Immobilized microalgae biofertilizer and its application in alleviating salt stress of crops

Abstract

The application discloses a kind of immobilized microalgae biological fertilizer preparation and its preparation method and application, belong to microbial fertilizer technical field.The application provides a kind of immobilized microalgae biological fertilizer preparation, the immobilized microalgae biological fertilizer preparation includes microalgae cell and embedding carrier, the embedding carrier is composed of sodium alginate and polyvinyl alcohol.The application further provides the preparation method of the immobilized microalgae biological fertilizer preparation, and its application in relieving crop salt stress.The immobilized microalgae biological fertilizer preparation of the application can effectively adsorb rhizosphere Na + , protect the structural integrity of photosynthetic system, improve antioxidant enzyme activity, significantly relieve the oxidative damage and growth inhibition of crop under salt stress.

Description

Technical Field

[0001] This invention belongs to the field of microbial fertilizer technology, specifically relating to immobilized microalgae bio-fertilizer and its application in alleviating crop salt stress. Background Technology

[0002] Soil salinization is the process by which soluble salts from the subsoil or groundwater rise to the surface via capillary action, accumulate in the soil after water evaporation, and lead to undesirable soil properties and a decline in soil quality. This severely impacts agricultural production, food security, and ecological stability in arid and semi-arid regions. In recent years, global warming, environmental degradation, the development of facility agriculture, and improper irrigation have exacerbated soil salinization. Statistics show that the total area of ​​saline soil in my country is 3.69 × 10⁻⁶. 7 hm 2 It accounts for 5.01% of the country's usable land area.

[0003] Salt stress hinders crop reproduction and growth by suppressing seed germination, slowing down physiological metabolism, and impeding nutrient absorption. High concentrations of salt stress induce a dual pressure of osmotic stress and ion toxicity through ion toxicity and plant tissue dehydration, severely damaging the integrity of plant cell membrane systems, inhibiting photosynthesis, hindering plant growth, and even causing plant death, thus threatening the sustainable and healthy development of regional agriculture.

[0004] Microalgae bio-fertilizer is a novel type of green natural fertilizer, mainly composed of nitrogen-fixing cyanobacteria, green algae, diatoms, and extracts from large seaweeds. It is rich in essential plant nutrients such as nitrogen, phosphorus, and potassium, as well as various vitamins, amino acids, fatty acids, extracellular polysaccharides, and plant hormones. It can promote plant growth and development, improve plant stress resistance and growth performance, and simultaneously repair desertified and saline-alkali soils, improving soil water and fertilizer retention capacity. Current research reports that a compound microalgae fertilizer containing *Scenedesmus obliquus* and *Navicula* can alleviate oxidative damage and growth inhibition caused by salt stress by regulating the content of antioxidant enzymes and endogenous hormone levels in cucumber cells. The extracellular polymers secreted by cyanobacteria have a tendency to chelate cations, effectively reducing the accumulation and toxicity of sodium ions, thereby reducing soil salinization.

[0005] Currently, microalgae bio-fertilizers are widely used in agricultural production in various forms, including algae powder, active algae liquid, and microalgae extracts. However, existing microalgae bio-fertilizer formulations still have the following shortcomings: (1) Although fresh algae (algae mud) is rich in active substances such as extracellular polysaccharides, plant hormones, vitamins and active enzymes, it can immediately provide nutrients and signaling substances to crops and soil. However, direct application is susceptible to the influence of environmental factors and algae activity. Long-term storage at room temperature will lead to algae cell death, produce odor and lose fertilizer effect. (2) Although dried algae powder has the characteristics of small size, good stability, long storage time and high dry matter content, the drying process destroys most of the heat-sensitive active substances and reduces the effect of biostimulants. (3) Existing immobilized microalgae technology is mostly focused on wastewater treatment. Research on applying immobilized microalgae to alleviate salt stress in agriculture is still blank, and there is a lack of systematic comparison of the salt stress relief efficacy of different forms of microalgae fertilizer preparations.

