Method for inducing and improving microalgae grease accumulation by using mannose

Abstract

The invention belongs to the technical field of microalgae oil biosynthesis, and discloses a method for improving microalgae oil accumulation through mannose induction. The invention relates to application of mannose in culturing microalgae to improve oil accumulation of the microalgae. The concentration of the mannose in a culture medium is not lower than 0.5 g / L. Specifically, the method comprises the following steps: inoculating a microalgae seed solution into a culture medium containing mannose for inducing grease accumulation culture; or firstly inoculating the microalgae seed solution into the culture medium for culture, and then adding mannose into the culture medium for continuous culture in the culture process, so that the accumulation of the microalgae grease is improved by utilizing the mannose. The invention reveals that the grease accumulation of the microalgae can be improved when the mannose is applied to microalgae culture for the first time, and the grease content of the microalgae under different environmental conditions and growth states can be simply, flexibly and quickly induced and improved by using the concentration of the mannose which is not lower than 0.5 g / L.

Description

Technical Field

[0001] The invention belongs to the technical field of microalgae oil biosynthesis, and more specifically, relates to a method for inducing and improving microalgae oil accumulation by using mannose. Background Art

[0002] Since fossil fuel resources are limited, non-renewable and cause environmental pollution, exploring environmentally friendly and renewable energy paths has become an important global issue. Among many renewable energy sources, biomass energy, such as biofuels, has won wide attention from researchers at home and abroad due to its rich raw material resources and clean and low-carbon production process. Microalgae, as a potential source of third-generation biofuels, has attracted much attention due to its significant advantages such as fast growth rate, high oil content, small and flexible space requirements, and the ability to grow using wastewater and waste gas. Although the use of microalgae to produce biofuels has broad prospects, its commercial application is still limited by high culture costs, low biomass yield and low oil yield. In order to improve the oil yield of microalgae, researchers mainly use abiotic stress conditions such as high light, high salt, nitrogen and phosphorus nutrient element limitation or add inducers to achieve it. Although these methods are effective to a certain extent, there are some problems. For example, although the nitrogen starvation strategy can promote oil accumulation, the culture conditions need to be precisely controlled during application, including the addition and removal of nitrogen sources, which increases the complexity and cost of the operation. Therefore, finding more easy-to-operate induction and regulation methods to increase 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 defects or improvement needs of the prior art, the purpose of the present invention is to provide a method for inducing and improving the oil accumulation of microalgae using mannose, which reveals for the first time that mannose can improve the oil accumulation of microalgae 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 the increase of the oil content of microalgae under different environmental conditions (e.g., different initial cell densities, addition of different carbon sources and addition of different concentrations of nitrogen sources) and growth states (e.g., different growth periods of autotrophic or polyculture).

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

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

[0006] As a further preferred embodiment of the present invention, the microalgae is Chlorella sorokinensis, Chlorella microalgae, or Haematococcus pluvialis.

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

[0008] Alternatively, the method is to first inoculate the microalgae seed liquid into a culture medium for culturing, and then add mannose to the culture medium during the culturing process to continue culturing, thereby utilizing mannose to increase the microalgae lipid accumulation; the amount of mannose added is such 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 is to inoculate the microalgae seed liquid into a medium containing mannose to induce lipid accumulation culture:

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

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

[0012] The culture meets the following requirements: the culture temperature is 28°C, the light-dark cycle is 12 / 12h, and the light intensity is 70μmol m -2 s -1 The culture time is 1-10 days, the culture atmosphere is air, and the flow rate is 0.2-0.7L / min.

[0013] As a further preferred embodiment of the present invention, when the method is to inoculate the microalgae seed liquid into a medium containing mannose to induce lipid accumulation culture:

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

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

[0016] The culture meets the following requirements: the culture temperature is 28°C, the light-dark cycle is 12 / 12h, and the light intensity is 70μmol m -2 s -1 The culture time is 1-10 days, the culture atmosphere is compressed air with a CO2 concentration of 5 vol%, and the flow rate is 0.2-0.7 L / min.

