Combined treatment method for algae

Abstract

The invention provides an algae combined treatment method, which relates to the technical field of bioengineering, and comprises the following steps: selecting microalgae seeds; inoculating the microalgae seeds into a conical flask of a Basal culture medium; culturing under a dark condition; and after the microalgae seeds are cultured to a plateau stage, supplementing plant hormones and a nitrogen source, and culturing to obtain the microalgae. The algae combined treatment method provided by the invention can effectively stimulate microalgae in a plateau phase, so that the total carotenoid content and the xanthophyll content can be effectively increased.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to an algae combined processing method. Background Art

[0002] Microalgae are considered to be a very promising source of lutein because, compared with marigold, the only natural source of lutein available, they grow very fast, have a high lutein yield, are highly adaptable to various environments, and are not restricted by seasons. Microalgae can be cultivated in three culture modes: autotrophic, heterotrophic, and polytrophic. Combining the three culture modes, the heterotrophic mode is the most conducive to large-scale cultivation of microalgae for lutein production. In addition, the conditions suitable for microalgae growth and lutein production are often not consistent. For some conditions where the suitable growth and lutein accumulation are quite different, in order to obtain the maximum lutein yield, the two-step method has become the most commonly used method.

[0003] However, the two-step cultivation processes commonly used for lutein production are often complicated. For example, they require heterotrophic cultivation to accumulate a large amount of biomass followed by light induction, mixed cultivation or photoautotrophic cultivation. Although these two-step cultivation strategies usually obtain good results under laboratory conditions, they are difficult to apply in large-scale production. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide an algae combined processing method for solving the problem that the two-step cultivation process used in the prior art for lutein production can obtain good results under laboratory conditions but is difficult to apply in large-scale production.

[0005] To achieve the above-mentioned purpose and other related purposes, the present invention provides a method for combined algae treatment, comprising the following steps: selecting microalgae seeds; inoculating the microalgae seeds into a conical flask of Basal culture medium; culturing under dark conditions; and after the microalgae seeds reach a plateau stage, supplementing plant hormones and nitrogen sources to obtain microalgae.

[0006] In one embodiment of the present invention, the selected microalgae seeds are C. protothecoides CS-41.

[0007] In one embodiment of the present invention, the microalgae seeds are inoculated into a conical flask of Basal culture medium, comprising: inoculating the microalgae seeds at 10% (v / v) into a 500 mL conical flask containing 200 mL of fresh modified Basal culture medium, and placing the flask in a shaking fermentation culture at 28° C. and 180 rpm.

[0008] In one embodiment of the present invention, the improved Basal medium includes KH2PO4, MgSO4·7H2O, EDTA·Na2, H3BO3, Mg CaCl2·2H2O, FeSO4·7H2O, ZnSO4·7H2O, MnCl2·4H2O, CuSO4·5H2O, Co(NO3)2·6H2O, and MoO3.

[0009] In one embodiment of the present invention, the mass concentration ratio of KH2PO4, MgSO4·7H2O, EDTA·Na2, H3BO3, mgCaCl2·2H2O, FeSO4·7H2O, ZnSO4·7H2O, MnCl2·4H2O, CuSO4·5H2O, Co(NO3)2·6H2O, and MoO3 is 1.25:1.0:0.5:0.1142:0.111:0.0498:0.0882:0.0142:0.0157:0.0049:0.0071.

[0010] In one embodiment of the present invention, the initial pH of the improved Basal medium is 6.1.

[0011] In one embodiment of the present invention, the number of days of culturing under dark conditions is at least three days.

[0012] In one embodiment of the present invention, the supplementation of plant hormones and nitrogen sources comprises: treating the microalgae seeds with IAA and high nitrogen combined with the microalgae seeds after the microalgae seeds reach the plateau stage.

[0013] In one embodiment of the present invention, the microalgae seeds that have reached the stationary phase through the IAA high nitrogen combined treatment include: stimulating the microalgae seeds that have reached the stationary phase through the combined use of urea and IAA.

[0014] In one embodiment of the present invention, the IAA is added at 200 mM, and the urea is added at 3.6 g / L.

