CN107189946B - Method for improving astaxanthin yield by avoiding microalgae photoinhibition - Google Patents
Method for improving astaxanthin yield by avoiding microalgae photoinhibition Download PDFInfo
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Abstract
The invention relates to a novel method for avoiding photoinhibition in a microalgae culture process so as to improve the yield of astaxanthin. The method for culturing the microalgae to produce the astaxanthin generally adopts a two-stage method, wherein the first stage realizes mass propagation of cells and accumulation of biomass in a heterotrophic, autotrophic or mixotrophic mode, and the second stage realizes the accumulation of the astaxanthin in the cells by methods of light stress, nutrition stress and the like. However, the conversion from the first stage to the second stage often causes phenomena such as photoinhibition, which seriously affect the yield of astaxanthin. The present invention is therefore based on the control of the culture conditions during the transformation, such as: the cell state is adjusted by means of controlling the composition of a culture medium, shading sun, carrying out weak light transition, improving the initial inoculation density, gradually diluting, adjusting outdoor inoculation time and the like, so that the cell has the capability of resisting strong light, the problem of photoinhibition of microalgae under strong light is fully solved, the efficiency of producing astaxanthin by the microalgae can be greatly improved, and the astaxanthin can be produced by the microalgae through low-cost, high-efficiency and large-scale culture.
Description
Technical Field
The invention belongs to the technical field of microalgae biology, and particularly relates to a method for adjusting the cell state of microalgae and improving the yield of astaxanthin.
Background
Astaxanthin (Astaxanthin), chemical name 3,3 ' -dihydroxy-4, 4 ' -diketo-beta, beta ' -carotene, molecular formula C40H52O4, relative molecular mass 596.86, Astaxanthin or lobster shell pigment, is a keto carotenoid. The color is pink, fat soluble, insoluble in water, and easily soluble in organic solvents such as chloroform, acetone, benzene and carbon disulfide. The chemical structure of astaxanthin is a six-section ring structure formed by connecting 4 isoprene units in a conjugated double bond mode and forming 2 isopentene units at two ends. Astaxanthin contains a long conjugated unsaturated double bond system in its chemical structure, and thus, it is easily destroyed by the action of light, heat, an oxide, or the like. The chemical structure of astaxanthin is shown in the following formula:
astaxanthin is one kind of carotenoid and the highest-grade product of carotenoid synthesis, and beta-carotene, lutein, canthaxanthin, lycopene and the like are all intermediate products of carotenoid synthesis. Therefore, astaxanthin has the strongest oxidation resistance in nature. Natural astaxanthin is the strongest antioxidant found in nature by human beings so far, and the antioxidant activity of the natural astaxanthin far exceeds that of the existing antioxidant, so that the natural astaxanthin is known as a super oxidant. The astaxanthin has wide application value, can be used as a feed additive for aquaculture and a human food additive, and also has great application potential in the fields of medicines, cosmetics, nutritional health products and the like.
In comparison, the method for producing astaxanthin by utilizing microalgae has obvious advantages. Among them, Haematococcus pluvialis contains astaxanthin in an amount of about 1 to 5% by dry weight of cells, and is a natural species having the highest content of astaxanthin in nature. Firstly, astaxanthin exists in microalgae mainly in the form of monoester, and the structure of astaxanthin is a trans-structure, so that the bioavailability is higher than that of a chemically synthesized cis-structure; secondly, the growth period of the microalgae is short, the occupied area of production equipment is small, and the product quality and the yield are relatively stable; finally, microalgae (such as haematococcus pluvialis and the like) are high-value products, contain a large amount of active ingredients such as protein, grease, polysaccharide and the like, and can be separated and extracted to realize comprehensive utilization of microalgae cells.
At present, according to the process of producing astaxanthin by microalgae, taking haematococcus pluvialis as an example, the microalgae culture process is generally divided into two stages of microalgae culture and astaxanthin accumulation. The first stage (microalgae culture) is mainly the culture of haematococcus pluvialis to make it grow rapidly, and can be photoautotrophy, heterotrophy and polyculture. In the second stage (astaxanthin accumulation), haematococcus pluvialis is promoted to be transformed into chlamydospores in a severe living environment through a series of stress means such as high illumination, high temperature, high salt, nutrient salt starvation and the like, so that the aim of accumulating astaxanthin is fulfilled.
In the two stages, the nutrition and environmental conditions required by microalgae are different, and at present, domestic and foreign researches mainly focus on the aspects of condition selection and control, influence of environmental factors and the like in the two stages. In general, the first stage does not aim at the accumulation of astaxanthin but at increasing the number and weight of the cells, by using relatively mild culture conditions, such as: low light, low salt; and then, the astaxanthin is transferred to a second stage, stress conditions such as strong light, high temperature and high salt are supplemented at the moment, a nitrogen and phosphorus deficient culture medium is added, the accumulation of the astaxanthin is promoted, the number of cells in the stage is not increased any more, sometimes, the number of cells is reduced along with the increase of the severity of the stress conditions, but the weight of the cells is slowly increased due to sporulation and expansion of the cells, and compared with the initial stage, the weight of the cells in a unit volume culture solution is increased by 2-4 times to reach about 2-3g/L after the stress culture is finished. The astaxanthin accumulation phase culture medium and the microalgae culture medium are not identical, the latter N, P is rich and requires reasonable proportion of elements (necessary elements such as carbon, nitrogen, phosphorus, sulfur, sodium, calcium, potassium, magnesium and the like), and the former only needs to be added with a few salt substances such as calcium salt and the like and generally lacks nitrogen and phosphorus.
