Vegetable-algae symbiosis method based on fish manure concentrated clear liquid

Abstract

The invention discloses a fish manure concentrated clear liquid-based vegetable-algae symbiosis method, which is characterized in that diluted fish manure concentrated clear liquid is placed in a transparent planting tank, the concentration of the diluted fish manure concentrated clear liquid is 25-50% of the concentration of the original fish manure concentrated clear liquid, chlorella pyrenoidosa is cultured in the planting tank, and meanwhile, the chlorella pyrenoidosa is cultured in the planting tank. Leaf vegetables are planted in the planting grooves through floating plates, planting cups for planting the leaf vegetables are arranged on the floating plates, and the leaf vegetables are planted in the planting cups. A culture wastewater microalgae treatment system and a vegetable water culture system are coupled to construct a vegetable-algae symbiotic system, oxygen is supplied to vegetable roots through O2 generated by microalgae photosynthesis, vegetable growth is promoted, and nutrient absorption efficiency is improved. Finally, the purpose of reducing the concentration of pollutants in the fish manure concentrated clear liquid is achieved, meanwhile, vegetables are produced, and the purpose of resource utilization of waste of the recirculating aquaculture system is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of resource utilization of fish manure concentrate, and particularly relates to a method for symbiotic cultivation of vegetables and algae based on fish manure concentrated supernatant. Background Art

[0002] A Recirculating Aquaculture System (RAS) can comprehensively apply technologies such as physics, biology, and chemistry in a factory workshop to adjust and control the living environment of aquaculture aquatic animals and plants, and achieve the recycling of treated aquaculture tail water. Compared with traditional pond aquaculture technology, RAS has the advantages of water saving, pond saving, high aquaculture density, and little influence from the external environment, and is the main development direction of aquaculture.

[0003] The baits used in aquaculture mainly use soybeans, corn, wheat bran, etc. as the main raw materials, and the crude protein content is mostly greater than 50%. The bait coefficient of RAS is generally 1.0 - 1.3, resulting in a large amount of unabsorbed nutrients being released into the aquaculture pond body. As a highly intensive aquaculture mode, the high-density and high feeding amount in RAS lead to more obvious fecal pollution with high nitrogen content in the water body compared with traditional pond aquaculture. Some studies have shown that for every 1.0 kg of feed fed, 0.25 - 0.50 kg of fish manure (including uneaten baits, etc.) and 0.02 - 0.04 kg of NH3 and NH4 + -N will remain in the water body.

[0004] Fish manure has the characteristics of being easily dispersed when soaked, floating easily, and having a small particle size, making it difficult to separate from the aquaculture water body, resulting in relatively few studies on RAS fish manure resource utilization technology. The applicant previously developed a combined process of "vertical flow sedimentation tank + high-pressure air flushing quantitative spraying concentrator" to achieve the efficient concentration of low-concentration fish manure, obtaining fish manure concentrated sediment (FS, TN > 2.57 ± 0.13 g / kg, C / N = 8 - 10:1, water content is 65 ± 2.3%) and fish manure concentrated supernatant (FW, TN > 350 mg / L, C / N = 5 - 7:1).

[0005] Using organic wastewater generated during agricultural production as an alternative medium required for microalgae cultivation can achieve the recycling of nutrients in the wastewater and reduce resource waste. FW is a typical nitrogen-rich organic wastewater and a potential alternative medium for the growth of Chlorella pyrenoidosa. However, the too high NH4 + -N concentration in FW will inhibit the growth process of microalgae.

[0006] Hydroponics is a planting technique that provides the water and nutrients required for plant growth by placing the roots of plants in water and supplying an appropriate amount of nutrient solution. The growth rate of hydroponic plants is generally 30%-50% faster than that of soil plants. It is the main development direction of future factory farming and also the main method for the resource treatment of aquaculture wastewater in a recirculating aquaculture system. Research shows that by configuring an aeroponics system during the hydroponic cultivation of vegetables and supplementing oxygen through tides or flow, the utilization efficiency of nutrients by hydroponic vegetables can be improved, enabling the rapid growth of hydroponic crops. However, due to the high cost of the air supplementation system equipment and the large amount of energy consumption during operation, it has hindered the development and application of hydroponic technology. Summary of the Invention

[0007] The present invention aims to solve the technical problems existing in the prior art and particularly innovatively proposes a method for symbiotic cultivation of vegetables and algae based on concentrated fish manure supernatant. It can achieve symbiotic cultivation of vegetables and algae while treating the pollutants in FW.

[0008] To achieve the above object, the present invention provides a method for symbiotic cultivation of vegetables and algae based on concentrated fish manure supernatant, which is characterized in that diluted concentrated fish manure supernatant is placed in a transparent planting tank, and the concentration of the diluted concentrated fish manure supernatant is 25-50% of the concentration of the original concentrated fish manure supernatant. Chlorella pyrenoidosa is cultured in the planting tank. At the same time, leafy vegetables are planted in the planting tank using a floating plate. The floating plate is provided with planting cups for planting leafy vegetables, and the leafy vegetables are planted in the planting cups.

[0009] In the above solution: the leafy vegetable is lettuce, and the daily light-dark ratio is controlled at 12-16:12-8h in the planting workshop.

[0010] In the above solution: a planting cotton for fixing the leafy vegetable is placed in the planting cup.

[0011] In the above solution: the temperature of the planting workshop is controlled at 25±2°C.

[0012] In the above solution: the entire planting and breeding cycle is 35 days. After every 35 days, the concentration of the liquid in the planting tank decreases. Part of the liquid in the planting tank is discharged, and then the corresponding new concentrated fish manure supernatant is supplemented to make the concentration of the liquid in the planting tank 25%-50% of the concentration of the original concentrated fish manure supernatant. Lettuce can be harvested after 35 days. After changing the water, new lettuce is planted.

[0013] In the above solution: the initial inoculation density of microalgae is 1.2×10 5 CFU / m.

[0014] As an important primary producer in the aquatic ecosystem, microalgae can absorb nitrogen-containing substances in aquaculture wastewater through photosynthesis, achieve wastewater treatment, and release a large amount of O2 at the same time. The present invention couples the microalgae treatment system for aquaculture wastewater and the vegetable hydroponic system to construct a vegetable-algae symbiotic system, which supplies oxygen to the vegetable roots through the O2 produced by microalgae photosynthesis, promotes vegetable growth, eliminates the need for an aeroponic system, and improves the nutrient absorption efficiency. Finally, it achieves the purpose of reducing the pollutant concentration in the fish manure concentrated supernatant, and at the same time co-produces lettuce, realizing the resource utilization of waste in the recirculating aquaculture system. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a schematic structural diagram of the vegetable-algae symbiotic system based on fish manure concentrated supernatant of the present invention.

[0016] Figure 2 For the changes in the concentrations of TN (A), NH4 + -N (B), TP (C), and COD (D) in FW in Example 1.

[0017] Figure 3 For the change in the oxygen content of the culture system in Example 1.

[0018] Figure 4 For the microalgae biomass in the culture systems with different FW concentrations in Example 1.

[0019] Figure 5 For the changes in Fv / Fm (A), Fv / F0 (B), and fluorescence parameter (C) in Example 1.

[0020] Figure 6 The changes in the concentrations of TN (A), NH4+-N (B), TP (C), and COD (D) in FW in Example 2.

[0021] Figure 7 For the change in the dissolved oxygen content of the vegetable-algae symbiotic system.