[0015] As described above, the algae combined treatment method of the present invention has the following beneficial effects: when microalgae seeds, especially C.protothecoides CS-41, are cultured in a culture medium, plant hormones and additional nitrogen sources can be used to stimulate the growth of microalgae and pigment accumulation, thereby stimulating lutein accumulation. IAA is beneficial to the production of lutein by C.protothecoides CS-41, but does not increase the total carotenoid content; the growth of C.protothecoides CS-41 is not affected by the concentration of nitrogen sources, but the lutein content increases with the increase of nitrogen source concentration. Therefore, the high nitrogen combined treatment of IAA and urea can effectively stimulate lutein accumulation and total carotenoid content, and in particular, 200mM IAA and 3.6g / L urea are the most favorable conditions for C.protothecoides CS-41. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shown is a schematic flow chart of the algae combined treatment method of the present invention.

[0017] Figure 2 Shown is a schematic diagram of the distribution of the number of differentially expressed genes in algae cells in one embodiment of the present invention.

[0018] Figure 3 It is a schematic diagram showing the transcription level of genes in the carotenoid biosynthesis pathway according to one embodiment of the present invention.

[0019] Figure 4 Shown is a schematic diagram of the transcription level of photosynthesis-related genes in a preferred embodiment of the present invention.

[0020] Figure 5 It is a schematic diagram showing the transcription level of central carbon metabolism related genes in a preferred embodiment of the present invention.

[0021] Figure 6 It is a schematic diagram showing the transcription level of genes related to the urea metabolic pathway in a preferred embodiment of the present invention.

[0022] Figure 7 A schematic diagram showing the growth of C. protothecoides CS-41 after adding different plant hormones in a preferred embodiment of the present invention.

[0023] Figure 8 A schematic diagram showing the lutein content of C. protothecoides CS-41 after adding different plant hormones in a preferred embodiment of the present invention.

[0024] Fig. 9A schematic diagram showing the chlorophyll and total carotenoid contents of C. protothecoides CS-41 after adding different plant hormones in a preferred embodiment of the present invention.

[0025] Fig.10 A schematic diagram showing the effect of indoleacetic acid on the percentage of lutein in total carotenoids in C. protothecoides CS-41 in a preferred embodiment of the present invention.

[0026] Fig.11 A schematic diagram showing the growth curve of C. protothecoides CS-41 with different concentrations of IAA added in a preferred embodiment of the present invention.

[0027] Fig.12 A schematic diagram showing the lutein content of C. protothecoides CS-41 added with different concentrations of IAA in a preferred embodiment of the present invention.

[0028] Fig.13 A schematic diagram showing the chlorophyll and total carotenoid contents of C. protothecoides CS-41 added with different concentrations of IAA according to a preferred embodiment of the present invention.

[0029] Fig.14 A schematic diagram showing the percentage of lutein in total carotenoids in C. protothecoides CS-41 to which different concentrations of indoleacetic acid are added according to a preferred embodiment of the present invention.

[0030] Fig.15 A schematic diagram showing the growth curve of C. protothecoides CS-41 with different concentrations of urea added in a preferred embodiment of the present invention.

[0031] Fig.16 A schematic diagram showing the glucose consumption curve of C. protothecoides CS-41 with different concentrations of urea added in a preferred embodiment of the present invention.

[0032] Fig.17 A schematic diagram showing the urea consumption curve of C. protothecoides CS-41 with different concentrations of urea added in a preferred embodiment of the present invention.

[0033] Fig.18 A schematic diagram showing the lutein content of C. protothecoides CS-41 with different concentrations of urea added according to a preferred embodiment of the present invention.

[0034] Fig.19A schematic diagram showing the chlorophyll and total carotenoid contents of C. protothecoides CS-41 with different concentrations of urea added according to a preferred embodiment of the present invention.

[0035] Fig. 20 It is a schematic diagram showing the biomass change of C. protothecoides CS-41 after IAA and high nitrogen combined treatment in a preferred embodiment of the present invention.

[0036] Fig.21 It is a schematic diagram showing the change of lutein content in C. protothecoides CS-41 after IAA and high nitrogen combined treatment in a preferred embodiment of the present invention.

[0037] Fig. 22 It is a schematic diagram showing the changes in carotenoid content of C. protothecoides CS-41 after IAA and high nitrogen combined treatment in a preferred embodiment of the present invention.

[0038] Fig.23 It is a schematic diagram showing the biomass changes of C. protothecoides CS-41 under different fermentation modes after IAA and high nitrogen treatment in a preferred embodiment of the present invention.

[0039] Fig.24 It is a schematic diagram showing the change of lutein content after IAA and high nitrogen combined treatment of C. protothecoides CS-41 under different fermentation modes in a preferred embodiment of the present invention.

