Use of offshore sponges for enrichment of seawater dissolved inorganic phosphorus

Abstract

The application discloses application of offshore sponges in enriching dissolved inorganic phosphorus in seawater. The application can enrich dissolved inorganic phosphorus in seawater well, and provides a new idea and new biological material for ecological restoration of offshore polluted water bodies (such as aquaculture tail water).

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of seawater eutrophication control, and particularly relates to the application of offshore sponges in enriching dissolved inorganic phosphorus in seawater. BACKGROUND

[0002] Phosphorus (P) element is essential for all life organisms since it is directly involved in numerous physiological and biochemical processes of organisms. However, the change in water body nutrition status caused by phosphorus nutrients has widely existed in coastal waters. Agricultural fertilizer discharge, livestock breeding, industrial production and domestic sewage discharge, change in freshwater input and aquaculture are typical factors causing eutrophication process in coastal waters, and the phosphorus over-standard / pollution in China has not been controlled far away. High concentration of dissolved inorganic phosphorus (DIP) not only causes eutrophication and potential harm to the environment and aquatic ecosystems, but also makes marine animals extremely sensitive to water quality change. When the concentration exceeds a certain value, it will cause poisoning reaction and cause huge economic losses. The concentration of phosphate of >0.02 mg / L or ~0.65 mM is considered to be the evaluation value of eutrophication. Nearly one fourth of the coastal cities in China may experience eutrophication, and the phosphate content in some cities does not meet the second-class seawater standard, and the phosphate content is seriously over-standard. Therefore, effective control of seawater phosphorus over-standard / pollution is one of the important tasks of ecological environment water pollution prevention and control. Due to the chemical properties of dissolved inorganic phosphorus, the enrichment and removal of phosphorus in offshore eutrophic water have been a historical problem. SUMMARY

[0003] The present application aims to overcome the defects of the prior art and provide the application of offshore sponges in enriching dissolved inorganic phosphorus in seawater.

[0004] The technical solution of the present application is as follows:

[0005] The application of offshore sponges in enriching dissolved inorganic phosphorus in seawater.

[0006] In a preferred embodiment of the present application, the offshore sponges are bathylinosella sp. or haliclona sp.

[0007] Further preferably, the offshore sponges are bathylinosella sp.

[0008] In a preferred embodiment of the present application, the offshore sponges are cultured in seawater in need of enrichment of dissolved inorganic phosphorus.

[0009] Further preferably, the culture conditions are as follows: the temperature is not more than 30℃, the dissolved oxygen is 3-3.5 mg / L, the salinity of seawater is 28-29‰, and the DIP concentration is 1-4 mM.

[0010] A method for enriching dissolved inorganic phosphorus in seawater, wherein a near-shore sponge is cultured in seawater in need of enrichment of dissolved inorganic phosphorus.

[0011] In a preferred embodiment of the present application, the near-shore sponge is Spongia officinalis or Tedania sp.

[0012] Further preferably, the near-shore sponge is Spongia officinalis.

[0013] In a preferred embodiment of the present application, the culture conditions are as follows: the temperature is not more than 30℃, the dissolved oxygen is 3-3.5 mg / L, the salinity of seawater is 28-29‰, and the DIP concentration is 1-4 mM.

[0014] The present application has the advantages that the present application can well enrich dissolved inorganic phosphorus in seawater, and provides a new idea and new biological material for ecological restoration of near-shore polluted water bodies (such as culture tail water). BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 FIG. 1 is a diagram of the effect difference of different sponges in Example 1 of the present application. The yellow mark indicates that Spongia officinalis and Tedania sp. show very high enrichment rate in the high-phosphorus coastal area of Dongshan, Fujian. The enrichment rate is the ratio of PolyP in each gram of sponge tissue to DIP in the surrounding marine environment.

[0016] Figure 2 FIG. 2 is a confocal fluorescence microscopic observation of the effect difference of different sponges in Example 1 of the present application. The sponge tissue is dyed by DAPI dye, and the difference is distinguished by the different emission wavelengths of DAPI combined with nucleic acid and PolyP. The green color is PolyP fluorescence, and the blue color is nucleic acid fluorescence. Among them, a: Tedania sp.; b: Mycale sp.; c: Haliclona sp.; d: Spongia officinalis.

[0017] Figure 3 FIG. 3 is an electron microscope image of the epiphyte microorganisms in the four sponges in Example 1 of the present application.

[0018] Figure 4 FIG. 4 is the monthly change of the degree of phosphorus accumulation of the near-shore sponge in Example 1 of the present application. Among them, (a) is the monthly change of the proportion of PolyP accumulated by different sponges to the dry weight of the sponges; (b) is the monthly change of the DIP enrichment rate of different sponges in seawater.

