Spirodela- composite bacteria symbiotic system, and construction method and use thereof

Abstract

The present application relates to a kind of Spirodela-compound bacteria symbiotic system and its construction method and purposes, belong to wastewater treatment and resource utilization technical field;The construction method includes the following steps: S1: after domestication culture, Spirodela is accessed in aquaculture wastewater, and culture system is obtained;S2: Bacillus subtilis, Bacillus tequilensis, pseudomonas fluorescens are activated respectively, and after culture to OD 600 =1.0±0.1, three kinds of bacteria are mixed, and compound bacteria bacterial liquid is obtained;S3: compound bacteria bacterial liquid is accessed to culture system, and the Spirodela-compound bacteria symbiotic system is obtained.The Spirodela-compound bacteria symbiotic system constructed by the present application can efficiently remove nitrogen, phosphorus and other pollutants in livestock and poultry breeding wastewater, and significantly improve the crude protein content in duckweed biomass.The present application does not require complex equipment or high energy consumption process, and is simple and easy to operate, low in cost, suitable for on-site application in livestock and poultry farms.

Description

Technical Field

[0001] This invention relates to a duckweed-complex bacteria symbiotic system for treating aquaculture wastewater and enhancing duckweed protein, as well as its construction method and applications, belonging to the field of wastewater treatment and resource utilization technology. Background Technology

[0002] Livestock and poultry farming is a core pillar of the agricultural economy, playing an irreplaceable role in ensuring a stable supply of agricultural products such as meat, eggs, and milk, and promoting the implementation of the rural revitalization strategy. However, with the transformation of farming models towards large-scale and intensive operations, the discharge of large amounts of livestock and poultry wastewater has become increasingly prominent, posing a key challenge to the prevention and control of agricultural non-point source pollution. Statistics show that my country's annual discharge of livestock and poultry wastewater exceeds 10 billion tons. This wastewater is rich in pollutants such as nitrogen, phosphorus, and chemical oxygen demand (COD). If discharged directly without effective treatment, it will cause a series of ecological problems, including eutrophication of water bodies, soil degradation, and greenhouse gas emissions, severely restricting the green and sustainable development of the livestock and poultry industry. At the same time, the feed industry has long faced the pain points of protein raw material shortages and high dependence on imports (such as the high dependence on imported soybean protein). Developing new and sustainable protein feed raw materials has become a key breakthrough in ensuring the security of the livestock and feed industry chain.

[0003] Aquatic ecological restoration technology, with its dual benefits of environmental purification and resource recovery, has gradually become one of the mainstream directions for aquaculture wastewater treatment. Among them, Spirodela polyrhiza (S. polyrhiza, also known as purple duckweed), a plant in the Lemnaaceae family, has outstanding advantages such as rapid growth, strong nitrogen and phosphorus accumulation capacity, and convenient harvesting. Studies have shown that this duckweed can remove 40%–70% of total nitrogen (TN) and total phosphorus (TP) from aquaculture wastewater (Li et al., 2016), making it an excellent material for aquatic restoration. Simultaneously, its biomass is rich in nutrients such as protein and starch, possessing the potential for utilization as feed resources. However, the protein content of S. polyrhiza grown under natural conditions is generally only 15%–20%, far lower than conventional protein feed raw materials such as soybean meal (40%–50%), making it difficult to meet the nutritional requirements of large-scale feed production (Yosef et al., 2022). Therefore, how to simultaneously enhance the wastewater purification efficiency and nutritional quality of duckweed has become a core scientific issue in promoting the industrial application of duckweed.

[0004] In recent years, the synergistic effect of microbial-plant symbiosis has been considered an effective strategy to improve water remediation efficiency and promote plant nutrient accumulation. Functional microorganisms can form a mutually beneficial symbiotic system with plants by participating in nitrogen and phosphorus transformations (such as ammonification, nitrification, denitrification, and phosphorus dissolution) and secreting growth-promoting substances (such as plant hormones and siderophores), thereby significantly improving pollutant removal efficiency and promoting the increase of plant biomass and protein content (Adesemoye & Kloepper, 2009; Stottmeister et al., 2003). Based on this, constructing a duckweed-microbial symbiotic system for the treatment of livestock and poultry breeding wastewater has the dual potential of enhancing water purification and improving the feed value of duckweed.