[0040] Fig.25 It is a schematic diagram showing the change of chlorophyll content after IAA and high nitrogen combined treatment of C. protothecoides CS-41 under different fermentation modes in a preferred embodiment of the present invention.

[0041] Fig.26 It is a schematic diagram showing the change of total carotenoid content after IAA and high nitrogen combined treatment of C. protothecoides CS-41 under different fermentation modes in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0042] The following is an explanation of the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the features in the following embodiments and the embodiments can be combined with each other without conflict. It should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods for which specific conditions are not specified in the following examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturers.

[0043] See also Figures 1 to 26 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention, so they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0044] Auxin is a type of endogenous hormone containing an unsaturated aromatic ring and an acetic acid side chain. It is abbreviated as IAA in English. Its chemical nature is indoleacetic acid. In addition, 4-chloro-IAA, 5-hydroxy-IAA, naphthylacetic acid (NAA), indolebutyric acid, etc. are auxin-like substances. The most important chemical substance in auxin is 3-indoleacetic acid. Auxin has the functions of regulating the growth rate of stems, inhibiting lateral buds, and promoting rooting. It is used in agriculture to promote rooting of cuttings with remarkable results.

[0045] Auxin is widely distributed in plants, almost in every part, but not evenly distributed. The content of a specific part at a certain time is affected by several factors. Most of it is concentrated in the parts with vigorous growth (coleoptile, meristem of bud and root tip, cambium, ovary after fertilization, young seeds, etc.), while it is very rare in tissues and organs that tend to age.

[0046] Urea, also known as carbamide, has a chemical formula of CH4N2O or CO(NH2)2. It is a white crystal, tasteless and odorless, easily soluble in water, ethanol and benzene, and slightly soluble in ether and chloroform. Urea is one of the simplest organic compounds and is the main nitrogen-containing end product of protein metabolism and decomposition in mammals and some fish. It can be used as fertilizer, animal feed, explosives, glue stabilizer and chemical raw material. Because this substance is contained in human urine, it is named urea. As a neutral fertilizer, urea is suitable for various soils and plants. It is easy to store, easy to use, and has little destructive effect on the soil. It is a chemical nitrogen fertilizer with a large usage. Urea contains 46% nitrogen (N), which is the highest nitrogen content among solid nitrogen fertilizers. In industry, ammonia and carbon dioxide are used to synthesize urea under certain conditions.

[0047] See also Figure 1 In one embodiment of the present invention, the present invention provides an algae joint treatment method, comprising the following steps:

[0048] Step S10: selecting microalgae seeds;

[0049] Step S20: inoculating the microalgae seeds into a conical flask of Basal culture medium;

[0050] Step S30: culturing under dark conditions;

[0051] Step S40: After the microalgae seed culture reaches a plateau phase, plant hormones and nitrogen sources are supplemented to culture microalgae.

[0052] It is not difficult to find from the above content that, through step S10: selecting microalgae seeds, it can be seen that in order to increase the lutein yield of microalgae, in the process of seed selection, algae species with higher lutein content can be selected as an indicator for algae species selection. Through steps S20-S40: inoculating the microalgae seeds in a conical flask of Basal culture medium; culturing under dark conditions; after the microalgae seeds reach the plateau stage, supplementing plant hormones and nitrogen sources to cultivate microalgae, it can be seen that after the algae species selection is completed, by inoculating the microalgae seeds in a conical flask of Basal culture medium, the maximum specific growth efficiency of the microalgae seeds can be improved, which is beneficial to the growth of the microalgae seeds, so that the microalgae seeds can reach the plateau stage as soon as possible when cultivated under dark conditions. And after reaching the plateau stage, additional nitrogen source supplementation and further cultivation can effectively regulate the lutein synthesis ability of the microalgae, making the lutein synthesis ability stronger, thereby significantly improving the total carotenoid content and lutein content of the microalgae. Moreover, this method of additional nitrogen source supplementation will not affect the biomass of the microalgae. In addition, by adding plant hormones after the plateau period, microalgae can be effectively stimulated to accumulate lutein.