However, there is a major problem in going from the previous stage (microalgae cultivation) to the second stage (astaxanthin accumulation), namely: when the cells are adapted to the mild environment of the first stage, the environment is changed and the harsh stress condition is changed when the cells are shifted to the second stage, so that the conditions that the cells are difficult to adapt to, death, damage and the like occur easily. For example: when the cells are cultured in the first stage, the cells are adapted to the weak light environment, and when the cells are switched to the second stage, the photosynthetic system is exposed to strong light and is easily damaged, so that the phenomena of cell death, photoinhibition and the like which are not beneficial to astaxanthin accumulation are caused.
Although there are many improvements in this method in the art, the analysis of the relevant patents filed at home and abroad shows that most of these patents are focused on the culture of photobioreactors and devices, haematococcus pluvialis culture media, novel methods for extracting astaxanthin, and the like. However, patents on astaxanthin-producing microalgae such as Haematococcus pluvialis (Haematococcus pluvialis), Chlorella (Chlorella zofingiensis) and the like for transition processes between culture and stress have not been retrieved.
Therefore, in response to the situation, there is a need in the art to develop a new culture method to overcome this defect and improve the yield of astaxanthin.
Brief description of the invention
In view of the above problems, the present application provides an effective solution. In order to overcome the defect that the two-stage culture process is not beneficial to astaxanthin production when the cell growth condition is switched to the astaxanthin accumulation condition, the invention designs a method for regulating the cell state of haematococcus pluvialis to improve the yield of astaxanthin, and the method has three modes: (1) directly inducing the cultured high-density cells with strong light, and then switching to a normal second stage for induction; (2) diluting the cultured high-density cells, performing weak light induction, and then performing normal second-stage induction; (3) the high density of cells obtained from the culture is transferred to the normal second stage induction, but the cells are adapted to the stress environment by shading (including shading with transparent or opaque material) and optimizing the time of inoculation (i.e., inoculation is performed at a time point of day where the light intensity is weak), and then shading is canceled.
The method of the invention has the following advantages:
(1) the astaxanthin output of the algae cells is improved;
(2) the method is various, and the most convenient mode can be selected according to the actual situation;
(3) the transition process can directly utilize the first stage or the second stage induced reactor without additional equipment.
According to one aspect of the present application, there is provided a novel method for avoiding photoinhibition during microalgae culture to increase astaxanthin production, the method comprising obtaining microalgae cells by a heterotrophic, photoautotrophic, or mixotrophic culture method; adjusting the cell state of microalgae; and performing light induction or photoautotrophic culture on the microalgae cells after the cell state is adjusted to accumulate the astaxanthin.
According to certain embodiments of the present application, the modulation of the microalgae cell state is achieved by controlling the culture conditions.
According to some embodiments of the present application, the adjusting of the microalgae cell state is directly inducing the cultured high-density cells with strong light, and then turning to the normal second-stage induction; diluting the cultured high-density cells, then carrying out weak light induction, and then turning to a normal second stage for induction; or transferring the high-density cells obtained by culture to a normal second stage for induction, enabling the cells to adapt to a stress environment in a shading mode and optimizing the inoculation time, and then eliminating shading, wherein the shading mode comprises shading with a transparent material or an opaque material; and the time for optimizing inoculation is the time point with weak light intensity in one day, wherein the high-density cells mean that the cell density is 0.5-5g/L, and the weak light intensity means that the light intensity is lower than 10 klux.
According to certain embodiments of the present application, the controlling culture conditions comprises: adjusting the culture medium of the microalgae cells, controlling the pH of the culture medium to be between 4 and 10, controlling the concentration of a carbon source (selected from sodium acetate and the like) to be 0 to 60mM, the concentration of a nitrogen source (selected from sodium nitrate and the like) to be 0 to 100mM and/or the concentration of phosphorus (selected from disodium glycerophosphate and the like) to be 0 to 10mM, and controlling the temperature to be 5 to 50 ℃; carrying out illumination on microalgae cells: the illumination intensity can be gradually increased along with the adjustment process, the range is 0-200 klx, the illumination is preferably continuous or intermittent illumination, the light source of the illumination can be natural light or artificial light, and the light quality can be red light, blue light, yellow light or white light; the manner of controlling the light intensity: when the artificial light source is used, the light intensity of the artificial light source is changed; when using the sunlight, the reactor can be shielded by a curtain, a piece of sun-shading cloth and a plastic film; regulating the density of the cells, e.g., such that the cell density is between 0.1g/L and 10 g/L; or to optimize the inoculation time, for example, to control the inoculation time between 3 pm and 12 pm on a sunny day.
According to some embodiments of the present application, the culture time for adjusting the state of the microalgae cell is 0.1-300 hours, and preferably, the concentration of nutrients such as carbon, nitrogen and/or phosphorus in the culture medium is controlled to be low or even zero, i.e., less than 10mM, at the end of the adjustment of the state of the microalgae cell.
According to certain embodiments of the present application, the microalgae are selected from the group of microalgae that can synthesize astaxanthin, such as Haematococcus pluvialis (Haematococcus pluvialis), Chlorella vulgaris (Chlorella zofingiensis).
According to certain embodiments of the present application, the modulating the microalgae cell state comprises: adjusting the size of the cells: e.g., from between 0.1ng to 50ng per cell to between 0.01ng to 5ng per cell; increase of chlorophyll: for example, the content of chlorophyll is increased from 0.04 to 2 percent to 0.1 to 5 percent by mass; and (3) adjusting the morphology of the cells: the number of the motor cells is as much as possible, which can be more than 10 percent and can reach 100 percent to the maximum; or increasing the number of cells and/or increasing the dry weight of cells.
