CN106978383B - Method for culturing microalgae using amino clay, microalgae obtained by the method, and method for recycling microalgae culture solution - Google Patents
Method for culturing microalgae using amino clay, microalgae obtained by the method, and method for recycling microalgae culture solution Download PDFInfo
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Abstract
The present invention relates to a method for culturing microalgae using amino clay, microalgae cultured by the method, and a method for recycling microalgae culture solution, wherein the method for culturing microalgae using amino clay comprises the steps of: the first step is as follows: a step of preparing an amino clay; the second step is as follows: a step of adding the amino clay to a culture medium containing microalgae, mixing, and culturing; the third step: a step of recovering the microalgae.
Description
Technical Field
The present invention relates to a method for culturing microalgae using an amino clay, microalgae cultured by the method, and a method for recycling a microalgae culture solution, which enables continuous culture of microalgae by collecting the culture solution of the method.
Background
Fossil fuels account for about 80% of the world's energy demand, but due to the limited amount of fossil fuels buried, energy safety issues arise, and due to greenhouse gases such as carbon dioxide generated and emitted when fossil fuels are burned, environmental issues such as global warming that threaten climate change, and other various issues, discussions are being actively made around the world to reduce the amount of fossil fuels used.
Accordingly, biodiesel attracts attention as an alternative fuel to fossil fuels. The biodiesel is widely used as a biofuel together with bioethanol, and as a biofuel made from vegetable oil such as soybean oil, together with bioethanol. Separating glycerol from triglyceride obtained by bonding glycerol to fatty acid of 3-methyl with common alcohol, and preparing into fatty acid ester by transesterification method. The biodiesel produced at this time was Fatty Acid Methyl Ester (FAME).
However, since biodiesel is mainly produced using vegetable oils extracted from edible plants such as beans, there occur problems that the price of grains is increased and that the grain is damaged for use as a cultivation place for grains in tropical rainforests.
In order to solve the above problems, attention is being paid to a technique of using microalgae as a raw material rather than conventional extraction from edible plants, and there is a strong demand for a method of purifying the microalgae and a method of culturing the microalgae capable of increasing the lipid content of the microalgae.
The prior art documents related thereto include the recombinant vectors for increasing the biomass of microalgae and the productivity of lipids and the use thereof disclosed in Korean laid-open patent No. 2015-0084148 (published: 2015.07.22).
Disclosure of Invention
Accordingly, an object of the present invention is to provide a microalgae cultivation method, in which the production efficiency of biodiesel is improved by increasing the lipid and size of microalgae by changing the cultivation conditions, through a non-shape-conversion method.
Further, another object of the present invention is to provide a process which is advantageous for commercialization of a microalgae cultivation process by providing a reusable method of a microalgae cultivation solution which is used in large quantities.
The technical problems to be solved by the present invention are not limited to the above technical problems, and other technical problems not mentioned above will be clearly understood by those skilled in the art to which the present invention pertains from the following description.
In order to solve the above technical problems, the present invention provides a method for culturing microalgae using an amino clay, comprising the steps of: the first step is as follows: a step of preparing an amino clay; the second step is as follows: a step of adding the amino clay to a culture medium containing microalgae, mixing, and culturing; the third step: a step of recovering the microalgae.
The present invention provides microalgae which are increased in lipid and size by preparing an amino clay, adding the amino clay to a culture medium containing the microalgae, mixing, and recovering the mixture after culturing, based on the culture method.
Further, according to another aspect of the present invention, there is provided a method for recycling a microalgae culture solution, comprising the steps of: step a: preparing amino clay, adding the amino clay into a culture medium containing microalgae, mixing, and supplying carbon dioxide for culture; step b: a step of terminating the supply of carbon dioxide to the culture medium and recovering the microalgae; step c: recovering the first-class culture medium and reusing the first-class culture medium as a culture medium for microalgae.
The present invention provides a microalgae cultivation method for greatly increasing the content of fatty acid methyl ester (fat acid methyl ester) which is one of lipids by increasing the size of microalgae used in biodiesel production, so that the biodiesel can be produced by using the obtained microalgae, and the production efficiency of the biodiesel can be greatly increased.
In addition, the method of using the nanoclay added with nanoparticles as a culture condition, instead of the shape-to-mass conversion method of microalgae, can increase the lipid content of microalgae, and thus can more easily increase the lipid of microalgae.
Further, by culturing microalgae in a medium and adjusting the supply amount of carbon dioxide, collecting the obtained microalgae and the first-class liquid on the upper layer of the medium, and repeatedly culturing microalgae several times and reusing water, the efficiency of the microalgae purification process can be greatly increased.
Drawings
FIG. 1 is a process flow diagram illustrating a method for culturing microalgae using an amino clay according to an embodiment of the present invention.