[0053] In step S10, the selected microalgae seeds are C.protothecoides CS-41. In this embodiment, in the process of seed selection, in order to increase the lutein production, the microalgae seeds can select C.protothecoides CS-41 which is more suitable for producing lutein. Moreover, the growth of C.protothecoides CS-41 is not affected by the nitrogen source concentration, but the lutein content increases with the increase of the nitrogen source concentration. Of course, in order to achieve the purpose of additionally supplementing the nitrogen source to increase the lutein production, the microalgae seeds can also select other types of algae such as P.kessleri HH2.

[0054] See also Figure 2 , showing the distribution of the number of differentially expressed genes in cells. In order to determine the mechanism of additional nitrogen stimulating lutein synthesis in algal cells at the transcriptional level, and to analyze the difference in urea addition time, three experimental groups were set up, and their transcriptional levels were compared in pairs, namely, the initial addition of two times urea (2X) and control 1 (C1), the initial addition of one times urea and the additional supplementation of one times urea after the plateau period (1+1X) and control 2 (C2), and the initial addition of two times urea (2X) and the additional supplementation of one times urea after the plateau period (1+1X). The figure shows that the urea 2X and urea 1+1X groups have the most differentially expressed genes, 4900 up-regulated and 4486 down-regulated. Urea 2X and control 1 have the least differentially expressed genes, only 39 up-regulated and 25 down-regulated. The comparison found that the additional supplementation of nitrogen source after the plateau period had a greater difference in cell transcriptional levels and more differential genes compared with the control group.

[0055] See also Figure 3 , showing the transcription levels of genes in the carotenoid synthesis pathway. It was found that there was no significant difference between 2X and C1, while 1+1X significantly upregulated the transcription levels of CMS, CRTISO and LCYB in the carotenoid synthesis pathway compared with C2, and downregulated the transcription levels of ZEP and VDE. This shows that the addition of additional urea after the plateau phase better regulates the transcription levels of genes in the lutein synthesis pathway. At the same time, CMS, CRTISO, LCYB, ZEP and VDE may be key genes that can increase lutein content by adding additional urea. Comparing 2X and 1+1X, it was found that PSY had great differences in different culture stages, and PSY was significantly upregulated in the logarithmic growth phase (2X) compared with the plateau phase (1+1X). This shows that there is more flux to carotenoid synthesis in the logarithmic growth phase.

[0056] See also Figure 4, showing the transcription levels of photosynthesis-related genes. It was found that there was no significant difference between 2X and C1. Compared with C2, all photosynthesis-related genes in 1+1X were downregulated, indicating that additional nitrogen supplementation would reduce the photosynthetic activity of algal cells. Compared with 1+1X, 2X was almost all upregulated, which may be caused by the different growth stages of the two groups of algal cells. 2X is in the logarithmic growth phase, and its photosynthetic activity is stronger than 1+1X after the plateau phase.

[0057] See also Figure 5 , showing the transcription levels of central carbon metabolism-related genes. The results showed that there was no significant difference between 2X and C1, and most of the central carbon metabolism-related genes were significantly downregulated compared with C2 in 1+1X, indicating that additional nitrogen supplementation would reduce the carbon efficiency of algal cells. Among them, the significant upregulation of PYC and PEPCK enhanced the efficiency of pyruvate metabolism, which may provide more substrates for carotenoid synthesis. Since the two groups of algal cells are at different stages of growth, it is meaningless to compare the differences in the transcription levels of central carbon metabolism-related genes between 2X and 1+1X.

[0058] See also Figure 6 , showing the transcription levels of genes in the urea metabolism-related pathway. Urea-active transporters have been found in many bacteria and phytoplankton. Genome sequencing shows that marine unicellular photosynthetic eukaryotes possess the gene DUR3, which encodes a high-affinity urea-active transporter that is part of the larger sodium:solute symporter family (SSSF). GS is a key gene for urea catabolism. As can be seen from the figure, urea metabolism-related genes in 1+1X are significantly downregulated compared with C2, indicating that the supplementation of additional nitrogen sources reduces the ability of urea transport and catabolism. However, in addition to active transport through transporters, urea can also enter the cell by free diffusion. Additional urea in the cytoplasm regulates the metabolism of algal cells as a kind of environmental stress, thereby increasing their lutein synthesis.

[0059] In one embodiment of the present invention, the microalgae seeds are inoculated into a conical flask of Basal medium, comprising:

[0060] The microalgae seeds are inoculated into a 500 mL conical flask containing 200 mL of fresh modified Basal medium at 10% (v / v), and placed in a shaking table at 28° C. and 180 rpm for shaking fermentation. In this embodiment, the shaking table can be a constant temperature shaking table of model DHZ-DA, and the oscillator can be a vortex oscillator of model 88880018. In addition, when the culture medium is cultured, the microalgae seeds can also be inoculated into a fermentation tank of Basal medium, such as a fermentation tank of model BioFlo 115.