According to some embodiments of the present disclosure, when obtaining the microalgae cells by the heterotrophic, photoautotrophic, or mixotrophic culture method, the microalgae cells can be directly, or after being concentrated, or after being diluted, used for adjusting the state of the microalgae cells.
According to some embodiments of the present disclosure, the microalgae culture obtained after adjusting the state of the microalgae cells may be directly photo-induced or cultured, or diluted and then added with a culture medium required for normal induction for photo-induction or culture.
According to some embodiments of the present application, the adjusting of the state of the microalgae cell is performed in any device that can be used for culturing microalgae, such as a shake flask, a stirring type or airlift type or bubbling type fermentation tank, a raceway pond, a round pond, a flat plate type photobioreactor, a pipeline type photobioreactor, a column type photobioreactor, a film bag and a hanging bag, or a wall-attached method that the microalgae cell is coated on a solid state such as a solid film surface, and preferably, the light induction or light culture performed after the adjusting of the state of the microalgae cell can be replaced by a new culture device or performed in the same culture device.
According to one aspect of the present application, there is provided a medium for regulating the state of microalgae cells, said medium comprising or consisting of a nitrogen source, an organic carbon source (selected from sodium acetate and the like), an inorganic carbon source (selected from carbon dioxide and the like), a plant growth hormone, inorganic salts, trace elements and water.
According to certain embodiments of the present application, the cell status adjusting medium is used for status adjustment of microalgae cells that can synthesize astaxanthin.
In conclusion, the method provided by the invention supplements and perfects the traditional two-stage method for producing the microalgae astaxanthin, further improves the efficiency of astaxanthin production, and provides an important technical means for solving the problem of large-scale industrialization of the microalgae astaxanthin.
Drawings
FIG. 1 shows a process for obtaining Haematococcus pluvialis first stage cells by heterotrophic means;
FIG. 2 shows the process of the transient culture of H.pluvialis in a 1L bioreactor, after 144h of transition, the cells changed from thick parietal spores to green cells, but the dry weight of the single cells decreased, as indicated by the cells becoming smaller;
FIG. 3 shows the procedure of Haematococcus pluvialis induction culture in a 1L bioreactor (cell induction after the cells have undergone the transient process of the present invention, cell induction data comparison without the transient process); after the light induction culture is carried out for 10 days, the dry weight of the cells subjected to the transition process reaches 1.4g/L, the astaxanthin is increased to 4.6 percent, the yield of the astaxanthin reaches 64mg/L and far exceeds the cells directly induced without the transition process; and
FIG. 4 shows the details of the cellular photosynthetic system after transition under different light intensity and nitrogen source.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The method can improve the survival rate of cells during photoinduction, promote the rapid accumulation of astaxanthin in the cells, obviously improve the production efficiency, reduce the production cost and provide high-quality astaxanthin.
The method of the invention comprises a process for obtaining microalgae cells by a heterotrophic, photoautotrophic or mixotrophic culture method; a transition process for adjusting the state of microalgae cells; and a process for culturing the microalgae cells after the cell state is adjusted by photoinduction or photoautotrophy to accumulate astaxanthin.
According to some embodiments of the present application, the process for obtaining microalgae cells is a culture method using heterotrophy, photoautotrophy, or mixotrophy of microalgae. According to certain embodiments of the invention, the culture conditions and media under which the microalgae cells are obtained are known in the art.
The application provides a method for improving astaxanthin yield by regulating haematococcus pluvialis cell state. The method has three modes: (1) performing direct strong light induction on the high-density cells obtained by culture, and then switching to a normal second stage for induction; (2) diluting the cultured high-density cells, performing weak light induction, and then performing normal second-stage induction; or (3) transferring the high-density cells obtained by culturing to a normal second stage for induction, but adapting the cells to the stress environment in a shading mode (comprising shading by using a transparent material or an opaque material), and then removing shading.
According to certain embodiments of the present application, high density cells refers to cells having a density of 0.3-5g/L, for example, 0.3-4 g/L, 0.3-3g/L, 0.3-2g/L, 0.3-1g/L, 0.3-0.5g/L, 0.5-5g/L, 0.5-4g/L, 0.5-3g/L, 0.5-2g/L, 0.5-1g/L, 1-5g/L, 1-4g/L, 1-3g/L, 1-2g/L, 2-5g/L, 2-4g/L, 2-3g/L, 3-5g/L, 3-4g/L, or 4-5 g/L.
According to certain embodiments of the application, the normal second-stage induced cell density is 0.01-5g/L, e.g., 0.01-3g/L, 0.01-1g/L, 0.01-0.5g/L, 0.01-0.1g/L, 0.1-5g/L, 0.1-3g/L, 0.1-1g/L, 0.1-0.5 g/L, 0.5-5g/L, 0.5-3g/L, 0.5-1g/L, 1-5g/L, 1-3g/L, or 3-5 g/L.
According to certain embodiments of the present application, the modulation of the microalgae cell state is achieved by controlling the culture conditions.
According to certain embodiments of the present application, the controlling culture conditions comprises adjusting the culture medium of the microalgae cells; illuminating the microalgae cells; controlling the light intensity or regulating the density of the cells.
According to certain embodiments of the present application, the modulation of microalgae cell state is achieved by optimizing the time of inoculation. According to some embodiments of the present application, the time of the optimized inoculation is to perform inoculation at a time point of the day when the light intensity is weak, for example, the inoculation time is controlled to be between 3 pm and 12 pm on a sunny day, for example, between 5 pm and 12 pm, or between 7 pm and 12 pm.
