Preparation method of rare earth doped fertilizer for increasing microalgae growth and metabolite

Abstract

The invention relates to the technical field of algae fertilizers, and discloses a preparation method of a rare earth doped fertilizer for increasing microalgae growth and metabolites, the rare earth doped fertilizer comprises a rare earth additive and a carrier material; the rare earth additive is a solution containing lanthanum and / or cerium ions; the carrier material comprises the following components: potassium phosphate, potassium diphosphate, ferrous sulfate, zinc sulfate, boric acid, ammonium molybdate, copper sulfate and manganese sulfate. According to the invention, lanthanum and / or cerium rare earth elements are introduced into a composite nutrition carrier system, so that the cell density of chlorella, the content of photosynthetic pigments and the accumulation level of functional lipid metabolites are remarkably improved, and synergistic enhancement of microalgae growth and metabolism is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of algae fertilizers, and in particular to a method for preparing a rare earth-doped fertilizer capable of increasing the growth of microalgae and producing metabolites. Background Art

[0002] Microalgae have attracted considerable attention due to their high photosynthetic efficiency, short growth cycle, and ability to synthesize a variety of high-value-added metabolites. To increase microalgae biomass and the yield of specific products, researchers have explored culture conditions, nutrient ratios, and exogenous stimulants.

[0003] In microalgae cultivation technology, conventional culture media are mostly constructed based on inorganic salts such as phosphates, sulfates, and trace metal ions, but this type of culture system has limited regulatory capabilities in promoting the efficient proliferation of Chlorella or inducing specific metabolites. Some studies have attempted to improve the growth state of microalgae by adding metal ion trace regulators, but related research has mostly focused on conventional nutrients such as iron, zinc, and manganese. On the other hand, rare earth elements are believed to have certain growth regulation potential in the field of plant physiology, but their application in microalgae culture systems is still in the exploratory stage. At present, there is no mature solution to introduce and embed rare earth elements into a controlled-release compound fertilizer system to improve the proliferation efficiency and metabolic activity of microalgae. Summary of the Invention

[0004] In view of this, the present invention proposes a method for preparing a rare earth-doped fertilizer that increases the growth of microalgae and metabolites, aiming to solve the problems mentioned above.

[0005] In one aspect, the present invention provides a rare earth-doped fertilizer for increasing the growth and metabolites of microalgae, comprising:

[0006] The microalgae is Chlorella vulgaris, and the rare earth-doped fertilizer comprises: a rare earth additive and a carrier material;

[0007] The rare earth additive is a solution containing lanthanum and / or cerium ions;

[0008] The carrier material comprises the following components: potassium monophosphate, potassium diphosphate, ferrous sulfate, zinc sulfate, boric acid, ammonium molybdate, copper sulfate and manganese sulfate.

[0009] Preferably, the carrier material comprises the following components in parts by weight:

[0010]

[0011] Preferably, the rare earth additive is in the form of an aqueous solution of nitrate corresponding to lanthanum and / or cerium ions.

[0012] Preferably, the volume ratio of the carrier material to Chlorella is 10:1.

[0013] On the other hand, the present invention also provides a method for preparing the above-mentioned rare earth-doped fertilizer for increasing the growth of microalgae and metabolites, comprising the following steps:

[0014] Mix potassium monophosphate, potassium diphosphate, ferrous sulfate, zinc sulfate, boric acid, ammonium molybdate, copper sulfate and manganese sulfate, adjust the pH to 6.8-7.2, prepare a carrier material, and sterilize under high pressure for later use;

[0015] Adding rare earth additives to the carrier material and mixing them evenly to obtain rare earth-doped fertilizer.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] Lanthanum and / or cerium ions are introduced into the nutrient system in the form of water-soluble nitrates within a specific concentration range (e.g., 10 -3 mM) significantly increased the cell density of Chlorella vulgaris, among which the combination of lanthanum and cerium with a ratio of 1:2 and 1:5 showed an extremely significant proliferation-promoting effect.

