Algal co-processing method

Abstract

The application provides an algal combined treatment method, and 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 dark conditions; after the microalgae seeds reach the plateau phase, supplementing plant hormones and a nitrogen source, and culturing to obtain microalgae. The algal combined treatment method can effectively stimulate the microalgae in the plateau phase, and then can effectively improve the total carotenoid content and the lutein content.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioengineering, in particular to an algal combined treatment method. BACKGROUND

[0002] Microalgae are considered as a very promising source of lutein, because they have a very fast growth rate, high lutein yield, strong adaptability to various environments, and are not limited by seasons, compared with the only natural source of lutein, marigold. Microalgae can be cultured in three cultivation modes, i.e., autotrophic cultivation, heterotrophic cultivation and mixotrophic cultivation. Among the three cultivation modes, the heterotrophic cultivation is the most suitable cultivation mode for large-scale cultivation of microalgae for lutein production. In addition, the conditions suitable for the growth of microalgae and the production of lutein are often inconsistent. For some conditions with a large difference between the growth and lutein accumulation, in order to obtain the maximum lutein yield, a two-step method has become the most commonly used method.

[0003] However, the two-step cultivation process commonly used for lutein production is often very complex. For example, they need to be induced by light after heterotrophic cultivation of a large amount of biomass, mixotrophic cultivation or light autotrophic cultivation. Although these two-step cultivation strategies usually obtain good results under laboratory conditions, they are difficult to apply in large-scale production. SUMMARY

[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide an algal combined treatment method, which solves the problem that the two-step cultivation process for lutein production in the prior art 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 application provides an algal combined treatment method, comprising the following steps: selecting a microalgae seed; inoculating the microalgae seed into a conical flask of a Basal medium; culturing in the dark; after the microalgae seed culture reaches the plateau phase, supplementing plant hormones and a nitrogen source, and culturing to obtain microalgae.

[0006] In an embodiment of the present application, the selected microalgae seed is C.protothecoides CS-41.

[0007] In an embodiment of the present application, the microalgae seed is inoculated into a conical flask of a Basal medium, comprising: inoculating the microalgae seed into a 500mL conical flask containing 200mL of fresh modified Basal medium at 10%(v / v), and placing it in a 28℃, 180rpm shaking incubator for fermentation culture.

[0008] In an embodiment of the present application, the modified Basal medium comprises KH2PO4, MgSO4·7H2O, EDTA·Na2, H3BO3, mg CaCl2·2H2O, FeSO4·7H2O, ZnSO4·7H2O, MnCl2·4H2O, CuSO4·5H2O, Co(NO3)2·6H2O, MoO3.

[0009] In an embodiment of the present application, the mass concentration ratio of KH2PO4, MgSO4·7H2O, EDTA·Na2, H3BO3, mg CaCl2·2H2O, FeSO4·7H2O, ZnSO4·7H2O, MnCl2·4H2O, CuSO4·5H2O, Co(NO3)2·6H2O, 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 an embodiment of the present application, the initial pH of the modified Basal medium is 6.1.

[0011] In an embodiment of the present application, the culturing time under dark condition is at least three days.

[0012] In an embodiment of the present application, the supplement of plant hormones and nitrogen source comprises: the microalgae seed after reaching the plateau stage by IAA high nitrogen combined treatment.

[0013] In an embodiment of the present application, the microalgae seed after reaching the plateau stage by IAA high nitrogen combined treatment comprises: the microalgae seed after reaching the plateau stage by urea and IAA combined stimulation.

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

[0015] As described above, the algal combined treatment method of the present application has the following beneficial effects: through the cultivation of microalgae seeds, especially C. protothecoides CS-41 in the culture medium, the growth and pigment accumulation of microalgae can be effectively regulated by the stimulation of plant hormones and additional nitrogen sources, thereby stimulating the accumulation of lutein. 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 the concentration of nitrogen source. Therefore, through the high-nitrogen combined treatment of IAA and urea, the accumulation of lutein and the total carotenoid content can be effectively stimulated, and in particular, 200 mM IAA and 3.6 g / L urea are the most favorable conditions for C. protothecoides CS-41. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 A flowchart showing the algal combined treatment method of the present application.

