is 25 ⁇ 5 °C.
  • culture temperature is 25 ⁇ 5 °C.
  • a mixture of air and C0 2 was introduced through the aeration head, the aeration amount was 2.5 L/min, the percentage of C0 2 was 5%, and the continuous culture time was 11 days. The specific growth is shown in Figure 5.
  • semi-continuous culture is used.
  • the biomass of algae cells can be multiplied by 1-3 times per day, which is a traditional culture (traditional culture method refers to the culture conditions of the control group). Consistent with the primary reactor, the culture method is a traditional batch culture method that performs 2.133 times of the complete cell lag phase, log phase, stabilization and decay phase in one reactor.
  • the tee When the concentration of the algae cells reaches 750 nm and the absorbance is 1.0-1.5, the tee is opened. Under the action of gravity, 20-80% by volume of the algae liquid flows into the secondary reactor, and then under the action of a separate submersible pump, Change the same volume A good f/2 medium was placed in the primary reactor.
  • the algae solution in the primary reactor is inoculated into the secondary reactor by the action of gravity through the 4 tee in Fig. 3, and the culture solution of the secondary reactor is still improved f/ 2.
  • the illumination is natural light and an artificial light source that passes through the first-stage reactor, and the temperature, the aeration, and the C0 2 content are in the same level.
  • the solution was concentrated by filtration, and the volume of the culture solution was concentrated to 50% of the original volume.
  • the obtained algal solution was all placed in the runway cell reactor (ie, the tertiary reactor), and the original volume was added.
  • the /2 nitrogen-deficient medium was subjected to nitrogen stress at a temperature of 30 ⁇ 10 ° C.
  • the light was an artificial light source and stirred by a propeller to achieve rapid accumulation.
  • the total lipid content was determined by differential method.
  • the total lipid content of the nitrogen stress group was 14.38%, which was 2.169 times higher than that of the control group, 6.169 times, in the tertiary reactor.
  • the total lipid content of the nitrogen stress group reached 29.84 ° /. .
  • the specific results are shown in Figure 6.
  • M 1 mass of glass bottle and total fat, g;
  • M 2 quality of dry algal flour
  • g Example 2 three-step culture method and application of nitrogen stress in the culture of Alternaria serrata.
  • the deserted algae Scenedesmus deserticolcd was purchased from Jinan University.
  • the desert Scenedesmus access to a reactor, with BG11 medium, inoculated algae solution logarithmic growth phase (inoculated ratio of 5: 1), an initial OD 750 of 0.583.
  • the medium was irradiated under continuous illumination, and the light was the lamp bundle designed by the invention, and the light intensity was 126 ⁇ 10 umol/(m2.s), and the culture temperature was 25 ⁇ 5 ⁇ .
  • the first-stage reactor From the bottom of the first-stage reactor, a mixture of air and C0 2 was introduced through the aeration head, the aeration amount was 2.5 L/min, the percentage of C0 2 was 5%, and the continuous culture time was 10 days.
  • the specific growth situation is shown in Figure 7.
  • semi-continuous culture is used. By monitoring the OD value at 750 nm, it can be seen that the biomass of algae cells can be multiplied by 1-3 times per day, which is a traditional culture (traditional culture method refers to the culture conditions of the control group). Consistent with the primary reactor, the culture method is 1.754 times the traditional batch culture method in which one cell is subjected to the lag phase, log phase, stable phase and decay phase of the intact cell.
  • the tee When the concentration of algae cells reaches 750 nm and the absorbance is 1.0-2.0, the tee is opened. Under the action of gravity, 50% of the algae liquid flows into the secondary reactor, and then under the action of a separate submersible pump, the same volume The BG11 culture solution is placed in the primary reactor.
  • the algae solution in the first-stage reactor is inoculated into the secondary reactor by gravity under the action of gravity, and the culture solution of the secondary reactor is still BG11, and the illumination is The natural light and the artificial light source through the primary reactor, the temperature, the aeration, the C0 2 content in the same level of reaction.
  • the volume of the culture solution is concentrated to 50% of the original volume
  • the obtained algal liquid is all put into the runway pond reactor (ie, the tertiary reactor), and added to the original
  • the volume of 1/2 of the nitrogen-deficient BG11 medium was subjected to nitrogen stress at a temperature of 30 ⁇ 10 ° C.
