A method for inducing improved lipid accumulation in microalgae using mannose

Abstract

The application belongs to the technical field of microalgae oil biosynthesis, and discloses a method for improving microalgae oil accumulation by using mannose induction. The application of mannose in culturing microalgae to improve microalgae oil accumulation, the concentration of mannose in the culture medium is not less than 0.5g / L. Specifically, the microalgae seed liquid can be inoculated into the culture medium containing mannose for induction of oil accumulation culture; or the microalgae seed liquid can be inoculated into the culture medium for culture, and then mannose is added to the culture medium for continuous culture, so as to improve the microalgae oil accumulation by using mannose. The application discloses for the first time that the application of mannose in microalgae culture can improve the microalgae oil accumulation, and by using the concentration of mannose not less than 0.5g / L, the oil content of microalgae under different environmental conditions and growth states can be simply, flexibly and quickly induced and improved.

Description

Technical Field

[0001] This invention belongs to the field of microalgal lipid biosynthesis technology, and more specifically, relates to a method for using mannose to induce and enhance microalgal lipid accumulation. Background Technology

[0002] Due to the limited, non-renewable nature of fossil fuel resources and their environmental pollution, exploring environmentally friendly and renewable energy pathways has become a crucial global issue. Among numerous renewable energy sources, biomass energy, such as biofuels, has garnered widespread attention from researchers both domestically and internationally due to its abundant raw material resources and clean, low-carbon production process. Microalgae, as a potential source of third-generation biofuels, has attracted considerable attention due to its significant advantages, including rapid growth, high oil content, small and flexible space requirements, and ability to utilize wastewater and waste gas for growth. Despite the broad prospects for biofuel production using microalgae, its commercial application still faces limitations such as high cultivation costs, low biomass yield, and low oil yield. To improve the oil yield of microalgae, researchers mainly employ abiotic stress conditions such as high light, high salinity, and nitrogen and phosphorus nutrient restriction, or by adding inducers. While these methods are effective to some extent, they also present some challenges. For example, while nitrogen starvation strategies can promote oil accumulation, precise control of cultivation conditions, including the addition and removal of nitrogen sources, is required during application, increasing operational complexity and cost. Therefore, finding more easy-to-operate methods to induce and regulate the oil yield of microalgae is of great significance for the advancement of microalgae oil production technology and the promotion of commercial development and utilization. Summary of the Invention

[0003] In view of the above-mentioned defects or improvement needs of the existing technology, the purpose of this invention is to provide a method for inducing and improving the accumulation of microalgal lipids using mannose. It reveals for the first time that mannose can improve the accumulation of microalgal lipids when applied to microalgae culture. By using a mannose concentration of not less than 0.5 g / L, it is possible to simply, flexibly and quickly induce and improve the lipid content of microalgae under different environmental conditions (e.g., different initial cell densities, different carbon sources, and different concentrations of nitrogen sources) and growth states (e.g., different growth stages in autotrophic or polyculture).

[0004] To achieve the above objectives, according to one aspect of the present invention, the application of mannose in culturing microalgae to enhance microalgal lipid accumulation is provided, characterized in that the concentration of mannose in the culture medium is not less than 0.5 g / L.

[0005] As a further preferred embodiment of the present invention, the culture is an autotrophic culture, a heterotrophic culture, or a mixed culture.

[0006] As a further preferred embodiment of the present invention, the microalgae are *Chlorella sorokinense*, *Chlorella* microalgae, or *Haematococcus pluvialis*.

[0007] According to another aspect of the present invention, the present invention provides a method for inducing and enhancing lipid accumulation in microalgae using mannose, characterized in that the method involves inoculating microalgae seed culture into a mannose-containing culture medium for inducing lipid accumulation; wherein the concentration of mannose in the mannose-containing culture medium is not less than 0.5 g / L;

[0008] Alternatively, the method involves first inoculating the microalgal seed culture into a culture medium for cultivation, and then adding mannose to the culture medium during the cultivation process to continue cultivation, thereby utilizing mannose to enhance the accumulation of microalgal lipids; the amount of mannose added is sufficient to ensure that the concentration of mannose in the culture medium is not less than 0.5 g / L.