[0017] As a further preferred embodiment of the present invention, when the method is to first inoculate the microalgae seed liquid into the culture medium for culturing, and then add mannose to the culture medium during the culturing process to continue culturing, thereby utilizing mannose to improve the microalgae lipid accumulation:

[0018] The amount of mannose added is such that the concentration of mannose in the culture medium is 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 before and after the addition of mannose both meet the following conditions: the culture temperature is 28°C, the light / dark cycle is 12 / 12h, and the light intensity is 70 μmol m -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 liquid is obtained by inoculating microalgae into a pre-culture medium and concentrating by centrifugation after pre-culture;

[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 meets the following requirements: the culture temperature is 28°C, the light-dark cycle is 12 / 12h, and the light intensity is 70μmol m -2 s -1 The culture time is 3-4 days.

[0024] As a further preferred embodiment of the present invention, the microalgae is Chlorella sorokinensis, Chlorella microalgae, Haematococcus pluvialis; the microalgae seed liquid is inoculated to meet the final OD 680 =0.1-4.

[0025] As a further preferred embodiment of the present invention, the method further comprises: collecting algal cells after the culturing is completed, and extracting oil in the algal cells using an organic solvent after freeze-drying; preferably, the collection is specifically performed by centrifugation; after the collection, washing with distilled water for 2-3 times, then freezing at -80°C for 12 hours, and then vacuum freeze-drying for 48 hours to collect 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;

[0026] More preferably, the method further comprises: after the algae cells are collected after the cultivation is completed, the lost inorganic salts and carbon and nitrogen sources are added to the remaining culture medium, and then the microalgae seed liquid can be re-inoculated for a new round of inducing oil accumulation culture.

[0027] Through the above technical scheme conceived by the present invention, compared with the prior art, the present invention discloses for the first time that mannose can improve the accumulation of microalgae oil when applied to microalgae culture (of course, the concentration of mannose in the culture medium needs to be not less than 0.5g / L); taking the following embodiments as an example, the oil content of microalgae 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, quickly and persistently significantly improve the microalgae lipid content under different environmental conditions (such as different initial cell densities, adding different carbon sources and adding different concentrations of nitrogen sources) and growth states (such as autotrophic or polyculture at different growth periods) in a short period of time, reduce the cost of microalgae cultivation, and maximize the potential and efficient production approach to achieve the economy of microalgae biodiesel.

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

[0029] (1) Mannose, as a non-nutrient-limiting inducer, can significantly increase the lipid content of microalgae without relying on traditional methods such as nitrogen starvation, and the lipid 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 between Example 5 and Comparative Example 5 below, the amount of sodium nitrate added in both is 1.5 g / L), and the lipid content can be as high as more than 40% (as shown in Example 15 below);

[0030] Of course, the mannose strategy for improving microalgae lipid accumulation in the present invention can also be combined with nitrogen starvation operation. Taking the following examples as an example, under the same nitrogen starvation conditions, compared with Comparative Example 14, Example 12 obtains a higher lipid content under mannose induction (Comparative Example 14 corresponds to the traditional nitrogen starvation operation for improving microalgae lipid content, and Example 12 is an operation of adding mannose induction on the basis of nitrogen starvation);

[0031] (2) The induction effect of mannose is rapid, flexible and sustained. In different growth stages of microalgae autotrophic or mixed culture, in microalgae heterotrophic culture, or at different initial cell densities, the addition of mannose can quickly induce and promote microalgae lipid accumulation in a short period of time;

[0032] (3) Adding additional carbon sources can simultaneously increase the biomass of microalgae while mannose induction promotes the increase of microalgae lipid content;

[0033] (4) Mannose is one of the components of lignocellulose, a renewable resource with abundant content. It is widely present in industrial and agricultural wastes, biorefining wastewater, and a variety of natural plants and microorganisms. It is easy to obtain and has a wide range of application potential. In addition, in the method of the present invention, mannose is not significantly consumed during the culture period. After the algae cells are collected after the culture is completed, the lost inorganic salts and carbon and nitrogen sources can be added to the remaining culture medium, and the microalgae seed liquid can be re-inoculated for a new round of inducing oil accumulation culture.