  • the light was natural light and stirred by a propeller to achieve rapid accumulation.
  • the total lipid content was determined by differential method.
  • the total lipid content of the nitrogen stress group was 35.43%, which was 2.21 times higher than that of the control group 16.035%.
  • BG11 medium The formulation of BG11 medium is as follows:

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Description

一种微藻培养方法及其光生物反应器系统 技术领域
本发明涉及微藻养殖领域， 具体而言， 涉及一种用新型光生物反应器系统来培养 微藻的方法以及所述方法中使用的新型光生物反应器系统。 背景技术
在当前资源和环境面临双重危机的情况下， 找到一种高效的可再生资源的同时实 现 C02减排就变得尤为重要。 微藻是光合效率最高的原始植物之一， 与农作物相比， 单位面积的产率可高出数十倍。 且微藻富含大量蛋白质、 多糖、 色素等活性物质， 可 以广泛应用于词料、 食品、 医药保健品、 化妆品、 可再生燃料等领域。 进入 21世纪以 后， 石油价格快速飙升， 对微藻的研究迅速成为一个热点， 微藻生物柴油技术也取得 了长足的进步。 在美国、 澳洲、 日本、 西欧、 印度和南非， 无论是政府还是企业， 都 投入大量资金来进行产油微藻的开发。
在微藻的规模化培养中， 传统方法有人工控制光照、 温度、 相对无菌条件等， 但 成本均较高。 这些方法中采用较多的是控制光照。 现有的光照控制技术， 有的采用添 加内置人工光源补光， 但要求使用防水材料， 成本较高； 有的采用外置的单排或双排 光照， 但是占地面积大， 且光照利用率偏低。 这些限制因素大大降低了生物柴油的市 场竞争力。
微藻高效培养首先要有一个高效的光生物反应器； 其次， 藻种要抗逆性较强、 生 长速率高、 总脂含量高。 符合上述条件的微藻藻种， 主要有小球藻、 硅藻、 栅藻等。 一般采用两步培养法： 第一步， 在指数培养期保证最佳的生长条件和营养条件， 让其 生物量快速累积； 第二步， 在指数末期和稳定期前期开始转化培养条件， 从而在体内 积累油脂或色素等物质。
与本发明最相近的实现方案为 CN200410017249.X, 该发明选用的是多级气升式 反应器， 在第一级反应器中添加培养液， 通过重力作用来转移藻液， 并添加了一个人 工光源， 创造较佳条件， 可以生产大量的对数生长期的高活性的藻种。 但该发明的技 术方案没有补光系统， 因而很难保证一级反应器中的微藻快速生长， 且反应器较多， 反应器之间的液液输送繁琐， 能耗较大。 发明内容
本发明针对微藻的规模化培养中， 高密度培养和产品的快速累积」直制约规模化 培养的问题， 提供了一种新型的微藻光生物反应器系统及采用该反应器系统的培养工 艺。 本发明为微藻的规模化培养提供了新的工艺和装置。
一方面， 本发明提供一种生产油脂的培养藻种的方法， 所述方法包括下列步骤-
(a) 在一级反应器中将微藻生长保持在对数期，优选通过半连续培养将微藻生长保 持在对数期；
(b)将步骤 (a)中收获的藻液在二级反应器中继续进行微藻的对数期和稳定期生长； (c)对步骤 (b)中得到的藻液进行浓缩， 然后在三级反应器中进行营养和 /或环境胁 迫诱导， 以促进微藻中的油脂积累， 优选所述营养胁迫是在缺氮的培养基中进行氮胁 迫培养来进行的。