[0005] At present, domestic and foreign research on duckweed-microbe symbiotic systems mostly focuses on the removal efficiency of single functional strains on a certain pollutant (such as ammonia nitrogen or phosphate) (Hemalatha & Mohan, 2022). The following core research gaps still exist: (1) There is a lack of research on the screening and construction of duckweed-complex bacteria symbiotic systems for the characteristics of high-load livestock and poultry breeding wastewater. The synergistic potential of different strain combinations, the inoculation ratio of bacteria and plants (dose-effect relationship), and the optimal process parameters under different application scenarios (such as wastewater concentration, hydraulic retention time, light and temperature) have not been clarified (Song et al., 2026); (2) Most existing studies only focus on the single goal of wastewater purification efficiency and fail to simultaneously achieve the synergistic regulation of efficient pollutant removal and duckweed feed quality (especially crude protein content), resulting in the treated duckweed biomass still being difficult to directly serve as a high-quality protein feed raw material (Hemalatha & Mohan, 2022). In summary, developing a duckweed-microorganism symbiotic system and its application method that can simultaneously enhance the purification efficiency of livestock and poultry breeding wastewater and the nutritional quality of duckweed protein has significant practical implications and industrial value. Summary of the Invention

[0006] This invention provides a duckweed-complex bacteria symbiotic system, its construction method, and its uses to solve the technical problems existing in the prior art as described above.

[0007] The technical solution provided by this invention is as follows: One objective of this invention is to provide a method for constructing a *Azolla*-complex microorganism symbiotic system, comprising the following steps: S1: After domesticating and cultivating duckweed, it is introduced into aquaculture wastewater to obtain a culture system; S2: Bacillus subtilis, Bacillus tekirae, and Pseudomonas fluorescens are activated and cultured separately, then mixed to obtain a compound bacterial solution; S3: Inoculate the compound bacterial solution into the culture system to obtain the duckweed-compound bacterial symbiotic system.

[0008] Based on the above technical solution, the present invention can be further improved as follows: Further, in step S1, the specific steps for the domestication of duckweed are as follows: the duckweed is rinsed clean with sterile water to remove attached impurities and plankton, and then domesticated and cultured in a culture medium.

[0009] Furthermore, the domestication conditions for the *Lemna minor* are: temperature 22±2℃, light intensity 4000±1000 lx, light-dark cycle 2:1, and pH 7.0~7.5. Furthermore, in step S1, the fresh weight of the duckweed introduced into the aquaculture wastewater is 0.5~0.7g / L, and the duckweed coverage rate on the water surface is 40~60%. Furthermore, in step S2, the volume ratio of the three types of bacteria is 1:1:1.

[0010] Further, in step S2, the three types of bacteria are cultured to OD levels respectively. 600 Mix after setting the concentration to 1.0 ± 0.1. Furthermore, in step S3, the volume ratio of the compound bacterial solution to the culture system is 5~10:1.

[0011] Furthermore, the aquaculture wastewater is biogas slurry from livestock and poultry farming.

[0012] The second objective of this invention is to provide a duckweed-complex bacteria symbiotic system constructed using the construction method described above.

[0013] The third objective of this invention is to provide the application of the above-mentioned duckweed-compound bacteria symbiotic system in the treatment of aquaculture wastewater and the enhancement of duckweed protein.

[0014] The technical solution provided by this invention has the following advantages compared with the prior art: 1. The duckweed-compound bacteria symbiotic system constructed in this invention can significantly increase the crude protein content in duckweed biomass while efficiently removing pollutants such as nitrogen and phosphorus from livestock and poultry breeding wastewater. After treatment, the crude protein content of duckweed can reach or approach the level of conventional protein feed raw materials, thereby achieving the resource utilization goal of "treating waste with waste and turning waste into treasure," overcoming the technical defects of existing technologies that only focus on the single purification effect and neglect the improvement of the nutritional quality of duckweed.

[0015] 2. This invention reveals the synergistic mechanism between the compound microbial system and duckweed in terms of nitrogen and phosphorus conversion, secretion of growth-promoting substances and protein accumulation. It provides new theoretical support for a deeper understanding of plant-microbe interactions to enhance water body restoration and resource conversion, and promotes research progress in the interdisciplinary field of water body ecological restoration and feed protein development.

[0016] 3. This invention requires no complex equipment or high-energy-consuming processes, is easy to operate, and has low cost, making it suitable for on-site application in livestock and poultry farms. This construction method not only reduces the pressure of treating livestock and poultry wastewater, but also simultaneously produces high-protein duckweed biomass, which can be used as raw material for aquatic or livestock and poultry feed, forming a green circular chain of "wastewater treatment - biomass production - feed application", providing practical and feasible technical support for the green and sustainable development of the livestock and poultry farming industry.