[0009] As a further preferred embodiment of the present invention, when the method involves inoculating microalgae seed culture into a mannose-containing culture medium for inducing lipid accumulation:

[0010] The concentration of mannose in the mannose-containing culture medium is 0.5-8 g / L;

[0011] Preferably, the mannose-containing culture medium is a modified BG11 culture medium, which is obtained by adding mannose to the original BG11 culture medium, and optionally adding glucose and sodium nitrate; the concentration of glucose in the mannose-containing culture medium is 0-5 g / L, and the concentration of sodium nitrate is 0-3 g / L.

[0012] The culture conditions were: a culture temperature of 28℃, a light / dark cycle of 12 / 12h, and a light intensity of 70 μmol / m². -2 s -1 The incubation period is 1-10 days, the incubation atmosphere is air, and the flow rate is 0.2-0.7 L / min.

[0013] As a further preferred embodiment of the present invention, when the method involves inoculating microalgae seed culture into a mannose-containing culture medium for inducing lipid accumulation:

[0014] The concentration of mannose in the mannose-containing culture medium is 0.5-8 g / L;

[0015] The mannose-containing culture medium is a modified BG11 culture medium, which is obtained by adding mannose to the original BG11 culture medium.

[0016] The culture conditions were: a culture temperature of 28℃, a light / dark cycle of 12 / 12h, and a light intensity of 70 μmol / m². -2 s -1 The incubation period is 1-10 days, and the incubation atmosphere is compressed air with a concentration of 5 vol% CO2 and a flow rate of 0.2-0.7 L / min.

[0017] As a further preferred embodiment of the present invention, when the method involves first inoculating the microalgal seed culture into a culture medium for cultivation, and then adding mannose to the culture medium during the cultivation process to continue cultivation, thereby utilizing mannose to enhance the accumulation of microalgal lipids:

[0018] The amount of mannose added is sufficient to make the concentration of mannose in the culture medium 0.5-8 g / L;

[0019] Preferably, the culture medium is a modified BG11 culture medium, which is obtained by optionally adding glucose and sodium nitrate to the original BG11 culture medium; the concentration of glucose in the culture medium is 0-5 g / L, and the concentration of sodium nitrate is 0-3 g / L.

[0020] More preferably, the culture conditions before and after the addition of mannose both meet the following requirements: culture temperature of 28℃, light / dark cycle of 12 / 12h, and light intensity of 70 μmol / m². -2 s -1 The culture time after adding mannose is 1-10 days.

[0021] As a further preferred embodiment of the present invention, the microalgae seed solution is obtained by inoculating microalgae into a pre-culture medium, pre-culturing, and then centrifuging and concentrating.

[0022] Preferably, the pre-culture medium is a modified BG11 medium, which is obtained by adding glucose and yeast extract to the original BG11 medium; in the pre-culture medium, the concentration of glucose is 5 g / L and the concentration of yeast extract is 2 g / L.

[0023] The pre-culture conditions are: culture temperature of 28℃, light / dark cycle of 12 / 12h, and light intensity of 70 μmol / m³. -2 s -1 The incubation period is 3-4 days.

[0024] As a further preferred embodiment of the present invention, the microalgae are *Chlorella sorokinense*, *Chlorella* species, or *Haematococcus pluvialis*; the microalgae seed culture, after inoculation, meets the final OD... 680 =0.1-4.

[0025] As a further preferred embodiment of the present invention, the method further includes: collecting algal cells after cultivation, freeze-drying them, and then extracting the oil from the algal cells using an organic solvent; preferably, the collection is specifically performed by centrifugation; after collection, the algal cells are first washed 2-3 times with distilled water, then frozen at -80°C for 12 hours, followed by vacuum freeze-drying for 48 hours, and the freeze-dried powder is collected; the organic solvent is a chloroform-methanol mixed solution obtained by mixing chloroform and methanol in a volume ratio of 1:1.

[0026] More preferably, the method further includes: after collecting algal cells, by adding the lost inorganic salts and carbon and nitrogen sources to the remaining culture medium, the microalgal seed solution can be re-inoculated for a new round of induced lipid accumulation culture.

[0027] Compared with the prior art, the present invention, through the above-described technical solution, reveals for the first time that the application of mannose in microalgae cultivation can increase the accumulation of lipids in microalgae (of course, the concentration of mannose in the culture medium needs to be no less than 0.5 g / L); in the examples below, the lipid content of microalgae cultivated using the method of the present invention is increased. The cultivation method of the present invention is simple and easy to operate, and can flexibly, rapidly and sustainably increase the lipid content of microalgae under different environmental conditions (e.g., different initial cell densities, different carbon sources, and different concentrations of nitrogen sources) and growth states (e.g., different growth stages in autotrophic or polyculture) in a short period of time, thereby reducing the cost of microalgae cultivation and maximizing the potential for efficient and economical production of microalgae biodiesel.