在一个实施方案中， 一级反应器为柱式反应器、 板式反应器或袋式反应器， 其中 心位置为空心， 空心处设置有人工光源。 在一个实施方案中， 二级反应器为空心的柱 式反应器、 板式反应器或袋式反应器。 在一个实施方案中， 一级反应器和二级反应器 呈同心圆分布， 二级反应器内径大于一级反应器内径， 优选在所述一级反应器和二级 反应器之间的间隙中设置有另外的人工光源。 在一个实施方案中， 二级反应器和一级 反应器的体积比为 8-6: 1。
在一个实施方案中， 三级反应器为开放式反应器。 在一个实施方案中， 所述三级 反应器为跑道形状的反应器 （跑道池反应器）、 板式反应器或袋式反应器。
在一个实施方案中， 在一级反应器中进行半连续培养， 每天补入按体积计 20-80% 的新鲜培养液， 并收集按体积计 20-80%的藻液， 其中按体积计 20-80%的量通过一级 反应器中初始藻液和剩余藻液的高度差来实现。
在一个实施方案中， 在一级反应器的培养过程中， 同时通入 C02， 调节 pH值至 适合微藻生长的范围， 并补充生长所需的碳源。
在一个实施方案中， 藻液在二级反应器中培养 3-5天。
在一个实施方案中， 藻液从一级反应器到二级反应器的转移是通过三通在重力的 作用下进行的。
在一个实施方案中，转移藻液的量是通过初始藻液和剩余藻液的高度差来实现的。 在一个实施方案中， 所述的藻硅藻门、 绿藻门、 金藻门和红藻门等多种藻， 优选 为小球藻、 硅藻或栅藻。 在一个实施方案中， 所述藻选自牟氏角毛藻、 三角褐指藻、 小球藻、 雨生红球藻、 拟微绿球藻、 荒漠栅列藻等多种藻。
在一个实施方案中， 其中所述人工光源在培养过程中连续打开， 或根据接种的藻 株和藻液的浓度差异选择部分或全部将人工光源关闭。 在一个实施方案中， 在一级反 应器的接种初期， 将人工光源全部关闭， 单纯依靠自然光生长； 在接种的 3-6小时后， 部分打开人工光源； 在接种 7-10小时后， 全部打开人工光源。
另一方面， 本发明提供一种光生物反应器系统， 如图 1和图 3所显示， 所述光生 物反应器系统包括 级反应器和二级反应器， 其中一级反应器为柱式反应器、 板式反 应器或袋式反应器， 其中心位置为空心， 空心处设置有人工光源， 二级反应器也为空 心的柱式反应器、板式反应器或袋式反应器， 一级反应器和二级反应器呈同心圆分布， 一级反应器和二级反应器通过三通连接， 其中二级反应器内径大于一级反应器外径， 优选在所述一级反应器和二级反应器之间的间隙中设置有另外的人工光源以补充微藻 生长所需要的光源。 在一个实施方案中， 所述光生物反应器系统还包括独立设置的三 级反应器。 在一个实施方案中， 所述三级反应器为开放式反应器。在一个实施方案中， 所述三级反应器为跑道形状的反应器 （跑道池反应器） 或者是板式反应器。 在一个实 施方案中， 所述一级反应器的内径为 50-70mm， 外径为内径 + ( 10-20mm)， 二级反应 器的内径为一级反应器的外径 +10mm， 外径为二级反应器的内径 +(50-100mm)。 在一 个实施方案中， 二级反应器和一级反应器的体积比为 8-6: 1。 在一个实施方案中， 人工 光源、 一级反应器和二级反应器之间的高度顺序为： 人工光源>一级反应器 >二级反应 器， 一级反应器和二级反应器的高度差异， 可以避免光遮挡， 最大限度地利用自然光 和人工光源。 在一个实施方案中， 所述一级反应器的中心位置设置有人工光源， 所述 人工光源为多个排列为圆束状的灯管。 在一个实施方案中， 其中所述人工光源可以根 据微藻的细胞密度来调节光照强度。 在一个实施方案中， 所述人工光源在培养过程中 连续打开或根据接种的藻株和藻液的浓度差异选择部分或全部将人工光源关闭。 在一 个实施方案中， 在一级反应器的接种初期， 将人工光源全部关闭， 单纯依靠自然光生 长； 在接种的 3-6小时后， 部分打开人工光源； 在接种 7-10小时后， 全部打开人工光 源， 这样能达到人工光源的最有效地利用， 同时也节约成本。 在一个实施方案中， 其 中一级反应器采用易透光的玻璃材质， 这样既可以清洗方便， 又能够充分地透光， 让 二级反应器也接受部分人工光源， 促进细胞生长。 该反应器的主要优势为: (1) 一级反应器的中空处添加一独立的人工光源， 该人工光源既能达到反应器内 置光源的效果， 防止中心处因光源衰减而造成光强不足， 同时因为是独立放置， 人工 光源材料可选用一般材料， 成本比使用防水材料的反应器内置光源低很多。
