A culture method for increasing the content of lemna starch

Abstract

This invention discloses a cultivation method for increasing the starch content of duckweed, relating to the field of plant culture technology, comprising the following steps: taking pig farm biogas slurry, adjusting ammonia nitrogen to 10 mg / L~20 mg / L, total phosphorus to 0.1 mg / L~2.0 mg / L, and pH to 5.0~6.0, then inoculating initial duckweed into it and culturing for 5~7 days to obtain acclimatized duckweed; taking pig farm biogas slurry, adjusting ammonia nitrogen to 40~50 mg / L, total phosphorus to 0.4 mg / L~5.0 mg / L, and pH to 5.0~6.0, then adding sodium pyrophosphate and Bacillus amyloliquefaciens, then inoculating the acclimatized duckweed into it and culturing for 2~3 days to obtain high-starch duckweed. This invention constructs a two-step cultivation system of acclimatization and stress through culture solutions with differentiated nitrogen and phosphorus concentrations, and with the synergistic effect of sodium pyrophosphate and Bacillus amyloliquefaciens, achieves efficient and stable accumulation of duckweed starch.

Description

Technical Field

[0001] This invention relates to the field of plant cultivation technology, and specifically to a cultivation method for increasing the starch content of duckweed. Background Technology

[0002] With the continued growth of global energy demand, the depletion of fossil fuels, and increasing pressure on the ecological environment, the development of sustainable renewable energy has become an urgent priority. Biofuels, especially fuel ethanol, have become an important alternative to petroleum due to their wide availability of renewable feedstocks and good compatibility with existing engine systems. However, current fuel ethanol production mainly relies on terrestrial food or cash crops such as corn, sugarcane, and cassava, which is not only costly but also faces the dilemma of competing with humans for food and land. Therefore, there is an urgent need to find low-cost, non-food, sustainable alternative feedstocks that do not occupy valuable arable land.

[0003] Duckweed (belonging to the family Lemnaceae), an aquatic energy plant, has emerged as a highly promising candidate due to its unique advantages. Duckweed is the world's smallest and simplest flowering plant, widely distributed (found globally except in extremely cold regions), and boasts an astonishing growth rate—under ideal conditions, its biomass can double within 16 to 48 hours, with a theoretical annual dry matter yield reaching up to 55 tons per hectare, far exceeding that of most higher plants. Crucially, duckweed can grow in eutrophic waters and even treated wastewater, efficiently absorbing pollutants such as nitrogen and phosphorus, thus achieving an organic combination of pollution control and biomass production, significantly reducing raw material costs and environmental footprint.

[0004] While duckweed possesses the aforementioned significant advantages, a key bottleneck for its use as a feedstock for fuel ethanol lies in its starch accumulation level. Starch is the primary source of fermentable sugars in fuel ethanol production. However, under conventional natural or cultured conditions, although duckweed exhibits rapid biomass growth, its starch content is typically low, highly fluctuating, and difficult to control. Existing duckweed cultivation technologies primarily focus on increasing biomass yield or pollutant removal efficiency, exhibiting significant shortcomings in the systematic and efficient induction and accumulation of starch. This low and unstable starch content directly restricts the conversion efficiency and commercial competitiveness of duckweed as a high-efficiency bioenergy feedstock. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art. This invention provides a cultivation method to increase the starch content of duckweed. By constructing a two-step cultivation system of acclimatization and stress through a culture medium with different nitrogen and phosphorus concentrations, and with the synergistic effect of sodium pyrophosphate and Bacillus amyloliquefaciens, the efficient and stable accumulation of duckweed starch is achieved.

[0006] This invention provides a cultivation method for increasing the starch content of duckweed, comprising the following steps: S1. Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 10mg / L~20mg / L, the total phosphorus concentration to 0.1mg / L~2.0mg / L, and the pH value to 5.0~6.0. After sterilization, the basic acclimatization culture medium is obtained. S2. The initial duckweed is inoculated into the basic acclimatization culture solution and statically cultured for 5 to 7 days under the conditions of light intensity of 8000 LUX~12000 LUX, temperature of 21℃~25℃, and light-dark ratio of 11h:13h~13h:11h to obtain acclimatized duckweed. S3. Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 40mg / L~50mg / L, the total phosphorus concentration to 0.4mg / L~5.0mg / L, and the pH value to 5.0~6.0. After sterilization, the basic stress culture medium is obtained. S4. Add sodium pyrophosphate at a dosage of 10 mg to 50 mg per liter and Bacillus amyloliquefaciens at a dosage of 4 mg to 6 mg per liter to the basic stress culture medium, and stir and mix for at least 15 minutes to obtain a heterogeneous stress culture medium. S5. The domesticated duckweed is inoculated into the heterogeneous stress culture medium and statically cultured for 2 to 3 days under the conditions of light intensity of 8000 LUX~12000 LUX, temperature of 21℃~25℃, and light-dark ratio of 11h:13h~13h:11h to obtain high starch duckweed.

[0007] Specifically, the basic physicochemical properties of the pig farm biogas slurry include: The total nitrogen concentration ranges from 500 mg / L to 1200 mg / L, the total phosphorus concentration ranges from 10 mg / L to 50 mg / L, the ammonia nitrogen concentration ranges from 500 mg / L to 1000 mg / L, the chemical oxygen demand ranges from 650 mg / L to 850 mg / L, and the pH value ranges from 7.5 to 8.5.

[0008] Specifically, in steps S1 and S3, an aqueous filter membrane with a pore size of 5μm to 8μm is used to filter and remove impurities from the pig farm biogas slurry.

[0009] Specifically, in steps S1 and S3, the ammonia nitrogen and total phosphorus concentrations of the filtered and impurity-free pig farm biogas slurry are adjusted using clean water, and the pH value of the pig farm biogas slurry after dilution with clean water is adjusted using citric acid or acetic acid.

[0010] Specifically, in steps S1 and S3, the sterilization process is as follows: heating to 65°C and holding for 30 minutes.

[0011] Specifically, the initial duckweed is sparse-veined duckweed, or duckweed with few roots, or duckweed with purple back.

[0012] Specifically, the surface area of ​​the basic acclimatization culture medium is A, the inoculation area of ​​the initial duckweed in the basic acclimatization culture medium is B, and the constraint relationship between A and B is: 0.8A≤B≤A.

[0013] Specifically, the surface area of ​​the heterogeneous stress culture medium is E, the inoculation area of ​​the domesticated duckweed in the heterogeneous stress culture medium is F, and the constraint relationship between E and F is: 0.95E≤F≤E.

[0014] Specifically, before inoculating the initial duckweed into the basic acclimatization culture solution, the initial duckweed is rinsed with clean water 2 to 3 times, and then drained for 30 to 40 minutes. Before inoculating the domesticated duckweed into the heterogeneous stress culture medium, rinse the domesticated duckweed with clean water 2 to 3 times, and then drain for 30 to 40 minutes.