[0028] Specifically, the present invention has the following beneficial effects:

[0029] (1) Mannose, as a non-nutritive limiting inducer, can significantly increase the oil content of microalgae without relying on traditional methods such as nitrogen starvation. The oil content of microalgae can be increased by up to 1.82 times compared with that before the addition of mannose (as shown in the comparison of Example 5 and Comparative Example 5 below, both of which have a sodium nitrate addition of 1.5 g / L), and the oil content can exceed 40% (as shown in Example 15 below).

[0030] Of course, the strategy of using mannose to increase the accumulation of microalgal lipids in this invention can also be combined with nitrogen starvation. For example, in the following examples, under the same nitrogen starvation conditions, compared with Comparative Example 14, Example 12 obtained a higher lipid content under mannose induction (Comparative Example 14 corresponds to the traditional nitrogen starvation operation to increase the lipid content of microalgae, while Example 12 is an induction by adding mannose on the basis of nitrogen starvation).

[0031] (2) Mannose induces rapid, flexible and continuous effects. In different growth stages of microalgae autotrophic or polytrophic culture, in microalgae heterotrophic culture, or at different initial cell densities, the addition of mannose can rapidly induce and promote the accumulation of microalgae lipids in a short period of time.

[0032] (3) Adding an additional carbon source can simultaneously increase the biomass of microalgae while mannose induces and promotes the increase of microalgae lipid content.

[0033] (4) Mannose, as a component of lignocellulose, a rich renewable resource, is widely found in industrial and agricultural waste, biorefining wastewater, and various natural plants and microorganisms, making it easy to obtain and possessing broad application potential. Furthermore, in the method of this invention, mannose is not significantly consumed during cultivation. After the algal cells are collected upon completion of cultivation, the lost inorganic salts and carbon and nitrogen sources can be added to the remaining culture medium, allowing for re-inoculation of microalgal seed culture for a new round of induced lipid accumulation cultivation.

[0034] (5) Mannose induction helps overcome the limitations of traditional methods, increases the oil content of microalgae, and provides a new technical approach for the commercial application of microalgae biofuels. Attached Figure Description

[0035] Figure 1 The biomass and lipid content of *Chlorella sorokinense* after cultivation in Examples 1-5 and Comparative Examples 5-6 are shown.

[0036] Figure 2 The curves show the changes in mannose content in the supernatant during the culture period in Examples 1-5 and Comparative Example 6.

[0037] Figure 3 The morphological and structural characterization results of *Chlorella sorokinense* W1 in Examples 12-14 and Comparative Examples 13-15 are shown; among them, Figure 3 In the image, 'a' corresponds to the confocal microscopy imaging of Nile River-stained lipid fluorescence within algal cells; Figure 3 In the diagram, 'b' corresponds to the structural imaging within algal cells using transmission electron microscopy (L: lipid droplets; S: starch granules). Examples 12-14 represent cultures with added mannose (4 g / L in all cases), labeled M-induce; Comparative Examples 13-15 represent cultures without added mannose, labeled control; "0N5C" indicates cultures with 0 g / L sodium nitrate and 5 g / L glucose, corresponding to Example 12 and Comparative Example 13, respectively; "03N5C" indicates cultures with 0.3 g / L sodium nitrate and 5 g / L glucose, corresponding to Example 13 and Comparative Example 14, respectively; "3N5C" indicates cultures with 3 g / L sodium nitrate and 5 g / L glucose, corresponding to Example 14 and Comparative Example 15, respectively. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0039] In summary, this invention provides the application of mannose in culturing microalgae to enhance microalgal lipid accumulation, and correspondingly provides a method for using mannose to induce and enhance microalgal lipid accumulation, which may include, for example, the following steps:

[0040] S1, Microalgae are inoculated into the culture medium, pre-cultured, and then concentrated by centrifugation to obtain microalgae seed solution;

[0041] S2, inoculate the microalgae seed culture into a fresh culture medium (e.g., BG11 medium), and add mannose to induce lipid accumulation culture (e.g., culture at 28℃ under light for 3-4 days).

[0042] S3: After cultivation, algal cells are harvested, freeze-dried, and then the oil in the algal cells is extracted using organic solvents.

[0043] The microalgae used in Examples 1-20 below is *Chlorella sorokiniana* W1; the original BG11 medium used was commercially available.

