Microalgae preparation group for greenhouse gas emission reduction in rice field and method for greenhouse gas emission reduction in rice field

Abstract

The application discloses a microalgae preparation group for greenhouse gas emission reduction in a rice field and a greenhouse gas emission reduction method in a rice field, and relates to the technical field of agricultural greenhouse gas emission reduction. In view of the technical problems that photosynthesis oxygen release of a single algae species is limited by a temperature window, it is difficult to cover the whole process during a methane double-peak emission period in the rice field, and nitrous oxide cannot be reduced simultaneously, the application provides a microalgae preparation group, which comprises a first preparation and a second preparation, both of which comprise chlorella vulgaris, atropteris varians and chlorella pyrenoidosa; the concentration ratio of chlorella pyrenoidosa to chlorella vulgaris in the first preparation is higher, the concentration ratio of chlorella vulgaris to chlorella pyrenoidosa in the second preparation is higher, and the total concentration is 0.5*10 7 to 5*10 7 million cells / mL; and the method is to apply the first preparation at 2.5-3.5 L / mu after the rice field is rewatered after returning green, and to apply the second preparation at 1.5-2.5 L / mu after the tillering ends. The application can significantly reduce the cumulative emission amount of methane and nitrous oxide, and has a yield-increasing effect.

Description

Technical Field

[0001] This invention relates to the field of agricultural greenhouse gas emission reduction technology. More specifically, this invention relates to a microalgae preparation for greenhouse gas emission reduction in paddy fields and a method for greenhouse gas emission reduction in paddy fields. Background Technology

[0002] Rice cultivation is a significant anthropogenic source of methane emissions from agriculture, accounting for approximately 10% of global anthropogenic methane emissions. The anaerobic environment created by prolonged flooding in rice paddies provides ideal conditions for methanogenic bacteria, whose methane production is released into the atmosphere in large quantities through the aerenchyma of rice plants and gas diffusion. Methane emissions from rice paddies exhibit a distinct bimodal pattern, with peak emissions occurring after rice transplanting and re-flooding, and again after tillering. The water temperature conditions during these two emission peak periods differ significantly: after re-flooding, during the early growth stage of rice, water temperatures are relatively low and there is frequent overcast and rainy weather; after tillering, during the middle growth stage of rice, water temperatures are higher.

[0003] Currently, methane emission reduction in paddy fields mainly employs methods such as water management, optimization of straw return to the field, and application of chemical inhibitors. Intermittent irrigation can inhibit the activity of methanogenic bacteria by altering the soil redox potential, but it may affect rice yield and is difficult to implement in water-scarce areas; chemical inhibitors are costly and may have adverse effects on soil microbial communities.

[0004] Microalgae, as photosynthetic autotrophic microorganisms, can increase the dissolved oxygen content in paddy field water through photosynthetic oxygen release, thereby inhibiting the activity of methanogenic bacteria and promoting the oxidation and consumption of methane by methanogenic bacteria. Chinese patent document CN112005826A discloses a method for increasing dissolved oxygen in paddy field water by applying Chlorella. After rice seedling transplantation, Chlorella mother liquor is transferred to the paddy field water, increasing dissolved oxygen and reducing methane emissions through Chlorella photosynthesis. Chinese patent document CN122081077A discloses a high-photosynthetic-efficiency, fast-growing Chlorella mutant strain and its application in paddy field carbon reduction. Mutagenesis and selection are used to improve the photosynthetic oxygen release performance of Chlorella to inhibit the formation of methane and nitrous oxide. Chinese patent document CN114788485A discloses a rice-algae symbiotic management method to promote straw decomposition. Chlorella is inoculated in the paddy field to form a symbiotic system to accelerate the aerobic decomposition of straw. However, the aforementioned existing technologies all use a single algal species, namely Chlorella. The photosynthetic oxygen release activity of a single algal species is limited by the temperature window, making it difficult to maintain a stable oxygen release capacity under different temperature and light conditions throughout the entire growth period of rice. The photosynthetic activity decreases significantly during low temperature or cloudy / rainy periods, and it cannot simultaneously cover the two periods with different temperature conditions corresponding to the bimodal peaks of methane emissions from paddy fields. At the same time, a single algal species only inhibits methane through a single oxygen release pathway, and its inhibitory effect on nitrous oxide emissions from paddy fields is limited and unstable. In fact, the consumption of oxygen due to algal decay can even exacerbate the anaerobic environment, making it impossible to achieve synergistic emission reduction of both methane and nitrous oxide, two greenhouse gases.

[0005] Therefore, it is necessary to design a technical solution that can overcome the above-mentioned defects. Summary of the Invention

[0006] One objective of this invention is to provide a microalgae formulation and method for greenhouse gas emission reduction in paddy fields, aiming to overcome the shortcomings of existing single-microalgae technologies, which suffer from temporal and spatial misalignment in oxygen release and supply, and difficulty in achieving synergistic emission reduction of methane and nitrous oxide. Methane emissions from paddy fields exhibit a bimodal characteristic, with emission peaks occurring after greening and re-irrigation and after tillering. The water temperature conditions differ significantly between these two stages, with relatively lower temperatures after greening and re-irrigation and higher temperatures after tillering. The photosynthetic oxygen release activity of a single algae species is limited by the temperature window, making it impossible to simultaneously cover the two emission peak periods with different temperature conditions.

[0007] To achieve the above objectives, according to one aspect of the present invention, a microalgae formulation for greenhouse gas emission reduction in paddy fields is provided, comprising a first formulation and a second formulation; both the first and second formulations comprise *Hymenochrysis galbana*, *Anabaena variegata*, and *Chlorella proteoglycans*; the concentration of *Chlorella proteoglycans* in the first formulation is higher than that of *Hymenochrysis galbana*, and the concentration of *Hymenochrysis galbana* in the second formulation is higher than that of *Chlorella proteoglycans*; the first formulation is applied after the rice has turned green and been re-irrigated, and the second formulation is applied after the rice tillering stage; the total concentration of microalgae in both the first and second formulations is 0.5 × 10⁻⁶. 7 Up to 5×10 7 Cells / mL.