[0018] The rare earth-doped fertilizer of the present invention not only increases the number of algal cells, but also significantly improves the chlorophyll a and b contents of Chlorella vulgaris and enhances its photosynthetic efficiency, indicating that rare earth elements have a synergistic activation effect on algal photosynthesis at a specific ratio.

[0019] Under the same culture cycle conditions, rare earth doping treatment significantly increased the dry matter content of algal cells per unit volume, indicating that this fertilizer can be used to increase the biomass production capacity of Chlorella and has engineering application value in amplifying culture efficiency.

[0020] The content of polyunsaturated fatty acids such as α-linolenic acid, EPA and DHA in Chlorella treated with this fertilizer increased significantly. In particular, the 1:5 lanthanum-cerium group showed extremely significant induction ability in the enrichment of EPA and DHA, and has potential value in the development of biological functional lipid products.

[0021] The present invention uses a composite carrier material of multiple trace elements and phosphate to not only meet the basic growth requirements of microalgae but also serve as a stable dispersion matrix for rare earth additives. After high-pressure sterilization, it is easy to promote and apply in laboratories or production systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Various other advantages and benefits will become apparent to those skilled in the art upon reading the detailed description of the preferred embodiment below. The accompanying drawings are for illustration purposes only and are not to be considered as limiting the present invention. The same reference symbols are used throughout the drawings to represent the same components. In the drawings:

[0023] Figure 1This is a bar graph showing the effects of different concentrations of lanthanum and cerium on the density of Chlorella vulgaris provided in an embodiment of the present invention.

[0024] Figure 2 A bar graph showing the effects of different concentrations of lanthanum and cerium on the density of Chlorella provided in an embodiment of the present invention.

[0025] Figure 3 This is a bar graph showing the effects of different concentrations of lanthanum and cerium combinations on the chlorophyll content of Chlorella vulgaris provided in an embodiment of the present invention.

[0026] Figure 4 This is a bar graph showing the effects of different concentrations of lanthanum and cerium combinations on the dry matter content of algal cells provided in an embodiment of the present invention.

[0027] Figure 5 This is a bar graph showing the effects of different concentrations of lanthanum and cerium combinations on α-linolenic acid content provided in an embodiment of the present invention.

[0028] Figure 6 A bar graph showing the effects of different concentrations of lanthanum and cerium combinations on EPA content provided in an embodiment of the present invention.

[0029] Figure 7 This is a bar graph showing the effects of different concentrations of lanthanum and cerium combinations on DHA content provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0030] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0031] It should be noted that Figure 1-7 “Control” on the horizontal axis represents the control group.

[0032] Example

[0033] Algae species: Chlorella vulgaris FACHB-1067 was purchased from the Algae Seed Bank of the Institute of Hydrobiology, Chinese Academy of Sciences;

[0034] Preparation of carrier materials:

[0035] Potassium monophosphate (KH2PO4) 75 mg;

[0036] Potassium diphosphate (K2HPO4) 75 mg;

[0037] Ferrous sulfate (FeSO4·7H2O) 4.98 mg;

[0038] Zinc sulfate (ZnSO4·7H2O) 1.57 mg;

[0039] Boric acid (H3BO3) 11.42 mg;

[0040] Ammonium molybdate ((NH4)6Mo7O 24 )0.71mg;

[0041] Copper sulfate (CuSO4·5H2O) 1.57 mg;

[0042] Manganese sulfate (MnSO4·H2O) 0.49mg;

[0043] pH adjustment: neutral (6.8-7.2);

[0044] After preparation, sterilize under high pressure (121°C, 15 min) and store in a refrigerator at 4°C.

[0045] 1. Effects of rare earth elements on the growth of Chlorella

[0046] Test Example 1

[0047] This test case investigates the effects of different concentrations of lanthanum and cerium on the density of Chlorella vulgaris. The specific steps are as follows:

[0048] (1) Inoculation: Add 100 mL of algae culture medium to a 300 mL conical flask and mix the algae cells in the logarithmic growth phase at a ratio of 1:10 to make the initial concentration 10 6 cells / mL.