[0044] Example 1

[0045] A method for using mannose to induce and enhance lipid accumulation in microalgae includes the following steps:

[0046] Microalgae pre-culture: Chlorella sorokinense was transferred to a modified BG11 medium (obtained by adding glucose and yeast extract to the original BG11 medium; the concentration of glucose in the modified BG11 medium was 5 g / L, and the concentration of yeast extract was 2 g / L). The culture was carried out at 28℃ with a 12 / 12 h light / dark cycle and a light intensity of 70 μmol / m². -2 s -1 The microalgae were cultured in an air atmosphere at a flow rate of 0.5 L / min for 3-4 days, and then concentrated by centrifugation to obtain the microalgae seed culture.

[0047] Lipid accumulation culture: Microalgal seed culture was inoculated into BG11 medium (containing 1.5 g / L sodium nitrate) to achieve the final OD... 680 =0.4, and at the beginning of the culture, 1 g / L mannose was added (mannose was prepared as a sterile stock solution, then added to the culture medium at the required final concentration and mixed thoroughly), at 28℃, with a light / dark cycle of 12 / 12 h and a light intensity of 70 μmol m. -2 s -1 Autotrophic culture was carried out. The culture atmosphere was air, and the flow rate was 0.5 L / min.

[0048] Microalgal biomass harvesting and oil extraction after cultivation: After 3 days of cultivation, all biomass was collected by centrifugation, washed 2-3 times with distilled water, and then frozen at -80℃ for 12 hours, followed by vacuum freeze-drying for 48 hours. The freeze-dried powder was collected, and the weight of the harvested biomass was determined by gravimetric analysis. The organic solvent used was a chloroform-methanol mixture, with a volume ratio of chloroform to methanol of 1:1. Oil was extracted by shaking at 140 rpm for at least 4 hours at room temperature. The oil yield was determined by gravimetric analysis, and the oil content was obtained by comparing it with the weight of the obtained biomass.

[0049] Comparative Examples 1-4

[0050] The only difference between Comparative Examples 1-4 and Example 1 is that:

[0051] Comparative Examples 1-4 were cultured for lipid accumulation without the addition of mannose, but with the addition of 1 g / L glucose, 1 g / L xylose, 1 g / L arabinose and 1 g / L galactose, respectively.

[0052] Comparative Example 5

[0053] The only difference between Comparative Example 5 and Example 1 is that:

[0054] Comparative Example 5 did not add mannose (that is, no monosaccharides were added) during the lipid accumulation culture.

[0055] The biomass and lipid content of the microalgae after induction culture according to Example 1 and Comparative Examples 1-5 are shown in Table 1.

[0056] Table 1. Effects of autotrophic culture and different sugar cultures on biomass and lipid content of Chlorella sorokin.

[0057]

[0058]

[0059] Depend on Figure 1 As shown in Table 1, compared to the autotrophic culture of Comparative Example 5, the addition of mannose in Example 1 had no significant effect on biomass, but it significantly increased the lipid content of microalgae. Compared to the cultures of Comparative Examples 1-4 with the addition of glucose, xylose, arabinose, or galactose, the microalgae cultured with mannose in Example 1 achieved the highest lipid content, demonstrating the significant promoting effect of this invention on the accumulation of lipids in microalgae.

[0060] Examples 2-5

[0061] The only difference between Examples 2-5 and Example 1 is that:

[0062] In Examples 2-5, the amount of mannose added during the lipid accumulation culture was 0.5, 2, 4, and 8 g / L, respectively.

[0063] Comparative Example 6

[0064] The only difference between Comparative Example 6 and Example 1 is that:

[0065] In Comparative Example 6, the amount of mannose added during the lipid accumulation culture was 0.2 g / L.

[0066] Examples 6-9

[0067] The only difference between Examples 6-9 and Example 4 is that:

[0068] Examples 6-9: Initial cell density (OD) during lipid accumulation culture. 680 The values ​​are 0.1, 1, 2, and 4 respectively.

[0069] Comparative Examples 7-10

[0070] Comparative Examples 7-10 correspond one-to-one with Examples 6-9. The only difference between Comparative Examples 7-10 and Examples 6-9 is that:

[0071] Comparative Examples 7-10 were cultured for lipid accumulation without the addition of mannose (that is, without the addition of any monosaccharides).