FIG. 2: (a) is a schematic diagram illustrating the structure of an nanoclay according to an embodiment of the present invention, and (b) is a transmission electron micrograph of an nanoclay according to an embodiment of the present invention.
FIG. 3 is a graph illustrating lipid contents of microalgae cultured by the microalgae culturing method using the nanoclay according to the embodiment of the present invention.
Fig. 4 is a graph illustrating the size and cell number of microalgae according to the microalgae cultivation method using the nanoclay according to the embodiment of the present invention.
FIG. 5 is a graph illustrating fluorescence intensity showing the relative intensity of the amount of Reactive Oxygen Species (ROS) produced as an index of pressure in cells when microalgae were cultured using an amino clay according to an embodiment of the present invention.
FIG. 6 is an optical microscope photograph of Nile-red (Nile-red) stained lipid of microalgae cultured by the microalgae culturing method using the amino clay according to the embodiment of the present invention.
FIG. 7 is a graph showing the optical density of the number of microalgae cells in a culture solution based on a method for recycling a microalgae culture solution according to another embodiment of the present invention.
FIG. 8 is a schematic diagram showing an embodiment of a method for recycling a microalgae culture solution according to another embodiment of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
The advantages and features of the present invention and its embodied method will become more apparent with reference to the embodiments and the accompanying drawings described later.
However, the present invention is not limited to the embodiments described below, and can be implemented in various forms, and the embodiments of the present invention are provided only to make the description of the present invention more complete, so that those skilled in the art to which the present invention pertains will fully understand the scope of the present invention, and the present invention is defined only based on the scope of the claims.
In addition, in explaining the present invention, if it is considered that explanations of related well-known technologies and the like may obscure the gist of the present invention, detailed explanations thereof will be omitted.
The present invention will be described in detail below with reference to the accompanying drawings.
The microalgae cultivation method using the amino clay according to the present invention comprises: the first step is as follows: a step of preparing an amino clay; the second step is as follows: a step of adding the amino clay to a culture medium containing microalgae, mixing, and culturing; the third step: a step of recovering the microalgae.
FIG. 1 is a process flow diagram illustrating a method for culturing microalgae using an amino clay according to an embodiment of the present invention.
Referring to fig. 1, the nanoclay may be Mg2+、Ca2+、Al3+、Fe3+、Ni2+、Co2+、Cu2+、Mn2+And Ce3+One positively charged metal particle arbitrarily selected from the group consisting of as an amino group (-NH)2) Substituted organic molecular units.
In addition, the nanoclay forms nanoparticles, which can wrap and cover the surfaces of the microalgae, and in this case, an environment can be formed that stimulates the microalgae, thereby limiting the exchange of substances between the cells and the culture solution, increasing the content of Reactive Oxygen Species (ROS) in the cells, and thus changing the lipid content in the microalgae.
In the step of preparing the nanoclay, magnesium chloride hydrate (MgCl)2.6H2O) is dissolved in the solution, and 3-Aminopropyltriethoxysilane (3-Aminopropyltriethoxysilane) is added to the solution, and the mixture is stirred, wherein magnesium (Mg) of the magnesium chloride hydrate can be added in a molar ratio of 1:1 to 1.34 with respect to silicon element (Si) of the 3-Aminopropyltriethoxysilane (S100).
The molar ratio indicates an appropriate equivalent ratio when synthesizing the amino clay based on Mg and Si, and when the equivalent ratio is 1 to 1.34, all precursors participating in the reaction are reacted.
The amino clay is pulverized into a powder form and becomes transparent when dissolved in water.
When the amino clay is insoluble in water, the amino clay is not charged by cations, and the water-soluble cation particles are self-assembled and induced to form a nano structure.
When the amino clay is not charged by the cation, an environment in which the amino clay coats the surface of the microalgae and is irritating is not formed, which may cause a problem in that the size and lipid content of the microalgae are not affected.
The culture medium containing microalgae of the second step may be added with the amino clay and mixed, thereby performing cultivation (S200).
The amino clay is added to the medium at 0.25 to 1.0 g/L.
If the range is not satisfied, the lipid content of the cultured microalgae does not change so much, and if the range is exceeded, the nanoclay exhibits toxicity to the microalgae, and inhibits cell growth, but rather causes a problem of a decrease in lipid content.
In addition, when the amino clay is provided based on the range, although the concentration of the entire cells is decreased, the size of the cells is increased, and the lipid content in the cells can be greatly increased without deformation of the cells.
