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WO2012019539A1 - Procédé de culture en milieu semi-sec et semi-solide pour la production industrialisée de micro-algues - Google Patents

Procédé de culture en milieu semi-sec et semi-solide pour la production industrialisée de micro-algues Download PDF

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Publication number
WO2012019539A1
WO2012019539A1 PCT/CN2011/078198 CN2011078198W WO2012019539A1 WO 2012019539 A1 WO2012019539 A1 WO 2012019539A1 CN 2011078198 W CN2011078198 W CN 2011078198W WO 2012019539 A1 WO2012019539 A1 WO 2012019539A1
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microalgae
cells
culture
concentration
carbon source
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PCT/CN2011/078198
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Chinese (zh)
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刘天中
王俊峰
张维
陈晓琳
彭小伟
陈昱
陈林
高莉丽
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中国科学院青岛生物能源与过程研究所
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Publication of WO2012019539A1 publication Critical patent/WO2012019539A1/fr

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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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof

Definitions

  • the invention belongs to the field of industrial production of microalgae and microalgae products, in particular to a semi-dry solid culture method for large-scale production of microalgae biomass and secondary metabolites. Background technique
  • Microalgae are aquatic planktonic algae that are capable of photosynthesis. Some microalgae are rich in protein and can be used as aquatic bait or livestock feed (such as spirulina). More importantly, some microalgae can synthesize secondary metabolites such as oils, carotenoids, polysaccharides, etc. under certain conditions. These substances are often bioactive substances with extremely high economic value and can be used in functional foods, food additives, pharmaceuticals, bioenergy and other fields. In particular, the large-scale cultivation of microalgae oil by microalgae, which is converted into biodiesel, is considered to be one of the most important ways to solve bioenergy production and carbon sequestration. At present, on a global scale, microalgae biotechnology has rapidly formed a large-scale complete industrial chain, in which scale cultivation is an important link.
  • PBR is generally a thin structure made of a light-transmitting material (such as glass, plexiglass, plastic film, etc.). Since the light path is small and the illumination area/volume of the culture system is relatively large, the cell illumination is sufficient. At the same time, the carbon-fixing gas has a long contact time with the liquid, and the culture solution dissolves the C0 2 concentration higher, so the cell growth rate and the culture density are higher than those of the open culture tank.
  • PBR is often expensive, expensive to operate, difficult to maintain, and difficult to scale.
  • microalgae Compared with terrestrial plants, microalgae have high photosynthetic efficiency and high growth rate, which is an important advantage of microalgae as one of the most promising new biomass resources. It is also the development of microalgae industry (food, feed, chemicals, biology). The basis of energy, etc.).
  • the photosynthetic efficiency of microalgae is theoretically about 10 times higher than that of terrestrial plants, even with the most efficient PBR, the highest biomass concentration can only reach lOgL with natural light and no external light source. / 1 or so, if considering the floor space and actual conditions, the annual maximum biomass production is generally less than 200 tons / Hectares, close to higher plants. Therefore, the photosynthesis potential of microalgae under traditional culture methods is far from being fully utilized.
  • the portion which accounts for the largest specific gravity of the culture liquid is water.
  • the role of water in microalgae culture is mainly as follows: 1) As a solvent and transmission medium for various nutrients (including co 2 and inorganic salts), it promotes effective contact between microalgae cells and nutrients; 2) as a regulating environment Buffer system, stable environmental parameters such as pH, temperature and osmotic pressure of culture solution. 3) As a supporting system of microalgae cells, expand the living space of the microalgae cells, so that the cells can fully receive the light. In principle, the amount of water required to complete the first two functions is small, as long as the water layer keeps the algae cells moist.
  • water exhibits more harm than good because of its own nature. It is mainly 1) When light passes through a water body, energy attenuation occurs, and the larger the light path, the more severe the attenuation. 2) Most of the energy consumed during the temperature control process is used for the water body, not the algae cells themselves. 3) Under normal circumstances, the density of microalgae is greater than that of water, so it is necessary to constantly stir the water to avoid sedimentation of algae cells. This process consumes a lot of energy. At the same time, the agitation of water will cause the light environment of the cells to fluctuate drastically, which may affect biomass. accumulation. 4) Excessive water body leads to only increase nutrient salt and C0 2 consumption In order to maintain the necessary concentration.
  • microalgae biologically mediated oxidative stress
  • metabolites such as oils and fats
  • cell growth requires a high nitrogen environment
  • oil accumulation requires a low nitrogen and other stress environment.