[0008] Preferably, the concentration ratio of Alternaria monofilata, Alternaria variegata, and Chlorella proteoglycans in the first formulation is 1:(0.6-1.0):(1.2-1.6), and the concentration ratio of Alternaria monofilata, Alternaria variegata, and Chlorella proteoglycans in the second formulation is 1:(0.6-1.0):(0.6-0.9).

[0009] Preferably, the concentration ratio of *Chlorella vulgaris* to *Chlorella proteoglycans* in the second formulation is greater than that in the first formulation; the *Chlorella vulgaris* in the second formulation includes free *Chlorella vulgaris* and *Chlorella vulgaris* encapsulated in a sustained-release carrier, and the mass of the sustained-release carrier accounts for 10%-30% of the total mass of the second formulation.

[0010] Preferably, the concentration ratio of *Houttuynia cordata* in the second formulation is greater than that in the first formulation; the mass fraction of sodium nitrate in the second formulation is lower than that in the first formulation, and the mass fraction of sodium nitrate in the second formulation is 40%-60% of that in the first formulation.

[0011] Preferably, both the first and second formulations are liquid formulations. The liquid formulation contains a culture medium carrier, which is BG11 liquid culture medium containing sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, ferric ammonium citrate, and a mixture of A5 trace elements. The liquid formulation also contains sodium carboxymethyl cellulose, with a mass fraction of 0.1%-0.5%. The mass fraction of sodium carboxymethyl cellulose is positively correlated with the total microalgae concentration; for every 1×10⁻⁶ increase in total microalgae concentration... 7 Cells / mL, the mass fraction of sodium carboxymethyl cellulose increases by 0.05%-0.1%.

[0012] Preferably, both the first and second formulations are microalgae-coated granular fertilizers, with each kilogram of granular fertilizer containing 50-200 mL of microalgae formulation; the coating of the coated granular fertilizer has a double-layer structure, with an inner layer of algae containing microalgae formulation and an outer layer of slow-release coating; the slow-release coating disintegrates within 12-48 hours after the granular fertilizer is added to water, and the inner algae layer releases the microalgae formulation after the slow-release coating disintegrates; or both the first and second formulations are microalgae compound liquid fertilizers, which are composed of microalgae formulations mixed with amino acid water-soluble fertilizer or humic acid liquid fertilizer at a volume ratio of 1:2 to 1:5, and the pH value of the compound liquid fertilizer is 6.0-7.5.

[0013] Preferably, the total microalgae concentration of the first formulation is higher than that of the second formulation; the total microalgae concentration of the first formulation is 2 × 10⁻⁶. 7 Up to 3×10 7 Cells / mL, the total microalgae concentration of the second formulation is 1×10⁻⁶. 7 Up to 1.9×10 7 Cells / mL.

[0014] According to another aspect of the present invention, the present invention provides a method for reducing greenhouse gas emissions from paddy fields, comprising: applying a first preparation of the aforementioned microalgae preparation group to the paddy field after the transplanted rice seedlings have recovered and been rehydrated, and applying a second preparation of the aforementioned microalgae preparation group to the paddy field after tillering has ended; the application rate of the first preparation is 2.5-3.5 L / mu, and the application rate of the second preparation is 1.5-2.5 L / mu.

[0015] Preferably, the criteria for determining the period after the seedlings have returned to green and been re-irrigated are when the field is irrigated to a water depth of 3-5 cm and the new roots of the seedlings begin to sprout, which is 5-10 days after transplanting; the criteria for determining the period after the tillering ends are when the total number of leaves on the main stem reaches the predetermined leaf age and the number of tillers in the field population begins to decrease, which is the period of young panicle differentiation.

[0016] The present invention has at least the following beneficial effects:

[0017] This invention involves applying a first formulation to rice paddies after transplanting and re-irrigating, with a higher concentration of *Chlorella protozoa* than *Alternaria monotypica*. A second formulation, with a higher concentration of *Alternaria monotypica* than *Chlorella protozoa*, is then applied after tillering. During the early growth stage of rice, from re-irrigation to peak tillering, the paddy field water temperature is relatively low, and there is frequent overcast and rainy weather. *Chlorella protozoa*, which constitutes a higher proportion in the first formulation, maintains its photosynthetic oxygen-emitting activity within this temperature range. After tillering, during the middle growth stage, the paddy field water temperature rises. *Alternaria monotypica*, which constitutes a higher proportion in the second formulation, maintains a strong photosynthetic oxygen-emitting rate under high-temperature conditions. The phased application of these two formulations ensures effective dissolved oxygen supply in the paddy field water during both low- and high-temperature periods, eliminating oxygen-emitting windows for single algal species during low- or high-temperature periods. This also inhibits the anaerobic metabolic activity of methanogenic bacteria throughout the entire growth cycle, significantly reducing cumulative methane emissions. The filamentous forms of *Anabaena variegata* entwine and attach to the base of rice stems and the soil surface, extending photosynthetic oxygen-emitting sites to the water-soil interface and compensating for the difficulty of dissolved oxygen in water penetrating the water layer to reach the soil reducing layer. Its heterocellular nitrogen fixation fixes atmospheric nitrogen into ammonium nitrogen and secretes it extracellularly, providing nitrogen nutrition for rice and symbiotic algae. The photosynthetic processes of the three algae simultaneously absorb ammonium and nitrate nitrogen from the water, compressing the substrate concentration in the nitrification-denitrification process and inhibiting nitrous oxide formation at its source, thus achieving synergistic emission reduction of both methane and nitrous oxide, two greenhouse gases. This invention employs two phased additions—one after greening and re-watering and the other after tillering—to align the algal colonization window with the bimodal phase of methane emissions. This establishes a photosynthetic oxygen-emitting barrier before the peak methane emission flux arrives and repairs the algal community weakened by high temperatures and nutrient competition in the later stages of growth, achieving full-coverage emission reduction precisely corresponding to the methane emission timeline. Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0018] Figure 1 This is a table showing the daily variation of CH4 emission flux in each treatment group of Experiment 1.