[0061] In one embodiment of the present invention, the improved Basal medium includes KH2PO4, MgSO4·7H2O, EDTA·Na2, H3BO3, Mg CaCl2·2H2O, FeSO4·7H2O, ZnSO4·7H2O, MnCl2·4H2O, CuSO4·5H2O, Co(NO3)2·6H2O, and MoO3.

[0062] Preferably, the mass concentration ratio of KH2PO4, MgSO4·7H2O, EDTA·Na2, H3BO3, mgCaCl2·2H2O, FeSO4·7H2O, ZnSO4·7H2O, MnCl2·4H2O, CuSO4·5H2O, Co(NO3)2·6H2O, and MoO3 is 1.25:1.0:0.5:0.1142:0.111:0.0498:0.0882:0.0142:0.0157:0.0049:0.0071.

[0063] In this embodiment, the modified Basal medium ( / L): 1.25g KH2PO4, 1.0g MgSO4·7H2O, 0.5g EDTA·Na2, 114.2mg H3BO3, 111mg CaCl2·2H2O, 49.8mg FeSO4·7H2O, 88.2mg ZnSO4·7H2O, 14.2mg MnCl2·4H2O, 15.7mg CuSO4·5H2O, 4.9mg Co(NO3)2·6H2O, 7.1mg MoO3. Compared with Kuhl medium and BG11 medium, the modified Basal medium is more conducive to the growth of C.protothecoides CS-41 algae species. The modified Basal medium in this embodiment is three times concentrated than the basic Basal medium (boric acid is the initial concentration), the initial glucose concentration is 50g / L, and the initial urea concentration is 4.5g / L.

[0064] Preferably, the initial pH of the improved Basal medium is 6.1.

[0065] Preferably, the number of days of culturing under dark conditions is at least three days. In this embodiment, generally, when the microalgae are cultured in a culture medium, a plateau phase can be reached after 36 hours of culture.

[0066] In a preferred embodiment of the present invention, when C. protothecoides CS-41 microalgae seeds are cultured in the improved Basal medium, the plateau phase of C. protothecoides CS-41 microalgae can be reached after 36 hours of culture.

[0067] In one embodiment of the present invention, the supplementation of plant hormones and nitrogen sources comprises:

[0068] The microalgae seeds reached the plateau phase after being treated with IAA and high nitrogen.

[0069] Preferably, the microalgae seeds reaching the plateau phase through IAA high nitrogen combined treatment include:

[0070] The microalgae seeds were stimulated to reach the plateau phase by the combined use of urea and IAA.

[0071] Experimental Example 1

[0072] Plant hormone screening culture: C. protothecoides CS-41 microalgae seeds were inoculated into 250 mL conical flasks containing 100 mL fresh modified Basal medium at 10% (v / v) and cultured at 28°C, 180 rpm in the dark for 4 days. Except for the control group, 100 mM indoleacetic acid, α-naphthylacetic acid, salicylic acid, abscisic acid and gibberellin were added.

[0073] Experimental Example 2

[0074] Plant hormone concentration selection and cultivation: C. protothecoides CS-41 microalgae seeds were inoculated into 250 mL conical flasks containing 100 mL fresh modified Basal medium at 10% (v / v) and cultured at 28°C, 180 rpm in the dark for 5 days. 0, 50, 100, and 200 mM indoleacetic acid were added respectively. Another experimental group was set up in which indoleacetic acid was not added initially, and 200 mM indoleacetic acid was added on the 3rd day.

[0075] Experimental Example 3

[0076] Nitrogen source concentration selection culture: C. protothecoides CS-41 microalgae seeds were inoculated into 100 mL of fresh modified Basal medium in a 250 mL conical flask at 10% (v / v), and cultured at 28°C, 180 rpm in the dark for 4 days. 0.45, 0.9, 1.8, 3.6 g / L urea were added respectively.