[0017] Figure 2 A schematic diagram showing the number distribution of differentially expressed genes of algal cells in an embodiment of the present application.

[0018] Figure 3 A schematic diagram showing the transcription level of carotenoid synthesis pathway genes in an embodiment of the present application.

[0019] Figure 4 A schematic diagram showing the transcription level of photosynthesis-related genes in a preferred embodiment of the present application.

[0020] Figure 5 A schematic diagram showing the transcription level of central carbon metabolism-related genes in a preferred embodiment of the present application.

[0021] Figure 6 A schematic diagram showing the transcription level of urea metabolism pathway-related genes in a preferred embodiment of the present application.

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

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

[0024] Figure 9Figure 6 shows the effect of different plant hormones on chlorophyll and total carotenoid content of C. protothecoides CS-41 in accordance with a preferred embodiment of the present application.

[0025] Figure 10 Figure 7 shows the effect of indole acetic acid on the percentage of lutein to total carotenoid of C. protothecoides CS-41 in accordance with a preferred embodiment of the present application.

[0026] Figure 11 Figure 8 shows the growth curve of C. protothecoides CS-41 with different concentrations of IAA in accordance with a preferred embodiment of the present application.

[0027] Figure 12 Figure 9 shows the lutein content of C. protothecoides CS-41 with different concentrations of IAA in accordance with a preferred embodiment of the present application.

[0028] Figure 13 Figure 10 shows the chlorophyll and total carotenoid content of C. protothecoides CS-41 with different concentrations of IAA in accordance with a preferred embodiment of the present application.

[0029] Figure 14 Figure 11 shows the percentage of lutein to total carotenoid of C. protothecoides CS-41 with different concentrations of indole acetic acid in accordance with a preferred embodiment of the present application.

[0030] Figure 15 Figure 12 shows the growth curve of C. protothecoides CS-41 with different concentrations of urea in accordance with a preferred embodiment of the present application.

[0031] Figure 16 Figure 13 shows the glucose consumption curve of C. protothecoides CS-41 with different concentrations of urea in accordance with a preferred embodiment of the present application.

[0032] Figure 17 Figure 14 shows the urea consumption curve of C. protothecoides CS-41 with different concentrations of urea in accordance with a preferred embodiment of the present application.

[0033] Figure 18 Figure 15 shows the lutein content of C. protothecoides CS-41 with different concentrations of urea in accordance with a preferred embodiment of the present application.

[0034] Figure 19Figure 6 shows the changes in chlorophyll content of C. protothecoides CS-41 after treatment with different concentrations of urea in accordance with a preferred embodiment of the present application.

[0035] Figure 20 Figure 7 shows the changes in biomass of C. protothecoides CS-41 after treatment with IAA and high nitrogen in accordance with a preferred embodiment of the present application.

[0036] Figure 21 Figure 8 shows the changes in lutein content of C. protothecoides CS-41 after treatment with IAA and high nitrogen in accordance with a preferred embodiment of the present application.

[0037] Figure 22 Figure 9 shows the changes in carotenoid content of C. protothecoides CS-41 after treatment with IAA and high nitrogen in accordance with a preferred embodiment of the present application.

[0038] Figure 23 Figure 10 shows the changes in biomass of C. protothecoides CS-41 after treatment with IAA and high nitrogen under different fermentation modes in accordance with a preferred embodiment of the present application.

[0039] Figure 24 Figure 11 shows the changes in lutein content of C. protothecoides CS-41 after treatment with IAA and high nitrogen under different fermentation modes in accordance with a preferred embodiment of the present application.

[0040] Figure 25 Figure 12 shows the changes in chlorophyll content of C. protothecoides CS-41 after treatment with IAA and high nitrogen under different fermentation modes in accordance with a preferred embodiment of the present application.

[0041] Figure 26 Figure 13 shows the changes in total carotenoid content of C. protothecoides CS-41 after treatment with IAA and high nitrogen under different fermentation modes in accordance with a preferred embodiment of the present application. DETAILED DESCRIPTION

[0042] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. It should also be understood that the terminology used in the embodiments of the present invention is for describing specific implementation schemes and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers.