[0034] (5) Mannose induction helps overcome the limitations of traditional methods, increase the lipid content of microalgae, and provide a new technical approach for the commercial application of microalgae biofuels. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The biomass and oil content of Chlorella sorokinensis after the cultivation in Examples 1-5 and Comparative Examples 5-6.

[0036] Figure 2 The mannose content change curve of the supernatant during the culture period in Examples 1-5 and Comparative Example 6 is shown.

[0037] Figure 3 The morphological structure characterization results of Chlorella sorokinensis W1 in Examples 12-14 and Comparative Examples 13-15; wherein, Figure 3 a in the figure corresponds to the confocal microscopy imaging of the fluorescence of Nile-stained lipids in algal cells; Figure 3 b in the figure corresponds to the imaging of the structure in the algae cell by transmission electron microscopy (L in the figure: lipid droplets; S: starch granules). Examples 12-14 are cultured with the addition of mannose (the amount of mannose added is 4 g / L), marked as M-induce; Comparative Examples 13-15 are cultured without the addition of mannose, marked as control; "0N5C" means the addition of 0g / L sodium nitrate and 5g / L glucose, corresponding to Example 12 and Comparative Example 13, respectively; "03N5C" means the addition of 0.3g / L sodium nitrate and 5g / L glucose, corresponding to Example 13 and Comparative Example 14, respectively; "3N5C" means the addition of 3g / L sodium nitrate and 5g / L glucose, corresponding to Example 14 and Comparative Example 15, respectively. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0039] In general, the present invention provides an application of mannose in culturing microalgae to increase the lipid accumulation of microalgae, and accordingly, a method for inducing and increasing the lipid accumulation of microalgae using mannose can be obtained. The method may include the following steps:

[0040] S1, inoculating microalgae into culture medium, pre-culturing and concentrating by centrifugation to obtain microalgae seed liquid;

[0041] S2, inoculating the microalgae seed solution into a fresh culture medium (e.g., BG11 culture medium), and adding mannose to induce lipid accumulation culture (e.g., culturing at 28°C under light for 3-4 days);

[0042] S3, after the cultivation is completed, the algal cells are harvested, freeze-dried, and the oil in the algal cells is extracted using an organic solvent.

[0043] The microalgae used in Examples 1-20 below is Chlorella sorokiniana W1; the original BG11 culture medium used is purchased from the market:

[0044] Example 1

[0045] A method for using mannose to induce and increase microalgae lipid accumulation, the specific steps are as follows:

[0046] Pre-culture of microalgae: Chlorella sorokinensis was selected and placed in modified BG11 medium (modified 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), at 28 °C, a light / dark cycle of 12 / 12 h, and a light intensity of 70 μmol m -2 s -1 , cultured for 3-4 days in an air atmosphere with a flow rate of 0.5 L / min, and concentrated by centrifugation to obtain the microalgae seed liquid.

[0047] Oil accumulation culture: Inoculate the microalgae seed solution into BG11 medium (containing 1.5 g / L sodium nitrate) to make the final OD 680 =0.4, and 1 g / L mannose was added at the beginning of the culture (mannose was prepared into a mother solution and sterilized, then added to the culture medium at the required final concentration and then mixed evenly), at 28 °C, with a light / dark cycle of 12 / 12 h and a light intensity of 70 μmol m -2 s -1 The culture atmosphere was air with a flow rate of 0.5 L / min.

[0048] Harvesting of microalgae biomass and oil extraction after cultivation: After 3 days of cultivation, all biomass was collected by centrifugation, washed with distilled water 2-3 times, and placed in -80℃ freezer for 12 hours, followed by vacuum freeze drying for 48 hours, and the freeze-dried powder was collected and the weight of the harvested biomass was measured by weighing. The organic solvent used was a chloroform-methanol mixed solution, in which the volume ratio of chloroform to methanol was 1:1. The oil was extracted by shaking at 140rpm at room temperature for more than 4 hours, and the oil yield was measured by weight, and the oil content was obtained by comparing it with the weight of the biomass obtained.