[0077] See also Figure 7, Growth of C.protothecoides CS-41 after adding different plant hormones. In order to screen suitable plant hormones to stimulate the accumulation of lutein in C.protothecoides CS-41, 100mM indoleacetic acid, α-naphthylacetic acid, salicylic acid, abscisic acid and gibberellin were added to the culture medium before the start of culture. Except for α-naphthylacetic acid, the other four plant hormones did not affect the growth of microalgae. The experimental group with α-naphthylacetic acid grew more slowly and reached the maximum biomass 1 day later than the other groups. However, it had no effect on the final biomass.

[0078] See also Figure 8 , showing the lutein content of C. protothecoides CS-41 after adding different plant hormones. As can be seen from the figure, the lutein content of the experimental group with IAA added was significantly increased (p<0.01), an increase of 20.4% compared to the control group. The other four experimental groups had no significant difference from the control group. Interestingly, Fig. 9 It can be seen that there is no significant difference in the content of chlorophyll and total carotenoids between the five experimental groups and the control group. Calculation of the proportion of lutein to total carotenoids shows that IAA significantly increased the proportion of lutein to total carotenoids in C. protothecoides CS-41 from 54.1% to 79.2%. Fig.10 .

[0079] Preferably, the IAA added is 200 mM, and the urea added is 3.6 g / L.

[0080] See also Fig.11 In order to determine the effect of IAA concentration on lutein production by C.protothecoides CS-41, 0, 50, 100, and 200 mM IAA were added to the culture medium before inoculation. In addition, an experimental group was set up in which IAA was not added to the initial culture medium. After reaching the plateau phase (the third day), 200 mM IAA was added to the culture medium. As can be seen from the figure, although it did not affect the final biomass, the addition of IAA significantly reduced the growth rate of C.protothecoides CS-41, and the degree of influence increased with the increase of IAA concentration. Adding high concentrations of IAA after the plateau phase had no effect on the biomass of C.protothecoides CS-41.

[0081] See also Fig.12, the lutein content of all experimental groups with added IAA was higher than that without added (p<0.001), an increase of 20.4% compared with the control group. The other four experimental groups had no significant difference from the control group. Among them, the lutein content of the experimental group with high concentration added after the plateau period was the highest, at 2.73 mg / g, an increase of 82% compared with the control group (1.50 mg / g). Consistent with the previous results, there was no significant difference in the chlorophyll and total carotenoid content between the four experimental groups and the control group, such as Fig.13 Similarly, if Fig.14 As shown, the addition of IAA significantly increased the ratio of lutein to total carotenoids in C. protothecoides CS-41, among which the experimental group with high concentration added after the plateau period had the highest ratio of 85.8%, which was 78% higher than that of the control group.

[0082] See also Fig.15 , showing the growth curve of C.protothecoides CS-41 with different concentrations of urea. In order to obtain more carbon flux for the carotenoid synthesis pathway, nitrogen source can be added. As can be seen from the figure, increasing the nitrogen source concentration from 0.45 to 3.6 g / L has no effect on the growth of microalgae. Fig.16 and Fig.17 The consumption of glucose and urea by C.protothecoides CS-41 under different initial nitrogen source concentrations. Similar to the growth situation, different initial urea concentrations did not affect the consumption of glucose, while 0.9 g / L urea and glucose were consumed simultaneously, and 1.8 g / L and 3.6 g / L were both in a nitrogen excess state.

[0083] See also Fig.18 , is the lutein accumulation of C.protothecoides CS-41 under different initial nitrogen source concentrations. As can be seen from the figure, with the increase of nitrogen source concentration, the lutein content gradually increases. Among them, the lutein content of the two groups with excess nitrogen, i.e. 1.8g / L and 3.6g / L (2.33mg / g, 2.54mg / g) was significantly higher than that of the experimental group with an initial urea content of 0.45g / L (1.52mg / g). The total carotenoid and total chlorophyll contents of the low nitrogen group (0.45g / L) were significantly lower than those of the other three groups, such as Fig.19 .

[0084] From the above content, it can be seen that 200mM IAA and 3.6g / L urea are the optimal conditions for lutein production. Among them, high concentration of IAA inhibits the growth of algal cells, while adding it after the stationary phase has no effect on growth, and the additional nitrogen source after the stationary phase has a stronger ability to regulate lutein synthesis. Therefore, 200mM IAA and 3.6g / L urea were used in combination to stimulate C.protothecoides CS-41 cultured to the stationary phase.