The microalgae is selected from the group consisting of Chlorella (Chlorella vulgaris), Histoplasma (Analysis nidulans), Cellulomonas (Ankistrodesmus sp.), Euglenophyta (Biddulpha australis), Botryococcus braunii (Botryococcus braunii), Chaetoceros (Chaetoceros sp.), Chlamydomonas latus (Chlamydomonas applanata), Chlamydomonas reinhardtii (Chlamydomonas reinhardtii), Chlorella (Chlorella sp.), Chlorella mellea (Chlorella mellea) or Chlorella mellea (Chlorella sp.), Chlorella pumila (Chlorella emersonii), Chlorella protothecoides (Chlorella protothecoides), Chlorella pyrenoides (Chlorella pyrenoidosa), Chlorella (Chlorella sorokiniana), Chlorella sp.), Chlorella sp), Chlorella vulgaris (Chlorella sp), Chlorella viridis (Chlorella vulgaris (Chlorella sp), Chlorella vulgaris (Chlorella viridis (Chlorella sp), Chlorella vulgaris (Chlorella sp), Chlorella viridis (Chlorella sp), Chlorella vulgaris (Chlorella viridis), Chlorella viridis (Chlorella vulgaris (Chlorella sp), Chlorella viridis (Chlorella viridis), Chlorella viridis (Chlorella), Chlorella viridis (Chlorella viridis), Chlorella viridis (Chlorella viridis), Chlorella viridis (Chlorella viridis), Chlorella viridis (Chlorella viridis), Chlorella viridis (Chlorella viridis), Chlorella viridis (Chlorella, Chlorella viridis), Chlorella viridis (Chlorella viridis), Chlorella viridis), Chlorella viridis (Chlorella viridis ), Chlorella viridis), Chlorella viridis, Chlorella, Isochrysis galbana (Isochrysis galbana), Isochrysis galbana (Isochrysis sp.), Microcystis aeruginosa (Microcystis aeruginosa), Microcystis parvum (Micromonas pusilla), Allium sativum (Monodendron subterraneous), Microcystis microsphericium (Nannochlororis sp.), Micrococcus nanus (Nannochloropsis sp.), Nannochloropsis nandinensis (Nannochloropsis sp.), Micrococcus nanensis (Nannochloropsis genus), Micrococcus Nannochloropsis (Nannochloropsis sarlina), Nannochloropsis (Nannochloropsis pelliformis), Micrococcus rhodochrous (Nitzschia sp.), Micrococcus neoformans (Nitzschia closterium), Micrococcus fascicularis (Phanerochloa), Micrococcus fascicularis (Phaeococcus fasciata), Micrococcus fascicularis, Microcystis, Micrococcus fascicularia, Microcystis (Phaeococcus fascicularis sp.), Micrococcus fascicularis (Phaeococcus sp.), Micrococcus fascicularis sp.), Micrococcus sp), Micrococcus fascicularia sp), Microcystis sp (Phaeococcus fascicularia sp), Microcystis sp (Phaeococcus fascicularis sp.), Micrococcus (Phaeococcus sp), Micrococcus fascicularia sp), Microcystis sp), Phaeococcus (Phaeococcus fascicularia sp), Phaeococcus (Phaeococcus sp.), Microcystis sp), Phaeococcus (Phaeococcus fascicularia vulgaris (Phaeococcus sp), Phaeococcus (Phaeococcus sp), Phaeococcus (Phaeococcus sp), Phaeococcus (Phaeococcus sp), Phaeococcus (Phaeococcus sp), Phaeococcus (Phaeococcus sp), Phaeococcus (Phaeococcus sp), Phaeococcus (Phaeococcus sp), Phaeococcus (Phaeococcus sp), Phaeococcus (, Pleurotus cornucopiae (Synedra), Scenedesmus obliquus (Scenedesmus obliquus), crescent moon (Selenastrum gracile), Skeletonema costatum (Skeletonoma costatum), Platymonas sobria (Tetraselmis chui), Platymonas maculata (Tetraselmis maculosa), Tetraselmis curvatus (Tetraselmis sp.), Phytophyta plankton (Tetraselmis sueca), Thalassiosia (Thalassima pseudomona), Anabaena (Anabaena sp.), Euglena glauca (Calothrix sp.), ATCC 27169(Chaemis sp.), Anabaena (Chlorococcus sp.), Chroococcus sp.), Porphyridus (Chlorococcus sp.), Porphyromonas sp., Porphyridus (Pleurophyceae sp.), Porphyromonas sp., Porphyromonas (Pleurophyromonas sp.), Porphyromonas (Pleurophyromonas sp.), Porphyromonas sp., Porphyromonas (Pleurophyromonas sp.), Porphyromonas (Pleurotus), Porphyromonas sp., Porphyromonas (Phormidis), Porphyromonas sp.), Mycelosi (Phormis sp.), Mycelosi (Phormiana), Porphyromonas sp.), Mycelastrus (Photinus sp.), Mycelastrus sp., Porphyromonas (Phormidis), Mycelastrus (C (Photinus), Mycelastrus sp.), Mycelastrus (Photinus sp.), C (Photinus) and Mycelastrus sp.), Porphyromonas sp.), Mycelastrus sp., Porphyromonas (Photinus (P Synechococcus (Synechococcus), Synechocystis (Synechocystis sp.), Symbiodinium (Tolypophrix sp.) and Heterococcus (Xenococcus sp.).