[0022] Figure 8 is a schematic diagram showing the effect of the vegetable-algae symbiotic system on microalgae biomass.

[0023] Figure 9 For the microalgae photosynthetic performance parameters Fv / Fm and Fv / F0 (A), ΦPSII (B), Qp (C), and NPQ (D).

[0024] Figure 10 For the MDA content (A) and SOD activity (B).

[0025] Figure 11 For the contents of polysaccharide (A) and protein (B) and protein / polysaccharide (PN / PS).

[0026] Figure 12It is the composition of microalgae cells.

[0027] Figure 13 It is the analysis of the microbial community structure at the phylum level.

[0028] Figure 14 It is the analysis of the microbial community structure at the genus level.

[0029] Figure 15 Venn diagram and correlation heat map of the microbial community structure.

[0030] Figure 16 It is the PCoA analysis of KEEG function based on Bray-Curtis (A) and the relative abundance of KEEG pathways (B).

[0031] Figure 17 It is the difference in the relative abundance of functions related to the carbon (A) and nitrogen (B) element cycles. Specific implementation manners

[0032] The present invention will be further described below through examples in combination with the drawings:

[0033] The concentrated fish manure clear liquid of the present invention is taken from the Factory Agriculture R & D Center of the Chongqing Academy of Agricultural Sciences. Sea bass is cultured with an annual output of 160 t. The feed is from Chongqing Haida Feed Co., Ltd. (particle size: 2.0 mm, crude protein ≥ 49%, crude fiber ≤ 3.5%, crude ash ≤ 18.0%, calcium: 0.8 - 4.0%, total phosphorus ≥ 1.2%, crude fat ≥ 5.0%, lysine ≥ 3.0%, moisture ≤ 10.0%), which is a typical nitrogen-rich bait. It is fed at a daily bait feeding amount of 2% of the fish body weight, and 0.25 - 0.50 kg of solid suspended matter is generated per 1 kg of bait fed. After the fish manure is filtered, the obtained FS (fish manure) has a moisture content of about 65%, COD of 17.32 ± 0.32 g / kg, TN of 2.57 ± 0.13 g / kg, NH4 + -N of 0.34 ± 0.17 g / kg, C / N = 8 - 10:1.

[0034] FW (concentrated fish manure clear liquid), COD is 2576 ± 27 mg / L, TN is 378.82 ± 13.41 mg / L, NH4 + -N is 156.31 ± 7.32 mg / L, and C / N is 5 - 7:1.

[0035] Example 1 Selection of the optimal concentration for microalgae treatment of concentrated fish manure clear liquid

[0036] In this example, we only discuss the relationship between Chlorella pyrenoidosa and the FW concentration, NH4 +Excessive concentrations of nutrients such as -N can inhibit the growth of microalgae. Therefore, the effects of nutrient concentrations in FW at different dilution ratios on the growth of microalgae were studied to determine the optimal FW concentration for microalgae treatment. The experiment was conducted in 2.5-L glass bottles, and the experimental settings are shown in Table 1. Four treatment groups were set up: FW, 75% FW, 50% FW, and 25% FW. Before the experiment started, 20 mL of Chlorella pyrenoidosa was centrifuged at 4000 g for 5 min, washed three times with 0.85% sterile sodium chloride to remove residual medium and broken algal cells, and inoculated into the glass bottles. The microalgae inoculation density was 2.4×106 CFU / mL. The bioreactor used was placed in an illumination incubator, with a culture temperature of 25±2 °C, a photon flux density of 80±0.5 μmol / m 2 ·s, a light-dark ratio of 16 h:8 h, and shaken three times a day for 30 s each time to prevent microalgae from settling or accumulating and ensure uniform light exposure of microalgae. Every 2 days, 10 mL of the microalgae mixture was taken to measure TN, NH4 + -N, TP, and COD; the DO online monitoring system was used to continuously monitor the O2 content in the hydroponic system; every 2 days, 10 mL of the algal solution was taken, wrapped with tin foil for 15 min for dark reaction, and then monitored for microalgae photosynthetic parameters such as Fv / Fm and Fv / F0; every 2 days, 2 mL of the algal solution was taken, centrifuged at 10000 g for 5 min, and used to measure the microalgae photosynthetic pigment content; at the end of the experiment, 5 mL of the algal solution was taken, centrifuged at 8000 g at 4 °C for 10 min, and the supernatant was discarded for the determination of enzyme activities such as ROS and SOD. The 30 mL of microalgae was rinsed three times with sterile NaCl with the same concentration as the microalgae culture system and then resuspended, water-bathed at 80 °C for 30 min, and centrifuged at 5000 g for testing the EPS of microalgae. Through the monitoring of the above indicators, the optimal FW concentration for microalgae treatment was determined.

[0037] Table 1 Experimental design for FW microalgae treatment

[0038]

[0039] 1. Influence of initial FW concentration on microalgae treatment performance

[0040] The nitrogen element in FW mainly exists in the form of NH 4 + -N. When the concentration of NH 4 + -N exceeds 100 mg / L, it may inhibit the growth of microalgae and even cause microalgae death. To determine the optimal FW for Chlorella pyrenoidosa treatment, the changes in TN and NH 4 + -N concentrations during the treatment process with different concentrations of FW were monitored, and the results are shown in Figure 2 A of Figure 2 and B of Figure 2. FromFigure 2 As can be seen from A, the denitrification effect of 25% FW is the best, followed by 50% FW. The TN concentration of 25% FW decreased from 95.21 mg / L to 4.18 mg / L within 12 d, and the degradation rate was 95.61%. The degradation rate of NH 4 + -N was 94.29%. As the FW addition increased to 50%, the degradation rates of TN and NH 4 + -N slightly decreased to 93.54% and 90.42%. When the FW addition was 75%, the degradation rates of TN and NH 4 + -N significantly decreased to 72.05% and 66.98%. Some studies have shown that when NH 4 + -N is higher than 100 mg / L, the amount of free ammonia in balance with the NH 4 + -N ion significantly increases. The molecular weight of free ammonia is small, and it accumulates in the cytoplasm through cell diffusion, which has an adverse effect on the physiological metabolism of microalgae cells, resulting in the inhibition of microalgae growth and the reduction of nitrogen degradation efficiency. The NH 4 + -N concentration in the 75% FW treatment group was 112.42 mg / L. The higher NH 4 + -N concentration inhibited the growth of Chlorella pyrenoidosa, resulting in the lowest degradation rate of NH 4 + -N in 75% FW.

[0041] Phosphorus is mainly used for the synthesis of proteins, nucleic acids and phospholipids and is an important component of microalgae cells. The concentration changes of TP during the treatment with different concentrations of FW are shown in Figure 2 C. As can be seen from the figure, as the FW concentration increases, the degradation rate of TP gradually decreases. At the 12th d, the degradation rates of TP were 97.33%, 95.92% and 78.81% respectively. Among them, there was no significant difference in the degradation rate of TP by Chlorella pyrenoidosa between 25% FW and 50% FW (P>0.05), but there was an obvious difference with 75% FW (P<0.05), indicating that too high a concentration of FW would inhibit the degradation efficiency of Chlorella pyrenoidosa for TP.