[0043] Please see Figures 1 to 26 It should be understood that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes to aid those skilled in the art and to facilitate understanding. They are not intended to limit the scope of the invention and therefore have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to size, without affecting the effectiveness or purpose of the invention, should still fall within the scope of the technical content disclosed herein. Furthermore, the terms "upper," "lower," "left," "right," "middle," and "one" used in this specification are merely for clarity and not intended to limit the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0044] Auxins are a class of endogenous hormones containing an unsaturated aromatic ring and an acetic acid side chain, abbreviated as IAA, and their chemical nature is indoleacetic acid. Other auxin-like substances include 4-chloro-IAA, 5-hydroxy-IAA, naphthaleneacetic acid (NAA), and indolebutyric acid. The most important chemical substance among auxins is 3-indoleacetic acid. Auxins regulate stem growth rate, inhibit lateral buds, and promote rooting, and are used in agriculture to significantly promote rooting of cuttings.

[0045] Auxins are widely distributed in plants, present in almost every part, but not uniformly. The content in a specific part at a given time is affected by several factors. Most are concentrated in the parts with vigorous growth (coleoptile, bud and root tip meristem, cambium, fertilized ovary, young seeds, etc.), while very little is found in senescent tissues and organs.

[0046] Urea, also known as urea or carbamide, has the chemical formula CH4N2O or CO(NH2)2. It is a white crystalline solid, tasteless and odorless, readily 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 in mammals and some fish. It can be used as a fertilizer, animal feed, explosives, glue stabilizer, and chemical raw material. It is named urea because it is found in human urine. As a neutral fertilizer, urea is suitable for various soils and plants. It is easy to store, convenient to use, and has minimal impact on soil health, making it a widely used chemical nitrogen fertilizer. Urea contains 46% nitrogen (N), the highest nitrogen content among solid nitrogen fertilizers. Industrially, urea is synthesized from ammonia and carbon dioxide under specific conditions.

[0047] Please see Figure 1 In one embodiment of the present invention, the present invention provides a method for combined treatment of algae, comprising the following steps:

[0048] Step S10: Select microalgae seeds;

[0049] Step S20: Inoculate the microalgae seeds into an Erlenmeyer flask containing Basal medium;

[0050] Step S30: Cultivate in darkness;

[0051] Step S40: After the microalgae seed culture reaches the plateau stage, plant hormones and nitrogen sources are added to cultivate microalgae.

[0052] From the above, it is clear that step S10, selecting microalgae seeds, demonstrates that to increase lutein production in microalgae, algae species with high lutein content can be chosen as an indicator for seed selection. Steps S20-S40, involving inoculating the microalgae seeds into Erlenmeyer flasks of Basal medium and culturing in the dark, followed by supplementing with plant hormones and nitrogen after the seed culture reaches a plateau, show that inoculating the microalgae seeds into Erlenmeyer flasks of Basal medium after seed selection improves the maximum specific growth efficiency of the seeds, promoting their growth and enabling them to reach the plateau phase more quickly under dark conditions. Furthermore, supplementing with additional nitrogen after reaching the plateau phase and continuing cultivation effectively regulates the lutein synthesis capacity of the microalgae, resulting in stronger lutein synthesis and significantly increasing the total carotenoid and lutein content. Moreover, this method of supplementing with additional nitrogen does not affect the biomass of the microalgae. In addition, adding plant hormones after the plateau phase can effectively stimulate microalgae to accumulate lutein.

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

[0054] Referring to Figure 2 , the number of differentially expressed genes of cells is shown, in order to determine the mechanism of additional nitrogen stimulating lutein synthesis of algal cells at the transcriptional level, while the difference of urea addition time is analyzed, three experimental groups are set, and the transcriptional level is compared two by two, which are initial addition of double urea (2X) and control 1 (C1), initial addition of one time urea and additional one time urea after the plateau period (1+1X) and control 2 (C2), and initial addition of double urea (2X) and additional one time urea after the plateau period (1+1X) three groups. It is shown in the figure that the urea 2X and the urea 1+1X group have the most differentially expressed genes, 4900 up-regulated and 4486 down-regulated. The urea 2X and the control 1 have the least differentially expressed genes, only 39 up-regulated and 25 down-regulated. It is found by comparison that the additional nitrogen source after the plateau period has greater difference in the transcriptional level of cells and more different genes with the control group.