[0049] (2) Adding lanthanum and cerium: the final concentrations added to the algae culture medium are 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 mMLa(NO3)3·6H2O and Ce(NO3)3·6H2O, and the control group was sterile deionized water.

[0050] (3) Cultivation: Seal with four layers of sterile gauze and place in a light incubator for static cultivation. The culture conditions are temperature 24±1℃, light 4440-8880lux, and light-dark ratio 12:12h.

[0051] (4) Determination of absorbance: Samples were taken on the 7th day after culture, and the absorbance of the culture at 650 nm (A650) was measured using a spectrophotometer.

[0052] Figure 1 The results are shown in the figure below. Compared with the control group, 10-4 and 10 -1 The mM lanthanum group significantly increased the density of Chlorella vulgaris (p<0.01), -3 and 10 -2 The mM lanthanum group significantly increased the density of Chlorella vulgaris; compared with the control group, 10 -5 and 10 -1 The mM cerium group increased the density of Chlorella vulgaris (p<0.05), 10 -5 , 10 -4 and 10 -3 The mM cerium group significantly increased the density of Chlorella (p<0.001). Therefore, the effects of different ratios of lanthanum and cerium on the density of Chlorella will be explored.

[0053] Test Example 2

[0054] This test case investigates the effects of different concentrations of lanthanum and cerium on the density of Chlorella vulgaris. The specific steps are as follows:

[0055] (1) Inoculation: Add 100 mL of algae culture medium to a 300 mL conical flask and mix the algae cells in the logarithmic growth phase at a ratio of 1:10 to make the initial concentration 10 6 cells / mL.

[0056] (2) Add different proportions of lanthanum and cerium: the final concentrations added to the algae culture medium were 10 -3 mM lanthanum, 10 -3 The ratios of mM cerium, lanthanum and cerium were 10:1, 5:1, 2:1, 1:1, 1:2, 1:5 and 1:10, and the control group was sterile deionized water.

[0057] (3) Cultivation: Seal with four layers of sterile gauze and place in a light incubator for static cultivation. The culture conditions are temperature 24±1℃, light 4440-8880lux, and light-dark ratio 12:12h.

[0058] (4) Determination of absorbance: Samples were taken on the 7th day after culture, and the absorbance of the culture at 650 nm (A650) was measured using a spectrophotometer.

[0059] Figure 2The effects of different concentrations of lanthanum and cerium on the density of Chlorella are shown in the figure. Compared with the control group, the La and Ce group significantly increased the density of Chlorella (p < 0.001). Compared with the La and Ce group, the 5:1 lanthanum and cerium group increased the density of Chlorella (p < 0.05), the 1:1 lanthanum and cerium group significantly increased the density of Chlorella (p < 0.01), the 1:2 lanthanum and cerium group significantly increased the density of Chlorella (p < 0.001), the 1:5 lanthanum and cerium group significantly increased the density of Chlorella (p < 0.001), and the 1:10 lanthanum and cerium group significantly increased the density of Chlorella (p < 0.01). Therefore, the lanthanum and cerium ratios of 1:1, 1:2, and 1:5 will be selected for subsequent experiments.

[0060] Test Example 3

[0061] This test case investigates the effects of different concentrations of lanthanum and cerium on the chlorophyll content of Chlorella vulgaris. The specific steps are as follows:

[0062] (1) Inoculation: Add 100 mL of algae culture medium to a 300 mL conical flask and mix the algae cells in the logarithmic growth phase at a ratio of 1:10 to make the initial concentration 10 6 cel ls / mL.

[0063] (2) Add different proportions of lanthanum and cerium: the final concentrations added to the algae culture medium were 10 -3 mM lanthanum, 10 -3 The ratios of mM cerium, lanthanum and cerium were 1:1, 1:2 and 1:5, and the control group was sterile deionized water.

[0064] (3) Cultivation: Seal with four layers of sterile gauze and place in a light incubator for static cultivation. The culture conditions are temperature 24±1℃, light 4440-8880lux, and light-dark ratio 12:12h.