[0042] The degradation efficiency of Chlorella pyrenoidosa for COD under different FW dilution multiples is shown in Figure 2 D. As the initial COD increases, the degradation rate of COD by Chlorella pyrenoidosa gradually decreases. Among them, the degradation rates of COD in 25% FW and 50% FW were 72.24% and 67.94% respectively, while the COD degradation rate in 75% FW was only 47.7%. The decrease in COD degradation efficiency is mainly related to the high NH 4+ -N inhibited the growth of microalgae and was related to the reduction of microalgal physiological activity. At the same time, the FW used in this example was not strictly filtered. After adding high-concentration FW to the microalgae culture system, the turbidity of the system would increase, the light transmittance and light compensation point in the water body would decrease, thereby reducing the photosynthesis rate and resulting in a decrease in the degradation efficiency of Chlorella pyrenoidosa for nutrients. Therefore, 50% FW was the optimal concentration suitable for microalgae growth.

[0043] 2. Effects of Initial FW Concentration on Dissolved Oxygen in Microalgae Treatment System

[0044] Microalgae fix CO2 through photosynthesis and produce O2. Monitoring the O2 content can reflect the growth and photosynthetic activity of microalgae, and evaluate the physiological state and growth of microalgae. During the cultivation of Chlorella pyrenoidosa at different initial FW concentrations, the change process of DO was as Figure 3 shown. It can be seen from the figure that in the first 4 days of cultivation, the oxygen contents in the T1, T2, and T3 treatment groups all increased continuously, increasing from 1.45 mg / L before the reaction started to 2.32 mg / L, 2.69 mg / L, and 2.34 mg / L respectively. As the cultivation time extended, the oxygen contents in the T1 and T2 treatment groups continued to increase and reached the maximum values of 2.76 mg / L and 3.03 mg / L at the 6th day, while the oxygen concentration in the T3 treatment group continued to decrease. At the end of the reaction, the DO content was 0.69 mg / L, significantly lower than 2.23 mg / L and 2.44 mg / L at the end of the reaction in the T1 and T2 treatment groups. The DO concentration in the T3 treatment group first increased and then decreased sharply, indicating that microalgae had a certain adaptability to high-concentration FW in the early growth stage. As the cultivation time extended, toxic substances such as NH4 + -N accumulated in microalgal cells, resulting in the inhibition of microalgal physiological activities and the rapid death of microalgae; the DO concentrations in the T1 and T2 treatments first increased and then decreased, which was related to the sufficient nutrients in the early growth process of microalgae and the decrease in nutrient concentration in the later stage of the reaction, resulting in the gradual inhibition of microalgae growth.

[0045] 3. Effects of Initial FW Concentration on Microalgae Biomass

[0046] Photosynthetic pigments in microalgae, such as chlorophyll a and carotenoids, can guide the electron transfer in photosystem II by capturing light, participate in the regulation of physiological metabolism and gene expression, and are also important indicators for evaluating microalgae biomass. To evaluate the effects of different concentrations of FW on the photosynthetic performance of microalgae, the chlorophyll a and carotenoid contents of Chlorella pyrenoidosa in different concentration treatment groups were monitored, and the results were as Figure 4 shown. Figure 4The chlorophyll a content of microalgae under different FW concentrations was reflected. The chlorophyll a content in the 50% FW treatment group was the highest, reaching 7.02 mg / L on the 12th day, which was 1.36 and 3.03 times that of the 25% FW treatment group and the 75% FW treatment group, respectively. The decrease in chlorophyll a content in the 25% FW treatment group may be related to nutrient deficiency. It is worth noting that in the 75% FW treatment group, after 7 days of culturing Chlorella pyrenoidosa, the color of the microalgae in the culture system gradually changed from green to yellow, indicating that Chlorella pyrenoidosa could not carry out normal physiological activities in 75% FW, which may be related to the increase in the toxicity and turbidity of the culture medium at a higher FW concentration, resulting in a decrease in light penetration and photosynthesis. The inhibition of chlorophyll a synthesis reduced the potential of the microalgal photosynthetic reaction center and the ability to capture light energy, leading to impaired photosynthesis. As a non-enzymatic antioxidant, carotenoids have conjugated double bonds in their long molecular chains, which can absorb and consume the excitation energy of singlet oxygen, eliminate free radicals, protect cell membranes from damage, and thus protect the photosynthesis system. The change in carotenoid content is as Figure 4 shown. The content of carotenoids increased with the increase in FW concentration. At the end of the culture, the carotenoid contents under the culture conditions of 25% FW, 50% FW, and 75% FW were 3.77 mg / L, 3.94 mg / L, and 5.09 mg / L, respectively, indicating that under the 75% FW condition, more carotenoids were synthesized to resist oxidative stress, and the oxidative stress on Chlorella pyrenoidosa in 75% FW was the most obvious. The cumulative contents of chlorophyll a and carotenoids indicated that 50% FW was the ideal concentration for the growth of Chlorella pyrenoidosa.

[0047] 4. Effect of initial FW concentration on photosynthetic performance of microalgae

[0048] The Fv / Fm value is used to represent the maximum efficiency of chlorophyll to absorb and utilize light energy under light-saturated conditions, and is widely used to investigate the effects of light inhibition or various environmental stresses on light, and can quickly evaluate the adaptability of microalgae to the growth environment. During the whole culture process, the change process of Fv / Fm is as Figure 5As shown in A. During the entire cultivation process, the maximum value of Fv / Fm appeared under the cultivation condition of 50% FW, which was 0.663, while the maximum values of Fv / Fm for 25% FW and 75% FW were 0.614 and 0.332, respectively. Compared with the Fv / Fm (0.674) without adding FW, the Fv / Fm of 25% FW, 50% FW, and 75% FW decreased by 8.16%, 2.37%, and 45.8%, respectively. The Fv / Fm of Chlorella pyrenoidosa cultivated at 75% FW concentration decreased sharply in the early stage of cultivation, indicating a decrease in the energy conversion rate of the algae and inhibition of microalgae photosynthesis. In the later stage of cultivation, the Fv / Fm of Chlorella pyrenoidosa in 75% FW increased slowly, which was related to the fact that Chlorella pyrenoidosa had a certain resistance to the harsh environment and gradually adapted to the adverse environment. Fv / F0 is another important parameter for evaluating the change amplitude of chlorophyll fluorescence signal and can be used to characterize photosynthesis efficiency and plant physiological state. The results are as Figure 5 shown in B. During the cultivation process, Fv / F0 of each treatment group gradually increased. At the end of the cultivation, Fv / F0 in the 25% FW and 50% FW treatment groups were 2.53 and 2.30, respectively, while Fv / F0 in the 75% FW treatment group decreased to 1.75 at the end of the cultivation. The decrease in Fv / F0 indicates that the water-splitting complex on the donor side of Chlorella pyrenoidosa collapsed and the electron transport ability decreased. The effective photochemical quantum yield (ΦPSII) can reveal the physiological state of microalgae and the operating state of PSII under different environmental conditions. The change process of ΦPSII during the whole reaction process is as Figure 5 shown in C. With the increase of FW concentration, ΦPSII first increased and then decreased. At the end of the cultivation, ΦPSII of 25% FW, 50% FW, and 75% FW were 0.13, 0.26, and 0.06, respectively. The decreased ΦPSII may change the photochemical electron transport of PSII, which can be reflected in the changes of photochemical (Qp) and non-photochemical (NPQ) quenching coefficients. Qp is often used to evaluate the proportion of open PSII reaction centers