When the amino clay is added to the microalgae other than the microalgae under the same conditions, if the size and lipid content of the microalgae are changed, the species of the microalgae are not limited to the above ranges.
The medium of the second step may be formed by providing 0.375 to 0.75g/L of sodium nitrate in 300mL of the medium.
When microalgae are cultured under the condition of insufficient nitrogen content, the lipid content is increased. When the nitrogen component is added within the above range, the effect of increasing the lipid content of the amino clay can be confirmed without the nitrogen component being deficient.
Further, the medium of the second step is formed by supplying 0.05% to 2.0% v/v of carbon dioxide at 50mL/min to 150mL/min in 300mL of the medium.
When the supply of carbon dioxide is out of the range, grown microalgae cannot be gathered or it is difficult to precipitate and recover the microalgae.
The CO is2Concentration and supply (flow) rate are the lowest ranges that promote growth of microalgae, and if the range is not reached, growth of microalgae is not favored, and if the range is exceeded and supplied in excess, not only is CO supplied wastefully2Gas, and can cause agitation that is too aggressive, thereby causing problems in providing an environment unsuitable for the growth of microalgae.
The pH of the medium of the second step can be adjusted to 7.0 to 8.0 by adding 1.0N hydrochloric acid.
It is preferable to adjust the pH to the range according to the best embodiment of the present invention, but the nanoclay shows a cation at pH2.0 to 12, and thus has an advantage that the microalgae can be encapsulated without being limited by pH conditions.
After the cultivation is completed, the microalgae may be precipitated and recovered (S300).
According to another aspect of the present invention, there is provided a method for preparing an amino clay, adding the amino clay to a culture medium containing microalgae, mixing, and recovering lipids and microalgae having increased sizes after culturing.
The microalgae are characterized by a greatly increased intracellular lipid content and increased size through a change in culture conditions in the culture medium without morphological alteration.
The amino clay is added in the culture medium at 0.25 to 1.0g/L, and the carbon dioxide can be provided in the culture medium at 50 to 150mL/min of 300mL and 0.05 to 2.0% v/v.
In addition, in the microalgae, the content of fatty acid methyl ester (hereinafter referred to as 'FAME'), which is one of lipids, may be 150 to 240 mg.
The FAME content may vary depending on the kind of microalgae added to the medium, and is not necessarily limited to the range, which shows only the optimal conditions for the microalgae according to the embodiment of the present invention.
In addition, according to another aspect of the present invention, there is provided a method for recycling a microalgae culture solution.
The method for recycling microalgae comprises the following steps: step a: preparing amino clay, adding the amino clay into a culture medium containing microalgae, mixing, and supplying carbon dioxide for culturing; step b: a step of terminating the supply of carbon dioxide to the culture medium and recovering the microalgae; step c: recovering the first-class culture medium and reusing the first-class culture medium as a culture medium for microalgae.
The carbon dioxide provided in step a is 0.05 to 2.0% v/v carbon dioxide (CO) provided to 300mL of the medium at 50 to 150mL/min2)。
The lipid content of the microalgae can be increased by culturing the microalgae in the medium containing the microalgae when the carbon dioxide is supplied within the range, and the microalgae can be precipitated and recovered when the supply of the carbon dioxide is stopped after the carbon dioxide is supplied within the range.
After the microalgae are precipitated, the supernatant on the upper layer of the culture solution can be recovered and reused.
In the step a, when the amino clay is added to the culture medium in a range of 1.0g/L or less, the growth of cells in the reused culture solution is not affected, and when the amino clay is added beyond the range, the growth of microalgae is inhibited, and there is a possibility that the lipid content of microalgae is reduced.
Preferred embodiments are described below.
< example 1> microalgae culture Using Aminoplay
1. Preparing the material
3-aminopropyltriethoxysilane (3-aminopropyltriethoxysilane; APTES, ≧ 98%, 221.37g/mol), 2', 7' -dichlorofluoroyellow diacetate (2', 7' -dichlorofluorosceranitidinate; DCFDA, 487.29g/mol), dipyridamole (dipyridamole) and Dimethylsulfoxide (DMSO) were purchased from Sigma Aldrich (Sigma-Aldrich, St. Louis, USA). Magnesium chloride hexahydrate (Magnesium chloride hydrate, 98.0%, 203.30g/mol) was purchased from genuine Chemical company (Junsei Chemical co.ltd., Tokyo, Japan). Alcohol (18L, 95%) was purchased from Samchun Pure Chemicals, Pyungtack, Korea. All chemicals were used in the as purchased state without pretreatment. All experiments were performed using Distilled water [ Distilled deionized water (DI water; resistance >18m) ].