[0017] 4. The three strains in the composite bacteria of this invention form functional complementarity and metabolic coupling during wastewater purification: the ammonia nitrogen transforming strain (Bacillus subtilis) reduces the inhibition of free ammonia on phosphate-solubilizing bacteria and growth-promoting bacteria; the phosphate-solubilizing bacteria (Bacillus tekirae) release soluble phosphorus to promote the growth of growth-promoting bacteria and duckweed; and the extracellular polymers secreted by the growth-promoting bacteria (Pseudomonas fluorescens) provide microecological protection for the ammonia nitrogen transforming strain. The combined inoculation of these three bacteria has a significant synergistic effect, with significantly better pollutant removal rates and duckweed protein increases than single strains or combinations of two strains. Attached Figure Description

[0018] Figure 1 A comparison chart showing the removal efficiency of ammonia nitrogen by different symbiotic systems C-0, C-5, C-10, and C-20; Figure 2 A comparison chart showing the total nitrogen removal efficiency of different symbiotic systems C-0, C-5, C-10, and C-20; Figure 3 A comparison chart showing the removal efficiency of different symbiotic systems C-0, C-5, C-10, and C-20 for active phosphorus; Figure 4 A comparison chart showing the total phosphorus removal efficiency of different symbiotic systems C-0, C-5, C-10, and C-20; Figure 5 A comparison chart of crude protein content in *Lycoris radiata* in different symbiotic systems C-0, C-5, C-10, and C-20. Detailed Implementation

[0019] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0020] Example 1: Construction of the culture system The duckweed was rinsed three times with sterile water to remove attached impurities and plankton. It was then acclimatized and cultivated in aerated tap water for 7 days. The acclimatization conditions were controlled at a temperature of 22.0±2℃, a light intensity of 4000±1000 lx, a light-dark cycle of 16h:8h, and a pH of 7.2. After acclimatization, duckweed with consistent growth, uniform size, and no diseases or pests was manually selected.

[0021] The domesticated and selected duckweed was inoculated into biogas slurry for livestock and poultry farming at an initial fresh inoculation weight of 0.5914 g / L and an initial water surface coverage of about 50%, to obtain a culture system.

[0022] Example 2: Preparation of Compound Bacterial Solution Four *Lemna minor* plants were taken, and their roots were rinsed with about 10 ml of water. The rinsing solution was collected and diluted 100 times. 100 μL of the diluted solution was spread on nutrient agar (NA), lecithin-Tween 80 medium, and King's B (KB) medium, and incubated upside down at 37°C for 24 h (three replicates for each medium). Then, more than three single colonies were selected from each culture and purified twice by streak plating in four zones to obtain pure culture strains of *Bacillus subtilis*, *Bacillus tekirae*, and *Pseudomonas fluorescens*, respectively.

[0023] Three pure cultures of the bacterial strains were prepared into bacterial suspensions. The bacterial suspensions were cultured at 37℃ and 180 rpm for 24 h with shaking, and then cultured to the logarithmic growth phase, and adjusted to OD. 600 =1.0 (≈1×10) 8 The three bacteria were mixed in a volume ratio of 1:1:1 to prepare a compound bacterial solution (prepared immediately to ensure bacterial activity).

[0024] Example 3: Construction of the Azolla-Compound Bacterial Symbiotic System The compound bacterial solution obtained in Example 2 was inoculated into the culture system obtained in Example 1. The culture system and the compound bacterial solution were inoculated at a volume ratio of 5:1 and 10:1, respectively, to obtain the duckweed-compound bacterial symbiotic systems C-5 and C-10.

[0025] Comparative Example 1: The difference from Example 3 is that the culture system was not inoculated with the compound bacterial solution, resulting in system C-0.

[0026] Comparative Example 2: The difference from Example 3 is that the culture system and the compound bacterial solution were inoculated at a volume ratio of 20:1 to obtain the duckweed-compound bacterial symbiotic system C-20.

[0027] test: 10L of each of the symbiotic systems C-0, C-5, C-10, and C-20 obtained from Comparative Examples 1-2 and Example 3 were cultured in a light incubator for 14 days (water temperature 22±2℃, light intensity 4000±1000 lx, light-dark cycle 16h:8h, pH maintained at 8.2). During the experiment, ultrapure water was used daily to replenish the water lost by evaporation, and the system volume was kept constant.