[273] . The change process is as Figure 5 shown in C. At the end of the cultivation, compared with the 25% FW treatment group, Qp in the 50% FW and 75% FW groups decreased by 8.82% and 20.6%, respectively, from 0.34 to 0.31 and 0.27, respectively. The decrease in Qp indicates that high concentration of FW inhibits the photochemical conversion efficiency and reduces carbon assimilation and assimilation energy. NPQ can quantify the dissipation process of the extra light absorbed in the form of heat and reflects the potential heat dissipation ability of PSII. The smaller NPQ is, the weaker the heat loss ability of microalgae and the lower the photosynthetic efficiency. The change process of NPQ is as Figure 5As shown in Figure C. With the increase in FW concentration, the NPQ of the 25% and 50% FW treatment groups gradually increased. On the 12th day, the NPQ of the 50% FW group reached 0.34, while the NPQ of the 75% FW group dropped sharply to 0.16, indicating that an appropriate concentration of FW can stimulate the relevant protection mechanisms of the PSII reaction center, and most of the light energy is dissipated in the form of heat. Excessive FW concentration will inhibit the light energy dissipation of Chlorella pyrenoidosa. Generally speaking, the light energy utilization, photochemical conversion, and light energy dissipation abilities of Chlorella pyrenoidosa are inhibited under the culture conditions of 75% FW.

[0049] Example 2

[0050] The vegetable-algae symbiotic system based on concentrated fish manure supernatant was carried out in a rectangular transparent polypropylene planting tank with high light transmittance, as Figure 1 shown. In this example, the length, width, and height of the planting tank are 28 cm, 17 cm, and 20 cm respectively, the total volume is 9.5 L, and the effective reaction volume is 6.0 L. Lettuce was fixed on a planting cup with planting cotton, and the planting cup was placed on a foam floating board. The experimental device was placed in a constant temperature light incubator (25 ± 2 °C), and the light-dark ratio (0:24 - 24:0 h) and light quantum intensity (0 - 150 μmol / m 2 ·s) were controlled by a timer.

[0051] In this example, a total of 3 treatment groups were set up, namely "FW + lettuce (CK)", "FW + Chlorella pyrenoidosa (T1)", and "FW + Chlorella pyrenoidosa + lettuce (T2)". Both the CK and T1 groups were used as controls, as Figure 1 shown. Before the experiment, 100 mL of Chlorella pyrenoidosa was centrifuged at 5000 g for 5 min. After removing the supernatant, the centrifuge tube was rinsed with FW and added to the planting tank. To avoid competition for nutrients between microalgae and vegetables, the initial inoculation density of microalgae was 1.2×10 5 CFU / mL, the FW volume of the whole reaction system was 6.0 L, and it was diluted by 50%. The initial fresh weight of the above-ground part of lettuce was 18.31 ± 1.12 g, and the planting density was 40 - 50 plants / m 2 . The light quantum intensity was controlled at 100 ± 0.5 μmol / m 2·s, with a light-dark ratio of 16:8 and a temperature of 25 ± 2 °C, to ensure the stable growth of microalgae in a suitable environment. Before the experiment, the planting tank, planting cup, foam floating board, planting cotton, etc. were disinfected by the combined process of ultraviolet + ozone. To determine the changes in the microbial community structure of the vegetable-algae symbiotic system, samples of the microorganisms in the microalgae liquid culture system, lettuce roots, planting cotton, and the surface of the foam floating board were taken and analyzed on the 0th day, 15th day, and 35th day. Among them, CKC represents the microorganisms on the surface of vegetable roots on the 0th day; Z1 and Z2 represent the growth of microorganisms in the pure algae culture system on the 15th day and 35th day respectively; CZ1, CZ2, and CZ3 represent the growth of microorganisms in the microalgae system on the 0th day, 15th day, and 35th day respectively; CZC, CZM, and CZP represent the microorganisms on the surface of vegetable roots, planting cotton, and foam floating board on the 35th day. The collected samples were centrifuged and stored at -80 °C for submission for inspection. Every 5 days, 10 mL of the microalgae mixed solution was taken to test TN, NH4 + -N, TP, and COD to evaluate the degradation effect of microalgae on FW; the DO online monitoring system was used to continuously monitor the O2 content of the hydroponic system; every 5 days, 10 mL of the algal solution was taken, wrapped with tin foil for 15 minutes for dark reaction, and then the photosynthetic parameters of microalgae such as Fv / Fm and Fv / F0 were monitored; at the end of the experiment, 5 mL of the algal solution was taken, centrifuged at 8000 g and 4 °C for 10 minutes, and the supernatant was discarded for the determination of enzyme activities such as ROS, SOD, and CAT, microalgae biomass, and microalgae cell composition; the microalgae were rinsed 3 times with sterile NaCl with the same concentration as the microalgae culture system and then resuspended, and centrifuged at 5000 g after water bath at 80 °C for 30 minutes for the determination of EPS of microalgae; at the end of the experiment, lettuce in different treatment groups was taken, and the effects of the vegetable-algae symbiotic system on vegetable growth were evaluated by comparing plant height, stem diameter, total leaf area, above-ground fresh weight, and root length.

[0052] 1. Removal performance of nutrients in FW by the vegetable-algae symbiosis

[0053] The change in pH in the hydroponic system can affect the solubility and availability of nutrient elements and also has an important impact on the activity of microorganisms in the hydroponic system. Due to the interaction between microalgae, vegetables, and bacterial populations, the change in pH in the vegetable-algae symbiotic system may be more obvious than that in the traditional hydroponic system. During the FW degradation process, the changes in pH of different treatment groups are as shown in Figure 6 Figure A. The pH of each treatment group first decreased and then increased. On the 10th day, the pH of each treatment group reached the lowest value, which were 6.87, 7.11, and 7.18 respectively. Subsequently, the pH of each treatment group gradually increased, and at the end of the reaction, the pH of each treatment group increased to above 8.0. There was a significant difference in the lowest pH value between the CK group and the T1 and T2 treatment groups, which was related to the fact that lettuce usually absorbs nitrogen in the form of NO3 - -N, while microalgae absorb more NH4+ -N is related to the absorption of nitrogen. In the hydroponic system, microalgae consume inorganic carbon such as HCO3 in the water through photosynthesis - , resulting in the accumulation of OH - , thus increasing the pH. The consumption of NH4 + -N produces H + , leading to a decrease in pH. In the early stage of the reaction in the T1 and T2 treatment groups, nutrients were sufficient, and the consumption rate of NH4 + -N was relatively fast. The concentration of OH - produced by photosynthesis was lower than the H + produced by the degradation of NH4 + -N, resulting in a gradual decrease in pH. As the reaction proceeded, NH4 + -N was gradually consumed, and the OH - accumulated by the photosynthesis of microalgae was gradually higher than the H + produced by the degradation of NH4 + -N, causing the pH in the hydroponic system to continuously increase to 8.0. Generally, the pH tolerance range of hydroponic plants is 5.5 - 7.5. When the pH exceeds this range, the ability of plants to utilize nutrients will be reduced. However, in the present invention, the plants can grow normally within the range of pH > 7.5, which is due to the interaction between the roots of lettuce and bacteria in the water, improving the pH tolerance of lettuce, thereby realizing the absorption of nutrients and the normal growth of lettuce under high pH conditions.