[0072] Example 10

[0073] The only difference between Example 10 and Example 4 is that:

[0074] In Example 10, during the lipid accumulation culture, 5 g / L glucose was added to the BG11 medium for mixed culture.

[0075] Comparative Example 11

[0076] The only difference between Comparative Example 11 and Example 10 is that:

[0077] Comparative Example 11 did not add mannose during the oil accumulation culture.

[0078] Example 11

[0079] The only difference between Example 11 and Example 4 is that:

[0080] In Example 11, during the oil accumulation culture, the culture atmosphere was compressed air containing 5% (v / v) CO2 at a flow rate of 0.5 L / min.

[0081] Comparative Example 12

[0082] The only difference between Comparative Example 12 and Example 11 is that:

[0083] Comparative Example 12 did not add mannose during the oil accumulation culture.

[0084] Examples 12-14

[0085] The only difference between Examples 12-14 and Example 10 is that:

[0086] In Examples 12-14, the concentration of sodium nitrate added to the BG11 medium was adjusted to 0, 0.3, and 3 g / L, respectively, during the lipid accumulation culture.

[0087] Comparative Examples 13-15

[0088] Comparative Examples 13-15 correspond one-to-one with Examples 12-14. The only difference between Comparative Examples 13-15 and Examples 12-14 is that:

[0089] Comparative Examples 13-15 were cultured without the addition of mannose during the oil accumulation phase.

[0090] Table 2 shows the specific parameter settings for the culture conditions after adjustment during the initial stage of lipid accumulation culture in Examples 1-14 and Comparative Examples 5-15:

[0091] Table 2 shows the parameter adjustments for Examples 1-14 and Comparative Examples 5-15.

[0092]

[0093]

[0094] After inducing microalgae culture according to Examples 1-14 and Comparative Examples 5-15, the harvested biomass and lipid content are shown in Table 3 below (the detection method is the same as in Table 1).

[0095] Table 3 Biomass and lipid content of Chlorella sorokinae under different culture modes

[0096]

[0097]

[0098] A comparison of the data in Tables 1 and 3 clearly shows that the lipid content of the microalgae in Comparative Example 13 and Examples 1-14 is significantly higher than that in Comparative Examples 1-12 and 14-15. This fully demonstrates that, under the currently widely used nitrogen starvation-induced high lipid accumulation conditions in microalgae, i.e., the method used in Comparative Example 13, lipid accumulation in microalgae can indeed be effectively achieved. The mannose-induced method used in this invention, i.e., the method used in Examples 1-14, also achieves excellent results in promoting lipid accumulation in microalgae, and this induction method has good induction effects on microalgae under different environmental conditions (different initial cell densities, different carbon sources, and different concentrations of nitrogen sources).

[0099] For different amounts of mannose added, from Figure 1As can be seen from Examples 1-5 and Comparative Examples 5-6 in Table 3, the addition of mannose at a level of 0.5 g / L or higher can effectively induce high lipid accumulation in microalgae, and maintains a good induction effect within a wide range of 0.5-8 g / L. Figure 2 The results showing changes in mannose content in the supernatant during cultivation under different mannose addition levels indicate that mannose at different concentrations was not significantly consumed during cultivation. This suggests that mannose was not used by microalgae as a carbon source for lipid accumulation, but rather enhanced their lipid accumulation capacity through other pathways, such as induction. Furthermore, when algal cells were collected after cultivation, the mannose in the culture medium maintained an effective induction concentration because it was not significantly consumed. Figure 2 This indicates that by replenishing the lost inorganic salts and carbon and nitrogen sources to the remaining culture medium, the microalgal seed culture can be re-inoculated for the next round of lipid accumulation induction culture, making the use of this invention more economical.

[0100] For different initial cell densities (OD) 680 Compared with Comparative Examples 5 and 7-10, Examples 4 and 6-9 showed higher oil content, indicating that mannose maintained a good induction effect under a wide range of cell density variations. The results demonstrate that the present invention has good adaptability and anti-interference ability in use, is not sensitive to fluctuations in cell density and mannose concentration in large-scale microalgae culture (provided the mannose concentration in the culture medium is not less than 0.5 g / L), and can adapt to higher density cultures, achieving higher microalgae oil production.

[0101] As can be seen from Examples 10-11 and Comparative Examples 12-13, when adding additional carbon sources, the effect of mannose in inducing high lipid accumulation in microalgae remains stable. Compared with Example 4 without adding additional carbon sources, the biomass of Examples 10-11 with added carbon sources is increased, indicating that the present invention is not limited by nutrients and can maintain the induction of high lipid accumulation in microalgae while simultaneously increasing microalgal biomass when adding additional carbon sources, thereby obtaining higher lipid yields, which is beneficial for practical production applications.