(2)该人工光源可以根据藻细胞的浓度， 调节光照强度。 当藻细胞浓度很低， 在接 种初期， 可将人工光源全部或部分关闭。
(3)一级反应器中， 实行半连续培养， 从而保证微藻始终处于对数生长期， 生物 量积累迅速。
(4) 一级反应器和二级反应器的高度差异， 可以使光线得到最大限度地利用。 既 可以使一级反应器接受部分自然光， 同时， 一级反应器的人工光源也可以透过反应器 而照射到二级反应器中， 从而促进二级反应器中的藻细胞生长。
(5) 藻液从一级反应器到二级反应器的转移是通过三通在重力的作用下进行的， 操作简单方便， 节省能耗。
具体而言， 整个发明如图 4所示， 在一级反应器中实现半连续培养， 每天补入按 体积计 20-80%的新鲜培养液， 并收集按体积计 20-80%的藻液， 其中按体积计 20-80% 的量是通过一级反应器中初始藻液和剩余藻液的高度差来实现的， 这样可以保证微藻 一直处于对数生长期， 生长速度在 1-3倍 /天。 另外， 在培养过程中， 由于细胞生长较 迅速， 从而使 pH值上升很快， 在此同时通入 C02， 调节 pH值， 以保持最佳生长 pH 环境， 并补充生长所需的碳源。 将收获的藻液加入到二级反应器中， 以实现藻的对数 期和稳定期的生长， 培养 3-5天， 对藻液进行浓缩， 然后补入缺氮的培养液， 将其放 入三级反应器中， 三级反应器可选择板式或跑道池等开放式反应器， 本发明选用的是 敞开式的跑道池反应器， 这样受光面积大， 可以最大限度地利用自然光， 促进油脂的 快速累积。 本发明与现有技术相比的优势
1. 本发明在三步法培养微藻的不同阶段采用不同的光生物反应器，每一阶段反应器的 设计都合理地考虑到了该阶段的微藻最佳培养条件，以实现高密度培养和产品快速 积累。
2. 本发明采用三步培养法， 细胞生长和产品累积相结合。充分利用微藻在对数时期生 长较快的优点， 通过间歇式地补充营养液， 创造连续的对数生长期， 可以快速有效 实现生物量的积累。 再次， 在对数末期辅以氮胁迫， 来保证产物地快速转化， 减少 了污染几率， 且完全依靠自然光照等， 经济可行。
3. 本发明采用一种新型的光生物反应器系统， 可以实现藻种培育和微藻扩养相结合， 操作简单。 '
4. 本发明， 将补光系统圆束状排列， 与反应器结构相似， 可以最大限度地利用人工光 源， 且与普通的内置人工光源效果相近， 但无需防水材料， 大大地降低成本。
5. 在一级培养和二级培养之间， 靠三通在重力作用下进行藻液转移， 节约能耗， 降低 成本。
6. 选择硅藻类的牟氏角毛藻， 其生长快、 温度适用性广、 细胞内油脂含量高， 很适合 用来制备液态燃料或油脂。
7. 本发明一级培养用半连续培养法，夜间补光，使细胞维持在对数生长期，藻种优良。
8. 本发明通过浓缩藻液和重新补加缺氮培养液相结合， 既可以在产品转化期， 保证细 胞微生长， 同时又能消耗掉残余的氮源， 可以大大缩短转化时间， 同时也降低了污 染几率。 附图说明
图 1为本发明的光生物反应器系统中的一级反应器 （柱式反应器） 和二级反应器 (柱式反应器）：
1为放置人工光源区， 2为一级反应器区， 3为二级反应器区。
图 2为一级反应器中所使用的人工光源分布图。选的灯管为 T8，长度为 120cm, 功 率为 36瓦， 是培养微藻的专用灯管。 将其多个排列为圆束状， 可以使反应器最大限度 地利用该人工光源。
图 3为图 1的平视
1为一级反应器， 2为二级反应器， 3为人工光源区， 4为三通转移区。