[0028] Water samples were collected on days 0, 2, 4, 6, 8, 10, 12, and 14, with 50 mL collected each time. The samples were filtered through a 0.45 μm filter membrane and stored at 4°C. The tests were completed within 24 hours. Samples of duckweed were collected on days 0, 2, 5, 8, 11, and 14. The samples were rinsed three times with sterile water, the surface moisture was dried, and the fresh weight and dry weight were measured. The samples were then dried at 65°C to constant weight and tested.

[0029] The test monitors the ammonia nitrogen (NH4) in the water body periodically. + -N), nitrate nitrogen (NO) 3- -N), active phosphorus (PO4) 3- The pollutant removal rate is calculated based on the content of -P, using the following formula: R=[1-(C t / C0)]×100 Where: R is the corrected pollutant removal rate (%); C0 is the initial pollutant concentration in the treatment group (mg / L); C t The concentration of pollutants (mg / L) at time t in the treatment group.

[0030] 1. Removal efficiency of ammonia nitrogen in different symbiotic systems C-0, C-5, C-10, and C-20 The content of ammonia nitrogen was determined using Nessler's reagent method, and the results are as follows: Figure 1 As shown.

[0031] from Figure 1 The results show that during the entire 14-day culture period, the final removal rate of ammonia nitrogen in the compound bacterial treatment groups (C-5, C-10, and C-20) was over 95%, and was significantly higher than that in the sterile control group C-0 throughout the entire process (P<0.05). The removal rate of ammonia nitrogen in the control group was significantly lower than that in the compound bacterial treatment group (P<0.05), confirming that the compound bacterial system composed of Bacillus subtilis, Bacillus tekirae, and Pseudomonas fluorescens can significantly enhance the absorption and purification capacity of ammonia nitrogen in Duckweed.

[0032] In the compound bacterial treatment groups with different inoculation ratios, after 6 days of culture, the ammonia nitrogen removal rate of culture system C-5 rapidly increased to 56.4%. After 14 days of culture, the ammonia nitrogen removal rates of culture systems C-5, C-10, and C-20 reached over 95%, significantly higher than that of group C-0 (89%) (P<0.05). This indicates that the symbiotic bacteria and duckweed have formed a good balance and a virtuous cycle of mutual benefit and symbiosis.

[0033] 2. Total nitrogen removal efficiency of different symbiotic systems C-0, C-5, C-10, and C-20 The total nitrogen content was determined by alkaline potassium persulfate digestion and ultraviolet spectrophotometry. The results are as follows: Figure 2 As shown.

[0034] from Figure 2As can be seen, the total nitrogen purification rate of all treatment groups continued to increase with the progress of cultivation time, and there was no decline in the later stage. This is a direct manifestation of the characteristics of total nitrogen removal throughout the entire process.

[0035] The total nitrogen removal rate in the control group C-0 increased gradually throughout the treatment, reaching only 37.8% at day 8 and a final removal rate of 51.7% at day 14, the lowest among all groups. The treatment groups C-5 and C-10, inoculated with the compound symbiotic bacteria, showed significantly higher total nitrogen removal rates than the control groups C-0 and C-20, with final removal rates exceeding 60% at day 14, representing an increase of 21%–29% compared to the control group C-0. 3. Removal efficiency of different symbiotic systems C-0, C-5, C-10, and C-20 for active phosphorus The content of reactive phosphorus was determined by the ascorbic acid-molybdenum blue spectrophotometric method, and the results are as follows: Figure 3 As shown.

[0036] from Figure 3 As can be seen, the active phosphorus purification rate of all treatment groups continuously increased with the progress of cultivation time, without any decline in the later stage. The active phosphorus removal rates of treatment groups C-5, C-10, and C-20 inoculated with the compound symbiotic bacteria were significantly higher than those of the control group C-0 throughout the entire process from 0 to 10 days, showing a significant advantage.

[0037] 4. Total phosphorus removal efficiency of different symbiotic systems C-0, C-5, C-10, and C-20 The total phosphorus content was determined by ammonium molybdate spectrophotometry, and the results are as follows: Figure 4 As shown.

[0038] from Figure 4 It can be seen that the total phosphorus purification rate of all treatment groups showed a continuous linear upward trend with the progress of cultivation time, without any decline or rebound in concentration in the later stage, which reflects the irreversible removal characteristics of phosphorus with biological assimilation and fixation as the core.