[0019] Figure 2 This is a bar chart showing the cumulative CH4 emissions of each treatment group in Experiment 1.

[0020] Figure 3 This is a table showing the daily changes in N2O emission flux for each treatment group in Experiment 1.

[0021] Figure 4 This is a bar chart showing the cumulative N2O emissions of each treatment group in Experiment 1.

[0022] Figure 5 This is a table showing the daily variation of CH4 emission flux in each treatment group of Experiment 2.

[0023] Figure 6This is a table showing the daily changes in N2O emission flux for each treatment group in Experiment 2.

[0024] Figure 7 This is a bar chart showing the cumulative emissions of CH4 and N2O for each treatment group in Experiment 2.

[0025] Figure 8 This is a bar chart showing the rice yield of each treatment group in Experiment 2.

[0026] Figure 9 This is a daily variation curve of CH4 emission flux for each treatment group in Experiment 3.

[0027] Figure 10 This is a bar chart showing the cumulative CH4 emissions of each treatment group in Experiment 3.

[0028] Figure 11 This is a daily variation curve of N2O emission flux for each treatment group in Experiment 3.

[0029] Figure 12 This is a bar chart showing the cumulative N2O emissions of each treatment group in Experiment 3.

[0030] Figure 13 This is a bar chart showing the yield of each treatment group in Experiment 3.

[0031] Figure 14 This is a bar chart showing the rate of change in yield for each treatment group in Experiment 3. Detailed Implementation

[0032] Methane emissions from paddy fields exhibit a bimodal pattern, with peak emissions occurring after the rice seedlings have turned green and been re-irrigated, and again after tillering has ended. The water temperature conditions differ significantly between these two peak periods, and the photosynthetic oxygen release of a single algal species is limited by the temperature window and cannot simultaneously cover both periods with different temperature conditions. To address this technical challenge, a solution was adopted that involves applying two formulations with opposite ratios at different times. In Example 1, the first preparation consists of the following components in parts by weight: 1.0 part of *Alternaria monotypica* mud, 0.9 parts of *Auricularia auricula-judae* mud, 1.4 parts of *Chlorella proteoglycans* mud, 1.5 parts of sodium nitrate, 0.4 parts of dipotassium hydrogen phosphate, 0.8 parts of magnesium sulfate, 0.4 parts of calcium chloride, 0.1 parts of ferric ammonium citrate, 1.0 part of A5 trace element mixture, 0.36 parts of sodium carboxymethyl cellulose, with the remainder being water to bring the total to 100 parts; the second preparation consists of the following components in parts by weight: 1.0 part of *Alternaria monotypica* mud, 1.0 part of *Auricularia auricula-judae* mud, 0.7 parts of *Chlorella proteoglycans* mud, 0.8 parts of sodium nitrate, 0.4 parts of dipotassium hydrogen phosphate, 0.8 parts of magnesium sulfate, 0.4 parts of calcium chloride, 0.1 parts of ferric ammonium citrate, 1.0 part of A5 trace element mixture, 0.24 parts of sodium carboxymethyl cellulose, with the remainder being water to bring the total to 100 parts. In Example 2, the first formulation consists of the following components in parts by weight: 1.0 part of *Alternaria monotypica* mud, 0.7 parts of *Alternaria variegata* mud, 1.2 parts of *Chlorella proteoglycans* mud, 1.6 parts of sodium nitrate, 0.3 parts of dipotassium hydrogen phosphate, 0.7 parts of magnesium sulfate, 0.3 parts of calcium chloride, 0.1 parts of ferric ammonium citrate, 1.0 part of A5 trace element mixture, 0.39 parts of sodium carboxymethyl cellulose, with the remainder being water to bring the total to 100 parts; the second formulation consists of the following components in parts by weight: 1.0 part of *Alternaria monotypica* mud, 0.9 parts of *Alternaria variegata* mud, 0.8 parts of *Chlorella proteoglycans* mud, 0.7 parts of sodium nitrate, 0.3 parts of dipotassium hydrogen phosphate, 0.7 parts of magnesium sulfate, 0.3 parts of calcium chloride, 0.1 parts of ferric ammonium citrate, 1.0 part of A5 trace element mixture, 0.28 parts of sodium carboxymethyl cellulose, with the remainder being water to bring the total to 100 parts. In Example 3, the first preparation consists of the following components in parts by weight: 1.0 part of *Alternaria monotypica* mud, 0.8 parts of *Auricularia auricula-judae* mud, 1.6 parts of *Chlorella proteoglycans* mud, 1.4 parts of sodium nitrate, 0.5 parts of dipotassium hydrogen phosphate, 0.9 parts of magnesium sulfate, 0.4 parts of calcium chloride, 0.1 parts of ferric ammonium citrate, 1.0 part of A5 trace element mixture, 0.32 parts of sodium carboxymethyl cellulose, with the remainder being water to bring the total to 100 parts; the second preparation consists of the following components in parts by weight: 1.0 part of *Alternaria monotypica* mud, 1.0 part of *Auricularia auricula-judae* mud, 0.6 parts of *Chlorella proteoglycans* mud, 0.6 parts of sodium nitrate, 0.5 parts of dipotassium hydrogen phosphate, 0.9 parts of magnesium sulfate, 0.4 parts of calcium chloride, 0.1 parts of ferric ammonium citrate, 1.0 part of A5 trace element mixture, 0.22 parts of sodium carboxymethyl cellulose, with the remainder being water to bring the total to 100 parts.In the three embodiments, the concentration ratio of *Chlorella oocystiformis* in the first formulation was higher than that of *Anabaena variabilis*, the concentration ratio of *Anabaena variabilis* in the second formulation was higher than that of *Chlorella oocystiformis*, and the concentration ratio of *Anabaena variabilis* in the second formulation was higher than that in the first formulation. The mass fraction of sodium nitrate in the second formulation was 40% to 60% of that in the first formulation. The amounts of *Chlorella oocystiformis*, *Anabaena variabilis*, and sodium carboxymethyl cellulose varied among the three embodiments, and all embodiments fell within the formulation range of the microalgae formulation group of this application. Optionally, among the *Anabaena variabilis*, *Anabaena variabilis*, and *Chlorella pyrenoidosa* used in each embodiment, *Anabaena variabilis* and *Anabaena variabilis* were provided by the Freshwater Algae Culture Bank of the Chinese Academy of Sciences, and *Chlorella oocystiformis* was provided by Shanghai Guangyu Biotechnology Co., Ltd.