从一级反应器到二级反应器， 藻液的转移， 是通过三通在重力的作用下进入到二 级反应器的， 转移体积是通过一级反应器初始藻液和剩余藻液的高度差来调节的， 具 体情况见图 3， 在一级反应器中， 首先将培养体系接种到高度 A处， 将培养藻液放出 到刻度线 B处时， 即为 30%的量， 同理，将培养藻液放出到刻度线 C处时， 即为 50%。
图 4为用本发明的培养方法培养藻液的培养流程图。
图 5为一级反应器半连续培养与传统培养的牟氏角毛藻的生长曲线比对图。 图 6: 用本发明方法培养与传统培养法得到的牟氏角毛藻的总脂含量对照图。 图 7: 为一级反应器半连续培养与传统培养的荒漠栅列藻的生长曲线比对图。 图 8: 跑道形状反应器的俯视图。
跑道形状反应器通过搅拌转子实现藻液流动。
图 9: 为根据本发明另一个实施方案的光生物反应器系统中的一级反应器（内侧， 板式反应器） 和二级反应器 （外侧， 柱式反应器） 的俯视图：
其中 1为藻液培养区、 2为补光区、 3为空隙补光区。
图 10:为根据本发明另一个实施方案的光生物反应器系统中的一级反应器（内侧， 板式反应器） 和二级反应器 （外侧， 板式反应器） 的俯视图：
其中 1为藻液培养区、 2为补光区、 3为空隙补光区。
图 11 :为根据本发明另一个实施方案的光生物反应器系统中的一级反应器（内侧, 柱式反应器） 和二级反应器 （外侧， 柱式反应器） 的俯视图：
其中 1为藻液培养区、 2为补光区、 3为空隙补光区。
图 12:为根据本发明另一个实施方案的光生物反应器系统中的一级反应器的改进。 在一级反应器上， 安装上反光镜， 可以根据太阳光的位置和光强， 转动， 从而起到聚 光和给死角区加光的作用， 示例排布如图 12， 首先将太阳的照射角度输入计算机， 然 后设置转动角度， 已保证最佳光照， 这样可以最大限度地利用自然光源， 其中 1为支 撑柱、 2为反光镜、 3为转动轴。 具体实施方式
实施例 1三步培养法和氮胁迫在牟氏角毛藻培养中的应用
本发明所用的培养液配方: 改良的 f/2培养液配方:
营养物质 浓度 （mg/L)
NaN03 37.5
尿素 15
Na2Si03.9H20 30
NaH2P04.2H20 5.66
Na2HP04 5.15 FeCl3.6H20 3.162
Na2.EDTA 3.419
CuS04.5H20 0.01
ZnS04.7H20 0.023
CoCL2.6H20 0.012
MnCL2.4H20 0.18
Na2Mo04.2H20 0.07
VB1 0.1
VB12 0.5 χ ΐθ"3
生物素 0.5 x lO"3
MgCl2.7H20 7.5
海水晶 （是一种常用模拟海水的物质， 购自天津 30
中盐海洋生物科学有限公司 缺氮培养液配方： NaH2P04.2H20 5.66 mg/L, Na2HP04 5.15 mg/L, FeCl3.6H20 3.162 mg/L, Na2.EDTA 3.419 mg/L， 海水晶为 30 mg/L。 牟氏角毛藻 Chaetoce爾 muallerO (FACHB-862)购自武汉中科院水生所。 将所述 牟氏角毛藻接入到一级反应器中， 用改良的 f/2培养液， 接种对数生长期的藻液 （接种 的比率为 3: 1)， 初始 OD75Q为 0.512。 在前 3个小时， 人工光源全部关闭， 在 4-7个小 时， 人工光源开启一半， 剩余时间连续光照下培养， 光照为本发明设计的灯束， 连续 光照， 光强为 126±10umol/(m2.s)， 培养温度为 25±5 °C。 从一级反应器的底部通过曝气 头通入空气和 C02的混合气体， 通气量为 2.5L/min， C02的百分含量为 5% ， 连续培养 时间为 11天。 具体的生长情况见图 5。 在一级反应器中， 采用半连续培养， 通过监测 750nm下 OD值， 可以看出藻细胞的生物量可以每天倍增 1-3倍， 是传统培养 （传统 培养法， 是指和对照组培养条件和一级反应器一致， 只是培养方式采取传统的批次培 养法， 在一个反应器中进行完整的细胞的延滞期、 对数期、 稳定起和衰退期） 的 2.133 倍。