[0006] Therefore, developing a microalgae bio-fertilizer formulation that can maintain high microalgae activity, has good environmental adaptability, and can effectively alleviate crop salt stress has important practical significance and application value. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide an immobilized microalgae biofertilizer formulation, its preparation method, and its application. This invention prepares an immobilized algae ball formulation by embedding Chlorella cells in a sodium alginate-polyvinyl alcohol carrier. This formulation can effectively adsorb rhizosphere Na+. + It protects the structural integrity of the photosynthetic system, enhances the activity of antioxidant enzymes, and significantly alleviates the oxidative damage and growth inhibition of crops caused by salt stress. Its effect is superior to traditional forms of microalgae fertilizers such as algae mud and algae powder.

[0008] To achieve the above objectives, the present invention provides the following technical solution: This invention provides an immobilized microalgae bio-fertilizer formulation, which comprises microalgae cells and an embedding carrier, wherein the embedding carrier is composed of sodium alginate and polyvinyl alcohol.

[0009] This invention also provides a method for preparing the above-mentioned immobilized microalgae bio-fertilizer formulation, comprising the following steps: (1) Chlorella was cultured to the logarithmic growth phase, the algal cells were collected by centrifugation, washed with distilled water, and centrifuged again to obtain pure Chlorella algal mud; (2) Add sodium alginate and polyvinyl alcohol to distilled water, heat and stir until completely dissolved to obtain a carrier solution; (3) After the carrier solution cools down, add the Chlorella mud obtained in step (1), mix thoroughly, and obtain a mixed solution; (4) The mixture obtained in step (3) is dropped into calcium chloride solution to form gel balls. After static cross-linking, immobilized microalgae bio-fertilizer preparation is obtained.

[0010] Preferably, the microalgae cells are Chlorella cells.

[0011] Preferably, the mass ratio of sodium alginate to polyvinyl alcohol in step (2) is 1:(2~4).

[0012] Preferably, the heating temperature in step (2) is 75~85℃.

[0013] Preferably, the cooling temperature in step (3) is 35~45℃.

[0014] Preferably, the mass-volume concentration of the calcium chloride solution in step (4) is 1.5~3.0%.

[0015] Preferably, the static crosslinking time in step (4) is 18~30 h and the temperature is 2~8℃.

[0016] This invention also provides the application of the above-mentioned immobilized microalgae bio-fertilizer formulation in alleviating crop salt stress.

[0017] Preferably, the concentration of the immobilized microalgae bio-fertilizer preparation is 0.5~2.0 g / L.

[0018] It contains at least the following beneficial technical effects: (1) This invention is the first to systematically compare the efficacy of three forms of microalgae fertilizer preparations—algae mud, algae powder, and immobilized algae balls—in alleviating salt stress on crops. It found that the effect of immobilized algae balls is significantly better than that of algae mud and algae powder, filling the technological gap in the field of immobilized microalgae for alleviating agricultural salt stress.

[0019] (2) The immobilized microalgae bio-fertilizer preparation of the present invention uses sodium alginate and polyvinyl alcohol as composite encapsulation carriers. The constructed three-dimensional network structure can fix microalgae cells in a specific space, effectively prevent cell loss, maintain system stability, and enable the immobilized microalgae to maintain high activity.

[0020] (3) The sodium alginate-polyvinyl alcohol carrier in the immobilized microalgae bio-fertilizer formulation of the present invention has a porous structure and can effectively adsorb rhizosphere Na through physicochemical adsorption. + Reduce root resistance to Na + The absorption of these substances helps alleviate the toxicity of salt ions.

[0021] (4) The immobilized microalgae bio-fertilizer formulation of the present invention can protect the structural integrity of the photosystem II reaction center, especially protect the oxygen-evolving complex from salt ion attack, maintain the normal operation of the thylakoid membrane electron transport chain, and ensure the efficiency of photosynthesis.

[0022] (5) The immobilized microalgae bio-fertilizer formulation of the present invention can significantly enhance the antioxidant enzyme activity of crops under salt stress, reduce the abnormal activation of superoxide dismutase SOD and the accumulation of malondialdehyde (MDA), effectively remove reactive oxygen free radicals, and reduce membrane lipid peroxidation damage.

[0023] (6) The method for preparing the immobilized microalgae bio-fertilizer formulation of the present invention is simple, low in cost, and has good storage stability, making it suitable for industrial production and agricultural application. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the preparation process of the immobilized microalgae bio-fertilizer formulation of the present invention.