[0033] The three embodiments used the same preparation method. Water was added to the preparation vessel and stirred at 86.7 rpm. A mixture of sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, ferric ammonium citrate, and A5 trace elements was added and stirred continuously for 14.6 minutes until the solid was completely dissolved. Sodium carboxymethyl cellulose was slowly sprinkled into the liquid surface in three portions, and the temperature was raised to 63.4 degrees Celsius and stirred for 22.8 minutes until it was completely swollen. The resulting culture medium was autoclaved at 121.6 degrees Celsius for 19.4 minutes and then naturally cooled to 27.3 degrees Celsius. The three types of algal mud were inoculated according to the formula of each embodiment, and the mixture was made up to 100 portions. The mixture was then statically cultured at 28.6 degrees Celsius and a light intensity of 46.2 μmol / m² / s for 160.8 hours. The mixture was stirred at 38.5 rpm for 12.3 minutes to suspend the algal cells evenly. The mixture was then filled into light-proof containers and stored at 24.6 degrees Celsius for later use.

[0034] The application method of the above-mentioned microalgae preparations in paddy fields is as follows: The first preparation is applied to the paddy field after the transplanted rice seedlings have recovered and been re-irrigated. The criteria for recovery and re-irrigation are that the field is irrigated to a water depth of 3-5 cm and the new roots of the seedlings begin to sprout, which usually occurs 5-10 days after transplanting. The second preparation is applied to the paddy field after tillering has ended. The criteria for the end of tillering are that the total number of leaves on the main stem reaches the predetermined leaf age and the number of tillers in the field population begins to decrease, which is the panicle differentiation period. The application rate of the first preparation is 2.5-3.5 L / mu, and the application rate of the second preparation is 1.5-2.5 L / mu, for a total application rate of 4-6 L / mu. Before application, the concentrate of the preparation is diluted with irrigation water at a volume ratio of 1:5 to 1:50, and applied to the paddy field water layer using a plant protection drone or ground spraying machinery. The paddy field water layer is kept stable for 24 hours after application, and no drainage or irrigation operations are carried out.

[0035] The basic characteristics and emission reduction effects of the microalgae preparations obtained in the three examples were determined. Viscosity was measured using a rotational viscometer at 23.6°C; solid content was measured by drying at 105.3°C for 4.2 hours using a gravimetric method; pH was measured directly using a pH meter; algal cell density was obtained by counting algae cells under a microscope using a hemocytometer; and sedimentation rate was characterized by the volume ratio of the supernatant after standing in a graduated cylinder for 24.6 hours. Gas samples were collected using the static box method in the field plot as described in Experiment 1. Methane concentration was detected using an Agilent 8890 gas chromatograph equipped with an autosampler and a flame ionization detector, and nitrous oxide concentration was detected using an electron capture detector. The greenhouse gas emission flux (mg / m³) was then quantitatively determined. 2 / h), cumulative emissions (kg / hm) 2 The test results were as follows: the viscosity of the first formulation obtained in Example 1 was 8.6 mPa·s, the solid content was 1.2%, the pH value was 6.9, and the algal cell density was 2.6 × 10⁻⁶. 7 The algal cell density of the second formulation was 1.4 × 10⁻⁶ cells / mL, with a settling rate of 6.4%. 7 per milliliter, the cumulative emission reduction rate of methane is 30.2%, and the cumulative emission reduction rate of nitrous oxide is 48.1%; the viscosity of the first formulation obtained in Example 2 is 9.4 mPa·s, the solid content is 1.5%, the pH value is 7.2, and the algal cell density is 2.9 × 10⁻⁶. 7 The algal cell density of the second formulation was 1.8 × 10⁻⁶ cells / mL, with a settling rate of 5.8%. 7 per milliliter, the cumulative emission reduction rate of methane is 31.5%, and the cumulative emission reduction rate of nitrous oxide is 46.9%; the viscosity of the first formulation obtained in Example 3 is 7.9 mPa·s, the solid content is 1.3%, the pH value is 6.8, and the algal cell density is 2.2 × 10⁻⁶. 7 The algal cell density of the second formulation was 1.2 × 10⁻⁶ cells / mL, with a settling rate of 7.2%. 7 For each milliliter, the cumulative emission reduction rate for methane was 29.4%, and the cumulative emission reduction rate for nitrous oxide was 47.4%. The above three sets of test data are recorded on pages 36, 41, and 47 of the experimental logbook, respectively. The sample numbers are A-12-3, B-12-7, and C-12-9, and the test dates are June 12, 2025, June 19, 2025, and June 26, 2025, respectively.

[0036] The viscosity of the first formulation in the three examples ranged from 7.9 to 9.4 mPa·s, the solid content ranged from 1.2% to 1.5%, and the algal cell density ranged from 2.2 × 10⁻⁶. 7 Up to 2.9×10 7 The algal cell density of the second formulation was between 1.2 × 10⁻⁶ per milliliter.7 Up to 1.8×10 7 Between each milliliter, the cumulative emission reduction rate of methane was no less than 29%, and the cumulative emission reduction rate of nitrous oxide was no less than 46%, which is consistent with the measured results of the three-algae compound treatment in Experiment 1. This indicates that the microalgae preparation group of this application can maintain an effective dissolved oxygen supply during both low and high temperature periods, and achieve synergistic emission reduction of methane and nitrous oxide in paddy fields. All indicators meet the requirements for field application.