藻细胞的浓度达到 750nm吸光度为 1.0-1.5时， 将三通打开， 在重力的作用下， 按 体积计 20-80%的藻液流入二级反应器， 然后在独立的潜水泵的作用下， 将同体积的改 良的 f/2培养液放进一级反应器中。
如上所述， 通过图 3中的 4三通， 在重力的作用下， 将一级反应器中的藻液， 接 种到二级反应器中， 二级反应器的培养液依然为改良的 f/2, 光照为自然光和透过一级 反应器通过的人工光源， 温度、 通气量、 C02含量同一级反应。 在二级反应器培养 4 天后， 通过过滤浓缩， 培养液体积浓缩为原体积的 50%， 将得到的藻液全部放入跑道 池反应器 (即三级反应器)中， 并加入原体积 1/2 的缺氮培养基， 进行氮胁迫， 温度为 30±10°C , 光照为人工光源， 并通过螺旋桨进行搅拌， 从而实现快速累积。 在三级反应 器中， 牟氏角毛藻培养了 2 天后， 通过差重法测定总脂含量， 氮胁迫组总脂含量为 14.38% 比对照组 6.63%增加 2.169倍， 在三级反应器中培养 4天， 氮胁迫组总脂含量 达到 29.84°/。。 具体结果见图 6。
差重法： 取一定体积的藻液， 5000r/min离心 5min， 再用灭过菌的清水清洗 3次， 经冷冻干燥后， 用珠磨机破壁 5min, 采用氯仿： 甲醇提取法得出各微藻中总肪含量。 计算公式为-
X=(Mi-M0)/M2xl00%
其中 X：千藻粉中总脂的含量， ％;
M1 :玻璃瓶和总脂的质量， g;
Mo:玻璃瓶的质量， g;
M2:干藻粉的质量， g 实施例 2三步培养法和氮胁迫在荒漠栅列藻培养中的应用 荒漠栅列藻 Scenedesmus deserticolcd 购自暨南大学。 将所述荒漠栅列藻接入到 一级反应器中，用 BG11培养基，接种对数生长期的藻液 (接种的比率为 5: 1)，初始 OD750 为 0.583。 在连续光照下培养， 光照为本发明设计的灯束， 光强为 126±10umol/(m2.s)， 培养温度为 25±5 Ό。从一级反应器的底部通过曝气头通入空气和 C02的混合气体， 通 气量为 2.5L/min, C02的百分含量为 5% , 连续培养时间为 10天。 具体的生长情况见 图 7。 在一级反应器中， 采用半连续培养， 通过监测 750nm下 OD值， 可以看出藻细 胞的生物量可以每天倍增 1-3倍， 是传统培养 （传统培养法， 是指和对照组培养条件 和一级反应器一致， 只是培养方式采取传统的批次培养法， 在一个反应器中进行完整 的细胞的延滞期、 对数期、 稳定起和衰退期） 的 1.754倍。 藻细胞的浓度达到 750nm吸光度为 1.0-2.0时， 将三通打开， 在重力的作用下， 将 50%的藻液流入二级反应器， 然后在独立的潜水泵的作用下， 将同体积的 BG11 培养 液放进一级反应器中。
如上所述， 通过图 3中的 4三通， 在重力的作用下， 将一级反应器中的藻液， 接 种到二级反应器中， 二级反应器的培养液依然为 BG11， 光照为自然光和透过一级反应 器的人工光源， 温度、 通气量、 C02含量同一级反应。 在二级反应器培养 5天后， 通 过过滤浓縮（培养液体积浓缩为原体积的 50%)，将得到的藻液全部放入跑道池反应器 (即三级反应器)中， 并加入原体积 1/2 的缺氮 BG11 培养基， 进行氮胁迫， 温度为 30±10°C， 光照为自然光， 并通过螺旋桨进行搅拌， 从而实现快速累积。 在三级反应器 中，荒漠栅列藻培养了 7天后，通过差重法测定总脂含量，氮胁迫组总脂含量为 35.43% 比对照组 16.035%增加 2.21倍。
BG11培养基的配方如下表:
Figure imgf000010_0001

Claims

权 利 要 求
1. 一种生产油脂的微藻培养方法， 所述方法包括下列步骤：
(a) 在一级反应器中将微藻生长保持在对数期，优选通过半连续培养将微藻生长保 持在对数期；
(b)将步骤 (a)中收获的藻液在二级反应器中继续进行微藻的对数期和稳定期生长；