[0102] As seen in Examples 10 and 12-14, with different nitrogen concentrations after adding a carbon source, high nitrogen promotes increased microalgal biomass. Compared to Comparative Examples 11 and 13-15, the addition of mannose effectively induced higher lipid content in microalgae at different nitrogen concentrations. Under the same nitrogen starvation conditions, Example 12 achieved a higher lipid content under mannose induction compared to Comparative Example 13, indicating that the induction method of the present invention can be used synergistically with commonly used nitrogen starvation methods to promote microalgal lipid accumulation, further increasing the lipid content of microalgae. Figure 3Microscopic imaging characterization clearly shows that, under mannose induction in Examples 12-14, compared to Comparative Examples 13-15, the fluorescence of microalgal lipid staining was significantly enhanced, and the lipid droplets were significantly larger, demonstrating the effectiveness of the method of the present invention. The induction method of the present invention has a good ability to induce an increase in microalgal lipid content under the addition of additional carbon sources and different nitrogen concentrations, demonstrating the flexibility and stability of the method, which is not limited by nutritional conditions during cultivation and can be effectively used in large-scale microalgal cultivation.

[0103] Of course, in addition to the above-mentioned example of adding mannose at the beginning of cultivation, this invention also experimented with different timings for adding mannose:

[0104] Examples 15-17

[0105] The only difference between Example 15 and Example 4 is that:

[0106] In Example 15, the harvest time was the 4th day after the addition of mannose.

[0107] The only difference between Examples 16-17 and Example 4 is that:

[0108] In Examples 16-17, during the oil accumulation culture, no mannose was added at the beginning of the culture. Instead, 4 g / L of mannose was added on the 6th and 12th days, respectively. In addition, the harvest time in Examples 16-17 was the 4th day after the addition of mannose, that is, the harvest time was the 10th and 16th days, respectively.

[0109] Comparative Examples 16-18

[0110] Comparative Examples 16-18 correspond one-to-one with Examples 15-17. The only difference between Comparative Examples 16-18 and Examples 15-17 is that:

[0111] Comparative Examples 16-18 did not add mannose during the oil accumulation culture.

[0112] Examples 18-20

[0113] The only difference between Example 18 and Example 10 is that:

[0114] In Example 18, the harvest time was the 4th day after the addition of mannose.

[0115] The only difference between Examples 19-20 and Example 10 is that:

[0116] In Examples 19-20, during the oil accumulation culture, no mannose was added at the beginning of the culture. Instead, 4 g / L of mannose was added on the 3rd and 6th days of the culture, respectively. Meanwhile, the harvest time in Examples 19-20 was the 4th day after the addition of mannose, that is, the harvest time was the 7th day and the 10th day, respectively.

[0117] Comparative Examples 19-21

[0118] Comparative Examples 19-21 correspond one-to-one with Examples 18-20. The only difference between Comparative Examples 19-21 and Examples 18-20 is that:

[0119] Comparative Examples 19-21 did not add mannose during the oil accumulation culture.

[0120] After inducing microalgae culture according to Examples 15-20 and Comparative Examples 16-21, the harvested biomass and lipid content are shown in Table 4 below (the detection method is the same as in Table 1).

[0121] Table 4. Effects of different mannose addition times on biomass and lipid content of Chlorella sorokin.

[0122] Group Biomass (g / L) Oil content (%) Example 15 0.39±0.01 40.74±0.69 Example 16 0.53±0.03 37.47±2.82 Example 17 0.71±0.03 34.57±0.89 Example 18 1.29±0.05 32.74±0.55 Example 19 1.45±0.05 28.30±0.96 Example 20 1.56±0.02 31.11±0.91 Comparative Example 16 0.41±0.04 25.67±1.37 Comparative Example 17 0.57±0.01 28.75±0.58 Comparative Example 18 0.86±0.02 28.18±1.37 Comparative Example 19 1.53±0.03 25.83±0.43 Comparative Example 20 1.58±0.02 23.48±0.81 Comparative Example 21 1.60±0.02 23.57±0.84

[0123] As can be clearly seen from the data comparison in Table 4, the lipid content of microalgae in Examples 15-20, which were induced by adding mannose at different times in autotrophic or polyculture, was significantly higher than that in the corresponding comparative examples 16-21. This indicates that the induction method of the present invention has a good induction effect on microalgae in different growth states (autotrophic or polyculture at different growth stages).