[0037] In a further embodiment, the monogalactia in the second formulation includes free monogalactia and monogalactia embedded in a slow-release carrier. The slow-release carrier accounts for 10% to 30% of the total mass of the second formulation. After being applied to the paddy field, the slow-release carrier gradually disintegrates and continuously releases monogalactia, thus continuously replenishing the oxygenation capacity of the water during high-temperature periods. The first and second formulations can also be formulated into microalgae-coated granular fertilizers, with each kilogram of granular fertilizer containing 50 to 200 mL of microalgae formulation. The coating has a double-layer structure: the inner layer is an algal layer containing the microalgae formulation, and the outer layer is a slow-release coating layer. The slow-release coating layer disintegrates within 12 to 48 hours after the granular fertilizer is added to the water, and the inner algal layer immediately releases the microalgae formulation. Alternatively, they can be formulated into microalgae compound liquid fertilizers, which are made by mixing microalgae formulations with amino acid water-soluble fertilizers or humic acid liquid fertilizers at a volume ratio of 1:2 to 1:5. The pH value of the compound liquid fertilizer is 6.0 to 7.5. All of the above-mentioned formulations can be obtained by the aforementioned preparation methods and applied to the paddy field according to the aforementioned application methods.

[0038] The following specific experiments will further illustrate this point.

[0039] Experiment 1: Rice paddy microalgae methane and nitrous oxide emission reduction experiment

[0040] 1. Microalgae preparations and grouping arrangements

[0041] The three microalgae used in this experiment—*Houttuynia cordata*, *Anabaena variegata*, and *Chlorella pyrenoidosa*—were all commercially available species. The experimental site was located in the double-cropping rice region of the middle and lower reaches of the Yangtze River. The rice variety used was the locally dominant indica rice, planted via transplanting. The experiment included seven treatment groups: CK was the blank control (applied with an equal volume of water), CK1 was the culture medium control (applied with an equal volume of BG11 medium without microalgae), and treatment A was a single *Houttuynia cordata* species (concentration 1×10⁻⁶). 7 Treatment B consisted of a single, variable *Anabaena* algae (concentration 1×10⁻⁶ cells / mL). 7 Treatment C consisted of a combination of two algae, *Anabaena uniflora* and *Anabaena variegata* (total concentration 1×10⁻⁶ cells / mL). 7 Treatment D consisted of a single algae of Chlorella proteoglycans (concentration 1×10⁻⁶ cells / mL). 7 Treatment E consisted of a compound of three algae: *Hymenopterus uniflorus*, *Anabaena variegata*, and *Chlorella proteoglycans* (total concentration 1×10⁻⁶). 7 (cells / mL).

[0042] 2. Preparation method of microalgae preparations

[0043] *Hymenochrysis galbana*, *Anabaena variegata*, and *Chlorella proteoglycans* were inoculated into BG11 liquid medium and cultured in an airlift photobioreactor at 25°C under a 12 h:12 h light-dark cycle until the logarithmic growth phase. After determining the density of each algal species using a hemocytometer, for single-algae treatments, the corresponding stock solution was directly diluted to 1 × 10⁻⁶ with fresh BG11 medium. 7 Cells / mL. Two-algae combination treatment C: The mother liquor of *Hypericum monostellatum* and the mother liquor of *Anabaena variegata* were mixed at a 1:1 volume ratio, and the total concentration was adjusted to 1×10⁻⁶ using culture medium. 7 Cells / mL. Tri-algae compound treatment E: The three algal mother liquors were mixed at a volume ratio of 1:1:1, and the total concentration was adjusted to 1×10⁻⁶. 7 Cells / mL.

[0044] 3. Basic conditions of paddy fields and methods for applying microalgae

[0045] The experimental field soil was yellow-brown paddy soil with an organic matter content of approximately 2.8% and a pH of approximately 6.2. Rice was transplanted during the early rice season at a density of approximately 18,000 hills per mu (approximately 1,600 hectares per acre). The chemical fertilizer application scheme was consistent across all treatments: 25 kg / mu of compound fertilizer (15-15-15) was applied as basal fertilizer, and 10 kg / mu of urea was applied as topdressing during the tillering stage. Each microalgae preparation was applied evenly to the water layer of each treatment plot twice, once after greening and re-watering and again after tillering. The first application rate was 2.5-3.5 L / mu, and the second application rate was 1.5-2.5 L / mu. During application, the water layer was maintained at a depth of 3-5 cm. The water layer was kept stable for 48 hours after each application without drainage to ensure sufficient colonization of the microalgae cells.

[0046] 4. The test results data are shown in Tables 1, 2, and 3. Figures 1-4 .

[0047] Figure 1 The CH4 emission flux (mg / m³) for each treatment is shown. 2 (h) Daily variation data table. During the monitoring period, the CH4 emission flux of each treatment showed a trend of first increasing and then decreasing over time. The emission fluxes of the two control treatments, CK and CK1, reached 4.0 and 3.0 respectively on June 26, rose to 6.0 and 5.0 on July 1, and reached the peak of the entire monitoring period on July 5 (18.0 for CK and 17.0 for CK1), and then gradually declined, dropping to 0.5 by August 4. The emission flux of the tri-algae compound treatment E was 7.0 on the peak day, only 39% of the peak value of the control CK, and the rate of decline in emission flux was significantly faster than that of the control.

[0048] Figure 2 The cumulative CH4 emissions (kg / hm) for each treatment are shown.2 (Bar chart: CK = 110, CK1 = 106, Treatment A = 104, Treatment B = 109, Treatment C = 86, Treatment D = 79, Treatment E = 77. The cumulative CH4 emissions (77 kg / hm²) of the three-algae combination treatment E were the lowest among all treatments, about 30% lower than CK; the single-algae treatments A (104) and B (109) showed little difference from the control, with almost no emission reduction effect.)

[0049] Table 1. Ratios of CH4 emissions in each treatment group to CK and CK1 in Experiment 1

[0050]

[0051] Figure 3 The N2O emission flux (mg / m³) for each treatment is shown. 2 The table shows the daily variation data for each treatment. As can be seen from the table, the N2O emission flux of each treatment was low in the early stage of monitoring. Around July 29, the N2O emission flux of each treatment was ranked as A, CK, CK1, B, C, D, and E. The three-algae compound treatment E had the lowest value among all treatments, and then gradually returned to zero or remained at a low value.