2. Manufacture of amino clay
The nanoclay is MgCl mixed with 8.4g2.6H2After dissolving O in 200mL of an alcohol solution, magnetic Stirling refrigeration was performed for 1 minute. 13mL of APTES was then added to the mixture, at which time the Si of APTES was vs. MgCl2.6H2Molar ratio of Mg of O1: 1.34. the solution was then stirred for one day. The resulting diluted slurry was an amino clay obtained by a centrifugation process at 300rpm for 15 minutes. 100mL of the nanoclay isolated by the method was washed twice with alcohol.
Then, the nanoclay was dried at 60 ℃ for one night, and the granular nanoclay was made into a powder using a mortar before use. Then, in order to confirm whether or not the interlayer separation phenomenon occurs, 10mg/mL of the amino clay was put into a 100mL plastic bottle and treated in an ultrasonic bath for 5 minutes (bath-treatment), and whether or not it was dissolved in water and the degree of transparency was confirmed.
3. Culturing microalgae
Microalgae Chlorella (Chlorella vulgaris) (UTEX-265)]Is purchased from Ultes (UTEX Collection Center of the University of Texas at Austin, USA). Chlorella species were cultured by providing 300mL in a 500mL glass flask at 150mL/minModified BG-11 medium (sodium nitrate concentration 0.75g/L half of the original 1.5g/L medium) and 2% v/v carbon dioxide at 100. mu. mol/m2Low density in sec using a rotary-beater (orbital-shaker) at 125 rpm.
During the cultivation, in order to make the nutrient components especially NO3Complete exhaustion of NO3The supply conditions of (a) are changed differently from the existing conditions. The culture broth was adjusted to pH7.5 using 1.0N HCl. Culturing Chlorella vulgaris (Chlorella vulgaris) with amino clay in newly prepared BG-11 solution, adding amino clay with concentration of 0, 0.25, 0.5 and 1.0g/L to observe growth of microalgae based on amino clay content, and separating with and without 2.0% CO injection2The gas condition was cultured.
< example 2> reuse of culture solution
The reuse of water during the cultivation of microalgae is one of the important elements in the commercialization of biofuel production based on microalgae. After harvesting the amino clay based microalgae in said example 1, the first crop of liquid can be reused and the microalgae are continuously reused 3 times. Centrifugal separation was performed at 5000rpm for 10 minutes using 1.0g/L of amino clay, and after removing the remaining microalgae cells, a new inoculum was injected and the growth of the microalgae was observed. During the growth of the microalgae, the solution recycling experiment was continued until the buildup of the amino clay significantly inhibited the growth of the microalgae.
< Experimental example 1> culture results of micro algae based on amino clay
1. Analytical method
The morphology of the nanoclay (0.25mg/mL) dispersed in distilled water was observed using a field-emission transmission electron microscope (FE-TEM, 200kV, TecnaiF20, Philips). To confirm the effect of the amino clay, a Reactive Oxygen Species (ROS) analysis was performed.
Negative control (negative control) was performed in the case of 10. mu.M dipyridamole (dipyridamole) in 0mg/L of the amino clay and the amino clay present at various concentrations (0, 0.25, 0.5, and 1.0g/L), and ROS production was tested in 3 portions.
Samples were collected on 4, 8, and 12 days, respectively, and stained with DMSO containing 10. mu.MDFDA for 60 minutes at room temperature, and washed with phosphate-buffered saline (PBS, 50mM, pH 7.0).
The luminescence curve was recorded by an RF5301PC fluorescence spectrophotometer (Spectrofluorophotometer, 485nm excitation/530nm emission, 150W Xenon lamp, Shimadzu). The cell growth of the microalgae was observed by light microscope, the cell number was measured by hemocytometer, and the Optical Density (OD) of the microalgae cells was observed at 680nm by UV-VIS spectrometer (spectrophotometer). The biomass of the microalgae cells was measured by the weight of the cells using a Whatman filter paper and calculated.
The biomass samples were then vacuum-freeze-dried at-70 ℃ under a vacuum of 1.0mm torr. Of 10mg of the dried sample, lipid analysis studies were carried out by Gas Chromatography (GC), and sulfuric acid and methanol (MeOH) were used to convert chloroform (chloroform) into a mixture according to transesterification (trans esterification): methanol (methanol) was mixed at 2: the mixture was mixed at a ratio of 1v/v and used in the extraction. The size of the cells was measured using a Coulter counter (Beckman Coulter, Multisizer4, USA). Cell imaging of microalgae can be achieved by light microscopy (Leica dm25000, Leica Microsystems, Switzerland) divided between before and after Nile red (Nile red) staining.