According to certain embodiments of the present application, the light intensity is weaker with a light intensity below 10klux, such as below 8klux, below 5klux, below 3klux, below 1klux, below 0.5klux, or below 0.1 klux.
According to certain embodiments of the present application, the adjusting the culture medium of the microalgae cell comprises controlling the pH of the culture medium between 4 and 10, the concentration of the carbon source (selected from sodium acetate and the like) between 0 and 60mM, the concentration of the nitrogen source (selected from sodium nitrate and the like) between 0 and 100mM, and/or the concentration of the phosphorus (selected from disodium glycerophosphate and the like) between 0 and 10mM, and controlling the temperature between 5 and 50 ℃, preferably further comprising the concentration of magnesium within the range of 0.00001 to 0.001 mM.
In the method of the present invention, in the transition process for adjusting the state of microalgae cells, the pH is controlled to be constant, and the concentration of carbon, nitrogen and/or phosphorus is stabilized within a certain concentration range, and the concentration of the nutrient components such as carbon, nitrogen and/or phosphorus is controlled to be low or even zero, i.e., less than 10mM, for example, less than 9mM, 8mM, 7mM, 6mM, 5mM, 4mM, 3mM, 29mM, 1mM, 0.5mM, or 0.1mM, at the end of the transition.
In the transition process for adjusting the state of the microalgae cells, elements such as carbon, nitrogen and/or phosphorus are controlled to be stabilized in a certain concentration range by feeding. For example, the carbon content in the algal solution can be controlled within the range of 0-60mM, the nitrogen content within the range of 0-100mM, and the phosphorus content within the range of 0-10mM by feeding.
According to certain embodiments of the present application, in the transition process for adjusting the state of microalgae cells, the concentration of carbon source in the algal solution is controlled by feeding in the range of 0-60mM, such as 0-50mM, 0-40mM, 0-30mM, 0-20mM, 0-10mM, 10-60 mM, 10-50mM, 10-40mM, 10-30mM, 10-20mM, 20-60mM, 20-50mM, 20-40mM, 20-30mM, 30-50mM, 30-40mM, or 40-50mM, preferably 0-40 mM. According to certain embodiments of the present application, in the transition process for adjusting the state of microalgae cells, the nitrogen source concentration is controlled by feeding in the range of 0-100mM, such as 0-8mM, 0-5mM, 0-3mM, 0-1mM, 0-0.5mM, 0.5-10mM, 0.5-8mM, 0.5-5mM, 0.5-3mM, 0.5-1mM, 1-10mM, 1-8mM, 1-5mM, 1-3mM, 3-10 mM, 3-8mM, 3-5mM, 5-10mM, 5-8mM, or 8-10mM, preferably 0.5-50 mM. According to certain embodiments of the present application, in the transition process for adjusting the state of microalgae cells, the concentration of phosphorus is controlled by feeding in the range of 0-10mM, such as 0-0.5mM, 0-0.1mM, 0-0.5mM, 0-0.01mM, 0.01-1mM, 0.01-0.5mM, 0.01-0.1mM, 0.1-1mM, 0.1-0.5 mM, or 0.5-1mM, preferably 0-5 mM.
In one embodiment, the three elements of carbon, nitrogen and phosphorus are stabilized within a certain concentration range by feeding. For example, the carbon content in the algal solution can be controlled within the range of 0.5-60mM, the nitrogen content within the range of 0.5-50mM, and the phosphorus content within the range of 0.01-5mM by feeding.
In a specific embodiment, the method further comprises controlling the content of magnesium in the algal liquid to be in the range of 0.00001-0.001mM, for example, 0.00001-0.0005mM, 0.00001-0.0001mM, 0.00001-0.00005mM, 0.00005-0.001mM, 0.00005-0.0005mM, 0.00005-0.0001mM, 0.0001-0.001mM, 0.0001-0.0005 mM, or 0.0005-0.001mM, by feeding.
In one embodiment, the microalgae is selected from Haematococcus pluvialis (Haematococcus pluvialis), Chlorella vulgaris (Chlorella zofingiensis), and the like.
In one embodiment, the process of modulating the state of the microalgae cell comprises: inoculating microalgae solution into bioreactor, adding culture medium, intermittently introducing carbon dioxide to control pH to 5.0-10.0, culturing at 10-40 deg.C to control pH to less than 10.0, and controlling dissolved oxygen to be above 0.1%.
In one embodiment, in the process of adjusting the state of microalgae cells, the pH of the algal solution is controlled to a constant value within a range of 5.0-10.0 by introducing carbon dioxide, for example, pH of 5.0-9.0, 5.0-8.0, 5.0-7.0, 5.0-6.0, 6.0-10.0, 6.0-9.0, 6.0-8.0, 6.0-7.0, 7.0-10.0, 7.0-9.0, 7.0-8.0, 8.0-10.0, 8.0-9.0, or 9.0-10.0; for example, the pH may be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, preferably 7.5.
It will be appreciated that some variation in pH is permissible. For example, variations in pH of + -Y may be allowed, where Y.ltoreq.1.0, such as Y.ltoreq.0.2, Y.ltoreq.0.1. In certain embodiments, Y ═ 0. Therefore, in one embodiment, the pH of the algae solution is controlled to be X + -Y, wherein the pH is 7.0 + -X + -Y + -9.0. For example, in one embodiment of the present invention, the pH of the algal solution is controlled within a range of 8.0. + -. 0.3 by passing carbon dioxide.