[0039] The total phosphorus purification rate of the control group C-0 showed an increasing trend throughout the entire culture, with the rate of increase being slow at the beginning, rapid in the middle, and stable at the end. The purification rate was flat from 0 to 6 days, increased slightly from 6 to 10 days, decreased from 10 to 14 days, and began to stabilize. The final purification rate at 14 days was 57.2%, the lowest among all groups. At the end of 8 days of culture, the total phosphorus purification rates of the experimental groups C-5 and C-10, inoculated with the compound symbiotic bacteria, were significantly higher than those of the control groups C-0 and C-20 throughout the entire culture. At the end of 14 days of culture, the final total phosphorus purification rate of the C-10 group was the highest, reaching 59.5%, followed by C-5 at 58.9%, both higher than those of the control groups C-0 and C-20.

[0040] 5. Effects of different symbiotic systems (C-0, C-5, C-10, C-20) on the crude protein content of *Lemna minor*. The crude protein content of *Lemna minor* was determined using the Kjeldahl method, and the results are as follows: Figure 5 As shown.

[0041] from Figure 5 The results show that while purifying aquaculture wastewater, the nutritional quality of Duckweed was also improved. The crude protein content of Duckweed in all treatment groups showed a continuous linear increase with the progress of cultivation time, without any subsequent decline. This indicates that during the 14-day cultivation period, Duckweed maintained good growth and continuously assimilated nitrogen from the water into its own protein, resulting in a sustained improvement in nutritional quality over time. In all experimental groups inoculated with symbiotic bacteria, the crude protein content was significantly higher than that of the control group throughout the entire cultivation process, and the differences between groups continued to widen with the progress of cultivation time.

[0042] The crude protein content of the treatment groups inoculated with the compound symbiotic bacteria was significantly higher than that of the control group throughout the entire culture process, and the difference between the groups continued to widen as the culture time progressed. At 14 days, the crude protein content of groups C-5 and C-10 reached the highest of 38.9%, which was 8.4% higher than that of the control group C-0 (30.5%), and was comparable to the crude protein level of soybeans; it was also 3.3% higher than that of the control group C-20 (35.6%).

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a *Azolla*-complex microorganism symbiotic system, characterized in that, Includes the following steps: S1: After domesticating and cultivating duckweed, it is introduced into aquaculture wastewater to obtain a culture system; S2: Bacillus subtilis, Bacillus tekirae, and Pseudomonas fluorescens are activated and cultured separately, then mixed to obtain a compound bacterial solution; S3: Inoculate the compound bacterial solution into the culture system to obtain the duckweed-compound bacterial symbiotic system.

2. The method for constructing the duckweed-complex bacteria symbiotic system according to claim 1, characterized in that, In step S1, the specific steps for the domestication of duckweed are as follows: the duckweed is rinsed clean with sterile water to remove attached impurities and plankton, and then domesticated and cultured in a culture medium.

3. The method for constructing the duckweed-complex bacteria symbiotic system according to claim 2, characterized in that, In step S1, the domestication conditions for the duckweed are: temperature 22±2℃, light intensity 4000±1000 lx, light-dark cycle 2:1, and pH 7.0~7.

5.

4. The method for constructing the duckweed-complex bacteria symbiotic system according to claim 1, characterized in that, In step S1, the fresh weight of the duckweed introduced into the aquaculture wastewater is 0.5~0.7g / L, and the duckweed coverage rate on the water surface is 40~60%.

5. The method for constructing the duckweed-complex bacteria symbiotic system according to claim 1, characterized in that, In step S2, the volume ratio of the three bacteria is 1:1:

1.

6. The method for constructing the duckweed-complex bacteria symbiotic system according to claim 1, characterized in that, In step S2, the three types of bacteria are cultured to OD levels. 600 Mix after setting the concentration to 1.0 ± 0.

1.

7. The method for constructing the duckweed-complex bacteria symbiotic system according to claim 1, characterized in that, In step S3, the volume ratio of the compound bacterial solution to the culture system is 5~10:

1.

8. The method for constructing the duckweed-complex bacteria symbiotic system according to claim 1, characterized in that, The wastewater is biogas slurry from livestock and poultry farming.

9. A duckweed-complex bacteria symbiotic system, characterized in that, It is constructed using the construction method described in any one of claims 1 to 7.

10. The use of the duckweed-compound bacteria symbiotic system as described in claim 9 in the treatment of aquaculture wastewater and the enhancement of duckweed protein.

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