[0049] Comparative Examples 1-4

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

[0051] In comparative examples 1-4, during the lipid accumulation culture, no mannose was added, and 1 g / L glucose, 1 g / L xylose, 1 g / L arabinose and 1 g / L galactose were added respectively.

[0052] Comparative Example 5

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

[0054] In Comparative Example 5, no mannose was added during the oil accumulation culture (that is, no monosaccharide was added).

[0055] After the microalgae were induced and cultured according to Example 1 and Comparative Examples 1-5, the biomass and oil content of the harvested microalgae were shown in Table 1.

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

[0057]

[0058]

[0059] Depend on Figure 1 As can be seen from Table 1, compared with the autotrophic culture of Comparative Example 5, the addition of mannose in Example 1 has no significant effect on the biomass, but can significantly increase the lipid content of the microalgae. Compared with the addition of glucose, xylose, arabinose or galactose to culture in Comparative Examples 1-4, the addition of mannose to culture microalgae in Example 1 can obtain the highest lipid content, showing that the present invention significantly promotes the lipid accumulation of microalgae.

[0060] Embodiment 2-5

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

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

[0063] Comparative Example 6

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

[0065] In comparative example 6, during the oil accumulation culture, the amount of mannose added was 0.2 g / L.

[0066] Embodiment 6-9

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

[0068] Example 6-9: Initial cell density (OD 680 ) are 0.1, 1, 2, and 4 respectively.

[0069] Comparative Examples 7-10

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

[0071] In Comparative Examples 7-10, no mannose was added during the oil accumulation culture (that is, no monosaccharide was added).

[0072] Example 10

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

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

[0075] Comparative Example 11

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

[0077] In Comparative Example 11, no mannose was added during the oil accumulation culture.

[0078] Embodiment 11

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

[0080] Example 11 During the oil accumulation culture, the culture atmosphere is compressed air containing 5% (v / v) CO2, and the flow rate is 0.5L / min.

[0081] Comparative Example 12

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

[0083] In Comparative Example 12, no mannose was added during the oil accumulation culture.

[0084] Examples 12-14

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

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

[0087] Comparative Examples 13-15

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

[0089] In Comparative Examples 13-15, no mannose was added during the oil accumulation culture.

[0090] In the initial stage of oil accumulation culture, the specific parameter conditions of Examples 1-14 and Comparative Examples 5-15 after the culture conditions were adjusted are shown in Table 2:

[0091] Table 2 Related parameter adjustment of Examples 1-14 and Comparative Examples 5-15

[0092]

[0093]

[0094] After inducing and culturing the microalgae according to Examples 1-14 and Comparative Examples 5-15, the harvested biomass and oil content results are shown in Table 3 below (the detection method is consistent with Table 1).

[0095] Table 3 Biomass and oil content of Chlorella sorokinensis under different culture modes

[0096]

[0097]

[0098] It can be clearly seen from the data comparison of Table 1 and Table 3 that the oil content of microalgae in Comparative Example 13 and Examples 1-14 is significantly higher than that in Comparative Examples 1-12 and Comparative Examples 14-15. This fully demonstrates that under the widely used nitrogen starvation-induced high oil accumulation of microalgae, that is, the method used in Comparative Example 13, microalgae oil can indeed be effectively accumulated. The mannose induction method used in the present invention, that is, the method used in Examples 1-14, can also achieve an excellent effect of promoting microalgae oil accumulation, and this induction method has a good induction effect on microalgae under different environmental conditions (different initial cell densities, addition of different carbon sources, and addition of different concentrations of nitrogen sources).