[0015] Specifically, in step S2, the initial liquid level of the basic acclimatization culture medium is controlled to be ≥10cm; In step S5, the initial liquid level of the heterogeneous stress culture medium is controlled to be ≥5cm.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention constructs a two-step culture system of acclimatization and stress using culture media with differentiated nitrogen and phosphorus concentrations. Combined with the synergistic effect of sodium pyrophosphate and Bacillus amyloliquefaciens, it achieves efficient and stable accumulation of starch in duckweed. First, the duckweed was cultured in a basic acclimatization culture medium with relatively low concentrations of ammonia nitrogen and total phosphorus to adapt to the environment. This allowed the duckweed to gradually adapt to the weakly acidic substrate and relatively low nutrient environment of the basic acclimatization culture medium, forming a stable physiological state. This laid a solid physiological foundation for subsequent stress culture and avoided physiological damage to the duckweed caused by direct stress. Using a basal stress culture medium with relatively high ammonia nitrogen and total phosphorus concentrations as a base, sodium pyrophosphate was quantitatively added to precisely reduce the effective phosphorus concentration available to domesticated duckweed in the basal stress culture medium, thus constructing a heterogeneous stress environment with relatively high ammonia nitrogen concentration and relatively low effective phosphorus concentration. Combined with the metabolic regulation of Bacillus amyloliquefaciens, the conventional growth and metabolic balance of domesticated duckweed was disrupted in two ways. The relatively high ammonia nitrogen concentration provides sufficient nitrogen for the synthesis of chlorophyll and photosynthetic enzymes in domesticated duckweed, ensuring the efficient operation of the photosynthetic system and high production of photosynthetic products. The relatively low effective phosphorus concentration strictly inhibits the vegetative growth of domesticated duckweed. With the regulation and guidance of Bacillus amyloliquefaciens, photosynthetic products are converted into starch synthesis. From the dual dimensions of product synthesis guarantee and distribution regulation, the core problems of low starch content, large fluctuations and difficulty in control in conventional culture of duckweed are effectively solved, achieving a leapfrog increase in starch content with stable and repeatable results. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic flowchart of the cultivation method in an embodiment of the present invention; Figure 2 This is an electron microscope image of the duckweed sample from Example 1; Figure 3 This is an electron microscope image of the duckweed sample from Example 2; Figure 4 This is an electron microscope image of the duckweed sample from Example 3; Figure 5 This is an electron microscope image of the duckweed sample from Comparative Example 1. Figure 6 This is an electron microscope image of the duckweed sample from Comparative Example 2; Figure 7 This is an electron microscope image of the duckweed sample from Comparative Example 3; Figure 8 This is a schematic diagram of the glucose standard curve. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] This invention provides a cultivation method for increasing the starch content of duckweed. Figure 1 A schematic flowchart of a cultivation method according to an embodiment of the present invention is shown. The cultivation method includes the following steps: S1. Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 10mg / L~20mg / L, the total phosphorus concentration to 0.1mg / L~2.0mg / L, and the pH value to 5.0~6.0. After sterilization, the basic acclimatization culture medium is obtained. Filtration removes solid impurities from the pig farm biogas slurry, preventing them from adhering to the surface of duckweed and affecting its physiological metabolism; the relatively low ammonia nitrogen and total phosphorus concentrations provide a mild nutritional environment for the duckweed, and the slightly acidic pH is suitable for the substrate requirements of the duckweed's subsequent growth; sterilization reduces competition and damage to the duckweed from other bacteria in the culture medium, ensuring the purity of the acclimatization culture system and creating a stable and safe initial culture foundation for the initial duckweed.

[0021] Optionally, the ammonia nitrogen concentration in the basic acclimatization culture medium can be 10 mg / L, 11 mg / L, 13 mg / L, 16 mg / L, or 20 mg / L; the total phosphorus concentration in the basic acclimatization culture medium can be 0.1 mg / L, 0.5 mg / L, 1.0 mg / L, 1.5 mg / L, or 2.0 mg / L; and the pH value in the basic acclimatization culture medium can be 5.0, 5.1, 5.3, 5.6, or 6.0.

[0022] In some specific embodiments, the basic physicochemical properties of the pig farm biogas slurry include: total nitrogen concentration ranging from 500 mg / L to 1200 mg / L, total phosphorus concentration ranging from 10 mg / L to 50 mg / L, ammonia nitrogen concentration ranging from 500 mg / L to 1000 mg / L, chemical oxygen demand ranging from 650 mg / L to 850 mg / L, and pH value ranging from 7.5 to 8.5. It should be noted that the pig farm biogas slurry comes from Dongrui Food Group Co., Ltd., and has been fermented for more than 3 months; note that pig farm biogas slurry meeting these basic physicochemical properties can be used as a raw material for basic acclimatization culture medium.

[0023] In some specific embodiments, an aqueous filter membrane with a pore size of 5μm to 8μm is used to filter and remove impurities from the pig farm biogas slurry. Too small a pore size leads to low filtration efficiency and easy clogging of the membrane, while too large a pore size cannot effectively remove solid impurities from the pig farm biogas slurry. An aqueous filter membrane within this pore size range can effectively remove solid impurities that affect duckweed growth, while not filtering out trace small molecules beneficial to duckweed in the pig farm biogas slurry, achieving a balance between filtration efficiency and filtration effect, ensuring the cleanliness and basic nutritional value of the subsequent basic acclimatization culture medium. Optionally, the pore size of the aqueous filter membrane can be 5μm, 6μm, 7μm, or 8μm, specifically selected according to the particle size distribution of solid impurities in the pig farm biogas slurry.

[0024] In some specific embodiments, diluting and adjusting the ammonia nitrogen and total phosphorus concentrations of the filtered and impurity-free pig farm biogas slurry with clean water is a simple and inexpensive method that does not introduce harmful impurities into the pig farm biogas slurry and avoids secondary pollution caused by chemical regulators. It should be noted that if the basic acclimatization culture medium is over-diluted with clean water, resulting in low ammonia nitrogen and / or total phosphorus concentrations, pig farm biogas slurry (after filtration and impurity removal) can be added again to replenish the concentration. Alternatively, ammonium chloride, sodium dihydrogen phosphate, etc., can be added to replenish the corresponding element concentrations.