  • Large water cultures greatly increase the difficulty of switching between these two environments.
  • the conventional method waits until the original nitrogen source in the culture system is consumed, and then gradually converts into a nitrogen-deficient induction environment, and this process often takes 10 to 15 days. If you want to switch quickly, you only need to collect the algae cells from the high nitrogen medium before transferring them to low-nitrogen or nitrogen-free medium for lipid metabolism, which has a large workload and high energy consumption.
  • microalgal cells can grow without relying on water as a support system.
  • the use of agar solid medium to make a culture plate or a test tube bevel for the culture of microalgae has been used in the laboratory, but the method generally involves placing the culture dish or the test tube in a light incubator for cultivation. Screening or preservation.
  • the process generally only uses C0 2 in natural air, does not need to introduce artificial C0 2 gas environment, and does not use the method of changing medium composition, pH value, illumination, etc. to achieve biomass or secondary metabolite cooperation. It is regulated, so it is not suitable for large-scale microalgae biomass or secondary metabolites, nor is it suitable for industrial production of microalgae.
  • the cell layer is then periodically exposed/immersed in the liquid medium at a certain frequency by swinging the culture equipment to achieve biomass increase and oil accumulation.
  • the microalgae adhered to the surface of the support to facilitate harvesting, but since the culture material was still placed in a large amount of liquid medium, various problems caused by the large water bodies mentioned above were not excluded.
  • Even biomass production is lower than traditional culture methods. For example, in this paper, the biomass yield under the light intensity of 110 ⁇ 120 ⁇ 1 m is about 3.5 ⁇ 1 ⁇ 2 (1 is much lower than the average level of traditional liquid immersion culture 5-30gm under the same conditions).
  • An object of the present invention is to provide a semi-dry solid state culture method for industrial production of microalgae to solve the problem of the inability to industrially produce microalgae.
  • the culture method provided by the present invention mainly has the following two points: 1) semi-dry solid state culture 2) environmental control of the culture system.
  • the specific steps of the culture method provided by the present invention are: first inoculation of microalgae cells on the surface of the solid material, and keeping the cell population moist by supplementing the liquid; then, adding an inorganic carbon source to the cell population under illumination; then, passing Controlling the composition of the dampening solution, light intensity, and carbon source concentration regulates the growth and metabolism of microalgae cells, and realizes the accumulation of microalgae biomass and/or secondary metabolites.
  • the solid material in the present invention refers to a porous water absorbing material which is non-toxic or slightly toxic to microalgae cells and has a certain liquid storage capacity, and includes various types of filter paper, filter cloth, sponge, plastic foam, and fiber woven material.
  • the method of inoculation in the present invention may be any means, method, such as, but not limited to, immersion, spraying, filtration, smearing, injection, etc., which can cause algae cells to exist on the surface of the solid material.
  • the inoculation surface of the solid material in the present invention may be a flat surface or an arbitrary curved surface.
  • the solid material may be placed in a single layer, or may be arranged in a plurality of layers to form a combination, and the layer spacing may be adjusted to an arbitrary value according to requirements.
  • the liquid supplemented in the present invention may be pure water or a microalgae medium of various types, concentrations, components, or a solution containing various stresses and/or inducing factors.
  • the microalgae medium differs depending on the species of algae. For example, for freshwater algae, BG11 medium is generally used, and for seaweed, f/2 medium is generally used.
  • the stress and/or inducing factor may be a nitrogen content in the medium, a phosphorus content, a metal salt content (iron, magnesium, etc.), a pH, and the like.
  • the method of adding an inorganic carbon source to the cell population may be by increasing the gas environment in which the cells are located.
  • C0 2 concentration (Wv) pathway (0-100%), for example, continuously or intermittently fed co 2 / air mixture or artificial gas atmosphere containing C0 2 concentration is higher than the pure C0 2 gas, a flue Gas or the like; may also be a method of supplementing a solution containing an inorganic carbon source (including a carbonate solution containing carbonate and/or bicarbonate (carbonate concentration in the range of 0-1 mol/L, and/or bicarbonate) The concentration is in the range of 0-lmol / L), and the C0 2 solution), the process can be carried out separately or in combination with the rehydration process.
  • an inorganic carbon source including a carbonate solution containing carbonate and/or bicarbonate (carbonate concentration in the range of 0-1 mol/L, and/or bicarbonate)
  • the concentration is in the range of 0-lmol / L), and the C0 2 solution
  • the process can be carried out separately or in combination with the rehydration process.