[0099] For different mannose addition amounts, Figure 1It can be seen from Examples 1-5 and Comparative Examples 5-6 in Table 3 that when the addition amount of mannose is 0.5 g / L or more, high lipid accumulation of microalgae can be effectively induced, and a good induction effect is maintained within a wide range of 0.5-8 g / L. Figure 2 The results of the changes in mannose content in the supernatant during the culture period under different mannose additions showed that during the culture process, different concentrations of mannose were not significantly consumed. This shows that mannose is not used as a carbon source by microalgae for oil accumulation, but improves the ability of microalgae to accumulate oil through other pathways, such as induction. In addition, when the algal cells were collected after the culture, the mannose in the culture medium still maintained an effective induction concentration ( Figure 2 ). This indicates that, by supplementing the lost inorganic salts and carbon and nitrogen sources to the remaining culture medium, the microalgae seed solution can be re-inoculated for the next round of induction culture for oil accumulation, making the use of the present invention more economical.

[0100] For different initial cell densities (OD 680 ), compared with Comparative Example 5 and Comparative Examples 7-10, the oil content in Example 4 and Examples 6-9 is higher, indicating that mannose can maintain a good induction effect under a wide range of cell density changes. The results show that the present invention has good adaptability and anti-interference ability when used, is insensitive to fluctuations in different cell densities and mannose concentrations in large-scale microalgae culture (based on the concentration of mannose in the culture medium being not less than 0.5 g / L), and can adapt to higher density culture to achieve higher microalgae oil production.

[0101] As for the addition of additional carbon sources, it can be seen from Examples 10-11 and Comparative Examples 12-13 that when additional organic (glucose) or inorganic (CO2) carbon sources are added to cultivate microalgae, the effect of mannose inducing high lipid accumulation remains stable. Compared with Example 4 without adding an additional carbon source, the biomass of Examples 10-11 with the addition of an additional carbon source is increased, indicating that the present invention is not limited by nutrition, and can maintain the induction of high lipid accumulation in microalgae when adding an additional carbon source, and simultaneously increase the biomass of microalgae, thereby obtaining a higher lipid yield, which is beneficial to actual production applications.

[0102] For different nitrogen concentrations after adding carbon sources, it can be seen from Example 10 and Examples 12-14 that high nitrogen promotes the increase of microalgae biomass. Compared with Comparative Example 11 and Comparative Examples 13-15, the addition of mannose at different nitrogen concentrations effectively induced microalgae to obtain higher oil content. Under the same nitrogen starvation conditions, compared with Comparative Example 13, Example 12 obtained a higher oil content under mannose induction, indicating that the induction method of the present invention can be used in conjunction with the existing commonly used nitrogen starvation to promote microalgae oil accumulation, so that the microalgae oil content is further increased. Figure 3The microscopic imaging characterization shows that under the induction of mannose in Examples 12-14, the microalgae oil staining fluorescence is significantly enhanced and the lipid droplets are significantly enlarged compared with Comparative Examples 13-15, showing the effectiveness of the method of the present invention. The induction method of the present invention has a good ability to induce the increase of microalgae oil content under the addition of additional carbon sources and different nitrogen concentrations, reflecting the flexibility and stability of the method of the present invention, which is not limited by nutritional conditions during the culture period and can be effectively used in large-scale cultivation of microalgae.

[0103] Of course, in addition to the above embodiment of adding mannose at the beginning of the culture, the present invention also experiments with different timings of adding mannose:

[0104] Examples 15-17

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

[0106] The harvest time of Example 15 is the 4th day after the addition of mannose, that is, the harvest time is the 4th day.

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

[0108] In Examples 16-17, during the oil accumulation culture, no mannose was added at the beginning of the culture, but 4 g / L mannose was added on the 6th and 12th days, respectively; at the same time, the harvest time of 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 to Examples 15-17 one by one. The only difference between Comparative Examples 16-18 and Examples 15-17 is that:

[0111] In Comparative Examples 16-18, no mannose was added during the oil accumulation culture.

[0112] Examples 18-20

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

[0114] The harvest time of Example 18 is the 4th day after the addition of mannose, that is, the harvest time is the 4th day.