[0055] Referring to Figure 3 , the transcriptional level of genes in the carotenoid synthesis pathway is shown. It is found that 2X has no significant difference with C1, while 1+1X significantly up-regulates the transcriptional level of CMS, CRTISO and LCYB in the carotenoid synthesis pathway, and down-regulates the transcriptional level of ZEP and VDE compared with C2. This shows that the addition of additional urea after the plateau period is better for the regulation of the transcriptional level of lutein synthesis pathway genes, and CMS, CRTISO, LCYB, ZEP and VDE may be the key genes for increasing the content of lutein by adding additional urea. By comparing 2X and 1+1X, it is found that PSY has great difference in different culture stages, and PSY is significantly up-regulated in the logarithmic growth phase (2X) compared with the plateau period (1+1X). It is shown that the flux to carotenoid synthesis is more in the logarithmic growth phase.

[0056] Referring to Figure 4, which shows the transcription level of photosynthesis related genes. It is found that 2X has no significant difference with C1, and all photosynthesis related genes are down-regulated in 1+1X compared with C2, which indicates that the additional nitrogen source will reduce the photosynthesis activity of algal cells. However, 2X is almost up-regulated compared with 1+1X, which may be caused by the different growth stages of the two groups of algal cells. 2X is in the logarithmic growth phase, and the photosynthesis activity is stronger than that of 1+1X which is in the stationary phase.

[0057] Please refer to Figure 5 , which shows the transcription level of central carbon metabolism related genes. The results show that 2X has no significant difference with C1, and most of the central carbon metabolism related genes are significantly down-regulated in 1+1X compared with C2, which indicates that the additional nitrogen source will reduce the carbon efficiency of algal cells. The significant up-regulation of PYC and PEPCK enhances the efficiency of pyruvate metabolism, and may provide more substrates for carotenoid synthesis. Since the two groups of algal cells are in different stages of growth, it is meaningless to compare the transcription level differences of central carbon metabolism related genes between 2X and 1+1X.

[0058] Please refer to Figure 6 , which shows the transcription level of urea metabolism related pathway genes. Urea active transporters have been found in many bacteria and phytoplankton. Genome sequencing shows that marine single-cell photosynthetic eukaryotes have 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, the urea metabolism related genes are significantly down-regulated in 1+1X compared with C2, which indicates that the additional nitrogen source reduces the ability of urea transport and catabolism. However, in addition to active transport through transporters, urea can also diffuse freely into cells. The additional urea in the cytoplasm regulates the metabolism of algal cells as an environmental stress, which increases the synthesis of lutein.

[0059] In an embodiment of the present application, the microalgae seeds are inoculated in a conical flask of Basal medium, comprising:

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

[0061] In one embodiment of the present application, the modified Basal medium comprises KH2PO4, MgSO4·7H2O, EDTA·Na2, H3BO3, mg CaCl2·2H2O, FeSO4·7H2O, ZnSO4·7H2O, MnCl2·4H2O, CuSO4·5H2O, Co(NO3)2·6H2O, MoO3.

[0062] Preferably, the KH2PO4, MgSO4·7H2O, EDTA·Na2, H3BO3, mg CaCl2·2H2O, FeSO4·7H2O, ZnSO4·7H2O, MnCl2·4H2O, CuSO4·5H2O, Co(NO3)2·6H2O, MoO3are in a mass concentration ratio of 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) comprises 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 the Kuhl medium and the BG11 medium, the modified Basal medium is more conducive to the growth of the C. protothecoides CS-41 strain. The modified Basal medium in this embodiment is three times the concentration of 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 modified Basal medium is 6.1.

[0065] Preferably, the culture time in the dark condition is at least three days. In this embodiment, generally, the microalgae can reach the plateau phase after 36 hours of culture in the medium.