[0025] In some specific embodiments, citric acid or acetic acid is used to adjust the pH value of the pig farm biogas slurry after dilution with water. Both citric acid and acetic acid are weak acids, and the pH value of the biogas slurry changes gently during the adjustment process. The sudden change in pH value will not damage the biogas slurry matrix. Moreover, citric acid / acetic acid can be utilized by duckweed in small amounts or decomposed naturally in the basic acclimatization culture medium without residual pollution. At the same time, it can stably maintain the weakly acidic environment of 5.0~6.0 in the basic acclimatization culture medium, which is suitable for the growth needs of duckweed.

[0026] In some specific embodiments, the sterilization process involves heating to 65°C and holding for 30 minutes. Compared to high-temperature and high-pressure sterilization, this sterilization method is low-temperature moist heat sterilization, which does not damage the natural trace nutrients in the biogas slurry, avoids nutrient loss in the culture medium, and effectively kills bacteria, fungi, and other miscellaneous microorganisms in the biogas slurry, ensuring the purity and nutritional integrity of the culture medium, thus balancing sterilization effectiveness and culture medium quality.

[0027] S2. The initial duckweed is inoculated into the basic acclimatization culture solution and statically cultured for 5 to 7 days under the conditions of light intensity of 8000 LUX~12000 LUX, temperature of 21℃~25℃, and light-dark ratio of 11h:13h~13h:11h to obtain acclimatized duckweed. Precise light intensity, temperature, and light-dark ratio parameters create a suitable environment for duckweed growth, ensuring its basic photosynthetic and metabolic activities proceed normally. By allowing the initial duckweed to undergo static cultivation and acclimatization in a basic acclimatization culture solution for 5 to 7 days, the initial duckweed gradually adapts to the weakly acidic substrate and relatively low nutrient environment of the basic acclimatization culture solution, forming a stable physiological state. This lays a solid physiological foundation for subsequent stress cultivation and avoids physiological damage to duckweed caused by direct stress.

[0028] Optionally, the light intensity can be 8000 LUX, 9000 LUX, 10000 LUX, 11000 LUX, or 12000 LUX; the temperature can be 21℃, 22℃, 23℃, 24℃, or 25℃; and the light-dark ratio can be 11h:13h, 12h:12h, or 13h:11h. Static culture can be carried out for 5, 6, or 7 days. Preferably, the initial duckweed is inoculated into a basic acclimatization culture medium and statically cultured for 6 days under conditions of 10000 LUX light intensity, 23℃ temperature, and a light-dark ratio of 12h:12h to obtain acclimatized duckweed. The photosynthetic and metabolic activities of this acclimatized duckweed are stable and efficient throughout the process. The resulting acclimatized duckweed exhibits uniform physiological state and strong stress resistance, laying an optimal physiological foundation for subsequent stress culture, effectively avoiding physiological damage caused by direct stress, and simultaneously preparing for efficient starch accumulation.

[0029] In some specific embodiments, the initial duckweed is *Lysimachia foenum-graecum* (Duckweed). Lemna aequinoctialis ), or less rooted duckweed ( Landoltia punctata ), or purple-backed duckweed ( Spirodela polyrhiza These three types of duckweed are common species in the Lemnaceae family, with fast growth rates and strong adaptability to eutrophic water bodies.

[0030] In some specific embodiments, the surface area of ​​the basic acclimatization culture medium is A, and the inoculation area of ​​the initial duckweed in the basic acclimatization culture medium is B. The constraint relationship between A and B is: 0.8A≤B≤A. The high inoculation density allows the initial duckweed to quickly cover the surface of the basic acclimatization culture medium during the acclimatization stage, avoiding direct sunlight exposure that could lead to the growth of other algae and reducing nutrient competition between other algae and the initial duckweed. Simultaneously, an inoculation density of 0.8~1.0 is suitable for acclimatization environments with relatively low ammonia nitrogen and total phosphorus concentrations. It prevents a decrease in the photosynthetic efficiency of the initial duckweed due to excessively low density, and also prevents internal hypoxia and uneven growth due to excessively high density, ensuring synchronous growth and uniform physiological state of the duckweed population during the acclimatization stage.

[0031] In some specific embodiments, before inoculating the initial duckweed into the basic acclimatization culture medium, the initial duckweed is rinsed with clean water 2 to 3 times, and then drained for 30 to 40 minutes. Rinsing with clean water 2 to 3 times can remove dust, bacteria and other substances attached to the surface of the initial duckweed, preventing these substances from being introduced into the basic acclimatization culture medium and contaminating the system or affecting the absorption of substances by the initial duckweed. Draining for 30 to 40 minutes can remove excess water from the surface of the initial duckweed, preventing water from diluting the nitrogen and phosphorus concentrations of the basic acclimatization culture medium and ensuring the accuracy of the parameters of the basic acclimatization culture medium.

[0032] In some specific embodiments, the initial liquid level of the basic acclimatization culture medium is controlled to be ≥10cm. The minimum initial liquid level of 10cm can provide sufficient nutrients and water for the initial duckweed, ensuring the basic growth of duckweed during the acclimatization stage, while avoiding the temperature and nutrient concentration of the basic acclimatization culture medium from fluctuating due to environmental influences caused by a shallow liquid level.

[0033] S3. Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 40mg / L~50mg / L, the total phosphorus concentration to 0.4mg / L~5.0mg / L, and the pH value to 5.0~6.0. After sterilization, the basic stress culture medium is obtained. The filtration and sterilization processes are continued to ensure the purity of the stress culture system. Compared with the basic acclimatization culture medium, the ammonia nitrogen concentration is increased to 40 mg / L~50 mg / L to reserve sufficient nitrogen for the efficient operation of the duckweed photosynthetic system. The total phosphorus concentration is controlled at 0.4 mg / L~5.0 mg / L to lay the foundation for the subsequent addition of sodium pyrophosphate to reduce available phosphorus. The pH value is maintained at a slightly acidic level of 5.0~6.0, consistent with the substrate of the basic acclimatization culture medium, to avoid stress damage to the duckweed caused by sudden pH changes, and to achieve a smooth transition of substrate pH value from acclimatization to stress culture.

[0034] Optionally, the ammonia nitrogen concentration of the basal stress culture medium can be 40 mg / L, or 41 mg / L, or 43 mg / L, or 46 mg / L, or 50 mg / L; the total phosphorus concentration of the basal stress culture medium can be 0.4 mg / L, or 1 mg / L, or 2 mg / L, or 3 mg / L, or 4 mg / L, or 5 mg / L; and the pH value of the basal stress culture medium can be 5.0, or 5.1, or 5.3, or 5.6, or 6.0.

[0035] In some specific embodiments, the basic physicochemical properties of the pig farm biogas slurry include: total nitrogen concentration ranging from 500 mg / L to 1200 mg / L, total phosphorus concentration ranging from 10 mg / L to 50 mg / L, ammonia nitrogen concentration ranging from 500 mg / L to 1000 mg / L, chemical oxygen demand ranging from 650 mg / L to 850 mg / L, and pH value ranging from 7.5 to 8.5. It should be noted that the pig farm biogas slurry comes from Dongrui Food Group Co., Ltd., and has been fermented for more than 3 months; note that pig farm biogas slurry meeting these basic physicochemical properties can be used as a raw material for basic stress culture medium.