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

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

[0117] Comparative Examples 19-21

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

[0119] In Comparative Examples 19-21, no mannose was added during the oil accumulation culture.

[0120] After inducing and culturing the microalgae according to Examples 15-20 and Comparative Examples 16-21, the harvested biomass and oil content results are shown in Table 4 below (the detection method is consistent with Table 1).

[0121] Table 4 Effects of different addition times of mannose on biomass and oil content of Chlorella sorokinensis

[0122] Group Biomass (g / L) Oil content (%) Embodiment 15 0.39±0.01 40.74±0.69 Example 16 0.53±0.03 37.47±2.82 Embodiment 17 0.71±0.03 34.57±0.89 Embodiment 18 1.29±0.05 32.74±0.55 Embodiment 19 1.45±0.05 28.30±0.96 Embodiment 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] It can be clearly seen from the data comparison in Table 4 that the oil content of the microalgae in Examples 15-20, in which mannose was added at different times for induction in autotrophic or polyculture, was significantly higher than that in the corresponding Comparative Examples 16-21, indicating 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 periods).

[0124] In addition to the above-mentioned example of mannose induction using Chlorella sorokinensis W1 as the subject, the present invention also experiments on the effects of mannose induction on different microalgae:

[0125] Examples 21-26

[0126] Compared with Example 4, Examples 21-26 replaced the microalgae species Chlorella sorokiniana W1 used in the microalgae monoculture system; other operating steps and corresponding parameter conditions remained unchanged from those in Example 4; 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; Example 26 used Haematococcus pluvialis FACHB-874.

[0127] Comparative Examples 22-27

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

[0129] In Comparative Examples 22-27, no mannose was added during the oil accumulation culture.

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

[0131] Table 5 Biomass and lipid content of different microalgae induced by mannose

[0132]

[0133]

[0134] It can be clearly seen from the data comparison in Table 5 that, compared with Comparative Examples 22-27, the oil content of different microalgae in Examples 21-26 was significantly improved after adding mannose for induction without optimizing the culture conditions and induction conditions. The results show that the new induction method proposed in the present invention can be applied to different microalgae and has universal applicability.

[0135] In addition to the above embodiments of providing light for autotrophic or polytrophic mannose induction, the present invention also experiments the effect of heterotrophic culture on mannose induction:

[0136] Embodiment 27

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

[0138] In Example 27, no light was provided during the oil accumulation culture.

[0139] Comparative Example 28

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

[0141] In Comparative Example 28, no mannose was added during the oil accumulation culture.

[0142] After inducing the microalgae according to Example 27, 1.20±0.06 g / L of biomass and 33.51±2.61% of oil content can be harvested. After inducing the microalgae according to Comparative Example 28, 1.35±0.04 g / L of biomass and 24.28±1.23% of oil content can be harvested. From the comparison between Example 27 and Comparative Example 28, it can be seen that the method of inducing high oil accumulation of microalgae proposed by mannose in the present invention is also applicable to heterotrophic culture.

[0143] The above embodiments are only examples. For example, the microalgae basal culture medium may use other culture media suitable for the growth of microalgae (such as f / 2 culture medium, TAP culture medium and BBM culture medium, etc.); the seed liquid may be cultured using different culture conditions (autotrophic, heterotrophic or mixed culture, as well as different light intensities, temperatures, gas flow rates and light-dark cycles, etc.; for example, taking the gas flow rate as an example, other flow rates may also be used, such as other flow rate settings in the range of 0.2-0.7 L / min).

[0144] It will be easily understood by those skilled in the art that the above description is only 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 in the protection scope of the present invention.

Claims

1. The use of mannose in cultivating microalgae to improve the lipid accumulation of microalgae, characterized in that: The concentration of mannose in the culture medium is not less than 0.5 g / L.

2. The use according to claim 1, characterized in that: The culture is autotrophic culture, heterotrophic culture or mixed culture.