[0025] Figure 2 This is a photograph of the immobilized microalgae bio-fertilizer formulation (immobilized algae balls) of the present invention.

[0026] Figure 3 Comparative photographs showing the effects of different forms of microalgae fertilizer on the growth of white rice seedlings under salt stress.

[0027] Figure 4 Figure 1 shows the effects of different forms of microalgae fertilizer on the plant height and root length of white rice seedlings under salt stress.

[0028] Figure 5 The figure shows the effect of different forms of microalgae fertilizer on the biomass (fresh weight and dry weight) of white rice seedlings under salt stress.

[0029] Figure 6 Figure 1 shows the effects of different forms of microalgae fertilizer on the chlorophyll a, chlorophyll b and total chlorophyll content of white awn rice seedlings under salt stress.

[0030] Figure 7 Figure 1 shows the effects of different forms of microalgae fertilizer on the activities of CAT, POD, and SOD in the leaves of white rice seedlings under salt stress.

[0031] Figure 8 Figure 1 shows the effect of different forms of microalgae fertilizer on the MDA content in the leaves of white rice seedlings under salt stress.

[0032] Figure 9 The figure shows the effects of different forms of microalgae fertilizer on the Fv / Fm and OJIP fluorescence kinetic curves of leaves of white rice seedlings under salt stress. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0039] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0040] Example 1 I. Experimental Materials This experiment used white-awned rice as the research subject, donated by the Zhangye Academy of Agricultural Sciences. The microalgae were mainly Chlorella, isolated and preserved by the Gansu Provincial Microalgae Engineering Center. Preparation method of Chlorella algae mud: Chlorella cells were collected by centrifugation when the OD679 value reached about 10.0. The cells were rinsed with distilled water 2-3 times to remove the influence of the culture medium in the Chlorella. After thorough mixing, the cells were centrifuged again to obtain pure Chlorella algae mud. Preparation method of algae powder: The centrifuged algae mud was weighed and placed in a culture dish wrapped with tin foil. It was dried at 80 ℃ for 10 h and then ground to prepare the powder. Preparation method of immobilized algae balls: 3 g of sodium alginate (SA) and 9 g of polyvinyl alcohol (PVA) were weighed and added to 150 mL of distilled water. The mixture was heated to 80 ℃ in a water bath and stirred until completely dissolved. When the temperature dropped to about 40 ℃, 3 g of pure fresh algae mud was added and thoroughly mixed. The mixture was then dripped into a 2% (W / V) calcium chloride (CaCl2) solution at a uniform rate using a syringe with an inner diameter of 0.40 mm to form gel balls with a diameter of 2 mm. The gel balls were placed in a refrigerator at 4 ℃ for static cross-linking for 24 h. After cross-linking is completed, rinse 2-3 times with 0.9% (w / v) physiological saline for later use.

[0041] II. Experimental Design This experiment employed hydroponics. Plump, intact, and uniformly sized white rice seeds were placed in germination boxes, disinfected with 1% sodium hypochlorite solution for 30 minutes, rinsed three times with distilled water, and germinated for 24 hours in a 30℃ constant temperature incubator under darkness. Seeds with uniform sprouting were then transferred to hydroponic boxes with different treatments. An artificial climate incubator was used for cultivation, with the following parameters: a 12-hour light-12-hour dark cycle, light hours from 06:00 to 18:00 daily, and a light intensity of 12000 lx. Daytime temperature was controlled at (30±2)℃, while nighttime temperature was controlled at (25±2)℃, with a relative humidity of approximately 80%. To simulate salt stress, sodium chloride was used for treatment; specific treatment groups are shown in Table 1. The experiment was conducted in triplicate, with each treatment group randomly assigned. After 14 days of treatment, samples were collected and relevant indicators were measured.

[0042] Table 1 Experimental Design III. Indicator Measurement and Methods Morphological indicators were measured 14 days after sowing. Three plants were randomly selected from each experimental group for measurement.