In one embodiment, the culture temperature in the transition process for adjusting the state of the microalgae cells is 5-50 ℃, for example 5-40 ℃ and 10-50 ℃; preferably 10 to 40 ℃, such as 10 to 30 ℃, 10 to 20 ℃, 20 to 40 ℃, 20 to 30 ℃, or 30 to 40 ℃.
In one embodiment, the microalgae transition process is used for diluting the high-concentration microalgae solution by using a transition culture medium to dilute the heterotrophic/photoautotrophic microalgae solution to a cell density of 0.1-20 g/L and a pH of 5.0-9.0.
In a specific embodiment, the transition process comprises the steps of transferring the diluted algae liquid into a light induction device for low-light transition, continuously illuminating or intermittently illuminating, wherein the culture temperature is 5-50 ℃, the illumination intensity is 0.1-50 klx, and the transition period is 1-168 hours.
In one embodiment, the medium added to the transient process to adjust the state of the microalgae cells contains or consists of a nitrogen source, an organic carbon source, a small amount of inorganic salts, a plant growth hormone, trace elements and water.
In one embodiment, when the astaxanthin-producing algal species is Haematococcus pluvialis, the medium used in the transition process to adjust the state of the microalgae cells consists essentially of: 0.1-5.0g/L of sodium acetate, 0.05-1.5g/L of NaNO30.05-1.5 g/L of CaCl 2.7H 2O 0.05, 0.05-1.5g/L of KH2PO40.01, 0.01-1.0 g/L of MgSO 4.7H 2O0.01, 0.01-0.05 g/L of FeSO 4.7H 2O0.01, 0.001-35 mg/L of plant growth hormone, 0.5-4 ml of trace elements and water.
In a specific embodiment, the transition process can be performed in a shake flask, a mechanical stirring type, an airlift type or a bubbling type bioreactor, or can be performed in any device which can be used for photoautotrophic culture of microalgae, such as an open raceway pond or a round pond, a closed flat plate type photobioreactor or a pipeline type photobioreactor or a column type photobioreactor or a film bag and hanging bag photobioreactor, and the light source is natural light or various artificial lights.
According to certain embodiments of the present application, the modulation of the microalgal cell status comprises modulating the size of the cell, an increase in chlorophyll, modulating the morphology of the cell or increasing the number of cells and/or increasing the dry weight of the cell. According to certain embodiments of the present application, the resizing of the cells is such that between 0.1ng to 50ng per cell is reduced to between 0.01ng to 5ng per cell, for example between 1ng to 25ng per cell to between 0.05ng to 3ng per cell, between 5ng to 10ng per cell to between 0.1ng to 1ng per cell, between 5ng to 10ng per cell to between 0.5ng to 1ng per cell.
According to certain embodiments of the present application, the chlorophyll is increased in an amount from 0.04% to 2% by mass to 0.1% to 5% by mass, for example from 0.06% to 1.0% by mass to 0.6% to 2% by mass, or from 0.08% to 1.2% by mass to 0.8% to 2% by mass.
According to certain embodiments of the present application, the content of chlorophyll can be detected using methods known in the art. According to certain embodiments of the present application, the morphology of the cells is adjusted to make motile cells as many as possible, e.g., greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%, up to 100%.
According to some embodiments of the present disclosure, the microalgae culture obtained after adjusting the state of the microalgae cells can be directly subjected to light induction or culture. According to some embodiments of the present disclosure, the microalgae culture obtained after adjusting the state of the microalgae cells may be diluted and then added with a culture medium required for normal induction for light induction or culture.
According to some embodiments of the present application, the adjusting of the microalgae cell state is performed in any device that can be used for microalgae culture, such as a shake flask, a stirring or airlift or bubbling type fermentation tank, a raceway pond, a round pond, a flat plate type photobioreactor, a pipeline type photobioreactor, a column type photobioreactor, a thin film bag and a hanging bag, or by a semi-solid wall-attached method of coating microalgae cells on a solid film surface. According to some embodiments of the present application, the light induction or light culture performed after the microalgae cell state is adjusted may be replaced with a new culture apparatus. According to some embodiments of the present application, the light induction or light culture performed after the regulation of the state of the microalgae cell may be performed in the same culture apparatus.
Microalgae suitable for use in the present application include those that can synthesize astaxanthin, including but not limited to Haematococcus pluvialis (Haematococcus pluvialis), Chlorella vulgaris (Chlorella zofingiensis), and the like. In a preferred embodiment, the present invention employs Haematococcus pluvialis (Haematococcus pluvialis) to produce astaxanthin.
The medium for the transition process for adjusting the state of microalgae cells may generally contain a nitrogen source, an organic/inorganic carbon source, a plant growth hormone, a small amount of inorganic salts, trace elements and water.
Such media include C medium (Ichimura, T.1971Sexual cell division and conjugation-papilla formation in Sexual reproduction of Clostridium strigosum. in Proceedings of the Seventh International Seawewed Symposium, University of Tokyo Press, Tokyo, p.208-214.), medium (Borowitzka et al, 1991), BG-11 medium (Borssiba and Vonshak, 1991), BBM medium (Nichols and Bold, 1969), BAR medium (Barbera et al, 1993), KM medium (Kobayashi et al, 1991), Z8 medium (Renstrom et al, 1981), A9, Pircul et al, 1997, Gorghal et al, culture medium 3632, culture medium (Ma et al, culture medium, 3632, Maguy et al, culture medium, Kobayashi et al, 3632, culture medium, Kobayas et al, culture medium, Kobulra, 1981, Kobayashi et al, Kobulra 9, Legend, 1997), and Kobura et al, Kobulra 3632, Kobayas et al, culture medium, Kobayas, Kobulra 3632, Kobayas et al, Kobulra culture medium, Kobah, Kogya 3632, Kogya culture medium, Kogya, and Kogya, and Kogyo, Kogyo et al, Kogyo et al, Kogyo et al, Kogyo et al, Kogyo, No. culture medium, Kogyo et al, No. 3, No. K.35, No. K.32, No. 3.