3. The use according to claim 1, characterized in that: The microalgae are Chlorella sorokinensis, Chlorella microalgae, and Haematococcus pluvialis.

4. A method for using mannose to induce and increase microalgae lipid accumulation, characterized in that: The method comprises inoculating microalgae seed liquid into a culture medium containing mannose to induce oil accumulation culture; in the culture medium containing mannose, the concentration of mannose is not less than 0.5 g / L; Alternatively, the method is to first inoculate the microalgae seed liquid into a culture medium for culturing, and then add mannose to the culture medium during the culturing process to continue culturing, thereby utilizing mannose to increase the microalgae lipid accumulation; the amount of mannose added is such that the concentration of mannose in the culture medium is not less than 0.5 g / L.

5. The method according to claim 4, characterized in that: When the method is to inoculate the microalgae seed solution into a culture medium containing mannose to induce lipid accumulation culture: The concentration of mannose in the mannose-containing culture medium is 0.5-8 g / L; Preferably, the mannose-containing medium is a modified BG11 medium, which is obtained by adding mannose to the original BG11 medium, and optionally adding glucose and sodium nitrate to improve the medium; the concentration of glucose in the mannose-containing medium is 0-5 g / L, and the concentration of sodium nitrate is 0-3 g / L; The culture meets the following requirements: the culture temperature is 28°C, the light-dark cycle is 12 / 12h, and the light intensity is 70μmol m -2 s -1 The culture time is 1-10 days, the culture atmosphere is air, and the flow rate is 0.2-0.7L / min.

6. The method according to claim 4, characterized in that: When the method is to inoculate the microalgae seed solution into a culture medium containing mannose to induce oil accumulation culture: The concentration of mannose in the mannose-containing culture medium is 0.5-8 g / L; The mannose-containing medium is a modified BG11 medium, which is obtained by adding mannose to the original BG11 medium; The culture meets the following requirements: the culture temperature is 28°C, the light-dark cycle is 12 / 12h, and the light intensity is 70μmol m -2 s -1 The culture time is 1-10 days, the culture atmosphere is compressed air with a CO2 concentration of 5 vol%, and the flow rate is 0.2-0.7 L / min.

7. The method according to claim 4, characterized in that: When the method is to first inoculate the microalgae seed liquid into the culture medium for culturing, and then add mannose to the culture medium during the culturing process to continue culturing, thereby utilizing mannose to increase the microalgae lipid accumulation: The amount of mannose added is such that the concentration of mannose in the culture medium is 0.5-8 g / L; 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; More preferably, the culture before and after the addition of mannose both meet the following conditions: the culture temperature is 28°C, the light / dark cycle is 12 / 12h, and the light intensity is 70 μmol m -2 s -1 ; The culture time after adding mannose is 1-10 days.

8. The method according to claim 4, characterized in that: The microalgae seed liquid is obtained by inoculating microalgae into a pre-culture medium and concentrating by centrifugation after pre-culture; 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; The pre-culture meets the following requirements: the culture temperature is 28°C, the light-dark cycle is 12 / 12h, and the light intensity is 70μmol m -2 s -1 The culture time is 3-4 days.

9. The method according to claim 4, characterized in that: The microalgae are Chlorella sorokinensis, Chlorella microalgae, and Haematococcus pluvialis; the microalgae seed liquid is inoculated to meet the final OD 680 =0.1-4.

10. The method according to claim 4, characterized in that: The method further comprises: collecting algae cells after the cultivation is completed, and extracting oil in the algae cells using an organic solvent after freeze-drying; preferably, the collection is specifically performed by centrifugation; after the collection, washing with distilled water for 2-3 times, then freezing at -80°C for 12 hours, and then vacuum freeze-drying for 48 hours to collect 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; More preferably, the method further comprises: after the algae cells are collected after the cultivation is completed, the lost inorganic salts and carbon and nitrogen sources are added to the remaining culture medium, and then the microalgae seed liquid can be re-inoculated for a new round of inducing oil accumulation culture.

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