[0054] The change process of COD during the hydroponic process is as shown in Figure 6 B. At the end of the hydroponic experiment, the COD degradation rates of the CK, T1, and T2 treatment groups were 48.37%, 31.25%, and 83.66% respectively. The removal of COD mainly relies on the synergistic effect of bacteria and vegetables. Among them, root microorganisms play an important role in the absorption and degradation of organic pollutants. The degradation rate of COD in the T1 treatment group was the lowest because microalgae are typical autotrophic microorganisms that mainly use CO2 as a carbon source for photosynthesis and have limited ability to utilize organic matter. The degradation rate of COD in the T2 treatment group was the highest, which may be due to the formation of an efficient synergistic mechanism between microalgae and vegetables, promoting the growth of lettuce root microorganisms, thereby promoting the degradation of organic matter in the hydroponic system. The root length and the change of microbial community structure further confirmed the synergistic effect between lettuce and microalgae. The degradation of NH4 + -N in the hydroponic system is as shown in Figure 6 C. In the first 15 days, the degradation rates of NH4 + -N in the T1 and T2 treatment groups were 5.12 mg / L·d and 3.68 mg / L·d respectively, which were 4.37 times and 3.14 times that of the CK treatment group. This is because microalgae tend to absorb NH4 + -N, while vegetables tend to absorb NO3- -N is consistent with the conclusion. The TN removal rate is as Figure 6 shown in C. At the end of the hydroponics, the degradation rates of TN in the CK, T1, and T2 treatment groups were 60.76%, 53.52%, and 85.60% respectively. The removal of phosphorus is related to the precipitation of phosphorus and the fixation of microorganisms. At the end of the hydroponics, the removal rates of TP in the CK, T1, and T2 treatment groups were 75.02%, 78.68%, and 98.57% respectively ( Figure 7 in D). The above results indicate that a good synergy has been formed between vegetables and algae, improving the removal rates of main nutrients such as N and P, and achieving the efficient degradation ability and resource utilization of nutrients.

[0055] 2. Effects of vegetable-algae symbiosis on dissolved oxygen

[0056] Using the O2 produced by microalgae photosynthesis to supply oxygen to the lettuce roots and realizing the co-growth of lettuce and microalgae is the main goal of constructing a vegetable-algae symbiotic system. To determine the oxygen production of microalgae and oxygen consumption of lettuce in the vegetable-algae symbiotic system, the DO content at different growth stages of the vegetable-algae symbiotic system was monitored, and the results are as Figure 7 shown. It can be seen from the figure that in the first 10 days of the reaction, there was no significant difference in the DO content between the T1 and T2 treatment groups (P>0.05). As the time of vegetable-algae symbiosis extended, the DO content in the T1 treatment group continued to increase. At the 20th day, the DO content reached the highest value of 3.32 mg / L and then gradually decreased. Different from T1, from the 10th day to the 35th day, the DO content in the T2 treatment group first decreased and then increased slowly, and reached the lowest value of 1.54 mg / L at the 25th day. From the 5th day to the 35th day, the oxygen consumption of the vegetable-algae symbiotic system was 0.19 mg / L, 0.12 mg / L, 0.12 mg / L, 0.72 mg / L, 1.39 mg / L, 1.2 mg / L, 0.89 mg / L, and 0.65 mg / L respectively. The oxygen consumption of the vegetable-algae symbiotic system first increased and then decreased, indicating that the lettuce roots consumed a certain amount of oxygen during the growth process, and there were significant differences in the oxygen consumption at different growth stages.

[0057] 3. Effects of vegetable-algae symbiosis on the biomass of lettuce

[0058] Taking the plant height, stem diameter, total leaf area, above-ground fresh weight, and root length of lettuce as verification indicators respectively, the growth of lettuce in the algae-present / algae-absent treatment groups was compared. The growth of lettuce at the 35th day is shown in Table 2.

[0059] Table 2 Comparison of lettuce growth indicators

[0060]

[0061] Compared with the CK treatment group, the plant height, stem diameter, total leaf area, aboveground fresh weight, and root length of the T2 treatment group increased by 9.26%, 7.44%, 12.01%, 19.11%, and 6.07% respectively, indicating that there is a good synergistic effect between the vegetable and the microalgae, and the growth promotion effect on the total leaf area and aboveground fresh weight is the most significant.

[0062] The contents of soluble sugar, soluble protein, vitamin C, and NO3 - -N in the harvested lettuce on the 35th day were detected, and the results are shown in Table 3.

[0063] Table 3 Comparison of lettuce quality

[0064]

[0065] Compared with the CK treatment group, the contents of soluble sugar, soluble protein, and vitamin C in the lettuce of the T2 treatment group increased by 12.72%, 9.21%, and 27.78% respectively. Aerating the culture solution during the hydroponic process will significantly increase the lettuce leaf area, soluble sugar, yield, etc. Due to the photosynthesis of microalgae, the dissolved oxygen concentration in the T2 treatment group remained above 1.5 mg / L under light conditions, while the dissolved oxygen content in the CK treatment group was generally lower than 0.6 mg / L. The higher dissolved oxygen content in the T2 treatment group had a certain promoting effect on the rapid growth of lettuce. NO3 - -N content is an important indicator for evaluating the safety quality of vegetables. NO3 - -N is reduced to NO2 after entering the human body - -N. NO2 - -N can not only bind to hemoglobin and reduce the oxygen-carrying capacity of hemoglobin, but also react with amine compounds under the action of gastric acid to generate N-nitroso compounds, causing nucleic acid metabolism disorders and inducing organ cancer. The NO3 - -N concentration in the T2 treatment group decreased by 60.01% compared with the CK treatment group, indicating that the vegetable-microalgae symbiotic system can not only increase the nutrient concentration of vegetables, but also reduce the concentration of potential pathogenic substances in vegetables, improve the quality of lettuce, and be beneficial to human health.

[0066] 4. Effects of vegetable-microalgae symbiosis on microalgae biomass

[0067] The contents of photosynthetic pigments in microalgae, such as chlorophyll a, chlorophyll b, and carotenoids, play important roles in key photosynthetic reactions such as light energy capture, energy absorption, and energy conversion, and are important indicators for measuring microalgae biomass. During the entire hydroponic process, the change trends of microalgae photosynthetic pigments in the T1 and T2 treatment groups are as Figure 8As shown. During the entire hydroponic process, the photosynthetic pigments in the T1 and T2 treatment groups gradually increased. At the end of the hydroponics, the contents of chlorophyll a, chlorophyll b, and carotenoids in the T2 treatment group increased by 13.89%, 21.15%, and 18.31% respectively compared with the T1 treatment group. This may be because the respiratory process of lettuce roots and the formation of plant secretions increased the content of stable soluble carbon in the culture system, thus achieving the rapid growth of microalgae.