The culture medium C used in the invention basically consists of nitrate, sodium acetate, a small amount of inorganic salt, trace elements and water, and is added with some plant growth hormone.
The term "consisting essentially of … …" as used herein means that the composition of the present invention may contain, in addition to the main components such as nitrate, sodium acetate and small amounts of inorganic salts, trace elements and water, some components that do not substantially affect the basic or novel properties of the composition (i.e., maintain the cell density of microalgae at a higher level in a shorter culture period, while having a much higher content of active substances compared to conventional culture). The term "consisting of … …" as used herein means that the compositions of the present invention consist of the particular components indicated, are free of other components, but may carry impurities in amounts within the usual ranges.
In this medium, the components of the medium can be varied within certain limits without having a substantial effect on microalgae cell density and quality. Therefore, the amounts of these components should not be strictly limited by the examples. As is well known to those skilled in the art, small amounts of inorganic salts, such as magnesium sulfate, calcium chloride, ferrous sulfate, and phosphate, and the like, as well as small amounts of trace elements such as Mn, Zn, B, I, M, Cu, Co, and the like, and the addition of plant growth hormones, including a single hormone or a combination of hormones, may also be added to the medium. The amounts of inorganic salts and trace elements may be determined according to conventional knowledge.
In a specific embodiment, the high-density algae liquid obtained from the first stage of the two-step method (preferably, the high-density algae liquid does not contain organic carbon source during induction in an open reactor, so that excessive bacteria can be prevented from breeding in a light induction stage, but contains organic carbon source during induction in a closed photobioreactor, so that the cell amount is increased) is diluted, and the high-density algae liquid is diluted by using a transition culture medium, so that the cell density is maintained at 0.1-20 g/L, and the pH is 4.0-10.0. In some embodiments, the high density algae suspension is diluted with water and a medium without an organic carbon source to maintain a cell density of 0.1 to 10g/L and the pH is adjusted to 5.0 to 8.0. In another embodiment, the algal solution is diluted to maintain the cell density at 1 to 8g/L, and the pH is adjusted to 5.0 to 8.0. In a preferred embodiment, the cell density is maintained at 1.0-5.0 g/L, CO2 is introduced and the pH is adjusted to 5.0-8.0, and CO2 can be further introduced during the culture process to control the pH to 5.0-8.0.
In one embodiment, the transition medium that modulates the state of the microalgae cell comprises: MgCl 2.7. 7 H2O0.01-0.1 g/L, KCl 0.1-1g/L, CaCl 20.01-0.2 g/L, FeSO 4.7H 2O 0.01.01-0.06 g/L, EDTA 0.020-0.052 g/L, and plant growth hormone 0.001-35 mg/L.
In one embodiment, the plant growth hormone in the transition medium comprises: 0.001-5 mg/L of 2, 4-dichlorophenoxyacetic acid, 0.001-5 mg/L of benzylaminopurine, 0.001-5 mg/L of exogenous gibberellin, 0.001-5 mg/L of 3-indolebutyric acid, 0.001-5 mg/L of naphthylacetic acid and/or 0.001-5 mg/L of brassin.
In one embodiment, the plant growth hormone in the transition medium comprises: benzylaminopurine 0.001-5 mg/L and 3-indolebutyric acid 0.001-5 mg/L.
The culture medium adopted for dilution does not need high-pressure sterilization, and the pH is adjusted to 5.0-9.0 after preparation, so that the culture medium can be used.
It will be appreciated that in some embodiments, the algal cells obtained from the first stage of the two-step process do not need to be diluted, but rather are subjected to a light-intensive transition directly, depending on a reasonable match between cell density, broth composition and the actual transition conditions (e.g., light intensity, temperature, whether light-blocking measures are employed, etc.).
The transition process for adjusting the cell state of the microalgae aims to properly adjust cells of astaxanthin-producing microalgae before the microalgae enters a stress condition so as to be more suitable for a stress condition of photoinduction, so that the astaxanthin can be rapidly synthesized and accumulated in a large amount by the algal cells after the microalgae is subjected to photoinduction, and the concentration of the algal cells in a culture solution is properly increased.
According to some embodiments of the invention, the controlling culture conditions comprises illuminating the microalgae cells. According to some embodiments of the present invention, the illumination intensity can be gradually increased along with the adjustment process, and the range is 0 to 200klx, such as 0 to 150klx, 0 to 100klx, 0 to 50klx, 0.1 to 10klx, 0.1 to 5klx, 0.1 to 1klx, 1 to 200klx, 1 to 150klx, 1 to 100klx, 1 to 50klx, 1 to 10klx, 1 to 5klx, 5 to 200klx, 5 to 150klx, 5 to 100klx, 5 to 50klx, 5 to 10klx, 10 to 200klx, 10 to 100klx, 10 to 50klx, 50 to 200klx, 50 to 150klx, 50 to 100klx, 100 to 200klx, 100 to 150klx, or 150 to 150 klx.
According to some embodiments of the invention, the illumination is continuous or intermittent illumination. According to some embodiments of the invention, the light source of the illumination may be natural light or artificial light. According to some embodiments of the invention, the light quality of the illumination may be red, blue, yellow or white light.
According to some embodiments of the invention, the controlling of the light intensity is performed by varying a light intensity of the artificial light source when the artificial light source is used; when using sunlight, the reactor can be shielded by a curtain, a sun-shading cloth or a plastic film.