[0065] (4) Chlorophyll detection: On the 7th day after cultivation, the filter membrane used to collect algal cells was cut into pieces and placed in a 20 mL centrifuge tube. 10 mL of 90% ethanol solution was added and mixed evenly by shaking. The tube was wrapped with black cloth and placed in a 4°C refrigerator for extraction for 24 h. The tube was then extracted in a 75°C water bath for 15 min and centrifuged at 4000 rpm for 15 min. The supernatant was taken and the absorbance at 470 nm, 649 nm, and 665 nm was measured simultaneously using a spectrophotometer. The cytochrome concentration (mg / L) was calculated as follows:

[0066] Ca=13.95×A665-6.88×A649

[0067] Cb=24.96×A649-7.32×A665

[0068] Note: Ca and Cb are the concentrations of chlorophyll a and b, respectively; cytochrome content (mg / L) = [cytochrome concentration (mg / L) × extract volume (mL)] / culture volume (mL).

[0069] Figure 3 The effects of different concentrations of lanthanum and cerium combinations on the chlorophyll content of Chlorella vulgaris are shown in the figure. Compared with the control group, the lanthanum and cerium group extremely significantly increased the chlorophyll content (p<0.001); compared with the La and Ce group, the lanthanum and cerium 1:2 group extremely significantly increased the chlorophyll content (p<0.001), and the lanthanum and cerium 1:5 group extremely significantly increased the chlorophyll content (p<0.001).

[0070] 2. Effects of rare earth-doped fertilizers on the metabolites of Chlorella

[0071] Test Example 4

[0072] This test investigates the effects of different concentrations of lanthanum and cerium on the dry matter content of algae cells. The specific steps are as follows:

[0073] (1) Inoculation: Add 100 mL of algae culture medium to a 300 mL conical flask and mix the algae cells in the logarithmic growth phase at a ratio of 1:10 to make the initial concentration 10 6 cel ls / mL.

[0074] (2) Add different proportions of lanthanum and cerium: the final concentrations added to the algae culture medium were 10 -3 mM lanthanum, 10 -3 The ratios of mM cerium, lanthanum and cerium were 1:1, 1:2 and 1:5, and the control group was sterile deionized water.

[0075] (3) Cultivation: Seal with four layers of sterile gauze and place in a light incubator for static cultivation. The culture conditions are temperature 24±1℃, light 4440-8880lux, and light-dark ratio 12:12h.

[0076] (4) Determination of dry matter content of algal cells: On the 7th day after culture, the collected algal cells were dried at 105°C to a constant weight, weighed, and the dry matter was calculated using the following formula: dry matter per unit volume of algal cells (mg / L) = [weight of filter membrane after filtration (mg) - weight of filter membrane before filtration (mg)] / culture volume (L)

[0077] Figure 4 The effects of different concentrations of lanthanum and cerium combinations on the dry matter content of algae cells are shown in the figure. Compared with the control group, the lanthanum and cerium group increased the dry matter content of algae cells (p<0.05); compared with the La and Ce group, the lanthanum and cerium 1:2 group increased the dry matter content of algae cells (p<0.05), and the lanthanum and cerium 1:5 group increased the dry matter content of algae cells (p<0.05).

[0078] Test Example 5

[0079] This test case investigates the effects of different concentrations of lanthanum and cerium on the content of α-linolenic acid. The specific steps are as follows:

[0080] (1) Inoculation: Add 100 mL of algae culture medium to a 300 mL conical flask and mix the algae cells in the logarithmic growth phase at a ratio of 1:10 to make the initial concentration 10 6 cel ls / mL.

[0081] (2) Add different proportions of lanthanum and cerium: the final concentrations added to the algae culture medium were 10 -3 mM lanthanum, 10 -3 The ratios of mM cerium, lanthanum and cerium were 1:1, 1:2 and 1:5, and the control group was sterile deionized water.

[0082] (3) Cultivation: Seal with four layers of sterile gauze and place in a light incubator for static cultivation. The culture conditions are temperature 24±1℃, light 4440-8880lux, and light-dark ratio 12:12h.