  • Modification methods for achieving rapid accumulation of biomass and/or secondary metabolites in the present invention include, but are not limited to, adjustment of one or more of the following parameters: Wetting fluid composition and concentration (wetting fluid may be cultured by BG11 or f/2) Base composition, sodium nitrate content range of 0-1.5 g / liter, potassium hydrogen phosphate content range of 0-0.04 g / liter), light wavelength (visible light) and light intensity (in the range of 10-2500 umol / m 2 / s) , . 0 2 concentration 0) (in the range of 0-100%), temperature (in the range of 20-38 ° C, preferably 25-38 ° C), pH (in the range of 7,0-11.5, preferably 7.2-11.5) Wait.
  • Wetting fluid composition and concentration wetting fluid may be cultured by BG11 or f/2
  • Base composition sodium nitrate content range of 0-1.5 g / liter, potassium hydrogen phosphate content range of 0-0.04
  • the process of accumulating biomass and accumulating secondary metabolites in the present invention may be carried out separately or simultaneously or sequentially.
  • Microalgae suitable for use in the present invention include, but are not limited to, Scenedesmus, Haematococcus, Chlorella, Nannochloropsis, Trichophyton (Phaeodactylum), Dunaliella, Chrysophyta, Cyanophyta, etc.; induced secondary metabolites including, but not limited to, triglyceride (TG), astaxanthin (Astaxanthin) ), Carotenoids, etc.
  • the invention is characterized in that, in the premise of retaining a very small amount of aqueous solution as a cell growth and mass transfer medium, most of the water body as a cell support system in the traditional liquid immersion culture method is discarded, thereby greatly reducing water source, nutrient salt, and collection. Cost of the drying step: Since the solution of the present invention only keeps the cells moist, the light conduction does not need to be transmitted through a long water body, the path is greatly shortened, and the light energy transmission loss is greatly reduced.
  • the cells are also easily in contact with other nutrients (such as C0 2 , inorganic salt concentration, etc.), thereby greatly improving the utilization efficiency of light energy, C0 2 , and nutrient elements;
  • the kinetics are weakened, the position is relatively fixed, and the surface biofilm cells can be directly contacted with light.
  • the algae cells can be continuously subjected to efficient photosynthesis without excessive light intensity, and the light energy can be utilized.
  • the rate is high; in the present invention, the algae cells which are in the most vigorous stage of division and growth are always at the top of the group, and receive sufficient light and nutrient components to ensure rapid growth; in the present invention, since the water body is small, various stress conditions are easy to add. And releasing, thereby making the cell growth state easy to regulate; compared with the conventional method, the cell of the present invention adheres to the surface of the solid material to form a biofilm, and can directly collect the concentrated algae mud, and even obtain the dry algae cell by stopping the rehydration and naturally evaporating and drying.
  • the invention solves the problems of difficulty in harvesting and high energy consumption in drying of the conventional liquid immersion culture; the invention solves the limitation of the pressure of the conventional liquid immersion culture of a large amount of water on the enlargement and high-rise of the photoreactor, and the reactor weight involved Light, low material requirements, low cost, and can be placed in multiple layers, greatly improving the space utilization rate, thereby greatly improving the cell culture efficiency and unit area yield of microalgae, and helping to solve the industrialization of microalgae technology. bottleneck. detailed description
  • the culture method of the present invention is:
  • the microalgae cells are inoculated on the surface of the solid material.
  • the solid material refers to a porous water absorbing material which is non-toxic or toxic to microalgae cells and has a liquid storage capacity, and includes various types of filter paper, filter cloth, organic synthetic sponge, plastic foam, and fiber fabric material.
  • the method of inoculation may be any route, method, or method that enables algae cells to be present on the surface of the solid material, including but not limited to: immersion, spraying, filtration, vacuum filtration, smearing, injection, and the like.
  • the surface may be a flat surface or an arbitrary curved surface;
  • the solid material after inoculation can be placed in a single layer, or it can be arranged in a multi-layer arrangement to form a combination, and the layer spacing can be adjusted to an arbitrary value according to requirements.
  • the rehydration process may be in a batch, semi-continuous or continuous manner, and the specific methods include, but are not limited to, addition, dripping, spraying, etc.; the replenished liquid may be pure water or various types, concentrations, components of microalgae medium It may also be a solution containing various stresses and/or inducing factors (such as nitrogen content in the medium, phosphorus content, metal salt content (iron, magnesium, etc.), pH).