[0124] In addition to the above-mentioned examples of mannose induction using *Chlorella sorokinense* W1 as the subject, this invention also investigated the effects of mannose induction on different microalgae:

[0125] Examples 21-26

[0126] Compared with Example 4, Examples 21-26 changed the microalgae species Chlorella sorokiniana W1 used in the microalgae monoculture system; other operating steps and corresponding parameters remained unchanged from Example 4; Specifically, Example 21 used Chlorella sorokiniana W2; Example 22 used Chlorella sorokiniana W3; Example 23 used Chlorella sorokiniana W4; Example 24 used Chlorella sorokiniana W6; Example 25 used Chlorella sp. AC1; and Example 26 used Haematococcus pluvialis FACHB-874.

[0127] Comparative Examples 22-27

[0128] Comparative Examples 22-27 correspond one-to-one with Examples 21-26. The only difference between Comparative Examples 22-27 and Examples 21-26 is that:

[0129] Comparative Examples 22-27 did not add mannose during the oil accumulation culture.

[0130] After inducing and culturing microalgae according to Examples 21-26 and Comparative Examples 22-27, the harvested biomass and lipid content are shown in Table 5 below (the detection method is the same as in Table 1).

[0131] Table 5 Biomass and lipid content of different microalgae under mannose induction.

[0132]

[0133]

[0134] As can be clearly seen from the data comparison in Table 5, compared with Comparative Examples 22-27, the lipid content of different microalgae in Examples 21-26 was significantly increased after induction with the addition of mannose, even without optimization of culture and induction conditions. The results indicate that the novel induction method proposed in this invention is applicable to different microalgae and has universality.

[0135] In addition to the above-mentioned examples of mannose induction under autotrophic or polytrophic conditions with provided light, this invention also investigated the effect of heterotrophic culture on mannose induction:

[0136] Example 27

[0137] The only difference between Example 27 and Example 10 is that:

[0138] Example 27: No light was provided during the lipid accumulation culture.

[0139] Comparative Example 28

[0140] The only difference between Comparative Example 28 and Example 27 is:

[0141] Comparative Example 28 did not add mannose during the oil accumulation culture.

[0142] Following the induction culture of microalgae in Example 27, a biomass of 1.20 ± 0.06 g / L and an oil content of 33.51 ± 2.61% were harvested. Following the induction culture of microalgae in Comparative Example 28, a biomass of 1.35 ± 0.04 g / L and an oil content of 24.28 ± 1.23% were harvested. The comparison between Example 27 and Comparative Example 28 shows that the mannose-induced high oil accumulation method for microalgae proposed in this invention is also applicable to heterotrophic culture.

[0143] The above embodiments are merely examples. For instance, other suitable culture media for microalgae growth (such as f / 2 medium, TAP medium, and BBM medium) can be used for the microalgae basal culture medium. Seed culture can be carried out under different culture conditions (autotrophic, heterotrophic, or polytrophic, as well as different light intensities, temperatures, gas flow rates, and light-dark cycles, etc.). For example, regarding gas flow rate, other flow rates can also be used, such as other flow rate settings within the range of 0.2-0.7 L / min.

[0144] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of mannose in cultivating microalgae to enhance microalgal lipid accumulation, characterized in that, The mannose is used as a non-nutrient-limiting inducer, and the concentration of the mannose in the culture medium is not less than 0.5 g / L; The microalgae mentioned are *Chlorella sorokinense*, *Chlorella* microalgae, and *Haematococcus pluvialis*.

2. The application as described in claim 1, characterized in that, The culture is an autotrophic culture, a heterotrophic culture, or a mixed culture.

3. A method for inducing increased lipid accumulation in microalgae using mannose, characterized in that, The method involves inoculating microalgae seed culture into a mannose-containing culture medium to induce lipid accumulation; the concentration of mannose in the mannose-containing culture medium is not less than 0.5 g / L; wherein, mannose is used as a non-nutrient-limiting inducer, and the microalgae are *Chlorella sorokinense*, *Chlorella* microalgae, or *Haematococcus pluvialis*. Alternatively, the method involves first inoculating the microalgal seed culture into a culture medium for cultivation, and then adding mannose to the culture medium during the cultivation process to continue cultivation, thereby utilizing mannose to enhance the accumulation of lipids in the microalgae; the amount of mannose added is sufficient to ensure that the concentration of mannose in the culture medium is not less than 0.5 g / L; wherein, the mannose is used as a non-nutrient-limiting inducer, and the microalgae are *Chlorella sorokinense*, *Chlorella* microalgae, or *Haematococcus pluvialis*.