[0068] 5. Effects of Lettuce-Algae Symbiosis on the Photosynthetic Performance of Lettuce and Algae

[0069] Fv / Fm is the maximum photochemical efficiency of PSII, which reflects the potential maximum photosynthetic capacity (photosynthetic efficiency) of plants and is one of the main evaluation factors for studying the growth stress of plants under various environmental factors. During the entire hydroponic process, the changes in Fv / Fm of the T1 and T2 treatment groups are as Figure 9 shown in A. The Fv / Fm of the T1 treatment group always remained above 0.591, and the maximum value was 0.631. The maximum value of Fv / Fm in the T2 treatment group was 0.663, and the minimum value was 0.587. This indicates that the construction of the lettuce-algae symbiotic system does not have a significant impact on the growth of microalgae. The potential activity of the photosynthetic reaction center PSII is mainly represented by Fv / F0, and the change of this value is closely related to the photosynthetic electron transport ability. As can be seen from Figure 9 A, the Fv / F0 of the T1 and T2 treatment groups generally showed a trend of increasing first and then decreasing during the entire hydroponic process, and the maximum value (1.44) of Fv / F0 in the T2 treatment group was significantly higher than that in the T1 (1.26) treatment group (P < 0.05). At 30 - 35 d, the Fv / F0 of the T1 and T2 treatment groups were 1.21 and 0.93 respectively, and the Fv / F0 of the T2 treatment group was significantly lower than that of the T1 treatment group. This indicates that in the early stage of hydroponics, the lettuce-algae symbiotic system promoted the electron transport efficiency of microalgae. With the progress of the reaction, the gradual decrease in nutrient concentration and the change in the microbial community structure led to the inhibition of microalgae growth and the reduction of electron transport rate. The changes in ΦPSII, Qp, and NPQ of microalgae during the hydroponic process were further analyzed, as shown in Figure 9As shown in B of 9 and C and D of 9. It can be seen from the figure that ΦPSII in the T1 treatment group remained basically stable throughout the hydroponic process, with ΦPSII varying within the range of 0.159 - 0.176, while ΦPSII in the T2 treatment group first increased and then decreased during the whole hydroponic process, with the change range of 0.126 - 0.192, which was consistent with the change law of Fv / F0. Qp is often used to evaluate the proportion of open PSII reaction centers. Qp in the T1 treatment group gradually decreased, and Qp in the T2 treatment group first increased and then decreased, and the lowest value was lower than that of the T1 treatment group, indicating that the vegetable-algae symbiotic system promoted the light conversion efficiency of microalgae in the early stage of hydroponics and inhibited the light conversion process of microalgae in the later stage. NPQ is used to reflect the potential heat dissipation ability of PSII. The NPQ values of both the T1 treatment group and the T2 treatment group showed a trend of first increasing and then decreasing. Among them, the maximum value of NPQ in the T1 treatment group was 0.32, which was significantly lower than 0.368 of the T2 treatment group (P < 0.05), while the minimum value of NPQ in the T2 treatment group (0.229) was significantly lower than the minimum value of NPQ in the T1 treatment group (0.261) (P < 0.05), indicating that the vegetable-algae symbiotic system could stimulate the relevant protection mechanisms of PSII reaction centers in the early stage of hydroponics, and most of the light energy was dissipated in the form of heat. Generally speaking, the light energy utilization, photochemical conversion and light energy dissipation abilities of Chlorella pyrenoidosa under the vegetable-algae symbiotic system first increased and then decreased, and fluctuated greatly during the whole hydroponic process. At the same time, the maximum value of the photosynthetic efficiency of microalgae in the vegetable-algae symbiotic system was significantly higher than that in the pure-algae culture system, and the minimum value was significantly lower than that in the pure-algae culture system, indicating that the vegetable-algae symbiotic system had a greater impact on the growth of microalgae, but overall it showed a promoting effect.

[0070] 6. Effects of Vegetable-Algae Symbiosis on Microalgae Enzyme Activity

[0071] The dynamic balance of intracellular ROS generation and elimination plays an important role in maintaining the normal physiological functions of organisms. During the whole hydroponic process, the change processes of two typical antioxidant enzymes, MDA and SOD, in microalgae cells are as follows Figure 10as shown by A of [object] and B of 10. In the first 15 days of hydroponics, the contents of MDA and SOD in the T1 treatment group were always higher than those in the T2 treatment group. Within the 15th - 25th day, the contents of MDA and SOD in the T2 treatment group increased rapidly. Among them, MDA increased from 4.41 nmol / mgprot to 5.91 nmol / mgprot, and SOD increased from 3.23 nmol / mgprot to 4.42 nmol / mgprot. The growth rates reached 0.15 nmol / mgprot·d and 0.12 nmol / mgprot·d respectively, which were significantly higher than 0.034 nmol / mgprot·d and 0.016 nmol / mgprot·d of the T1 treatment group. MDA is an important indicator to evaluate the degree of cell lipid peroxidation and damage. The MDA value in the T2 treatment group increased significantly in the later stage of cultivation, indicating that the growth of microalgae cells was significantly inhibited in the later stage of cultivation, and microalgae secreted more SOD to cope with cell damage caused by oxidative stress.

[0072] 7. Effects of Vegetable - Algae Symbiosis on Microalgal Extracellular Polymeric Substances

[0073] EPS is a complex mixture secreted by microalgae outside the cells, mainly composed of substances such as polysaccharides and proteins. The ratio of protein to polysaccharide (PN / PS) in TB - EPS affects the hydrophobicity or viscosity of EPS. Generally speaking, a higher PN / PS is beneficial to the natural sedimentation of microalgae and improves the recycling efficiency of microalgae. The relatively high hydrophobicity on the surface of microalgae helps to promote the formation of bacteria - algae particles and improve the stability and rapid recycling of the bacteria - algae system. During the whole hydroponics process, the change processes of PN and PS in TB - EPS of the T1 and T2 treatment groups are as Figure 11As shown in the figure, with the extension of the hydroponic time, PN and PS in each treatment group gradually increased, and the protein content was always higher than the polysaccharide content. In the T1 treatment group, PN and PS increased from the initial 1.6 mg / L and 4.3 mg / L to 2.5 mg / L and 5.6 mg / L respectively, with growth rates of 0.026 mg / L·d and 0.037 mg / L·d respectively. While in the T2 treatment group, the PN and PS values increased to 2.11 mg / L and 6.55 mg / L respectively, with growth rates of 0.015 mg / L·d and 0.064 mg / L·d respectively. The growth rates of PN and PS in the T1 and T2 treatment groups showed opposite trends, that is, the growth rate of PN in the T1 treatment group was significantly higher than that in the T2 treatment group (P < 0.05), while the growth rate of PS in the T1 treatment group was significantly lower than that in the T2 treatment group (P < 0.05). This indicates that the co-culture of microalgae and vegetables will significantly change the proportion of the main components in the microalgae TB-EPS. At the same time, with the extension of the hydroponic time, the PN / PS in the T1 treatment group gradually decreased from 0.37 to 0.34, and the PN / PS in the T2 treatment group gradually increased from 0.38 to 0.44. The higher PN / PS value indicates that the microalgae in the vegetable-algae symbiotic system have stronger hydrophobicity, can improve the self-flocculation ability, and are conducive to the efficient recovery and resource utilization of microalgae in the follow-up. The total amounts of TB-EPS in the T1 and T2 treatment groups at the 35th day were 9.31 mg / L and 6.86 mg / L respectively, which further indicates that the vegetable-algae symbiotic system is conducive to the growth and biomass accumulation of microalgae.