[0036] In some specific embodiments, an aqueous filter membrane with a pore size of 5μm to 8μm is used to filter and remove impurities from the pig farm biogas slurry. Too small a pore size leads to low filtration efficiency and easy clogging of the filter membrane, while too large a pore size cannot effectively remove solid impurities from the pig farm biogas slurry. An aqueous filter membrane within this pore size range can effectively remove solid impurities that affect duckweed growth, while not filtering out trace small molecules beneficial to duckweed in the pig farm biogas slurry, achieving a balance between filtration efficiency and filtration effect, ensuring the cleanliness and basic nutritional value of the subsequent basic stress culture medium. Optionally, the pore size of the aqueous filter membrane can be 5μm, 6μm, 7μm, or 8μm, specifically selected according to the particle size distribution of solid impurities in the pig farm biogas slurry.

[0037] In some specific embodiments, diluting and adjusting the ammonia nitrogen and total phosphorus concentrations of the filtered and purified pig farm biogas slurry with clean water is a simple and inexpensive method that does not introduce harmful impurities into the pig farm biogas slurry and avoids secondary pollution caused by chemical regulators. It should be noted that if the pig farm biogas slurry used in the basal stress culture medium is over-diluted with clean water, resulting in low ammonia nitrogen and / or total phosphorus concentrations, the concentration can be replenished by adding more pig farm biogas slurry (after filtration and purification), or by adding ammonium chloride, sodium pyrophosphate, etc., to replenish the corresponding element concentrations.

[0038] In some specific embodiments, citric acid or acetic acid is used to adjust the pH value of the pig farm biogas slurry after dilution with water. Both citric acid and acetic acid are weak acids, and the pH value of the biogas slurry changes gently during the adjustment process. This will not damage the biogas slurry matrix due to sudden pH changes. Moreover, citric acid / acetic acid can be utilized by duckweed in small amounts or decomposed naturally in the subsequent heterogeneous stress culture medium without residual pollution. At the same time, it can stably maintain the weakly acidic environment of 5.0~6.0 in the subsequent heterogeneous stress culture medium, which is suitable for the growth requirements of duckweed.

[0039] In some specific embodiments, the sterilization process involves heating to 65°C and holding for 30 minutes. Compared to high-temperature and high-pressure sterilization, this sterilization method is low-temperature moist heat sterilization, which does not damage the natural trace nutrients in the biogas slurry, avoids nutrient loss in the culture medium, and effectively kills bacteria, fungi, and other miscellaneous microorganisms in the biogas slurry, ensuring the purity and nutritional integrity of the culture medium, thus balancing sterilization effectiveness and culture medium quality.

[0040] S4. Add sodium pyrophosphate at a dosage of 10 mg to 50 mg per liter and Bacillus amyloliquefaciens at a dosage of 4 mg to 6 mg per liter to the basic stress culture medium, and stir and mix for at least 15 minutes to obtain a heterogeneous stress culture medium. Sodium pyrophosphate significantly reduces the concentration of bioavailable phosphorus in the basal stress culture medium. This means that sodium pyrophosphate does not reduce the total phosphorus level, but rather, through a dual chemical action of complexation precipitation and co-precipitation, it efficiently converts the soluble available phosphorus (dihydrogen phosphate, hydrogen phosphate, etc.) that duckweed can absorb in the basal stress culture medium into insoluble phosphorus precipitate. This reduces the actual phosphorus concentration available to duckweed in the basal stress culture medium to an extremely low level of ≤0.1 mg / L, as detailed below: Sodium pyrophosphate is a water-soluble complex salt that rapidly dissociates upon dissolution in basal stress culture medium, releasing pyrophosphate ions. Pyrophosphate ions, as polydentate strong complexing ligands, exist in a protonated form (e.g., HP2O7) under weakly acidic conditions. 3- H2P2O7 2- ) and Ca, which is commonly found in biogas slurry 2+ Mg 2+ A complexation reaction occurs, generating stable, soluble metal pyrophosphate complex ions. With changes in pH and ion equilibrium, these soluble metal pyrophosphate complex ions further transform into water-insoluble acidic calcium pyrophosphate (such as CaH₂P₂O₇) and amorphous magnesium pyrophosphate precipitates. During the formation of these precipitates, the synergistic effect of surface adsorption and crystal encapsulation (co-precipitation) helps to contain free H₂PO₄ in the culture medium. HPO4 2 Soluble available phosphorus is simultaneously entrained into the precipitation system and eventually settles to the bottom of the culture medium with the precipitate. In this way, almost all the soluble available phosphorus in the basal stress culture medium that could originally be absorbed by duckweed is converted into unusable solid-phase precipitated phosphorus, achieving a significant and precise reduction in available phosphorus concentration and constructing a heterogeneous nutrient environment with relatively high ammonia nitrogen concentration and relatively low available phosphorus concentration.

[0041] Optionally, sodium pyrophosphate can be added to the basal stress culture medium at a dosage of 10 mg, 20 mg, 30 mg, 40 mg, or 50 mg per liter. The specific dosage depends on the effective phosphorus concentration in the basal stress culture medium, and should be sufficient to reduce the effective phosphorus concentration in the basal stress culture medium to ≤0.1 mg / L.

[0042] Among them, Bacillus amyloliquefaciens is a plant probiotic strain that can secrete a variety of secondary metabolites, including lipopeptide compounds (such as surfactants) and terpenoids. These metabolites are biologically active and can regulate the growth and metabolism of duckweed by affecting the balance of endogenous hormones in duckweed. These metabolites mainly reduce the level of endogenous gibberellin (GA) in duckweed, thereby significantly increasing the relative content of abscisic acid (ABA) in duckweed, reconstructing the GA / ABA hormone balance, and thus moderately inhibiting the vegetative growth of duckweed, activating the activity of key enzymes in starch synthesis, and promoting the allocation of photosynthetic products towards starch synthesis.

[0043] Optionally, Bacillus amyloliquefaciens can be added to the basal stress culture medium at a dosage of 4 mg, 4.5 mg, 5 mg, 5.5 mg, or 6 mg per liter. Preferably, adding Bacillus amyloliquefaciens to the basal stress culture medium at a dosage of 6 mg per liter ensures that the strain can rapidly colonize the basal stress culture medium and secrete sufficient amounts of secondary metabolites such as lipopeptides and terpenes. This avoids problems such as microbial competition and insufficient dissolved oxygen in the culture medium due to excessively high strain concentration, and also avoids the situation where insufficient secretion of metabolites and weak regulatory effect are caused by excessively low strain concentration.