[0043] Morphological index determination: After treatment, samples of white awned rice seedlings were taken. The samples were first washed three times with distilled water, and then the surface moisture was absorbed with filter paper. The plant height was manually measured with a tape measure, and the distance from the base of the main stem to the leaf tip was measured with a tape measure (accuracy 0.1 cm). The fresh weight of the above-ground and underground parts was weighed with an analytical balance (accuracy 0.00001 g). The dry weight was blanched at 105℃ for 30 min, and then the temperature was adjusted to 80℃ to dry until the sample reached constant weight. The sample was then taken out, weighed with an analytical balance, and the data was recorded.

[0044] Chlorophyll and antioxidant enzyme activity assays: Fully expanded functional leaves and intact roots (from root base to root tip) of white-awned rice were collected during the seedling stage for later use. Chlorophyll content was determined using the acetone-ethanol mixture extraction method.

[22] Catalase (CAT) activity was measured using a catalase (CAT) activity assay kit.

[23] Peroxidase (POD) activity was measured using a peroxidase (POD) activity assay kit.

[24] SOD activity was determined using a total SOD activity assay kit (NBT method).

[25] The malondialdehyde (MDA) content was determined using a malondialdehyde (MDA) detection kit (TBA colorimetric method).

[23] All of the above reagent kits were purchased from Beijing Solarbio Biotechnology Co., Ltd.

[0045] Fluorescence kinetic parameter determination: Fully expanded functional leaves from the plant apex were selected and allowed to undergo 20 minutes of dark adaptation in a dark environment. The dark-adapted leaves were then placed in the center of the instrument's measuring clip, ensuring the leaf surface completely covered the detection window (4 mm in diameter), avoiding the main vein area. The instrument's automatic detection program was then activated, with the system emission intensity set to 3000 μmol·m⁻¹. -2 ·s -1 Excitation was performed using saturated red light (peak 650 nm) for 1 second, and the complete fluorescence kinetic curve (time range 10 μs) from the initial fluorescence Fo to the maximum fluorescence Fm was recorded simultaneously. -1 Key parameters include photochemical quenching coefficient qP, non-photochemical quenching NPQ, and PSII maximum photochemical efficiency Fv / Fm (Fv=Fm-Fo). Each leaf was measured three times. The raw data of the OJIP curve were exported immediately after data acquisition.

[0046] IV. Data Processing and Analysis Microsoft Excel 2021, Graphpad Prism 10, and Origin 2024 were used for data processing, analysis, and graphing. SPSS 22 was used for one-way ANOVA and multiple comparisons (P < 0.05). The data are the mean ± standard error of three replicates.

[0047] V. Experimental Results 1. Effects of different forms of microalgae fertilizers on morphological indicators of white-awned rice under salt stress See Figure 1 Table 1 shows that under salt stress, the plant height and root length of *Rhizophora stylosa* were significantly inhibited. Compared with the control group (CK), the plant height of group T0 decreased by 61.1%, T1 by 51.7%, and T2 by 33.8%. The overall effect of T3 was significantly higher than that of T1 and T2, and even exceeded that of the CK group. Compared with the salt stress group, the treatment groups applying different forms of *Chlorella* bio-fertilizer all showed varying degrees of improvement. Among them, the overall effect of the T3 treatment group was the most significant, increasing by 234.3% compared with the T0 group. The T1 and T2 treatment groups increased by 23.9% and 70% compared with the T0 group, respectively. Root treatment was more sensitive. The root length of the T0 group was only 12.0% of that of the CK group, showing extremely significant inhibition. The root length of the T1, T2, and T3 groups recovered to 25.5%, 38.7%, and 85.1% of that of the CK group, respectively. The root length of the T3 group increased by 611.9% compared with the T0 group. The above results indicate that the addition of microalgae bio-fertilizer can significantly promote the growth and development of stems and roots of *Rhizophora stylosa* seedlings under salt stress.

[0048] Table 2. Effects of different forms of algal fertilizer on the growth of white rice under salt stress. Note: Plant height and root length are expressed as mean ± standard error. Different lowercase letters in the same column indicate significant differences between treatments (P<0.05), and the same applies below.

[0049] 2. Effects of different forms of microalgae fertilizer on the biomass of white-awned rice under salt stress like Figure 2 As shown, under salt stress, the biomass of each treatment group of white rice showed a gradient trend. The growth indicators of applying different forms of microalgae fertilizer were all better than those of salt stress treatment alone. Among them, the effect of algae balls was the most significant, even exceeding that of the CK group.