[0066] In a preferred embodiment of the present application, when the modified Basal medium is used to culture the C. protothecoides CS-41 microalgae strain, the culture can reach the plateau phase of the C. protothecoides CS-41 microalgae strain after 36 hours of culture.

[0067] In an embodiment of the present application, the additional plant hormone and nitrogen source comprises:

[0068] The microalgae seed after reaching the plateau stage is stimulated by IAA and high nitrogen combination treatment.

[0069] Preferably, the microalgae seed after reaching the plateau stage is stimulated by IAA and high nitrogen combination treatment, comprising:

[0070] The microalgae seed after reaching the plateau stage is stimulated by urea and IAA combination treatment.

[0071] Experimental Example 1

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

[0073] Experimental Example 2

[0074] Plant hormone concentration selection culture: C.protothecoides CS-41 microalgae seed was inoculated at 10% (v / v) into a 250 mL conical flask containing 100 mL of fresh modified Basal medium, and cultured at 28°C, 180 rpm in the dark for 5 days. 0, 50, 100, 200 mM indole acetic acid was added respectively. Another experimental group was not initially added indole acetic acid, and 200 mM indole acetic acid was added on the third day.

[0075] Experimental Example 3

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

[0077] Please refer to Figure 7The growth of *C. protothecoides* CS-41 was investigated after the addition of different plant hormones. To screen for suitable plant hormones to stimulate lutein accumulation in *C. protothecoides* CS-41, 100 mM indoleacetic acid (IAA), α-naphthaleneacetic acid (NAA), salicylic acid, abscisic acid (ABA), and gibberellin were added to the culture medium before the start of cultivation. Except for α-NAA, the other four plant hormones did not affect the growth of the microalgae. The experimental group with added α-NAA showed a slower growth rate and reached maximum biomass one day later than the other groups. However, it had no effect on the final biomass.

[0078] Please see Figure 8 The figure shows the lutein content of *C. protothecoides* CS-41 after the addition of different plant hormones. As can be seen from the figure, the lutein content in the experimental group with added IAA was significantly increased (p<0.01), increasing by 20.4% compared to the control group. The other four experimental groups showed no significant difference from the control group. Interestingly, the lutein content in the experimental group with added IAA was significantly increased (p<0.01), increasing by 20.4% compared to the control group. Figure 9 It can be seen that there were no significant differences in chlorophyll and total carotenoid content between the five experimental groups and the control group. Calculation of the proportion of xanthophyll to total carotenoids shows that IAA significantly increased the proportion of xanthophyll to total carotenoids in *C. protothecoides* CS-41 from 54.1% to 79.2%. Figure 10 .

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

[0080] Please see Figure 11 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. An additional experimental group had no IAA initially added, and 200 mM IAA was added to the culture medium after the plateau phase (day 3). As shown in the figure, although the final biomass was not affected, the addition of IAA significantly reduced the growth rate of *C. protothecoides* CS-41, and the degree of effect increased with increasing IAA concentration. Adding high concentrations of IAA after the plateau phase had no effect on the biomass of *C. protothecoides* CS-41.

[0081] Please see Figure 12, all the experimental groups with IAA addition had higher lutein content than the control group (p<0.001), which was 20.4% higher than the control group. The other four experimental groups had no significant difference with the control group. Among them, the lutein content of the experimental group with high concentration added after the plateau was the highest, which was 2.73 mg / g, which was 82% higher than the control group (1.50 mg / g). Consistent with the previous results, there was no significant difference in chlorophyll and total carotenoid content between the four experimental groups and the control group, as shown in Figure 13 . Similarly, as shown in Figure 14 , the addition of IAA significantly increased the proportion of lutein in total carotenoids of C.protothecoides CS-41, among which the ratio of the experimental group with high concentration added after the plateau was the highest, which was 85.8%, which was 78% higher than the control group.