2. Aminoplay-based microalgae cultivation
Fig. 2 (a) is a schematic diagram showing the structure of an nanoclay according to an embodiment of the present invention, and (b) is a transmission electron micrograph of an nanoclay according to an embodiment of the present invention.
Referring to the figure, an amino clay [ [ H ]2N(CH2)3]8Si8Mg6O12(OH)4]Octahedral brucite [ Mg (OH) ] in tetrahedral silica structure2]If sandwiched between, this forms a 2: 1 montmorillonite layerA phyllosilicate (small silicate). In the tetrahedral silicon dioxide layer, - (CH)2)3NH2The functional groups are densely formed into functionalization.
Referring to fig. 2 (b), the exfoliated amino clay layer obtained by transmission electron microscopy is amorphous and dispersed in a size of 30 to 100 nm.
FIG. 3 is a graph illustrating lipid contents of microalgae cultured by the microalgae culturing method using the nanoclay according to the embodiment of the present invention.
Referring to the figure, the results of culturing chlorella under atmospheric conditions and with 2.0% carbon dioxide injection based on various levels of nanoclay of 0, 0.25, 0.50, and 1.0g/L are shown.
When injected into the atmosphere, the fatty acid ester (FAME, dry biomass basis, mg/g) content showing a decrease in lipid content under the conditions of the amino clay charge was reduced. Values based on 0, 0.25, 0.5 and 1.0g/L nanoclay content are shown as 239, 167, 188 and 166mg/g, respectively. The results show that the presence of the nanoclay hinders the growth of microalgae and does not contribute to carbon dioxide (CO) due to the low solubility of carbon dioxide2) Conversion to bicarbonate (biocarbonate; HCO3 -) Ions.
In contrast, if 2.0% CO is filled2The FAME content of the gas was increased to 266, 289 and 275mg/g with the addition of 0.25, 0.50 and 1.0g/L of the amino clay, respectively, compared to 227mg/g without the amino clay.
At this time, the amino group converts carbon dioxide into bicarbonate ion (HCO) in a neutral or basic environment3 -)。
Fig. 4 is a graph illustrating the size and cell number of microalgae according to the microalgae cultivation method using the nanoclay according to the embodiment of the present invention.
Referring to the figure, the number of cells where the nanoclay was present was overall reduced. In particular, the number of cells (cells) in the state of normal growth (the static growth phase) after the culture to day 14 is 1273, 1054, 1021 and 532 × 106。
The reason why the density of cells is reduced but the lipid content and size of individual cells are increased is mainly because bicarbonate ions are supplied to microalgae, so that the concentration of cells is reduced, and a coating layer is formed on the surface of cells, thereby inhibiting the exchange between cells and a culture solution, which provides an environment having a pressure to the cells. The pressurized environment is capable of generating Reactive Oxygen Species (ROS).
FIG. 5 is a graph showing fluorescence intensity as a relative intensity of the amount of ROS produced as an index of pressure in cells when microalgae were cultured using an amino clay according to an embodiment of the present invention.
With increasing the content of the amino clay, the intracellular ROS content increased and the fluorescence intensity gradually increased during the 4, 8, and 12 day culture periods.
The results show that there is a stressful environment and HCO on the cell surface of microalgae3 -The high affinity conditions of the ions allow the size and lipid content of the cells to be increased.
3. Changes in the microalgae cultured on the basis of the Aminoplay
FIG. 6 is an optical microscope photograph of Nile-red (Nile-red) stained lipid of microalgae cultured by the microalgae culturing method using the amino clay according to the embodiment of the present invention.
[ TABLE 1 ]
The table 1 shows the average cell size of the microalgae based on the added amount of the nanoclay.
Referring to FIG. 6 and Table 1, the size of chlorella was increased from 3.524 μm to 4.175 μm based on the concentrations of the nanoclay, based on the case where the nanoclay was not added and the case where 1.0g/L of the nanoclay was added. The reason why the average cell size becomes smaller when 0.25g/L of the amino clay is added is that the number of cells increases in the region of 2 to 3 μm. The average cell size of the microalgae was significantly smaller compared to the size of the cells cultured without the addition of the amino clay (-3.524 μm).
In addition, a gradual increase in lipid content was observed in the yellow fluorescence photograph of the middle column.
The right column of FIG. 6 shows the respective culture conditions by a pattern.
As the amount of the nanoclay increases, the nanoclay having a positive charge is coated on the microalgae to coagulate the microalgae cells. As a result, the silica-coated microalgae cells can be considered as a system similar to the living cells with silica nail coat.