[0050] Salt stress significantly inhibited plant growth. Compared with the control group (CK), the fresh weight and dry weight of the T0 group decreased significantly, by 49.3% and 51%, respectively, indicating that salt stress severely inhibited the biomass accumulation of rice. The application of immobilized algal balls, algal mud, and algal powder all significantly alleviated salt stress and promoted the biomass accumulation of *Rhizophora stylosa*. The effects were as follows: the fresh weight recovery rate was T3 > T2 > T1. The fresh weight of the T3 treatment group reached 137.9% of the CK group, an increase of 272.0% compared to the T0 group, and the T2 group increased by 29.5% compared to the T0 group. Dry weight also showed a similar trend, with the dry weight of the T3, T2, and T1 groups recovering to 124.3%, 85.8%, and 72.0% of the CK group, respectively. The T3 group was significantly higher than the T1 and T2 groups. This indicates that the addition of the three algal fertilizers has a positive effect on the biomass accumulation of *Rhizophora stylosa* under salt stress.

[0051] 3. Effects of different forms of microalgae fertilizers on root growth of white-awned rice under salt stress Root scans under salt stress under different forms of microalgae fertilizer treatment, such as Figure 3 As shown in Table 3, after salt stress treatment, all root indicators of T0 showed significant decreases to varying degrees, with root surface area and branching number being the most significant. The root growth status of each treatment group improved significantly after adding different forms of microalgae fertilizer.

[0052] Salt stress significantly inhibited the root growth of *Rhizophora stylosa*. The total number of roots and branches in the T0 group decreased sharply, only 10.5% and 9.6% of the CK group, respectively, indicating that salt ions severely inhibited the elongation of lateral root primordia. The total root length and total root surface area also decreased significantly, to 27.5% and 26.4% of the CK group, respectively. The elongation of the average root diameter was also hindered, with cells only swelling without elongation. After adding microalgae fertilizer, the total number of roots and branches in the T3 treatment group were significantly higher than the CK group, 1.69 times and 1.71 times that of the CK group, respectively. This indicates that the immobilized algae balls greatly stimulated the development of lateral roots in *Rhizophora stylosa*. The total root length surpassed that of the CK group, but the thinner individual roots resulted in a slightly lower total surface area. All indicators in the T1 and T2 groups were significantly lower than those in the CK group, consistent with the aboveground growth pattern. In contrast, while algae mud and algae powder had some promoting effect, the increase in root number was far less than that of algae balls.

[0053] Table 3 Root scan data Note: Plant height and root length are expressed as mean ± standard error. Different lowercase letters in the same column indicate significant differences between treatments (P<0.05).

[0054] 4. Effects of different forms of microalgae fertilizers on chlorophyll content in white rice under salt stress like Figure 4 As shown in Figure A, after 14 days of salt stress treatment, the chlorophyll a content in the CK group was 1.628 mg·g⁻¹. - ¹. Compared with the CK group, the chlorophyll a content in the T0 group treated with a single salt decreased to 0.760 mg·g⁻¹. - ¹, the decrease was 53.3%, indicating that salt stress severely damaged the photosynthetic system function of *Rhizophora stylosa*. The application of algae balls, algae mud, and algae powder all effectively alleviated salt stress, with groups T2 and T3 showing the most significant effects. Chlorophyll a content increased by 281.8% and 288.9% respectively compared to group T0, significantly higher than group T1. Notably, the chlorophyll a content of groups T2 and T3 was 2.142 mg·g⁻¹. - ¹, 2.196 mg·g - ¹ There was no significant difference between the treatments, with recovery rates of 131.6% and 134.9%, respectively. This indicates that algae additives help maintain the photosynthetic capacity of white rice. This may be because algae additives provide the nutrients needed for chlorophyll synthesis, improve the physiological state of the plant, and promote chlorophyll synthesis and stabilization. The effect of algae powder is relatively weak.