[0083] (4) Determination of α-linolenic acid content: On the 7th day after cultivation, algae mud (about 50-100 mg wet sample) was transferred to a 15 mL brown screw-capped empty bottle with a silicone-lined screw cap, 2 mL of sodium methoxide (NaOMe, 0.5 mL) was added, and after vortex mixing, the mixture was placed in an ultrasonic cleaner in an 80°C water bath for 20 min. After cooling to room temperature, 800 μL of deionized water and 1200 μL of n-hexane were added, vortex mixing was performed, and the mixture was centrifuged at 1644 g for 3 min. The upper n-hexane layer containing fatty acid methyl esters was filtered through a 0.22 μM needle filter into a 2 mL injection bottle, and the fatty acid content was detected on a gas chromatograph.

[0084] Figure 5 The effects of different concentrations of lanthanum and cerium combinations on the α-linolenic acid content are shown in the figure. Compared with the control group, the lanthanum and cerium group significantly increased the α-linolenic acid content (p<0.01); compared with the La and Ce group, the lanthanum and cerium 1:2 group increased the α-linolenic acid content (p<0.05), and the lanthanum and cerium 1:5 group extremely significantly increased the α-linolenic acid content (p<0.05).

[0085] Test Example 6

[0086] This experimental example explores the effect of different concentrations of lanthanum and cerium combinations on the content of eicosapentaenoic acid (EPA). The specific steps are as follows: The experimental operation is the same as the effect of different concentrations of lanthanum and cerium combinations on the content of α-linolenic acid.

[0087] Figure 6The effects of different concentrations of lanthanum and cerium combinations on EPA content are shown in the figure. Compared with the control group, the lanthanum and cerium group increased EPA content (p<0.05). Compared with the La and Ce group, the lanthanum and cerium 1:2 group significantly increased EPA content (p<0.01), and the lanthanum and cerium 1:5 group significantly increased EPA content (p<0.01).

[0088] Test Example 7

[0089] This experimental example explores the effect of different concentrations of lanthanum and cerium combinations on the content of docosahexaenoic acid (DHA). The specific steps are as follows: The experimental operation is the same as the effect of different concentrations of lanthanum and cerium combinations on the content of α-linolenic acid.

[0090] The figure shows the effect of 7 different concentrations of lanthanum and cerium combination on DHA content. The results are shown in the figure. Compared with the control group, the lanthanum and cerium group increased the DHA content (p<0.05); compared with the La and Ce group, the lanthanum and cerium 1:2 group increased the DHA content (p<0.05), and the lanthanum and cerium 1:5 group significantly increased the DHA content (p<0.01).

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered by the scope of protection of the claims of the present invention.

Claims

1. A rare earth-doped fertilizer for increasing the growth and metabolites of microalgae, characterized in that: The microalgae is Chlorella vulgaris, and the rare earth-doped fertilizer comprises: a rare earth additive and a carrier material; The rare earth additive is a solution containing lanthanum and / or cerium ions; The carrier material comprises the following components: potassium monophosphate, potassium diphosphate, ferrous sulfate, zinc sulfate, boric acid, ammonium molybdate, copper sulfate and manganese sulfate.

2. The rare earth-doped fertilizer for increasing microalgae growth and metabolites according to claim 1, characterized in that: The carrier material comprises the following components in parts by weight:

3. The rare earth-doped fertilizer for increasing microalgae growth and metabolites according to claim 1, characterized in that: The rare earth additive is in the form of an aqueous solution of nitrate corresponding to lanthanum and / or cerium ions.

4. The rare earth-doped fertilizer for increasing microalgae growth and metabolites according to claim 1, characterized in that: The volume ratio of the carrier material to the Chlorella is 10:

1.

5. A method for preparing a rare earth-doped fertilizer for increasing microalgae growth and metabolites according to any one of claims 1 to 4, characterized in that: The following steps are involved: Mix potassium monophosphate, potassium diphosphate, ferrous sulfate, zinc sulfate, boric acid, ammonium molybdate, copper sulfate and manganese sulfate, adjust the pH to 6.8-7.2, prepare a carrier material, and sterilize under high pressure for later use; Adding rare earth additives to the carrier material and mixing them evenly to obtain rare earth-doped fertilizer.

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