4. The method as described in claim 3, characterized in that, When the method involves inoculating microalgae seed culture into a mannose-containing medium to induce lipid accumulation: The concentration of mannose in the mannose-containing culture medium is 0.5-8 g / L.

5. The method as described in claim 4, characterized in that, The mannose-containing culture medium is a modified BG11 medium, which is obtained by adding mannose to the original BG11 medium and optionally adding glucose and sodium nitrate; the concentration of glucose in the mannose-containing culture medium is 0-5 g / L and the concentration of sodium nitrate is 0-3 g / L. The culture conditions were: a culture temperature of 28℃, a light / dark cycle of 12 / 12 h, and a light intensity of 70 μmol / m². -2 s -1 The incubation period is 1-10 days, the incubation atmosphere is air, and the flow rate is 0.2-0.7 L / min.

6. The method as described in claim 3, characterized in that, When the method involves inoculating microalgae seed culture into a mannose-containing medium to induce lipid accumulation: The concentration of mannose in the mannose-containing culture medium is 0.5-8 g / L; The mannose-containing culture medium is a modified BG11 culture medium, which is obtained by adding mannose to the original BG11 culture medium. The culture conditions were: a culture temperature of 28℃, a light / dark cycle of 12 / 12 h, and a light intensity of 70 μmol / m². -2 s -1 The incubation period is 1-10 days, and the incubation atmosphere is compressed air with a concentration of 5 vol% CO2 and a flow rate of 0.2-0.7 L / min.

7. The method as described in claim 3, characterized in that, When the method involves first inoculating microalgal seed culture into a culture medium for cultivation, and then adding mannose to the culture medium during the cultivation process to continue cultivation, thereby utilizing mannose to enhance microalgal lipid accumulation: The amount of mannose added is sufficient to make the concentration of mannose in the culture medium 0.5-8 g / L.

8. The method as described in claim 7, characterized in that, The culture medium is a modified BG11 medium, which is obtained by optionally adding glucose and sodium nitrate to the original BG11 medium; the concentration of glucose in the culture medium is 0-5 g / L and the concentration of sodium nitrate is 0-3 g / L.

9. The method as described in claim 8, characterized in that, The culture conditions before and after the addition of mannose were met as follows: culture temperature 28℃, light / dark cycle 12 / 12 h, and light intensity 70 μmol / m². -2 s -1 The culture time after adding mannose is 1-10 days.

10. The method as described in claim 3, characterized in that, The microalgae seed solution is obtained by inoculating microalgae into a pre-culture medium, followed by centrifugation and concentration.

11. The method as described in claim 10, characterized in that, The pre-culture medium is a modified BG11 medium, which is obtained by adding glucose and yeast extract to the original BG11 medium; the concentration of glucose in the pre-culture medium is 5 g / L and the concentration of yeast extract is 2 g / L. The pre-culture conditions are: a culture temperature of 28℃, a light / dark cycle of 12 / 12 h, and a light intensity of 70 μmol / m². -2 s -1 The incubation period is 3-4 days.

12. The method as described in claim 3, characterized in that, The microalgae are *Chlorella sorokinense*, *Chlorella* species, and *Haematococcus pluvialis*; the seed culture of the microalgae meets the final OD standard after inoculation. 680 =0.1-4.

13. The method as described in claim 3, characterized in that, The method further includes: collecting algal cells after cultivation, freeze-drying them, and then extracting the oils from the algal cells using an organic solvent.

14. The method as described in claim 13, characterized in that, The collection process specifically involves centrifugation. After collection, the powder is washed 2-3 times with distilled water, then frozen at -80°C for 12 hours, followed by vacuum freeze-drying for 48 hours to collect the freeze-dried powder. The organic solvent is a chloroform-methanol mixed solution obtained by mixing chloroform and methanol in a volume ratio of 1:

1.

15. The method as described in claim 14, characterized in that, The method further includes: after collecting algal cells, by adding the lost inorganic salts and carbon and nitrogen sources to the remaining culture medium, microalgal seed liquid can be re-inoculated for a new round of induced lipid accumulation culture.

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