[0074] 8. Influence of Vegetable-Algae Symbiosis on Microalgae Cell Composition

[0075] To evaluate the application value of Chlorella pyrenoidosa under the co-culture mode of lettuce and Chlorella pyrenoidosa, the change process of the main high-value components (protein, lipid, and polysaccharide) of Chlorella pyrenoidosa during the whole hydroponic process was monitored, and the results are as Figure 12 shown. With the increase of the hydroponic time, the protein in the T1 treatment group ( Figure 12 A) gradually increased, from 51.9% on the 0th day to 55.5% on the 35th day, while in the T2 treatment group ( Figure 12B) The protein content first increased and then decreased during the overall hydroponic process, reaching the maximum value (57.4%) on the 15th day. The gradual decrease in the protein content of microalgae cells in the T2 treatment group during the later stage of hydroponics might be caused by nitrogen limitation or nitrogen starvation in the culture solution during the later stage of hydroponics. This is consistent with the change trend of TN concentration. At the end of hydroponics, the TN concentrations in the T1 and T2 treatment groups were 109.78 mg / L and 27.31 mg / L, respectively. The lipid content in the T1 treatment group increased slowly during the entire hydroponic process, while in the T2 treatment group, it rapidly increased from 13.4% to 15.9% from the 15th to the 35th day of hydroponics. Generally, when the growth of microalgae is under stress or limitation, the lipid content of microalgae increases instead, which is consistent with the conclusion that the microalgae in the vegetable-algae symbiotic system are under stress during the later growth stage. The polysaccharide content in the T1 and T2 treatment groups remained basically stable during the entire hydroponic process, indicating that adding a vegetable hydroponic unit to the microalgae cultivation system will have a significant impact on the protein and lipid contents of microalgae cells and has no significant impact on the polysaccharide content.

[0076] 9. Microbiome Analysis of Vegetable-Algae Symbiosis

[0077] 9.1 Relative Abundance of Microorganisms

[0078] Microorganisms are key components of the vegetable-algae symbiotic system and participate in important biogeochemical cycles in the system. To understand the functions and activities of different microorganisms, the microorganisms in the vegetable-algae symbiotic system were analyzed. Alpha diversity can reflect the diversity of species, as shown in Table 4. The coverage index in all samples in the vegetable-algae symbiotic system reached above 0.97, indicating that the sequencing results of bacteria in the samples were valid. CKC and CZC respectively reflect the changes in the microorganisms on the surface of vegetable roots on the 0th day and the 35th day. At the end of hydroponics, the OTUs, ACE, and Chao1 on the surface of vegetable roots increased from 13,090, 13,106, and 13,099 on the 0th day to 14,351, 14,371, and 14,358 on the 35th day, and the Shannon index increased from 6.13 to 6.80. This shows that the vegetable-algae symbiotic system is beneficial to increasing the number and species diversity of microorganisms on the surface of vegetable roots. The changes in the microbial diversity of microalgae, roots, colonization cotton, and foam floating boards at the end of the cultivation in the vegetable-algae symbiotic system were compared. At the end of cultivation, the number of microorganisms showed CZP > CZC > CZM > CZ3, and the species richness was CZM > CZC > CZP > CZ3, indicating that the foam floating board is more conducive to the increase in the number of microorganisms, and the colonization cotton is more conducive to improving the microbial diversity. Compared with the Z2 treatment group at the end of the pure algae cultivation system, CZ3 had an increase in both the number and diversity of microorganisms, indicating that the co-cultivation of vegetables and microalgae has a greater impact on the microbial community structure of the cultivation system.

[0079] Table 4 Microbial Diversity and Richness

[0080]

[0081] To determine the differences in microorganisms on the surface of microalgae culture solution, vegetable roots, and the colonization cotton and foam floating boards used for vegetable colonization in the vegetable-algae symbiotic system, samples were taken on the 0th day, 15th day, and 35th day of the culture to observe the change process of microorganisms. The changes in microorganisms at the phylum level are as follows Figure 13 shown. With the increase of culture time, the relative abundance of Proteobacteria in the microalgae culture system first decreased and then increased. On the 0th day, 15th day, and 35th day, the relative abundances of Proteobacteria were 10.75%, 5.63%, and 20.82% respectively; the relative abundance of Bacteroidetes gradually increased during the whole culture process, being 2.50%, 5.45%, and 14.37% respectively. The relative abundances of Proteobacteria and Bacteroidetes in the vegetable-algae symbiotic system continued to increase during the whole culture process. The relative abundances of Proteobacteria on the 15th day and 35th day were 12.90% and 35.17% respectively, and the relative abundances of Bacteroidetes on the 15th day and 35th day were 16.03% and 24.23% respectively. At the 35th day, the relative abundances of Proteobacteria and Bacteroidetes in the vegetable-algae symbiotic system increased by 14.35% and 9.86% respectively compared with the microalgae culture system. Proteobacteria and Bacteroidetes are closely related to the utilization of glucose, small molecule organic acids, etc., indicating that compared with the microalgae culture system, the vegetable-algae symbiotic system is more conducive to the degradation and utilization of organic matter. Compared with the microorganisms on the vegetable surface roots on the 0th day, on the 35th day, the relative abundances of Proteobacteria and Firmicutes on the vegetable root surface decreased from 58.94% and 10.28% to 23.73% and 0.89% respectively. There were significant differences in the attached microorganisms on the surfaces of CZM and CZP. Compared with CZM, the relative abundances of Proteobacteria, Bacteroidetes, and Chloroflexi on the CZP surface increased by 6.74%, 11.86%, and 1.70% respectively.

[0082] The changes in microorganisms at the genus level are as follows Figure 14As shown. Flavobacterium is a conditional pathogen that can cause fish gill rot disease in fish and is also the main microorganism for decomposing refractory organic matter. With the increase of culture time, the relative abundances of Flavobacterium in the microalgae culture system and the vegetable-algae symbiotic system both showed a trend of decreasing first and then increasing. Among them, the relative abundances of Flavobacterium in the microalgae culture system at 0 d, 15 d, and 35 d were 3.33%, 0.238%, and 2.43% respectively, and the relative abundances of Flavobacterium in the microalgae culture system in the vegetable-algae symbiotic system at 0 d, 15 d, and 35 d were 3.33%, 0.82%, and 5.99% respectively. This indicates that the vegetable-algae symbiotic system will increase the relative abundance of Flavobacterium. The water body after vegetable-algae symbiosis needs to go through a strict sterilization environment to avoid the impact of Flavobacterium on the organs such as the skin, fins, and gills of fish. Aequorivita is a microorganism that can improve the degradation ability of COD and nitrogen. At 0 d, the relative abundance of Aequorivita in the microalgae culture system was only 0.009%. At the end of the hydroponic culture, the relative abundances of Aequorivita in the microalgae culture system and the vegetable-algae symbiotic culture system increased to 0.24% and 3.52% respectively. The microbial community structures such as Pseudomonas, Malikia, and Arcobacter on the surface of the vegetable roots in the vegetable-algae symbiotic system changed significantly. Compared with CKC, at 35 d, the relative abundances of Pseudomonas, Malikia, Arcobacter, Fusobacterium, and Pelosinus in the CZC treatment group decreased by 2.47%, 9.40%, 8.11%, 5.96%, and 2.75% respectively, which indicates that the addition of microalgae has a significant impact on the microorganisms on the surface roots of vegetables. In each treatment group of the vegetable-algae symbiotic system, the relative abundance of Shewanella in the CZ3 treatment group was 11.75%, which was significantly higher than 1.73%, 0.78%, and 3.06% in the CZC, CZM, and CZP treatment groups. In the CZ2 treatment group, the relative abundance of Shewanella was extremely low (1.75×10-4%). Shewanella is a typical denitrifying microorganism

[287] . The significant increase in the relative abundance of the CZ3 treatment group indicates that the addition of microalgae increases the enrichment of Shewanella, a microorganism with high denitrification function, which is beneficial to the efficient utilization of nitrogen in the water body. At the end of the hydroponic culture, the Venn diagram of OTUs in the CZ3, CZC, CZP, and CZM treatment groups of the vegetable-algae symbiotic system is as Figure 15As shown in the figure. There are 1,883 OTUs in common among CZC, CZM, CZP, and CZ3, indicating a high similarity in species among the treatment groups. The correlation heatmap further analyzed the differences in the microbial community composition of different treatment groups in the vegetable-algae symbiotic system. The genera Pseudomonas, Aeromonas, Shewanella putrefaciens, Rheinheimera in the CZ3 treatment group, Pedobacter and Leadbetterella in the CZM treatment group, Comamonas and Flavobacterium in the CZP treatment group, and Simplicispira, Rubrivivax, Brevundimonas, Niabella, and Acidovorax in the CZC treatment group had the highest relative abundances. At the same time, correlation analysis showed that the microbial community structure compositions of the CZP and CZM treatment groups were more similar, and the differences in the microbial compositions of the CZ3 treatment group from those of CKC, CZP, and CZC were the most obvious.