[0055] The chlorophyll b content of rice in each treatment group under salt stress is as follows: Figure 4 As shown in Figure B, group T3 had the highest chlorophyll b content, reaching 0.929 mg·g⁻¹. - ¹, significantly better than other treatment groups. In the T0 group under single salt stress, chlorophyll b content decreased to 0.461 mg·g⁻¹. - ¹, compared to the CK group, it decreased by 18.55%. Chlorophyll b in plants mainly constitutes the light-harvesting antenna complex, regulating the stability of photosynthetic structures and environmental adaptability. The above results indicate that salt ions may have a targeted destructive effect on photosystem II. All three types of algal fertilizer preparations showed repair effects, with the T3 treatment group showing the most significant effect.

[0056] Under salt stress, the total chlorophyll content and chlorophyll a content of rice in each treatment group showed similar trends. The total chlorophyll content in the control group was 2.194 mg·g⁻¹. - ¹. Compared with the CK group, the total chlorophyll content in the T0 group treated with salt alone decreased to 1.221 mg·g⁻¹. -¹, the decrease was 44.3%, with the T3 group showing the most outstanding effect, with the total chlorophyll content increasing by 255.9% compared to the T0 group, which was significantly higher than the T1 and T2 groups.

[0057] The addition of algae effectively maintains or even increases chlorophyll content.

[0058] 5. Effects of different forms of microalgae fertilizers on the changes in Fv / Fm and OJIP curves of white-awned rice under salt stress Depend on Figure 5 It was found that salt stress severely impaired the function of photosystem II (PSII) in white-awned rice, resulting in a significant decrease in maximum photochemical quantum yield (Fv / Fm). After application of microalgae fertilizer, all fluorescence parameters were improved.

[0059] Fv / Fm is a core indicator for measuring the degree of photoinhibition and potential photosynthetic capacity of plants. Under 50 mM NaCl stress, the Fv / Fm of the T0 treatment was 0.549, a decrease of 13.7% compared to the control, indicating that salt stress alone severely damaged the PSII reaction center in white rice, resulting in the most severe damage to the photosynthetic system. After adding different forms of microalgae fertilizer, Fv / Fm gradually recovered, and the T1, T2, and T3 treatments all exceeded the CK group, showing significant differences. The results indicate that different forms of microalgae fertilizer protected the photosynthetic system from salt ion toxicity and could even enhance the potential efficiency of PSII to a level higher than the normal treatment level through activation and repair pathways.

[0060] The morphological variations in the OJIP fluorescence kinetics curves of *Rhizophora stylosa* leaves under salt stress reveal the essential characteristics of photosystem II functional impairment and its repair mechanism. For example... Figure 5 As shown, compared with the smooth OJIP rise trajectory of the control CK group, the T0 group, subjected to single salt stress, was severely stressed, and its OJIP curve showed phase K, indicating that salt stress not only inhibited photosynthesis but also damaged the oxygen evolution complex (OEC) of rice. At point J, a significant fluorescence increase occurred at approximately 2 ms, while at point I, there was a lag of approximately 30 ms, and at point P (maximum fluorescence), the fluorescence decreased. These three abnormal phases reflect the multi-level damage of PSII by salt ions: the increase at point J indicates that electron transfer from the first quinone receptor (QA) to the second quinone receptor (QB) is blocked (receptor-side damage), the lag at point I reflects the obstruction of PQ library reduction, and the decrease at point P corroborates the increased degree of closure of the reaction center. The effects of adding different forms of microalgae fertilizers varied significantly: the overall fluorescence kinetic curve of the algae ball treatment group shifted downward, the K phase disappeared, and the J point dropped, indicating that the algae balls not only protected the OEC but also optimized electron transport; the algae mud group showed an I-point depression, suggesting that the PQ library reduction process was blocked and electrons were not transported smoothly to the downstream chain; the effect of algae powder was relatively weak, which is related to the previous antioxidant data.