[0082] Please refer to Figure 15 , which shows the growth curve of C.protothecoides CS-41 with different concentrations of urea added. In order to obtain more carbon flux for carotenoid synthesis pathway, nitrogen source can be added. As can be seen from the figure, the concentration of nitrogen source increased from 0.45 to 3.6 g / L had no effect on the growth of microalgae. Figure 16 and Figure 17 are the consumption of glucose and urea of C.protothecoides CS-41 under different concentrations of initial nitrogen source. As with the growth, different concentrations of initial urea content do not affect the consumption of glucose, while 0.9 g / L urea and glucose are consumed synchronously, and 1.8 g / L and 3.6 g / L are nitrogen excess state.

[0083] Please refer to Figure 18 , which shows the lutein accumulation of C.protothecoides CS-41 under different concentrations of initial nitrogen source. 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 nitrogen excess group, i.e. 1.8 g / L and 3.6 g / L (2.33 mg / g, 2.54 mg / g), was significantly higher than that of the experimental group with initial urea content of 0.45 g / L (1.52 mg / g). The total carotenoid and total chlorophyll content of the low nitrogen group (0.45 g / L) was significantly lower than that of the other three groups, as shown in Figure 19 .

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

[0085] Referring to Figure 21 It can be seen that the treatment method has no effect on the biomass of C.protothecoides CS-41. Figure 22 and Figure 23 The changes of lutein content and total carotenoid content after two days of treatment are shown. Compared with the control group, the combination of IAA and urea significantly increases the total carotenoid content and lutein content. Among them, the lutein content after treatment can be as high as 47.6% higher than the control group.

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

[0087] In summary, the present application can effectively regulate the growth and pigment accumulation of microalgae by stimulating the microalgae seed, especially C.protothecoides CS-41, in the culture medium, 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 the concentration of nitrogen source. Therefore, by the combination of high nitrogen and IAA urea, lutein accumulation and total carotenoid content can be effectively stimulated, especially 200 mM IAA and 3.6 g / L urea are the most favorable conditions for C.protothecoides CS-41. Therefore, the present application effectively overcomes the shortcomings of the prior art and has high industrial utilization value.

[0088] The above embodiments are only illustrative of the principles of the present application and its efficacy, and are not intended to limit the present application. Any modification or change made by any person skilled in the art without departing from the spirit and scope of the present application shall be covered by the claims of the present application.

Claims

1. A method for combined treatment of algae, characterized in that, Includes the following steps: Select microalgae seeds; The microalgae seeds were inoculated into Erlenmeyer flasks containing Basal medium; Cultivate in dark conditions; After the microalgae seed culture reaches the plateau stage, plant hormones and nitrogen sources are added to cultivate microalgae. The selected microalgae seed was C. protothecoides CS-41; The plant hormones and nitrogen sources mentioned above include: The microalgal seeds after reaching the plateau phase through IAA high-nitrogen combined treatment.

2. The method for combined algae treatment according to claim 1, characterized in that: The microalgae seeds were inoculated into Erlenmeyer flasks containing Basal medium, including: The microalgae seeds were inoculated at 10% (v / v) into a 500 mL Erlenmeyer flask containing 200 mL of fresh modified Basal medium and placed in a shaker at 28 °C and 180 rpm for fermentation.

3. The method for combined algae treatment according to claim 2, 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.

4. The method for combined algae treatment according to claim 3, characterized in that: The following ingredients are added in a mass concentration ratio of 1.25:1.0:0.5:0.1142:0.111:0.0498:0.0882:0.0142:0.0157:0.0049:0.0071:KH2PO4,MgSO4·7H2O,EDTA·Na2,H3BO3,mgCaCl2·2H2O,FeSO4·7H2O,ZnSO4·7H2O,MnCl2·4H2O,CuSO4·5H2O,Co(NO3)2·6H2O,MoO3.

5. The method for combined algae treatment according to claim 3, characterized in that: The initial pH of the modified Basal medium was 6.

1.

6. The method for combined algae treatment according to claim 1, characterized in that: The incubation period under dark conditions is at least three days.

7. The method for combined algae treatment according to claim 1, characterized in that: The microalgal seeds that have reached the plateau phase after IAA high-nitrogen combined treatment include: The microalgal seeds were stimulated to reach the plateau phase by a combination of urea and IAA.

8. The method for combined algae treatment according to claim 7, characterized in that: The amount of IAA added was 200 mM, and the amount of urea added was 3.6 g / L.

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