(c)对步骤 (b)中得到的藻液进行浓缩，然后在三级反应器中进行营养和 /或环境胁迫 诱导， 以促进微藻中的油脂积累， 优选所述营养胁迫是在缺氮的培养基中进行氮胁迫 培养来进行的。
2. 权利要求 1的方法， 其中所述一级反应器其中心位置为空心， 空心处设置有人 工光源， 可包括所有形式的反应器， 优选所述一级反应器为空心的柱式反应器、 板式 反应器或袋式反应器等。
3. 权利要求 1的方法， 其中所述二级反应器包括所有种类的反应器， 优选空心的 柱式反应器、 板式反应器或袋式反应器。
4. 权利要求 2或 3的方法， 其中所述一级反应器和二级反应器呈同心圆分布， 二 级反应器内径大于一级反应器外径， 优选在所述一级反应器和二级反应器之间的间隙 中设置有另外的人工光源。
5. 权利要求 4的方法， 其中二级反应器和一级反应器的体积比为 8-6: 1。
6. 权利要求 1的方法， 其中所述三级反应器为开放式反应器。
7. 权利要求 6的方法， 其中所述三级反应器为跑道形状 （跑道池反应器） 或者是 板式反应器或是袋式反应器。
8. 权利要求 1 的方法， 在一级反应器中进行半连续培养， 每天补入按体积计 20-80%的新鲜培养液， 并将按体积计 20-80%的藻液转移至所述二级反应器。
9. 权利要求 8的方法， 其中按体积计 20-80%的量是通过一级反应器中初始藻液 和剩余藻液的高度差来实现的。
10. 权利要求 1或 8的方法， 其中在一级反应器的培养过程中， 同时通入 C02， 调节 pH值至适合微藻生长的范围， 并补充生长所需的碳源。
11. 权利要求 2的方法， 其中将藻液在二级反应器中培养 3-5天。
12. 权利要求 1或 5的方法， 其中藻液从一级反应器到二级反应器的转移是通过 三通在重力的作用下进行的。
13. 权利要求 2 的方法， 其中所述人工光源在培养过程中连续打开， 或根据接种 的藻株和藻液的浓度差异选择部分或全部将人工光源关闭。.
14. 权利要求 1、 2、 3、 5、 6、 7、 8、 9或 11 的方法， 所述藻选自硅藻门、 绿藻 门、 金藻门和红藻门等多种藻， 优选为小球藻、 硅藻或栅藻。
15. 权利要求 14的方法， 所述藻选自牟氏角毛藻、 三角褐指藻、 小球藻、 雨生红 球藻、 拟微绿球藻、 荒漠栅列藻等多种藻。
16. 一种用于权利要求 1-15任一项的方法的光生物反应器系统， 包括一级反应器 和二级反应器， 其特征在于： 一级反应器为柱式反应器、板式反应器或袋式反应器， 其 中心位置为空心的， 空心处设置有人工光源， 二级反应器为空心的柱式反应器、 板式 反应器或袋式反应器， 一级反应器和二级反应器呈同心圆分布， 一级反应器和二级反 应器通过三通连接， 其中二级反应器内径大于一级反应器外径。
17. 权利要求 16的光生物反应器系统， 还包括独立设置的三级反应器。
18. 权利要求 17的光生物反应器系统， 其中所述三级反应器为跑道形状的三级反 应器或者是板式反应器。
19. 权利要求 16-18任一项的光生物反应器系统，其中所述二级反应器和所述一级 反应器的体积比为 8-6:1。
20. 权利要求 16-18任一项的光生物反应器系统，其中所述人工光源、一级反应器 和二级反应器之间的高度顺序为： 人工光源>一级反应器 >二级反应器。
21. 权利要求 16-18任一项的光生物反应器系统，所述人工光源为多个排列为圆束 状的灯管。
22. 权利要求 21的光生物反应器系统， 其中根据微藻的细胞密度来调节所述人工 光源的光照强度。
23. 权利要求 16-18任一项、 或权利要求 22的光生物反应器系统， 其中一级反应 器、 二级反应器和 /或三级反应器采用易透光的玻璃材质。
24. 权利要求 16的光生物反应器系统，其中在所述一级反应器和二级反应器之间 的间隙中设置有另外的人工光源以补充微藻生长所需要的光源。
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