[0044] Stirring for ≥15 minutes ensures that sodium pyrophosphate and Bacillus amyloliquefaciens are evenly distributed in the culture medium, so as to give full play to their respective functions.

[0045] S5. The domesticated duckweed is inoculated into the heterogeneous stress culture medium and statically cultured for 2 to 3 days under the conditions of light intensity of 8000 LUX~12000 LUX, temperature of 21℃~25℃, and light-dark ratio of 11h:13h~13h:11h to obtain high starch duckweed. Maintaining the same environmental parameters such as light intensity, temperature, and light-dark ratio ensures the efficient operation of the duckweed photosynthetic system. High ammonia nitrogen concentration provides sufficient nitrogen for chlorophyll and photosynthetic enzyme synthesis, allowing for sustained high production of photosynthetic products. The relatively low effective phosphorus concentration strictly inhibits the vegetative growth of duckweed (such as the proliferation of branches and leaves), and in conjunction with the metabolic regulation of Bacillus amyloliquefaciens, it disrupts the normal growth and metabolic balance of duckweed, guiding photosynthetic products from vegetative growth to starch synthesis. Short-term stress of 2 to 3 days ensures efficient starch accumulation while avoiding damage to the duckweed photosynthetic system and a decrease in biomass caused by long-term stress, ultimately achieving a leapfrog increase in the starch content of duckweed.

[0046] Optionally, the light intensity can be 8000 LUX, 9000 LUX, 10000 LUX, 11000 LUX, or 12000 LUX, the temperature can be 21℃, 22℃, 23℃, 24℃, or 25℃, the light-dark ratio can be 11h:13h, 12h:12h, or 13h:11h, and the cells can be statically cultured for 2 or 3 days. Preferably, the domesticated duckweed is inoculated into a heterogeneous stress culture medium and statically cultured for 3 days under the conditions of 10000 LUX light intensity, 23℃ temperature, and 12h:12h light-dark ratio to obtain high-starch duckweed. The overall parameter combination maintains the optimal environmental adaptability of the domestication stage, allowing the nutrient stress of the heterogeneous stress culture medium, the biological regulation of Bacillus amyloliquefaciens, and the environmental culture parameters to form a triple synergy. It precisely exerts its efforts from three dimensions: photosynthetic energy production, metabolic regulation, and starch accumulation cycle, ultimately achieving efficient, stable, and sufficient accumulation of duckweed starch, while ensuring no significant loss of duckweed biomass, thus improving the raw material utilization value of high-starch duckweed.

[0047] In some specific embodiments, the surface area of ​​the heterogeneous stress culture medium is E, and the inoculation area of ​​the domesticated duckweed in the heterogeneous stress culture medium is F. The constraint relationship between E and F is: 0.95E≤F≤E. Allowing the domesticated duckweed to almost completely cover the surface of the heterogeneous stress culture medium maximizes the utilization of light for photosynthesis, increasing the yield of photosynthetic products. Simultaneously, high-density inoculation further enhances the population stress effect of the domesticated duckweed, allowing it to rapidly compete for nitrogen and phosphorus nutrients in the heterogeneous stress culture medium, exacerbating the stress effect of low available phosphorus, guiding photosynthetic products to more efficiently convert to starch synthesis, and avoiding the possibility of other algal growth, thus ensuring the efficiency of the stress culture.

[0048] In some specific embodiments, before inoculating the domesticated duckweed into the heterogeneous stress culture medium, the domesticated duckweed is rinsed with clean water 2 to 3 times, and then drained for 30 to 40 minutes. Rinsing with clean water 2 to 3 times can remove dust, bacteria, and other substances attached to the surface of the domesticated duckweed, preventing these substances from being introduced into the heterogeneous stress culture medium and contaminating the system or affecting the absorption of substances by the domesticated duckweed; draining for 30 to 40 minutes can remove excess water from the surface of the domesticated duckweed, preventing water from diluting the nitrogen concentration and additive concentration of the heterogeneous stress culture medium, and ensuring the accuracy of the parameters of the heterogeneous stress culture medium.

[0049] In some specific embodiments, the initial liquid level of the heterogeneous stress culture medium is controlled to be ≥5cm, and the minimum initial liquid level of 5cm is sufficient to meet the stress culture requirements of acclimatizing duckweed for 2 to 3 days.

[0050] This invention constructs a two-step culture system of acclimatization and stress using culture media with differentiated nitrogen and phosphorus concentrations. Combined with the synergistic effect of sodium pyrophosphate and Bacillus amyloliquefaciens, it achieves efficient and stable accumulation of starch in duckweed. First, the duckweed was cultured in a basic acclimatization culture medium with relatively low concentrations of ammonia nitrogen and total phosphorus to adapt to the environment. This allowed the duckweed to gradually adapt to the weakly acidic substrate and relatively low nutrient environment of the basic acclimatization culture medium, forming a stable physiological state. This laid a solid physiological foundation for subsequent stress culture and avoided physiological damage to the duckweed caused by direct stress. Using a basal stress culture medium with relatively high ammonia nitrogen and total phosphorus concentrations as a base, sodium pyrophosphate was quantitatively added to precisely reduce the effective phosphorus concentration available to domesticated duckweed in the basal stress culture medium, thus constructing a heterogeneous stress environment with relatively high ammonia nitrogen concentration and relatively low effective phosphorus concentration. Combined with the metabolic regulation of Bacillus amyloliquefaciens, the conventional growth and metabolic balance of domesticated duckweed was disrupted in two ways. The relatively high ammonia nitrogen concentration provides sufficient nitrogen for the synthesis of chlorophyll and photosynthetic enzymes in domesticated duckweed, ensuring the efficient operation of the photosynthetic system and high production of photosynthetic products. The relatively low effective phosphorus concentration strictly inhibits the vegetative growth of domesticated duckweed. With the regulation and guidance of Bacillus amyloliquefaciens, photosynthetic products are converted into starch synthesis. From the dual dimensions of product synthesis guarantee and distribution regulation, the core problems of low starch content, large fluctuations and difficulty in control in conventional culture of duckweed are effectively solved, achieving a leapfrog increase in starch content with stable and repeatable results.

[0051] The culture medium was prepared using biogas slurry from a pig farm, fermented for 3 months, from Dongrui Food Group Co., Ltd. Its basic physicochemical properties included: ammonia nitrogen concentration of 689.2 mg / L, total phosphorus concentration of 18.5 mg / L, and pH value of 8.1.