  • wetting fluid may consist of BG11 or f/2 medium, sodium nitrate content ranging from 0 to 1.5 g/L, Potassium hydrogen phosphate content in the range of 0-0.04 g / liter), light wavelength (visible light) and light intensity (in the range of 50-2500umol / m 2 / s), C0 2 concentration (in the range of 1-100%), temperature (in the range of 20-38, preferably 25-38 ° C), pH (in the range of 7.0-11.5, preferably 7.2-11.5); the process of accumulating biomass and accumulating secondary metabolites can be carried out separately or simultaneously or Go ahead.
  • Wetting fluid composition and concentration wetting fluid may consist of BG11 or f/2 medium, sodium nitrate content ranging from 0 to 1.5 g/L, Potassium hydrogen phosphate content in the range of 0-0.04 g / liter), light wavelength (visible light) and light intensity (in the range of 50-2500umol / m 2 / s
  • a plexiglass plate with a length X width of 0.5 mx 0.5 m and a thickness of 0.003 m was wrapped with 5 layers of medical gauze, and the surface was covered with an analysis filter paper, and then 1 L of BG11 medium (composed of: sodium nitrate 1.5 per liter of medium) Grams, 0.04 g of potassium hydrogen phosphate, 0.0375 g of magnesium sulfate 7 hydrate, 0.036 g of calcium chloride dihydrate, 0.006 g of citric acid, 0.006 g of ammonium ferric citrate, 0.001 g of EDTA disodium salt, 0.02 g of sodium carbonate, 0.00286 g of boric acid , 0.00186 g of manganese chloride 4, 0.00022 g of zinc sulfate 7-hydrate, 0.00039 g of sodium molybdate 2-hydrate, 0.00008 g of copper sulfate 5, 0.00005 g of cobalt nitrate 6 hydrate, the
  • the culture solution of Scenera enw 3 ⁇ 4tz. UTEX1237 (University of Texas University Algae Collection) was vacuum-filtered and attached to a cellulose acetate filter having a pore size of 0.45 ⁇ m, and the biomass density was 20 g m - 2 .
  • a cellulose acetate filter to which G. grisea cells are attached is spread over the filter paper and placed inside a glass case (length X width X height is 0.5 mx 0.5 mx 0.03 m, and the material is ordinary glass having a thickness of 0.003 m).
  • One side of the glass case (0.5mx0.03m) is open to facilitate the pick-and-place of the culture plate, and the other two sides of the opposite sides respectively have a circular opening with a diameter of 0.003m, and a silicone tube with an inner diameter of 0.003m is inserted into the opening as a feed.
  • Air port and exhaust port The intake air is compressed air mixed with 1.5% (V/V) carbon dioxide and passed into the tank at a flow rate of 1Lmin- 1 .
  • Ten identical glass boxes were stacked and placed on an angle steel frame with a length X width X height of 0.5 mx 0.5 mx 2 m, a layer height of 0.2 m, and a fluorescent lamp as a light source was placed on top of each layer.
  • the cell surface light intensity was adjusted to 40 ⁇ )1 ⁇ _ 2 3 - i, the ambient temperature was 28 ° C, and the culture was continued for 24 hours.
  • the BG11 medium was supplemented by spraying every day and the cell population was kept moist.
  • the spray rehydration was changed from BG11 medium to pure water, and the concentration of carbon dioxide in the aeration was increased to 10% (V/V), and the light intensity was increased to 200 ⁇ 1 m- 1 to induce accumulation of triglycerides in the cells of the gems.
  • the total lipid content of the algae cells reached 52% (dry weight) and the neutral lipid content reached 41.6% (dry weight).
  • the total lipid content increased by 8.3%
  • the neutral fat content increased by 73.3%
  • the induction time was shortened by 57%.
  • Example 2 An apparatus and conditions similar to those in Example 1 were utilized, but the artificial light source at the top of each layer was removed.
  • the next step was to cultivate Scenedesmus (Scewecfewnws dimorphus K tz. ⁇ /7 ⁇ : _ ⁇ ⁇ 7) (University of Texas Breeding Center) and induce triglyceride accumulation.
  • the daytime ventilation flow rate SL miif 1 carbon dioxide concentration 0.5% (V / V); in the evening filled the glass box filled with pure carbon dioxide, stop ventilation.
  • the cell population was sprayed with BG11 every 4 hours during the daytime period, and the nitrogen concentration of BG11 was reduced to zero within 5 days.
  • the average growth rate of cells in each layer of culture chamber was l Sg m ⁇ cT 1 , and the cell growth rate per unit area was 150 ⁇ 1 ⁇ 2 ⁇ .