[0061] 6. Effects of different forms of microalgae fertilizer on the antioxidant enzyme activity of white-awned rice seedlings under salt stress 6.1 Effects of different forms of microalgae fertilizer on catalase (CAT) activity in white-awned rice under salt stress Under salt stress, the dynamic changes in catalase (CAT) in rice characterize the response pattern of the plant's antioxidant defense system. CAT directly catalyzes the cleavage of hydrogen peroxide into water and oxygen, and its inhibition suggests that the enzyme protein structure is specifically damaged by salt ions. Figure 6 As shown in the results, 50 mM salt stress had a significant organ-specific effect on catalase (CAT) activity in white-awned rice seedlings. Compared with the control group (CK), the CAT activity in the leaves of the T0 group decreased from 33.66 U·g - ¹ decreased to 12.24 U·g - ¹, the decrease was 63.6%, reflecting that salt stress directly inhibited leaf enzyme activity. After adding different forms of algal fertilizer, CAT activity showed a mitigating effect: the T1 group restored leaf activity to 16.83 U·g. - ¹, the activity level in the leaves of the T2 group increased by 37.5% compared to the T0 group, but was still significantly lower than that of the CK group; the leaf activity in the T2 group further increased to 21.42 U·g - ¹, the activity increased by 75% compared to group T0, reaching 36.4% of the control (CK); group T3 showed the best effect, with leaf activity of 36.72 U·g. - ¹, exceeding the CK level, recovering to 109.1% of the CK, indicating that algal balls can differentially rebuild oxidative homeostasis through morphology-dependent mechanisms, and their special physical structure has a significantly better potential for salt stress remediation than algal mud and algal powder.

[0062] 6.2 Effects of different forms of microalgae fertilizer on peroxidase (POD) activity in white-awned rice under salt stress Under salt stress, the dynamic changes in peroxidase (POD) in rice characterize the response pattern of the plant's antioxidant defense system. POD participates in the oxidative coupling reaction of phenolic substances, alleviates membrane lipid peroxidation damage by decomposing hydrogen peroxide, and its increase reflects the activation of secondary detoxification pathways. Figure 7 As shown in the results, salt stress significantly altered the distribution of peroxidase activity in rice seedlings. Compared with the control group (CK), the POD activity in the leaves of the T0 group decreased from 638.66 U·g⁻¹. - ¹It dropped sharply to 323.17 U·g - ¹, the decrease was 49.40%, indicating that salt stress directly damaged the enzyme system in the leaves of white-awned rice. The addition of algal fertilizer showed a mitigating effect: in group T1, the leaf POD activity increased to 673.33 U·g. - ¹, the activity increased by 52% compared to group T0; the POD activity in group T2 further increased to 813.00 U·g - ¹, an increase of 60.25% compared to T0; the T3 group showed the best effect, with POD activity recovering to 885.33 U·g.- ¹, the increase was 63.50% compared to the T0 group, exceeding the 27.86% of the CK group. Significant differences were observed among the leaf treatment groups, indicating that sodium alginate algae balls reversed abnormal leaf activity by adsorbing salt ions. The results confirm that microalgae fertilizers can differentially regulate organ oxidative stress, with immobilized algae balls showing the best effect in alleviating salt ion toxicity.

[0063] 6.3 Effects of different forms of microalgae fertilizer on superoxide dismutase (SOD) activity in white-awned rice under salt stress Under salt stress, the dynamic changes in superoxide dismutase (SOD) in rice represent the response pattern of the plant's antioxidant defense system. SOD is the first line of antioxidant defense in plants, catalyzing the dismutation of superoxide anion free radicals into oxygen and hydrogen peroxide. Salt stress leads to increased SOD activity, which is a defensive response of plants to salt stress. Plants reduce oxidative damage by increasing SOD activity to scavenge superoxide anion free radicals. Figure 8 As shown, based on the experimental data analysis, 50 mM salt stress had a significant organ-specific effect on the SOD activity of white-awned rice seedlings. The SOD activity in the control group (CK) leaves was 143.667 U·g. - ¹, In the T0 group, leaf SOD activity surged to 489.333 U·g - ¹, the level increased by 240.6% compared to the control group, indicating that salt stress activated the leaf's antioxidant defense. After adding different forms of microalgae fertilizer, SOD activity showed a form-dependent repair: the T1 group reduced leaf SOD activity to 342.333 U·g. - ¹, compared to group T0, the SOD activity decreased by 30%; group T2 further reduced leaf SOD activity to 310.667 U·g. - ¹, a decrease of 36.5% compared to T0; group T3 showed the best effect, with leaf SOD activity of 234,000 U·g. - ¹, compared to group T0, the level decreased by 52.2%, recovering to 61.4% of the CK group. Significant differences were observed among the leaf treatment groups, indicating that the algal balls efficiently adsorbed salt ions through their porous structure, alleviating abnormal leaf activation by 61.4%. The results confirm that different forms of algal fertilizer can differentially regulate oxidative stress, with T3 showing significantly better repair efficacy on the leaf antioxidant enzyme system than T3 and T2.