According to some embodiments of the invention, the controlling of the culture conditions comprises modulating the density of the cells, e.g., such that the cell density is between 0.1g/L and 10g/L, e.g., 0.1g/L and 8g/L, 0.1g/L and 5g/L, 0.1g/L and 3g/L, 0.1g/L and 1g/L, 1g/L and 10g/L, 1g/L and 8g/L, 1g/L and 5g/L, 1g/L and 3g/L, 3g/L and 10g/L, 3g/L and 8g/L, 5g/L and 10g/L, 5g/L and 8g/L, Or 8g/L to 10 g/L.
Usually, the transition process is carried out at a temperature of 5-50 ℃, with an illumination intensity of 0.1-50 klx (during low cell density and weak light transition) or 50-200 klx (during high cell density and strong light transition and shading transition), with continuous illumination or intermittent illumination, a culture period of 0.1-300 hours and an air flow of 0.1-10.0 vvm. Wherein the reactors include all closed bioreactors (shake flasks, pipelines, flat plates, columns, vertical film bags, punching bags and the like) and all open bioreactors (raceway ponds, round ponds, bubbling basins and the like).
Usually, the culture temperature can be controlled within the range of 15-35 deg.C, such as 18-35 deg.C, 20-30 deg.C, etc. Generally, the illumination intensity is 0 to 200klx, for example, 0 to 30, 30 to 40, 0 to 40, 1 to 30, 1 to 20, 1 to 10klx, etc., depending on the specific production conditions. Generally, if the algal solution is sufficiently mixed by the gas, the aeration rate can be controlled to 0.1 to 2.0vvm, for example, 0.2 to 1.8, 0.5 to 1.5, 0.8 to 1.5, 1.0 to 1.5 vvm. At the same time, CO2 was introduced at a concentration to provide an inorganic carbon source and to control pH, e.g., 0.5% to 10% CO 2. In another embodiment, the culture temperature is controlled to 10 to 50 ℃, the illumination intensity is 1 to 10klx, and the ventilation volume is 0.05 to 2.0 vvm.
In other embodiments, the transition period is 0.1-300 hours, for example, the cultivation period can be 0.1-250 hours, 0.1-200 hours, 0.1-100 hours, 0.5-50 hours, 50-150 hours, 150-300 hours, 100-300 hours, 0.5-8 hours, and any time period within the range of 0.5-300 hours according to the actual weather conditions.
Light in this application, the term "transition period" includes the entire process of transition cultivation, for example, the transition period includes the time when there is no light at night when the cultivation is outdoors.
In the present application, "cultivation time" refers to the time during which the microalgae are subjected to the subculture using the illumination intensity described herein. Since the illumination intensity is as low as 0-200Lux, the time includes the time when there is no illumination at night (i.e., the illumination intensity is 0 Lux). The time is any time within the range of 0.1-300 hours.
The light needed by the transitional culture step can be artificially illuminated for light-induced culture, and the transitional culture can be carried out outdoors by utilizing natural illumination.
In a specific embodiment, for the microalgae cells in the first stage, after the cellular photosynthetic system in the culture solution is repaired, the transition process can be terminated, and the microalgae cells can enter the light-induced culture, wherein the repair of the cellular photosynthetic system comprises one or a combination of the following conditions: the chlorophyll of the cells is increased, the color of the cells is changed to green, and the efficiency of the photosynthetic system II is improved.
In a specific embodiment, for the microalgae cells in the first stage, the transition process can also be terminated and the microalgae cells can be subjected to light-induced culture when the astaxanthin particles are obviously appeared in the cells in the culture solution. In particular embodiments, the intracellular presence of astaxanthin particles in the cell is observed by microscopy or other methods known in the art.
When "about" is used in this application to modify a numerical value, it is meant that the numerical value may fluctuate within a range of ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2% or ± 1%.
The use of the terms "a" and "an" and "the" and similar referents in the context of describing the application (including the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order, as understood by those skilled in the art, unless otherwise indicated herein or otherwise clearly contradicted by context.
The present invention will be further described with reference to the following examples. It is to be understood that the meaning of "comprising", "including" in this application also includes "consisting of … …", "consisting of … …".
All patents, patent applications, and references cited in this application are incorporated by reference in their entirety to the same extent as if each reference were individually incorporated by reference. In the event of a conflict between the present application and the references provided herein, the present application shall control.
While preferred embodiments and examples have been described herein, those of ordinary skill in the art, having benefit of this disclosure, will appreciate that many changes can be made to the embodiments and examples described herein. Accordingly, this application is intended to cover all such modifications and alterations of the subject matter of the claims of this application as fall within the scope of the legal equivalents.
Examples
The present invention is described in detail below with reference to examples and the accompanying drawings. It will be understood by those of ordinary skill in the art that the following examples are for illustrative purposes and should not be construed as limiting the invention in any way. The scope of the invention is defined by the appended claims.
Example 1 transition Process causes the State of the microalgae cells to change
As shown in FIG. 1, in the first stage of cultivation, high-density Haematococcus pluvialis cells were obtained by heterotrophic cultivation. Adding a transition culture medium and water into a 1L bioreactor to 1L, performing steam sterilization, inoculating 12ml of cells obtained by heterotrophic haematococcus pluvialis with high density (26g/L) when the temperature is reduced to 25 ℃, starting transition culture, keeping the temperature at 28-38 ℃, controlling the air flow at 1vvm, and artificially illuminating for 24 hours, wherein the light intensity of each side is about 50 klx.