[0052] Example 1 (1) Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 20 mg / L, the total phosphorus concentration to 0.5 mg / L, and the pH value to 5.6. After sterilization, the basic acclimatization culture medium is obtained. (2) The initial duckweed (dwarf duckweed) was inoculated into the basic acclimatization culture medium and statically cultured for 6 days under the conditions of light intensity of 10000 LUX, temperature of 23℃ and light-dark ratio of 12h:12h to obtain acclimatized duckweed; (3) Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 40 mg / L, the total phosphorus concentration to 1.1 mg / L, and the pH value to 5.6. After sterilization, the basic stress culture medium is obtained. (4) Add sodium pyrophosphate at a dosage of 11 mg per liter and Bacillus amyloliquefaciens at a dosage of 6 mg per liter to the basic stress culture medium, stir and mix for 15 min to obtain heterogeneous stress culture medium; (5) The domesticated duckweed was inoculated into a heterogeneous stress culture medium and statically cultured for 3 days under the conditions of light intensity of 10000 LUX, temperature of 23℃ and light-dark ratio of 12h:12h to obtain high starch duckweed.

[0053] Example 2 Similar to Example 1, except that the rare vein duckweed was replaced with the less rooted purple duckweed.

[0054] Example 3 Same as in Example 1, except that *Lemna minor* is replaced with *Lemna minor*.

[0055] Comparative Example 1 (1) Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 20 mg / L, the total phosphorus concentration to 0.5 mg / L, and the pH value to 5.6. After sterilization, the basic acclimatization culture medium is obtained. (2) The initial duckweed (dwarf duckweed) was inoculated into the basic acclimatization culture medium and statically cultured for 6 days under the conditions of light intensity of 10000 LUX, temperature of 23℃ and light-dark ratio of 12h:12h to obtain acclimatized duckweed.

[0056] Comparative Example 2 Similar to Comparative Example 1, except that *Lemna minor* was replaced with *Lemna minor*.

[0057] Comparative Example 3 Similar to Comparative Example 1, except that *Lemna minor* was replaced with *Lemna minor*.

[0058] Comparative Example 4 (1) Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 20 mg / L, the total phosphorus concentration to 0.5 mg / L, and the pH value to 5.6. After sterilization, the basic acclimatization culture medium is obtained. (2) The initial duckweed (dwarf duckweed) was inoculated into the basic acclimatization culture medium and statically cultured for 6 days under the conditions of light intensity of 10000 LUX, temperature of 23℃ and light-dark ratio of 12h:12h to obtain acclimatized duckweed; (3) Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 40 mg / L, the total phosphorus concentration to 1.1 mg / L, and the pH value to 5.6. After sterilization, the basic stress culture medium is obtained. (4) The domesticated duckweed was inoculated into the basic stress culture medium and statically cultured for 3 days under the conditions of light intensity of 10000 LUX, temperature of 23℃ and light-dark ratio of 12h:12h to obtain the first duckweed.

[0059] Comparative Example 5 (1) Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 20 mg / L, the total phosphorus concentration to 0.5 mg / L, and the pH value to 5.6. After sterilization, the basic acclimatization culture medium is obtained. (2) The initial duckweed (dwarf duckweed) was inoculated into the basic acclimatization culture medium and statically cultured for 6 days under the conditions of light intensity of 10000 LUX, temperature of 23℃ and light-dark ratio of 12h:12h to obtain acclimatized duckweed; (3) Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 40 mg / L, the total phosphorus concentration to 1.1 mg / L, and the pH value to 5.6. After sterilization, the basic stress culture medium is obtained. (4) Add sodium pyrophosphate to the basic stress culture medium at a dosage of 11 mg per liter, stir and mix for 15 min to obtain heterogeneous stress culture medium; (5) The domesticated duckweed was inoculated into a heterogeneous stress culture medium and statically cultured for 3 days under the conditions of light intensity of 10000 LUX, temperature of 23℃ and light-dark ratio of 12h:12h to obtain the second duckweed.

[0060] Comparative Example 6 (1) Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 20 mg / L, the total phosphorus concentration to 0.5 mg / L, and the pH value to 5.6. After sterilization, the basic acclimatization culture medium is obtained. (2) The initial duckweed (dwarf duckweed) was inoculated into the basic acclimatization culture medium and statically cultured for 6 days under the conditions of light intensity of 10000 LUX, temperature of 23℃ and light-dark ratio of 12h:12h to obtain acclimatized duckweed; (3) Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 40 mg / L, the total phosphorus concentration to 1.1 mg / L, and the pH value to 5.6. After sterilization, the basic stress culture medium is obtained. (4) Add Bacillus amyloliquefaciens to the basic stress culture medium at a dosage of 6 mg per liter, stir and mix for 15 min to obtain the bacterial-enhanced stress culture medium; (5) The domesticated duckweed was inoculated into a bacterial stress-enhancing culture medium and statically cultured for 3 days under the conditions of light intensity of 10000 LUX, temperature of 23℃ and light-dark ratio of 12h:12h to obtain the third duckweed.

[0061] Comparative Example 7 (1) Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 20 mg / L, the total phosphorus concentration to 0.5 mg / L, and the pH value to 5.6. After sterilization, the basic acclimatization culture medium is obtained. (2) The initial duckweed (dwarf duckweed) was inoculated into the basic acclimatization culture medium and statically cultured for 6 days under the conditions of light intensity of 10000 LUX, temperature of 23℃ and light-dark ratio of 12h:12h to obtain acclimatized duckweed; (3) Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 40 mg / L, the total phosphorus concentration to 1.1 mg / L, and the pH value to 5.6. After sterilization, the basic stress culture medium is obtained. (4) Add xylose to the basic stress culture medium at a dosage of 2g per liter, stir and mix for 15 minutes to obtain xylose-enhanced stress culture medium; (5) The domesticated duckweed was inoculated into xylose-enhanced stress culture medium and statically cultured for 3 days under the conditions of light intensity of 10000 LUX, temperature of 23℃ and light-dark ratio of 12h:12h to obtain the fourth duckweed.

[0062] Comparative Example 8 (1) Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 20 mg / L, the total phosphorus concentration to 0.5 mg / L, and the pH value to 5.6. After sterilization, the basic acclimatization culture medium is obtained. (2) Add xylose to the basic acclimatization culture medium at a dosage of 2g per liter, stir and mix for 15 minutes to obtain xylose-enhanced acclimatization culture medium; (3) The initial duckweed (dwarf duckweed) was inoculated into xylose-enhanced acclimatization culture medium and statically cultured for 6 days under the conditions of light intensity of 10000 LUX, temperature of 23℃ and light-dark ratio of 12h:12h to obtain the fifth duckweed.

[0063] duckweed detection 1. Take duckweed samples from Examples 1-3 and Comparative Examples 1-3, wash them three times with distilled water, place them on filter paper for 5 minutes, and then randomly select a portion of the duckweed samples to package and preserve them before sending them to a biotechnology company for electron microscopy scanning.