[0083] 9.2 Metagenomic analysis

[0084] Based on the comparison with the KEGG database, the assembled metagenome was annotated and its functions were predicted, and the results are as Figure 16 shown in Figure A. Through the PCoA analysis of the functions of the KEGG pathways, it can be seen that there were significant differences in the functional genes of the Z2 treatment group in the microalgae culture system from those of the treatment groups in the vegetable-algae symbiotic treatment system, indicating that co-cultivation with vegetables can significantly change the expression of the functional genes of microalgae. There were significant differences in the functional genes between the CZ3 treatment group and the CKC, CZC, CZM, and CZP treatment groups, which may be related to the fact that the vegetable roots, foam board, and sponge as fixed carriers have rich surface functional groups and a large specific surface area, and some microorganisms can form a stable biofilm on the carrier surface through processes such as initial adhesion and secretion of EPS to enhance adhesion, resulting in differences in the functional genes of microorganisms in the microalgae suspension culture system. The relative abundances of the KEGG metabolic pathways are as Figure 16 shown in Figure B. The relative abundances of the genes related to energy metabolism, material transport, and catabolism in the CKC treatment group were significantly lower than those in other treatment groups, while the relative abundances of the membrane transport genes and signal transduction genes were significantly higher than those in other treatment groups. Compared with Z2, the relative abundances of the genes related to translation, transport, and catabolism in the CZ3 treatment group were significantly reduced.

[0085] To clarify the differences in the carbon and nitrogen metabolism potentials between the microalgae culture and the vegetable-algae co-culture system, the functional genes of the carbon and nitrogen element cycles at the end of the culture were analyzed. The results of the carbon cycle functional genes are as Figure 17As shown in A. The functional genes related to CKC and carbon element transformation are mainly anaerobic carbon fixation genes and two types of anaerobic-related genes, fermentation, indicating that the vegetable roots in the traditional aquaponics system are in an anaerobic state for a long time, resulting in the enrichment of anaerobic-related functional genes. The functional genes related to carbon fixation in Z2, CZ3, CZC, CZM, and CZP are mainly aerobic methane fixation genes, indicating that the culture system of the vegetable-algae symbiotic system is in an aerobic state, which is closely related to the oxygen produced by microalgae photosynthesis. Introducing a certain amount of oxygen during the hydroponic process can improve the utilization efficiency of nutrients by hydroponic vegetables and achieve the rapid growth of hydroponic crops. The construction of the vegetable-algae symbiotic system reduces the energy consumption demand of traditional mechanical aeration, increases the oxygen content in the hydroponic system, and changes the composition of carbon metabolism functional genes. The relative abundance differences of nitrogen metabolism functional genes are as Figure 17 shown in B. The relative abundances of functional genes related to nitrogen assimilation, nitrogen absorption, and dissimilatory nitrate reduction in the CKC treatment group are significantly higher than those in other treatment groups, and the denitrification functional genes are significantly lower than those in other treatment groups. Dissimilatory nitrate reduction and denitrification for nitrogen removal are two competing DNR pathways, which play important roles in the biogeochemical cycle of nitrogen. When the nitrogen source (electron acceptor) is relatively insufficient, ammonia is generally synthesized by dissimilatory nitrate reduction to amino acids and proteins for assimilation to achieve the growth of vegetables

[290] . The relative abundances of functional genes related to nitrogen mineralization in Z2, CZ3, CZC, CZM, and CZP increase significantly. The increase in nitrogen mineralization functional genes can improve the availability of nitrogen in the culture system, which is beneficial to the efficient utilization of nitrogen by microalgae and vegetables.

[0086] A good synergy can be formed between the vegetables and algae in the present invention, which improves the removal rates of main nutrients such as N and P, promotes the fresh weight of the above-ground part of vegetables, and reduces the content of potential pathogenic substances in vegetables. The relative abundances of genes related to energy metabolism and material transport and catabolism in the CKC treatment group. Microalgae increase the dissolved oxygen content in the culture system. The vegetable-algae symbiotic culture system changes from two types of anaerobic-related genes, anaerobic carbon fixation and fermentation, in the CKC treatment group to mainly aerobic methanogens related to carbon fixation in CZ3, CZC, CZM, and CZP, which helps to improve the utilization efficiency of nutrients. The nitrogen in the vegetable-algae symbiotic system is mainly caused by the increase in the abundance of nitrogen assimilation functional genes.

Claims

1. A method for symbiotic cultivation of vegetables and algae based on concentrated fish manure supernatant, characterized in that: Put the diluted concentrated fish manure solution into the transparent planting tank. The concentration of the diluted concentrated fish manure solution is 25-50% of the concentration of the original concentrated fish manure solution. Culture Chlorella pyrenoidosa in the planting tank. At the same time, grow leafy vegetables in the planting tank using a floating board. The floating board is provided with planting cups for planting leafy vegetables, and the leafy vegetables are planted in the planting cups.

2. The method for symbiotic cultivation of vegetables and algae based on concentrated fish manure supernatant according to claim 1, characterized in that: The leafy vegetable is lettuce. Control the daily light-dark ratio in the planting workshop to be 12-16:12-8 h.

3. The method for symbiotic growth of vegetables and algae based on concentrated fish manure supernatant according to claim 2, wherein: The planting cup is filled with planting cotton for fixing the leafy vegetable.

4. The method for symbiotic cultivation of vegetables and algae based on concentrated fish manure supernatant according to any one of claims 1 to 3, characterized in that: Control the temperature of the planting workshop to be 25±2 °C.

5. The method for symbiotic growth of vegetables and algae based on concentrated fish manure supernatant according to claim 4, characterized in that: The entire planting and breeding cycle is 35 days. After every 35 days, the concentration of the liquid in the planting tank decreases. Drain part of the liquid in the planting tank, and then supplement the corresponding new concentrated fish manure solution to make the concentration of the liquid in the planting tank 25%-50% of the concentration of the original concentrated fish manure solution.

6. The method for symbiotic growth of vegetables and algae based on concentrated fish manure supernatant according to claim 5, characterized in that: The light quantum intensity is controlled at 80 ± 0.5 μmol / m 2 ·s.

7. The method for symbiotic growth of vegetables and algae based on concentrated fish manure supernatant according to claim 5, wherein: The initial inoculation density of microalgae is 1.2×10 5 CFU / m.

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