[0052] Table 2. Ratios of N2O emissions in each treatment group to CK and CK1 in Experiment 1

[0053]

[0054] Figure 4 The cumulative N2O emissions (kg / hm) for each treatment are shown. 2 Bar chart: CK is 3.15, CK1 is 2.25, treatment A is 3.05, treatment B is 2.75, treatment C is 1.25, treatment D is 1.45, and treatment E is 1.65.

[0055] Table 3. Rice yield and yield increase rate in each treatment group of Experiment 1

[0056]

[0057] Table 1 shows the ratios of cumulative CH4 emissions for each treatment to the control (CK) and CK1. As shown in Table 1, the cumulative CH4 emissions from single-algae treatments A and B were close to the control level, showing almost no emission reduction effect. Single-algae treatment D significantly reduced emissions compared to the control. The two-algae combination treatment C further reduced emissions compared to single-algae treatments A and B, achieving a reduction rate of 22.0%, far exceeding the expected sum of the emission reduction effects of single-algae A and B. The three-algae combination treatment E had the lowest emission reduction rate among all treatments, reaching 29.8%, indicating that the three-algae combination had a synergistic effect on CH4 emission reduction. Table 2 shows the ratios of cumulative N2O emissions for each treatment to the control (CK) and CK1. As shown in Table 2, the single-algae treatments A and B had limited effect on N2O emission reduction. The cumulative N2O emissions from the three-algae combination treatment E increased slightly compared to the two-algae combination treatment C but were still significantly lower than the control, indicating that the three-algae combination maintained significant N2O emission reduction while ensuring the highest CH4 emission reduction rate, achieving synergistic emission reduction of both greenhouse gases.

[0058] 5. Conclusion

[0059] This experiment revealed the synergistic mechanism of trialbum combination through parallel comparisons of single algae, two-algae combination, and three-algae combination. The peak CH4 emission of the trialbum combination treatment E (7.0) was only 39% of that of the control CK (18.0), and its decay rate was significantly faster than that of the control. The cumulative CH4 emission of the trialbum combination treatment E (77) was the lowest among all treatments, while the single algae treatments A (104) and B (109) showed little difference from the control and had almost no emission reduction effect. The emission reduction rates of the single algae *Hylocereus undatus* and *Anabaena variegata* were extremely low, and the two were almost ineffective. However, the two-algae combination produced an emission reduction rate of 22.0%, and the trialbum combination reached 29.8%, far exceeding the simple summation of the effects of single algae. This indicates that the complementarity of the three algae in temperature niche is not a simple additive effect, but rather a nonlinear leap in synergistic effect.

[0060] Experiment 2: Greenhouse Gas Emission Reduction and Yield Increase Experiment of Microalgae in Early Rice in Southern Double-Crop Rice Region

[0061] 1. Microalgae preparations and grouping arrangements

[0062] The microalgae preparation used in this experiment was a compound nutrient solution containing three microalgae: *Houttuynia cordata*, *Anabaena variegata*, and *Chlorella proteoglycans*, all of which were commercially available. The experimental site was located in a double-cropping rice region in southern China, and the rice variety used was the locally dominant indica rice, planted by transplanting. The experiment included eight treatment groups: W1 was the control (applied with an equal volume of water), and W2 to W8 were treatment groups with different microalgae application rates, arranged from low to high, to examine the relationship between application rate and emission reduction and yield increase effects.

[0063] 2. Preparation method of microalgae preparations

[0064] Three microalgae were cultured separately in BG11 liquid medium at 25–30°C until the logarithmic growth phase. After counting, the algal solutions of the three algae were mixed at a volume ratio of 1:1:1, and the final concentration was adjusted to 1×10⁻⁶ with fresh medium. 7 Cells / mL were used to prepare a compound microalgae nutrient solution. This nutrient solution was stored in a light-protected container at 4°C for later use.

[0065] 3. Basic conditions of paddy fields and methods for applying microalgae

[0066] The experimental field soil was lateritic paddy soil with an organic matter content of approximately 2.5%. Rice was planted in the early rice season of 2022. The amount of chemical fertilizer used in each treatment was exactly the same. The microalgae preparation was applied twice, once after the rice had recovered from transplanting and been re-irrigated (7 days after transplanting) and once after tillering (30 days after transplanting). The first application was 60% of the total amount, and the second application was 40%. Before each application, the microalgae nutrient solution was diluted with water according to the corresponding treatment amount and then applied to the paddy field water layer by manual irrigation. During application, the water layer depth in the field was maintained at 3-5 cm, and the field was not drained within 48 hours after application.

[0067] 4. See the test results data. Figures 5-8 .

[0068] Table 4. CH4 and N2O emission reduction rates and yield performance of each treatment group in the Hainan trial.

[0069]

[0070] Figure 5 The daily variation data of CH4 emission flux for each treatment group are shown. It can be seen that during the monitoring period, the CH4 emission flux of each treatment group showed a trend of first increasing and then decreasing over time. All treatment groups reached their emission peak on March 9th. Subsequently, the emission flux of each treatment group began to decline on March 14th, and from March 18th onwards, the emission flux of most treatment groups dropped to near zero. On the emission peak day (March 9th), the emission flux of the microalgae treatment group was significantly lower than that of the control.

[0071] Figure 6 The table shows the daily variation of N2O emission flux for each treatment group. N2O emission flux for each treatment remained at a low level from February 18 to March 9, indicating that the microalgae treatment group had already begun to suppress N2O production before the peak emission period.

[0072] Figure 7The bar charts show the cumulative CH4 and N2O emissions for each treatment group. As can be seen from the bar charts, W1 (control) had the highest cumulative CH4 emissions, followed by W2, W3, and W4, with W4 showing the lowest emission reduction rate at 90.33%. Subsequently, W5, W6, W7, and W8 showed a fluctuating upward trend, indicating that the inhibitory effect of the tri-algae compound preparation on CH4 is not a strictly monotonic dose-response relationship, and the application rate with the highest emission reduction rate falls within the medium application rate range.