[0064] Figure 2 An electron micrograph of the duckweed sample from Example 1 is shown. Figure 3 Electron micrographs of the duckweed sample from Example 2 are shown. Figure 4 An electron micrograph of the duckweed sample from Example 3 is shown. Figure 5 An electron microscope image of the duckweed sample from Comparative Example 1 is shown. Figure 6 Electron micrographs of the duckweed sample from Comparative Example 2 are shown. Figure 7 Electron micrographs of the duckweed sample from Comparative Example 3 are shown.

[0065] Results: The irregular circular white parts in the electron micrographs are starch. Obviously, the starch content of the duckweed samples in Examples 1-3 is much higher than that in Comparative Examples 1-3, indicating that the stress culture treatment in Examples 1-3 can effectively increase the starch content in duckweed. Moreover, for stress culture treatment, the starch content improvement effect of Duckweed sparse veins and Duckweed sparse roots is much better than that of Duckweed purpurea.

[0066] 2. Initial samples of *Lemna minor*, *Lemna minor*, and *Lemna minor*, as well as samples from Examples 1-3 and Comparative Examples 1-7, were taken, washed three times with distilled water, placed on filter paper for 5 minutes, and then dried in an oven at 60°C until constant weight. Finally, they were ground into powder and subjected to quantitative starch detection. The results are shown in Table 1. (1) Sample processing preview: 60℃ drying → grinding into powder → ether defatting → ethanol sugar removal → acid hydrolysis of starch → DNS method to determine reducing sugar.

[0067] (2) Experimental procedure: Weigh approximately 0.1 g of duckweed powder sample, add 1 mL of anhydrous diethyl ether, shake thoroughly, centrifuge at 8000 g for 10 min at room temperature, discard the supernatant, and repeat the washing twice. Add 1 mL of 85% ethanol to the precipitate and wash until the microsaccharide test result is negative. Add 0.5 mL of distilled water to the precipitate, boil in a water bath for 15 min, shaking occasionally to ensure the sample is fully gelatinized. After cooling, add 0.35 mL of 6 mol / L hydrochloric acid, heat at 95 °C for 20 min (use iodine reagent to verify whether the starch has been completely hydrolyzed), centrifuge at 8000 g for 10 min at room temperature, and collect the supernatant for testing. After diluting with distilled water, take 50 μL of the supernatant into a 1.5 mL Eppendorf tube, add 50 μL of 6 mol / L NaOH solution to adjust the pH to alkaline, mix well, and then test. Take 50 μL of the test solution, add 50 μL of DNS reagent, mix well, boil in a water bath for 5 min, cool, add 200 μL of distilled water, mix well, and then pipette 200 μL into a 96-well plate and read the absorbance at 540 nm.

[0068] Microsaccharide test method: Take 2 mL of washing solution in a small test tube, add 4 drops of α-naphthol ethanol solution (10 g / L), and slowly add 1 mL of concentrated sulfuric acid along the tube wall. A purple ring at the water-acid interface indicates a positive result; a yellow-green ring at the water-acid interface indicates a negative result.

[0069] Calculation of results: Total starch (mg / g) = C × V × (F / M) × 0.9, where: C—sample concentration calculated from the sample absorbance value and input into the standard curve, mg / mL; V—extraction volume, 0.85 mL; F—dilution factor; M—sample mass, g; 0.9—coefficient of glucose to starch conversion. Construction of the standard curve: Dilute the 5 mg / mL glucose standard solution to a gradient of 1.5, 1.2, 1.0, 0.8, 0.6, 0.4, 0.2, and 0.1 mg / mL, and measure these solutions simultaneously with the sample. Plot the standard curve based on the relationship between concentration and absorbance (the standard curve plotted using the glucose standard solution is shown in the figure). Figure 8 As shown, the variance is 0.9996, indicating that the accuracy of this measurement is high and the detected data is reliable.

[0070]

[0071] A. Simple basic domestication and cultivation has almost no effect on increasing starch content. Comparative Examples 1-3 were duckweed (Leymus chinensis, Leymus chinensis, and Leymus chinensis) that underwent only basic acclimatization culture. Their starch contents were 248 mg / g, 232 mg / g, and 208 mg / g, respectively. Compared with the initial duckweed (Leymus chinensis, Leymus chinensis, and Leymus chinensis) of 247 mg / g, 227 mg / g, and 201 mg / g, there was almost no change. This indicates that basic acclimatization culture with relatively low ammonia nitrogen and total phosphorus concentrations cannot effectively increase the starch content of duckweed. It can only allow the duckweed to adapt to the culture environment and maintain its basic physiological state.

[0072] B. The two-step cultivation + dual-additive system of this invention achieves a leapfrog increase in starch content. Examples 1-3 employed a complete system of "basic domestication culture + heterogeneous stress culture containing sodium pyrophosphate and Bacillus amyloliquefaciens". The starch content of the three types of duckweed was significantly increased: 420 mg / g for *Lemna minor*, 415 mg / g for *Lemna minor*, and 346 mg / g for *Lemna minor*. Compared with the initial duckweed, the increases were approximately 70%, 82.8%, and 72.1%, respectively, confirming that this system is a core and effective means to increase the starch content of duckweed.

[0073] C. The synergistic effect of sodium pyrophosphate and Bacillus amyloliquefaciens is far superior to that of a single additive. Comparative Example 5 was a stress culture with only sodium pyrophosphate added (starch 372 mg / g, an increase of 124 mg / g compared to Comparative Example 1), and Comparative Example 6 was a stress culture with only Bacillus amyloliquefaciens added (starch 365 mg / g, an increase of 117 mg / g compared to Comparative Example 1). Both were much lower than the 420 mg / g in Example 1 (an increase of 172 mg / g compared to Comparative Example 1). The starch content of duckweed treated with sodium pyrophosphate and Bacillus amyloliquefaciens was 48 mg / g higher than that of the sodium pyrophosphate treatment alone and 55 mg / g higher than that of the Bacillus amyloliquefaciens treatment alone, respectively, and was much higher than the upper limit of the effect of the two single treatments. This confirms that the improvement effect of the synergistic treatment is significantly better than that of any single additive treatment.

[0074] D. Simple stress culture without additives has limited effect on increasing starch content. Comparative Example 4 was cultured under basic stress without additives and only under basic acclimatization. The starch content was 318 mg / g, which was higher than that under simple acclimatization (248 mg / g), but much lower than that under double additives in Example 1 (420 mg / g). This shows that high starch accumulation cannot be achieved by nutritional stress with relatively high ammonia nitrogen concentration and relatively moderate total phosphorus concentration. The regulation of additives is the key.