[0085] See also Fig.21 , it can be seen that this treatment method has no effect on the biomass of C. protothecoides CS-41. Fig. 22 and Fig.23 The changes in lutein content and total carotenoids two days after treatment are shown. Compared with the control group, the combination of IAA and urea significantly increased the total carotenoid content and lutein content. Among them, the lutein content after treatment can reach 47.6% higher than that of the control group.

[0086] See also Fig.23 , showing the effect of IAA high nitrogen combined with C. protothecoides CS-41 biomass under different fermentation modes. It can be found that this treatment method has no significant effect on biomass. At the same time, this IAA high nitrogen combined method can effectively increase the lutein content of C. protothecoides CS-41 under both fermentation modes (such as Fig.24 ), chlorophyll content (such as Fig.25 ) and total carotenoid content (e.g. Fig.26 ). In the batch fermentation group, the lutein content increased from 1.74mg / g to 3.13mg / g, an increase of 79.9%; in the exponential feeding group, the lutein content increased from 2.07mg / g to 3.27mg / g, an increase of 58%. The final lutein production of exponential feeding fermentation reached 163mg / L, an increase of 56.7% compared with the untreated 104mg / L; the final lutein yield was 20.38mg / L / d, which is the current maximum value for this algae species.

[0087] In summary, the present invention can effectively regulate the growth of microalgae and pigment accumulation by culturing microalgae seeds, especially C.protothecoides CS-41 in a culture medium through plant hormones and additional nitrogen source stimulation, thereby stimulating lutein accumulation. IAA is beneficial to the production of lutein by C.protothecoides CS-41, but does not increase the total carotenoid content; the growth of C.protothecoides CS-41 is not affected by the concentration of nitrogen source, but the lutein content increases with the increase of nitrogen source concentration. Therefore, the high nitrogen combined treatment of IAA and urea can effectively stimulate the accumulation of lutein and the total carotenoid content. In particular, 200mM IAA and 3.6g / L urea are the most favorable conditions for C.protothecoides CS-41. Therefore, the present invention effectively overcomes the various shortcomings of the prior art and has a high industrial utilization value.

[0088] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for combined algae treatment, characterized in that: The steps include: Selecting microalgae seeds; Inoculating the microalgae seeds into a conical flask containing Basal medium; Cultivate under dark conditions; After the microalgae seed culture reaches a plateau phase, plant hormones and nitrogen sources are supplemented to culture microalgae.

2. The algae combined treatment method according to claim 1, characterized in that: The selected microalgae seeds were C. protothecoides CS-41.

3. The algae combined treatment method according to claim 1, characterized in that: The microalgae seeds are inoculated in a conical flask of Basal medium, including: The microalgae seeds were inoculated into a 500 mL conical flask filled with 200 mL of fresh modified Basal medium at 10% (v / v), and placed in a shaking fermentation culture at 28° C. and 180 rpm.

4. The algae combined treatment method according to claim 3, characterized in that: The modified Basal medium includes KH2PO4, MgSO4·7H2O, EDTA·Na2, H3BO3, Mg CaCl2·2H2O, FeSO4·7H2O, ZnSO4·7H2O, MnCl2·4H2O, CuSO4·5H2O, Co(NO3)2·6H2O, and MoO3.

5. The algae combined treatment method according to claim 4, characterized in that: The mass concentration ratios of KH2PO4, MgSO4·7H2O, EDTA·Na2, H3BO3, mgCaCl2·2H2O, FeSO4·7H2O, ZnSO4·7H2O, MnCl2·4H2O, CuSO4·5H2O, Co(NO3)2·6H2O, and MoO3 are 1.25:1.0:0.5:0.1142:0.111:0.0498:0.0882:0.0142:0.0157:0.0049:0.0071.

6. The algae combined treatment method according to claim 4, characterized in that: The initial pH of the modified Basal medium was 6.

1.

7. The algae combined treatment method according to claim 1, characterized in that: The culture days were at least three days in dark conditions.

8. The algae combined treatment method according to claim 1, characterized in that: The plant hormones and supplemental nitrogen sources include: The microalgae seeds reached the plateau phase after being treated with IAA and high nitrogen.

9. The algae combined treatment method according to claim 8, characterized in that: The microalgae seeds after reaching the plateau phase through IAA high nitrogen combined treatment include: The microalgae seeds were stimulated to reach the plateau phase by the combined use of urea and IAA.

10. The algae combined treatment method according to claim 9, characterized in that: The IAA was added at 200 mM, and the urea was added at 3.6 g / L.

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