[0073] Figure 8 The bar charts showing the yield of rice in each treatment are shown. The yield of W1 (control) was about 400 kg / mu; the yield of W5 (6 L / mu) was the highest, which was the peak value among all treatments; the yield showed an inverted U-shaped curve with the increase of microalgae application rate, with medium application rate significantly increasing yield and high application rate inhibiting yield.

[0074] 5. Conclusion

[0075] Under strict control with completely consistent chemical fertilizer application, this experiment demonstrated that the tri-algae compound microalgae formulation can simultaneously achieve CH4 emission reduction (up to 90.33%), N2O emission reduction (up to 86.19%), and rice yield increase (up to 25%). The accelerated termination of CH4 emissions and the significant inhibition of N2O indicate that the formulation's effect is not simply to reduce emission peaks, but rather to achieve systematic greenhouse gas reduction from both the duration and recovery perspectives by aligning two applications with the emission timing. The optimal application rate falls within the effective emission reduction window, further validating the synergistic effect of the tri-algae compound formulation on both emission reduction and yield increase objectives.

[0076] Experiment 3: Greenhouse gas emission reduction and yield increase experiment of microalgae in the Japonica rice region of Northeast China (2023)

[0077] 1. Microalgae preparations and grouping arrangements

[0078] The microalgae preparation used in this experiment was a compound nutrient solution containing three microalgae: *Houttuynia cordata*, *Anabaena variegata*, and *Chlorella pyrenoidosa*, all of which were commercially available. The experiment was conducted in the Japonica rice-growing region of Northeast China by a local agricultural research institution. Seven treatments were included: a conventional fertilization control group, a culture medium control group, and a treatment with 5 million (5 × 10⁻⁶) microalgae. 6 10 million (cells / mL) 7 50 million (cells / mL) 7 Three concentration gradient treatment groups (cells / mL), a 15% nitrogen reduction treatment group, and a 15% nitrogen reduction + 10 million combined treatment group.

[0079] 2. Preparation method of microalgae preparations

[0080] Three microalgae were cultured in BG11 liquid medium at 25°C under a 12 h:12 h light-dark cycle until they reached the logarithmic growth phase. After determining the density of each algae species using a hemocytometer, the algal solutions of the three species were mixed at a 1:1:1 volume ratio and diluted with fresh medium to a concentration of 5 × 10⁻⁶. 6 1×10 7 and 5×10 7 Cells / mL were used to prepare three concentration gradients of compound microalgae nutrient solutions. The control medium used was BG11 medium without microalgae. All formulations were stored in light-protected containers at low temperatures, and removed to room temperature and thoroughly mixed before application.

[0081] 3. Basic conditions of paddy fields and methods for applying microalgae

[0082] The experimental field was a typical paddy field in the Northeast China japonica rice growing area, with dark brown paddy soil. The tested variety was the local main japonica rice variety, planted using the transplanting method. Chemical fertilizers were applied according to the protocol for each treatment: the control, culture medium control, and the three microalgae concentration treatments had the same amount of fertilizer applied; the 15% nitrogen reduction treatment reduced nitrogen fertilizer input by 15% compared to the control; the 15% nitrogen reduction + 10 million kcal / kg treatment applied an additional 1×1000 kcal / kg nitrogen fertilizer on top of the 15% nitrogen reduction. 7 Microalgae preparations at cell / mL. The microalgae were applied evenly to the water layer of each treatment plot twice, after the greening and re-watering and after tillering. The first application rate was 2.5-3.5 L / mu, and the second application rate was 1.5-2.5 L / mu. During application, a water layer of 3-5 cm was maintained in the field.

[0083] 4. See the test results. Figures 9-14 .

[0084] Figure 9 The daily CH4 emission flux variation curves for each treatment group are shown. The curves indicate that all treatments experienced varying degrees of CH4 emission peaks in mid-to-late July, but the peak shape and peak height differed significantly among the treatments. The CH4 emission peak in the conventional fertilization control group occurred in mid-to-late July, similar to that in the culture medium control group.

[0085] Figure 10 A bar chart showing the cumulative CH4 emissions for each treatment group is presented. The conventional fertilization control group had the highest cumulative emissions, followed by the culture medium control group. Emissions from all three microalgae concentration treatment groups were lower than those from the conventional fertilization control group. The 5 million CFU group had the lowest emissions, the 50 million CFU group was the second lowest but slightly higher than the 5 million CFU group, and the 10 million CFU group had the highest emissions among the three microalgae concentration treatment groups. This indicates that the effect of different microalgae concentrations on cumulative CH4 emissions is non-monotonic, with the low concentration (5 million CFU group) showing the best effect. The combined treatment of 15% nitrogen reduction + 10 million CFU had the lowest cumulative CH4 emissions among all treatments.

[0086] Figure 11 The daily variation curves of N2O emission flux for each treatment group are shown. The conventional fertilization control group showed a significant secondary emission peak in late July, while the microalgae treatment groups did not show a similar strong secondary peak during the same period, and their curves were relatively flat.

[0087] Figure 12 A bar chart showing the cumulative N2O emissions for each treatment is presented. The culture medium control group had the highest cumulative emissions.

[0088] Figure 13 The bar chart shows the yield of each treatment group. The yield of the three microalgae concentration treatment groups increased progressively with increasing concentration: the yields of the 5 million, 10 million, and 50 million groups increased sequentially, indicating that microalgae treatment has a concentration-dependent promoting effect on rice yield. The yield of the 15% nitrogen reduction group was lower than that of the conventional fertilization control group, while the yield of the 15% + 10 million nitrogen reduction group was slightly higher than that of the 15% nitrogen reduction group but still lower than that of the conventional fertilization control group.