< Experimental example 2> reuse of culture solution by recovering first-class solution
In order to reduce the amount of water used in the cultivation of microalgae, 3 experiments were performed to confirm whether water mixed with an amino clay can be reused in the cultivation of Chlorella (Chlorella vulgaris).
FIG. 7 is a graph showing the optical density of the number of microalgae cells in a culture solution based on a method for recycling a microalgae culture solution according to another embodiment of the present invention.
Referring to the drawing, the concentration of the reused nanoclay was 0.75g/L, which is the concentration of the supernatant after the first cultivation of microalgae. 0.25g/L of the amino clay was further injected, and the content of the amino clay was adjusted to 1.0g/L, followed by re-cultivation, and Optical Density (OD) measurement was performed at 680nm from the first pass of 5 times.
When the water mixed with the amino clay is reused, a little dead time occurs in an initial growth state before entering a growth stage at each reuse. Further, in order to observe the environment of the high concentration of the nanoclay, the experiment was repeated under the high concentration of the nanoclay. In the second experiment, the water containing the recycled nanoclay was 1.75g/L, having an OD of 6.83, which is compared to the 7.41OD value tested at the first recycle during the 10 day incubation period. In the third experiment, the nanoclay was reused at a concentration of 2.31g/L, indicating a 5.15OD value. However, 2.73g/L of the amino clay seriously hampers the chlorella culture due to brittle cell wall rupture caused by the application of thicker amino clay.
Therefore, when the chlorella is cultured for biological extraction by reusing the water containing the nanoclay, the concentration of the nanoclay is determined to be in the range of 0.5g/L to less than 2.0 g/L. Moreover, due to the antibacterial and antifungal effects of the amino clay in high concentrations, the inoculation (seeding) and re-cultivation of microalgae can be carried out in a competitive manner.
FIG. 8 is a schematic diagram showing an embodiment of a method for recycling a microalgae culture solution according to another embodiment of the present invention.
Based on the results, a micro-algal biorefinery based on amino-clays can be achieved, with reference to the attached figures.
The amino clay cultures Chlorella in the range of 0.5g/L to less than 2.0g/L, resulting in increased cell size and lipid content. In particular, in the environment of 1.0g/L of the amino clay, the supply of CO to the microalgae is stopped once the cultivation is finished2Precipitation proceeds spontaneously. By the reuse concept, the water containing the amino clay can be used in the later re-cultivation of microalgae. When re-culturing, the concentration of the amino clay was re-adjusted to 1.0g/L, and as a result, the re-cultured microalgae cells were prevented from contamination with amino clay bacteria and molds due to the formation of large amounts of HCO3 -Ionic environment, values are shown as similar results to the first cultured cell values. The microalgae biomass cultured on the basis of the amino clay showed higher lipid extraction efficiency due to the instability of the microalgae cells.
In addition, after the lipid extraction, the remaining amino clay and the microalgae biomass after the lipid extraction are heat-treated at 500 ℃ under the conditions of 4% hydrogen and 96% argon to fix magnesium oxide (MgO) into biocoke (biochar) due to the Bronsted basicity of magnesium oxide and the characteristic that the biocoke has a large number of carbonyl groups and hydroxyl groups, which can be used as an oil-biodiesel conversion catalyst. Using two other sizes of magnesium amino clay, iron amino clay, cerium amino clayCultivating chlorella in soil, heat treating the harvested microalgae biomass, mixing magnesium oxide and magnetic Fe2O3In this case, a biochar having excellent efficiency as an oil-biodiesel conversion catalyst can be produced.
Therefore, the method for culturing microalgae using amino clay according to the present invention can greatly increase the size and lipid content of cells by culturing microalgae using amino clay. Further, by recovering the first-class solution of the culture medium after completion of the culture and reusing the same, it is possible to re-culture the microalgae, and the biomass after the lipid extraction can be reused as a catalyst for biodiesel production or a fertilizer in the heat treatment process.
While the present invention has been described with reference to the specific examples of the method for culturing microalgae using nanoclay, the microalgae using the method for culturing microalgae, and the method for recycling a culture solution of microalgae, it is apparent that the present invention can be carried out in various modifications without departing from the scope of the present invention.
Therefore, the scope of the present invention is not limited to the embodiments described above, and should be defined by the scope of the claims to be described later and the equivalents of the claims.