[0075] E. There were significant differences in starch-enhancing effects among the tested duckweed varieties. Under the same culture system, the starch content and enhancement effect of *Lemna minor* and *Lemna minor* were significantly better than those of *Lemna minor*: in the examples, the starch content of *Lemna minor* and *Lemna minor* exceeded 400 mg / g, while that of *Lemna minor* was only 346 mg / g. This system is more suitable for the physiological characteristics of *Lemna minor* and *Lemna minor*, and is the preferred solution for enhancing the starch content of these two types of duckweed.

[0076] F. Xylose has no effect on increasing the starch content of duckweed, and may even have an inhibitory effect. Comparative Example 7, which involved stress culture with xylose added after acclimatization, showed a starch content of only 184 mg / g, lower than the 318 mg / g of Comparative Example 4 and also lower than the initial 247 mg / g of *Lemna minor*. This indicates that xylose inhibits the synthesis and accumulation of starch in *Lemna minor* under relatively high ammonia nitrogen concentrations. Comparative Example 8, which involved adding xylose during the acclimatization stage, showed a starch content of 257 mg / g, not significantly different from the initial 247 mg / g of *Lemna minor*. This confirms that in the basic culture system of this invention, xylose, whether added during acclimatization or stress stages, has no effective effect on increasing starch content.

[0077] G. The system of this invention has a stable effect on improving the starch content of duckweed. After initial cultivation in the complete system, the starch content of duckweed increased significantly from the range of 200mg / g to 250mg / g to the range of 346mg / g to 420mg / g. Moreover, the increasing trend was consistent among different varieties, and there were no fluctuations or sudden increases or decreases in starch content. This indicates that the system can stably and repeatedly achieve efficient accumulation of starch in duckweed, solving the problem of large fluctuations and difficulty in controlling starch content in conventional cultivation.

[0078] H. The key logic behind efficient starch accumulation lies in the dual role of nutritional stress and biological regulation. A comparison of the experimental groups reveals that the relatively high ammonia nitrogen concentration ensures the synthesis of photosynthetic products, sodium pyrophosphate reduces available phosphorus and inhibits vegetative growth, and Bacillus amyloliquefaciens regulates the distribution of photosynthetic products to starch. The combination of these three factors—"product synthesis assurance + vegetative growth inhibition + metabolic distribution regulation"—is the core mechanism for achieving a leapfrog increase in starch content. No single mechanism can achieve the same effect.

[0079] The above provides a detailed description of a cultivation method for increasing the starch content of duckweed according to embodiments of the present invention. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A culture method for increasing the content of Lemna starch, characterized by, Includes the following steps: S1. Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 10mg / L~20mg / L, the total phosphorus concentration to 0.1mg / L~2.0mg / L, and the pH value to 5.0~6.

0. After sterilization, the basic acclimatization culture medium is obtained. S2. The initial duckweed is inoculated into the basic acclimatization culture solution and statically cultured for 5 to 7 days under the conditions of light intensity of 8000 LUX~12000 LUX, temperature of 21℃~25℃, and light-dark ratio of 11h:13h~13h:11h to obtain acclimatized duckweed. S3. Take the biogas slurry from the pig farm, filter it to remove impurities, and adjust the ammonia nitrogen concentration to 40mg / L~50mg / L, the total phosphorus concentration to 0.4mg / L~5.0mg / L, and the pH value to 5.0~6.

0. After sterilization, the basic stress culture medium is obtained. S4. Add sodium pyrophosphate at a dosage of 10 mg to 50 mg per liter and Bacillus amyloliquefaciens at a dosage of 4 mg to 6 mg per liter to the basic stress culture medium, and stir and mix for at least 15 minutes to obtain a heterogeneous stress culture medium. S5. The domesticated duckweed is inoculated into the heterogeneous stress culture medium and statically cultured for 2 to 3 days under the conditions of light intensity of 8000 LUX~12000 LUX, temperature of 21℃~25℃, and light-dark ratio of 11h:13h~13h:11h to obtain high starch duckweed.

2. The culture method for increasing the content of Lemna starch according to claim 1, characterized by, The basic physicochemical properties of the biogas slurry from the pig farm include: The total nitrogen concentration ranges from 500 mg / L to 1200 mg / L, the total phosphorus concentration ranges from 10 mg / L to 50 mg / L, the ammonia nitrogen concentration ranges from 500 mg / L to 1000 mg / L, the chemical oxygen demand ranges from 650 mg / L to 850 mg / L, and the pH value ranges from 7.5 to 8.

5.

3. The culture method for increasing the content of Lemna starch according to claim 1, wherein In steps S1 and S3, an aqueous filter membrane with a pore size of 5μm to 8μm is used to filter and remove impurities from the pig farm biogas slurry.

4. The cultivation method for increasing the starch content of duckweed as described in claim 1, characterized in that, In steps S1 and S3, the ammonia nitrogen and total phosphorus concentrations of the filtered and impurity-free pig farm biogas slurry are adjusted by diluting with clean water, and the pH value of the pig farm biogas slurry after dilution with clean water is adjusted by citric acid or acetic acid.

5. The cultivation method for increasing the starch content of duckweed as described in claim 1, characterized in that, In steps S1 and S3, the sterilization process is as follows: heating to 65°C and holding for 30 minutes.

6. The cultivation method for increasing the starch content of duckweed as described in claim 1, characterized in that, The initial duckweed is sparse-veined duckweed, or duckweed with few roots, or duckweed with purple back.

7. The cultivation method for increasing the starch content of duckweed as described in claim 1, characterized in that, The surface area of ​​the basic acclimatization culture medium is A, and the inoculation area of ​​the initial duckweed in the basic acclimatization culture medium is B. The constraint relationship between A and B is: 0.8A≤B≤A.

8. The cultivation method for increasing the starch content of duckweed as described in claim 1, characterized in that, The surface area of ​​the heterogeneous stress culture medium is E, and the inoculation area of ​​the domesticated duckweed in the heterogeneous stress culture medium is F. The constraint relationship between E and F is: 0.95E≤F≤E.

9. The cultivation method for increasing the starch content of duckweed as described in claim 1, characterized in that, Before inoculating the initial duckweed into the basic acclimatization culture solution, rinse the initial duckweed with clean water 2 to 3 times, and then drain for 30 to 40 minutes. Before inoculating the domesticated duckweed into the heterogeneous stress culture medium, rinse the domesticated duckweed with clean water 2 to 3 times, and then drain for 30 to 40 minutes.

10. The cultivation method for increasing the starch content of duckweed as described in claim 1, characterized in that, In step S2, the initial liquid level of the basic acclimatization culture medium is controlled to be ≥10cm; In step S5, the initial liquid level of the heterogeneous stress culture medium is controlled to be ≥5cm.

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