[0089] Figure 14 The bar chart shows the yield change rate data for each treatment group. The conventional fertilization control group had a yield of 0.00%, the culture medium control group had a yield of +1.43%, the 5 million microalgae group had a yield of +3.52%, the 10 million microalgae group had a yield of +5.52%, the 50 million microalgae group had a yield of +6.21%, the 15% nitrogen reduction group had a yield of -2.16%, and the 15% nitrogen reduction + 10 million microalgae group had a yield of -0.75%. The yield increase rate of the three microalgae concentration treatments increased in a concentration-dependent manner. The 15% nitrogen reduction group had a yield reduction of 2.16%, but after adding microalgae (15% nitrogen reduction + 10 million microalgae group), the yield reduction narrowed to 0.75%, indicating that microalgae could partially compensate for the yield loss caused by nitrogen reduction.

[0090] 5. Conclusion

[0091] This experiment systematically verified the efficacy of the compound microalgae formulation from three dimensions: CH4 emission reduction, N2O emission reduction, and yield increase. Microalgae treatment reduced the peak CH4 emission level and accelerated its decline. The cumulative CH4 emissions of all concentration treatments were lower than those of the conventional fertilization control group. The cumulative CH4 emission curve of the combined treatment of 15% nitrogen reduction + 10 million N2O was the lowest among all treatments, and the combined inhibition effect far exceeded the expected sum of the effects of 15% nitrogen reduction and 10 million N2O alone, indicating a synergistic effect between microalgae and nitrogen reduction measures. Microalgae treatment alone effectively suppressed the late-stage surge in N2O. This experiment further verified the broad applicability of the three-algae compound formulation in paddy fields of different climatic zones and its potential for synergistic emission reduction with agronomic measures.

[0092] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A microalgae formulation for greenhouse gas emission reduction in paddy fields, characterized in that, The formulation includes a first preparation and a second preparation; both preparations contain *Hymenopterus uniflorus*, *Anabaena variegata*, and *Chlorella proteoglycans*; the concentration of *Chlorella proteoglycans* is higher in the first preparation than in the second preparation; the concentration of *Hymenopterus uniflorus* is higher in the second preparation than in the first preparation; the first preparation is applied after the rice has turned green and been re-irrigated, and the second preparation is applied after the rice has finished tillering; the total concentration of microalgae in both preparations is 0.5 × 10⁻⁶. 7 Up to 5×10 7 Cells / mL.

2. The microalgae preparation group according to claim 1, characterized in that, In the first formulation, the concentration ratio of Alternaria monofilata, Alternaria variegata, and Chlorella proteoglycans is 1:(0.6-1.0):(1.2-1.6), and in the second formulation, the concentration ratio of Alternaria monofilata, Alternaria variegata, and Chlorella proteoglycans is 1:(0.6-1.0):(0.6-0.9).

3. The microalgae preparation group according to claim 1, characterized in that, The concentration ratio of *Syngonium moniliforme* to *Chlorella proteoglycans* in the second formulation is greater than that in the first formulation. The *Syngonium moniliforme* in the second formulation includes free *Syngonium moniliforme* and *Syngonium moniliforme* encapsulated in a sustained-release carrier, with the sustained-release carrier accounting for 10%-30% of the total mass of the second formulation.

4. The microalgae preparation group according to claim 1, characterized in that, The concentration ratio of *Houttuynia cordata* in the second formulation is greater than that in the first formulation; the mass fraction of sodium nitrate in the second formulation is lower than that in the first formulation, and the mass fraction of sodium nitrate in the second formulation is 40%-60% of that in the first formulation.

5. The microalgae preparation group according to claim 1, characterized in that, Both the first and second formulations are liquid formulations. The liquid formulations contain a culture medium carrier, specifically BG11 liquid culture medium. BG11 liquid culture medium contains sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, ferric ammonium citrate, and a mixture of A5 trace elements. The liquid formulations also contain sodium carboxymethyl cellulose (CMC), with a CMC mass fraction of 0.1%–0.5%. Furthermore, the CMC mass fraction is positively correlated with the total microalgae concentration; for every 1 × 10⁻⁶ increase in total microalgae concentration, the concentration increases. 7 Cells / mL, the mass fraction of sodium carboxymethyl cellulose increases by 0.05%-0.1%.

6. The microalgae preparation group according to claim 1, characterized in that, Both the first and second formulations are microalgae-coated granular fertilizers, with each kilogram of granular fertilizer containing 50-200 mL of microalgae preparation. The coating of the coated granular fertilizer has a double-layer structure: the inner layer is an algal layer containing microalgae preparation, and the outer layer is a slow-release coating layer. The slow-release coating layer disintegrates within 12-48 hours after the granular fertilizer is added to the water, and the inner algal layer releases the microalgae preparation after the slow-release coating layer disintegrates.

7. The microalgae preparation group according to claim 1, characterized in that, Both the first and second formulations are microalgae compound liquid fertilizers. The compound liquid fertilizer is made by mixing microalgae formulations with amino acid water-soluble fertilizers or humic acid liquid fertilizers in a volume ratio of 1:2 to 1:

5. The pH value of the compound liquid fertilizer is 6.0-7.

5.

8. The microalgae preparation group according to claim 1, characterized in that, The total microalgae concentration of the first formulation was higher than that of the second formulation; the total microalgae concentration of the first formulation was 2 × 10⁻⁶. 7 Up to 3×10 7 Cells / mL, the total microalgae concentration of the second formulation is 1×10⁻⁶. 7 Up to 1.9×10 7 Cells / mL.

9. A method for reducing greenhouse gas emissions from paddy fields, characterized in that, include: After the rice seedlings have recovered from transplanting and been rehydrated, the first formulation of the microalgae formulation group according to any one of claims 1-8 is applied to the paddy field, and the second formulation of the microalgae formulation group is applied to the paddy field after tillering is completed; the application rate of the first formulation is 2.5-3.5 L / mu, and the application rate of the second formulation is 1.5-2.5 L / mu.

10. The method according to claim 9, characterized in that, The criteria for determining the seedlings after re-greening and re-watering are: the field is irrigated to a water depth of 3-5cm and the new roots of the seedlings begin to sprout. This period is 5-10 days after transplanting. The criteria for determining the seedlings after tillering is: the total number of leaves on the main stem reaches the predetermined leaf age and the number of tillers in the field population begins to decline. This period is the young panicle differentiation period.

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