[0064] 7. Effects of different forms of microalgae fertilizer on malondialdehyde (MDA) content in white-awned rice seedlings under salt stress Malondialdehyde (MDA) is a product of cell membrane lipid peroxidation, and its content reflects the degree of oxidative damage to the cell membrane. Under salt stress, the accumulation level of MDA in rice plants directly characterizes the severity of oxidative damage to the biomembrane. Figure 9As shown, single salt stress significantly induced MDA accumulation in white-awned rice seedlings. Compared with the control group (CK), after 14 days of stress, the MDA content in the leaves of the T0 group decreased from 4.700 μmol·g⁻¹. -1 Increased to 16.267 μmol·g -1 The increase was 246.1%, indicating that salt stress caused severe membrane lipid peroxidation damage.

[0065] Algal pellets, algal mud, and algal powder all showed significant alleviating effects on salt stress in *Rhizophora stylosa*. The overall alleviating effect was in the order T3 > T2 > T1, with the MDA content in group T1 being 11.485 μmol·g⁻¹. -1 The MDA content in group T2 was 6.122 μmol·g, a decrease of 29.4% compared to group T0. -1 The MDA content in group T3 was 5.382 μmol·g, a decrease of 62.4% compared to group T0. -1 The MDA content in the T3 group decreased by 66.9% compared to the T0 group. However, the MDA content in the leaves of the T3 group was only 14.5% higher than that of the CK group, showing no significant difference. These results indicate that 50 mM salt stress exacerbates oxidative damage in rice. Salt stress alone resulted in the highest MDA content and the most severe membrane damage. The MDA content decreased sequentially after adding different forms of microalgae fertilizer, and the degree of decrease corresponded to the effects of different algal morphologies. The MDA content in the T3 treatment group was close to that in the CK group, indicating that it almost completely protected the cell membrane from oxidative damage caused by salt stress.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An immobilized microalgae bio-fertilizer formulation, characterized in that, The immobilized microalgae bio-fertilizer formulation comprises microalgae cells and an embedding carrier, wherein the embedding carrier is composed of sodium alginate and polyvinyl alcohol.

2. The method for preparing the immobilized microalgae bio-fertilizer formulation according to claim 1, characterized in that, Includes the following steps: (1) Chlorella was cultured to the logarithmic growth phase, the algal cells were collected by centrifugation, washed with distilled water, and centrifuged again to obtain pure Chlorella algal mud; (2) Add sodium alginate and polyvinyl alcohol to distilled water, heat and stir until completely dissolved to obtain a carrier solution; (3) After the carrier solution cools down, add the Chlorella mud obtained in step (1), mix thoroughly, and obtain a mixed solution; (4) The mixture obtained in step (3) is dropped into calcium chloride solution to form gel balls. After static cross-linking, immobilized microalgae bio-fertilizer preparation is obtained.

3. The preparation method according to claim 2, characterized in that, The microalgae cells are Chlorella cells.

4. The preparation method according to claim 2, characterized in that, The mass ratio of sodium alginate to polyvinyl alcohol in step (2) is 1:(2~4).

5. The preparation method according to claim 2, characterized in that, The heating temperature in step (2) is 75~85℃.

6. The preparation method according to claim 2, characterized in that, In step (3), the cooling temperature is 35~45℃.

7. The preparation method according to claim 2, characterized in that, The mass-volume concentration of the calcium chloride solution in step (4) is 1.5~3.0%.

8. The preparation method according to claim 2, characterized in that, The static crosslinking time in step (4) is 18~30 h, and the temperature is 2~8℃.

9. The application of the immobilized microalgae bio-fertilizer preparation according to claim 1 or the immobilized microalgae bio-fertilizer preparation prepared by any one of claims 5-8 in alleviating crop salt stress.

10. The application according to claim 9, characterized in that, The concentration of the immobilized microalgae bio-fertilizer preparation is 0.5~2.0 g / L.

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