  • the spray rehydration solution was changed from BG11 medium to pure water, and the concentration of carbon dioxide in the aeration was increased to 10% (V/V), and the triglyceride was induced to accumulate in the algae cells under sunlight.
  • the total lipid content of the cells reached 55% (dry weight) and the neutral lipid content reached 49.5% (dry weight).
  • the growth rate increased by 275%, the total lipid content increased by 37.5%, and the neutral fat content increased by 518.8%.
  • the growth rate increased by 900%, the total lipid content increased by 57.1%, and the neutral fat content increased by 607%.
  • the rotating device structure is similar to a vertically placed conveyor belt.
  • two shaft structures with a diameter of 0.2 m are arranged along the center line, wherein the lower one has a lower cutting plane 0.6 m from the ground, and the upper and lower axes are two axes.
  • the distance between the two shafts is 1.3m, and the two shafts are connected to the motor through the shifting mechanism, and synchronous movement is achieved.
  • a track is connected to the two axes.
  • the track consists of 15 pieces of 0.5mx0.2m stainless steel plates, the long sides of which are joined together by a hinge structure. The surfaces of the two shafts have protruding teeth that just engage the hinges to drive the track to rotate.
  • a spray device is installed 0.5m from the ground. The liquid sprayed is a carbon dioxide-saturated microalgae medium (the medium with a sodium nitrate concentration of 1/4 of the BG11 medium is placed in a pressurized tank, and the tank is filled with pure C0.
  • the device can rotate the cells on both sides of the organic plate by the rotation of the crawler, supplement the carbon dioxide by spraying and maintain the cell population, and maintain the cell population temperature at 20 ⁇ 30 °C by evaporation of water.
  • the condition was continuously sprayed to the culture plate for 2 min every 1 ⁇ 10 min. The entire unit was shut down in the evening.
  • the algae liquid was collected to measure the growth and astaxanthin content of Haematococcus pluvialis. The results showed that the average biomass per unit area of the plant was about 75g m" 2 d" 1 , the production of carotenoids.
  • FACHB712 Concentrated Haematococcus pluvialis Pluvialis
  • FACHB712 purchased from the Freshwater Algae Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences in Wuhan
  • Wetting biomass concentration lg L" 1 , sodium nitrate concentration is 1/4 of BG11 medium.
  • the peristaltic pump is used to continuously replenish water.
  • the flow rate is controlled between 0.001L and 0.01Lmin- 1 (to keep the sponge block moist but not dripping), and the compression of carbon dioxide content is 1.5% (V/V).
  • the algae species used are replaced with Pseudomonas aeruginosa, Chlorella, Scenedesmus, Trichophyton, Algae, Dunaliella, Cyanobacteria, etc., or the gas introduced is changed to flue gas. Repeat the above experiment, the results are similar, but repeat the narrative from the simple.

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Abstract

Procédé de production industrialisé de micro-algues, comprenant les étapes spécifiques consistant à : inoculer d'abord des cellules de micro-algues sur la surface d'un matériau solide, tout en maintenant la population cellulaire humide par apport d'un liquide; puis, introduire une source de carbone inorganique dans la population cellulaire dans une condition d'éclairage artificiel; et, en outre, réguler la croissance et le métabolisme des cellules micro-algales par contrôle de paramètres tels que la composition du liquide d'humidification, l'intensité de l'éclairage, et les concentrations de la source de carbone pour obtenir une accumulation de la biomasse micro-algale et/ou de métabolites secondaires. La présente invention bannit la pratique consistant à utiliser une grande quantité d'eau comme le milieu de support utilisé dans le procédé de culture classique par immersion dans un liquide, et réduit le volume et le poids du système de culture, résolvant ainsi complètement le problème selon lequel on ne peut pas construire de photobioréacteurs à micro-algues de grande taille, l'utilisation de l'espace n'est pas maximisée, il est difficile de passer à une plus grande échelle et le rendement est bas vis-à-vis de l'utilisation de l'espace en raison de limitations sur la résistance des matériaux, ce qui en retour, réduit les coûts d'équipement et les coûts de fonctionnement de l'installation. Dans la présente invention, les micro-algues utilisent efficacement les nutriments, l'énergie lumineuse et les sources de carbone, et l'induction des métabolites secondaires est rapide, ce qui augmente ainsi significativement et de manière considérable les rendements de la biomasse et des métabolites secondaires par unité de surface.
PCT/CN2011/078198 2010-08-10 2011-08-10 Procédé de culture en milieu semi-sec et semi-solide pour la production industrialisée de micro-algues WO2012019539A1 (fr)

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