The above-described embodiments are to be considered in all respects as illustrative and not restrictive, the scope of the invention being determined based on the claims set forth below rather than the detailed description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (8)
1. A method for culturing microalgae using amino clay comprises the following steps:
the first step is as follows: a step of preparing an amino clay;
the second step is as follows: adding the amino clay to a culture medium containing microalgae, mixing the amino clay and the culture medium, supplying carbon dioxide, and culturing the microalgae;
the third step: a step of recovering the microalgae;
wherein the nanoclay of the second step is added to the medium at 0.25 to 1.0 g/L; and is
Providing 0.05 to 2.0% v/v of the carbon dioxide of the second step per 300mL of the medium at 50 to 150mL/min to supply bicarbonate ions HCO in the microalgae3 -Thereby providing a pressurized environment within the microalgae cells,
the amino clay is APTES-Mg2+、APTES-Ca2+、APTES-Al3+、APTES-Fe3+、APTES-Ni2+、APTES-Co2+、APTES-Cu2+、APTES-Mn2+And APTES-Ce3+One or more of (a) or (b),
the microalgae are selected from the group consisting ofChlorella vulgarisChlorella vulgaris (Chlorella vulgaris) with marrow circleChlorella ellipsoidea) Chlorella in water (A)Chlorella emersonii) Chlorella protothecoides (A) and (B)Chlorella protothecoides) Chlorella pyrenoidosa (C. pyrenoidosa) ((C. pyrenoidosa))Chlorella pyrenoidosa)、Chlorella sorokinianaAnd Chlorella minutissima (C)Chlorella minutissima) Any one of the group consisting of.
2. The method for culturing microalgae using nanoclay of claim 1, wherein the culture medium is a medium containing a culture medium,
said step of preparing the nanoclay is a magnesium chloride hydrate MgCl2·6H2Dissolving O in the solution, adding 3-aminopropyltriethoxysilane, and stirring, wherein the magnesium element Mg of the magnesium chloride hydrate is added in a molar ratio of 1: 1-1.34 relative to the silicon element Si of the 3-aminopropyltriethoxysilane.
3. The method for culturing microalgae using nanoclay of claim 1, wherein the culture medium is a medium containing a culture medium,
the amino clay is in a pulverized powder state and becomes transparent when dissolved in water.
4. The method for culturing microalgae using nanoclay of claim 1, wherein the culture medium is a medium containing a culture medium,
the medium of the second step is formed by providing 0.375 to 0.75g/L of sodium nitrate in 300mL of the medium.
5. The method for culturing microalgae using nanoclay of claim 1, wherein the culture medium is a medium containing a culture medium,
the medium of the second step was adjusted to pH7.0 to 8.0 by adding 1.0N hydrochloric acid.
6. A microalgae cultured with amino clay is prepared by preparing amino clay, adding the amino clay to a culture medium containing microalgae, mixing, supplying carbon dioxide, and culturing the microalgae; wherein
The amino clay is added to the medium at 0.25 to 1.0g/L,
supplying 0.05 to 2.0% v/v of said carbon dioxide per 300mL of culture medium at 50 to 150mL/min, supplying bicarbonate ions HCO in microalgae3 -Thereby providing an environment having a pressure in the microalgae cells for cultivation,
the amino clay is APTES-Mg2+、APTES-Ca2+、APTES-Al3+、APTES-Fe3+、APTES-Ni2+、APTES-Co2+、APTES-Cu2+、APTES-Mn2+And APTES-Ce3+One or more of (a) or (b),
the microalgae areChlorella vulgaris，
The microalgae have a fatty acid methyl ester content of 150 to 240mg per 1g of dry biomass.
7. A method for recycling a microalgae culture solution, comprising:
step a: preparing amino clay, adding the amino clay into a culture medium containing microalgae, mixing, supplying carbon dioxide, and culturing the microalgae;
step b: a step of terminating the supply of carbon dioxide to the culture medium and recovering the microalgae;
step c: recovering the first-class culture medium and reusing the first-class culture medium as a culture medium for microalgae; wherein
The nanoclay of step a is added at 0.25 to 1.0g/L,
supplying 0.05 to 2.0% v/v of the carbon dioxide of step a per 300mL of the culture medium at 50 to 150mL/min, and supplying bicarbonate ion HCO in microalgae3 -Thereby providing an environment having a pressure in the microalgae cells,
the amino clay is APTES-Mg2+、APTES-Ca2+、APTES-Al3+、APTES-Fe3+、APTES-Ni2+、APTES-Co2+、APTES-Cu2+、APTES-Mn2+And APTES-Ce3+One or more of (a) or (b),
the microalgae are selected from the group consisting ofChlorella vulgarisChlorella vulgaris (Chlorella vulgaris) with marrow circleChlorella ellipsoidea) Chlorella in water (A)Chlorella emersonii) Chlorella protothecoides (A) and (B)Chlorella protothecoides) Chlorella pyrenoidosa (C. pyrenoidosa) ((C. pyrenoidosa))Chlorella pyrenoidosa)、Chlorella sorokinianaAnd Chlorella minutissima (C)Chlorella minutissima) Any one of the group consisting of.
8. The method of recycling a microalgae culture solution according to claim 7, wherein the first-class solution recovered in the step c is used as the microalgae culture solution 3 to 5 times.
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