During the cultivation, 1% carbon dioxide was introduced intermittently to maintain the pH at a constant value of 8 to 9. Promoting the microalgae to adapt to the stress environment. After 144h, the cells changed from chlamydospores to green cells and the transition process was complete.
FIG. 2 shows the process of the Haematococcus pluvialis in a 1L bioreactor (the process used is the preferred process), and after 144h of transition, the cells are changed from chlamydospores to green cells, but the dry weight of the single cells is reduced, which is shown as the cells are smaller. As can be seen from FIG. 1, the state of the microalgae cells is changed during the transient process culture.
Example 2 cultivation method via transition Process leads to an increase in the Dry cell weight and an increase in the astaxanthin production
In this embodiment, after the microalgae cells are cultured in the first stage, the cells are induced by the transition process of the present invention, compared with the cells induced directly without the transition process of the present invention.
FIG. 3 shows a comparison of the 10-day culture conditions of Haematococcus pluvialis cells in a 1L bioreactor induction culture, with and without the transition process. As can be seen from FIG. 3, after 10 days of light induction culture, the dry weight of the cells after the transition process reached 1.4g/L, the astaxanthin content increased to 4.6%, and the astaxanthin yield reached 64 mg/L. After the haematococcus pluvialis cells which do not undergo the transition process are subjected to light induction culture for 10 days, the dry weight of the cells which undergo the transition process only reaches 0.3g/L, the astaxanthin is increased to 0.8%, and the yield of the astaxanthin reaches 2.4 mg/L.
Therefore, as can be seen from FIG. 3, the transition process of the present invention allows the dry weight of cells to be increased and the yield of astaxanthin to be increased, which far exceeds the direct induction without the transition process.
Example 3 Change in intracellular photosynthetic System of cells treated by the transitional Process
FIG. 4 shows the differences between the intracellular photosynthetic systems of the cells treated by the transition process and the control, which are the difference in the electron transfer Efficiency (ETR) of the intracellular photosynthetic system II (panel A), the difference in the non-photochemical quenching (NPQ) (panel B), the photosynthetic efficiency Y (II) of the photosynthetic system II (panel C) and the other non-photochemical energies (Y (NO), panel D) of the respective treated cells. The above results indicate that the efficiency of the intracellular photosynthetic system II is improved by the transition process, and particularly that the photosynthetic system is significantly restored by the increase of nitrogen element at the time of transition. The non-photochemical quenching moiety is relatively small, indicating that the cells are more resistant to intense light and damage to the cells due to photooxidation is minimized.
Embodiments of the present application are exemplarily described above with reference to the drawings. Those skilled in the art can easily conceive of the disclosure of the present specification that various embodiments can be appropriately modified and recombined according to actual needs without departing from the spirit of the present application. The protection scope of this application is subject to the claims of this application.
Claims (6)
1. A method for avoiding photoinhibition during microalgae cultivation to increase astaxanthin production, comprising:
obtaining microalgae cells by a heterotrophic, photoautotrophic or mixotrophic culture method; the microalgae cell is directly used for adjusting the state of the microalgae cell;
adjusting the state of microalgae cells by a transition process, wherein the transition process adopts a low-light transition process, the temperature is controlled to be 28-38 ℃, the illumination intensity is 50klx, the cell density is 0.1-10g/L, the microalgae cells are continuously illuminated for 24 hours artificially, and the culture time is 144-300 hours;
the microalgae cells are haematococcus pluvialis;
the pH value of the culture medium for adjusting the microalgae cell state is between 8 and 9, and the carbon source sodium acetate in the culture medium is 0.1 to 5.0g/L, the nitrogen source sodium nitrate is 0.05 to 1.5g/L and/or the glycerol disodium phosphate is 0 to 10 mM.
2. The method of claim 1, wherein the illumination is continuous or intermittent illumination, the illumination can be natural or artificial, and the light quality can be red, blue, yellow or white;
the manner of controlling the light intensity: when the artificial light source is used, the light intensity of the artificial light source is changed; when using the sunlight, the reactor can be shielded by a curtain, a piece of sun-shading cloth and a plastic film;
and optimizing the inoculation time, and controlling the inoculation time between 3 pm and 12 pm on a sunny day.
3. The method of claim 1, wherein: and controlling the concentration of nutrient components such as carbon, nitrogen and/or phosphorus in the culture medium to be lower or even zero, namely less than 10mM, at the end of the microalgae state adjustment.
4. The method of claim 1, wherein modulating the status of the microalgae cell comprises:
adjusting the size of the cells: from between 0.1ng to 50ng per cell to between 0.01ng to 5ng per cell;
increase of chlorophyll: the mass percentage of the chlorophyll is increased from 0.04 percent to 1.2 percent to 0.4 percent to 12 percent;
and (3) adjusting the morphology of the cells: the number of the motor cells is as much as possible, which can be more than 10 percent and can reach 100 percent to the maximum; or
Increasing the number of cells and/or increasing the dry weight of cells.
5. The method according to claim 1, wherein the microalgae culture obtained after the regulation of the state of the microalgae cells is diluted and then added with a culture medium required for normal induction for light induction or culture.
6. The method of claim 1, wherein the adjusting of the state of the microalgae cell is performed in any device that can be used for culturing microalgae, such as a shake flask, a stirred or airlift or bubbling fermenter, a raceway pond, a round pond, a flat plate photobioreactor, a pipeline photobioreactor, a column photobioreactor, a thin film bag and a hanging bag, or in a semi-solid state such as a solid film surface, and the culturing is performed by a wall-mounted method, and the light induction or light culturing performed after the adjusting of the state of the microalgae cell can be performed by a new culturing device or in the same culturing device.
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