[0019] Figure 5The influence of different conditions in Example 1 of the present application on the formation of PolyP in four sponges. Among them, (a) is the change of sponge PolyP content at different temperatures; (b) is the change of sponge PolyP content at different dissolved oxygen concentrations; (c) is the change of sponge PolyP content at different dissolved salinity; (d) is the change of sponge PolyP content at different DIP concentrations in seawater. DETAILED DESCRIPTION

[0020] The technical solutions of the present application are further described and explained through specific embodiments in combination with the accompanying drawings.

[0021] Example 1

[0022] I. Materials and methods

[0023] 1. Determination of PolyP content in sponges

[0024] (1) Preparation of PolyP concentration-fluorescence value standard curve

[0025] Dissolve the PolyP standard in 20 mM Tris-HCl buffer (pH 7.0) to prepare a PolyP standard solution with a concentration of 0.01 g / L.

[0026] Prepare the PolyP concentration-fluorescence value standard curve according to the following Table 1.

[0027] Table 1 Preparation of PolyP concentration-fluorescence value standard curve

[0028]

[0029] Note: The stock solution is a PolyP standard solution with a concentration of 0.01 g / L.

[0030] (2) Detection of sponge PolyP content

[0031] a) Freeze-dry the sponge tissue, cut it into small pieces, and grind it into powder. Record the mass of the sponge m;

[0032] b) Add 400 μL of 20 mM Tris buffer and vortex to mix well;

[0033] c) Ultrasonic treatment for 1.5 min and boiling water bath for 10 min;

[0034] d) Repeat step 3 once;

[0035] e) Ultrasonic treatment for 1.5 min;

[0036] f) Add 0.6 μL of DNase and 4 μL of RNase, and incubate at 37°C for 20 min;

[0037] g) Add 2 μL proteinase K, 37 °C water bath for 20 min; 15000 rpm centrifugal 2 min, collect supernatant as lysate 1;

[0038] h) Repeat steps 2-8 for the remaining precipitate until complete lysis, to obtain lysate 2, 3, 4…

[0039] i) Divide each lysate into 2, each 200 μL, add to a black 96-well plate; the first part adds 2 μL 1 mM DAPI dye; the second part does not do anything, as a control;

[0040] j) Set up multiple control groups: Tris group, PolyP standard solution group, enzyme group (Tris + 0.6 μL DNase + 4 μL RNase + 2 μL proteinase K) and DNA group, RNA group. Each group is also divided into 2, one with DAPI and the other without any treatment;

[0041] k) Keep in the dark for 5 min;

[0042] l) Use a multifunctional enzyme marker to detect fluorescence values. The excitation wavelength is set to 415 nm; the emission wavelength is set to 450 nm-620 nm, 5 nm slit;

[0043] m) The fluorescence value of the DAPI group minus the control group is considered as the PolyP fluorescence value. Use the 5.1.2.1 standard curve to convert the PolyP concentration c;

[0044] n) The percentage of PolyP in the sponge dry weight = c x V x 4652 ÷ m (Formula 4-1);

[0045] Where c is the PolyP concentration; V is the total volume of the lysate; m is the sponge mass; the constant 4652 is the molecular weight of the PolyP standard.

[0046] 2. DAPI fluorescence microscopic observation of PolyP in sponges

[0047] (1) Preparation of paraffin sections, as follows:

[0048] a) Elute the fixed tissue sample with a series of concentration gradient ethanol (70%, 80%, 90%, 100%), each rinse 2 times, 1-2 min each time;

[0049] b) Rinse with a volume ratio of 1:1 xylene / ethanol solution 2 times, 1-2 min each time;

[0050] c) Rinse with 100% xylene 2 times, 1-2 min each time;

[0051] d) Soak in liquid paraffin (previously melted) for 3-5 min, allowing the paraffin to fully penetrate the tissue sample;

[0052] e) Embed the tissue sample in paraffin, let it stand until solidification;

[0053] f) Cut the sponge tissue sample into 8-10 pm using a rotary microtome and place on a glass slide;

[0054] g) At 60°C, while the paraffin section is in a molten state, rinse gently 3 times with 100% xylene to remove paraffin;

[0055] h) Rinse gently 3 times with 100% ethanol to remove xylene;

[0056] i) Rinse several times with ultrapure water to remove ethanol.

[0057] (2) Confocal microscopy observation

[0058] a) Stain with 10 mM DAPI for 5 min;

[0059] b) Rinse 3 times with ultrapure water;

[0060] c) Cover with a cover glass;

[0061] d) Image using a confocal microscope (Zeiss LSM510 duo inverted confocal microscope); objective parameters: 40x Zeiss Plan Neofluar (1.3 N.A.); laser source: 405 nm; fluorescent emission signals are separated into two channels by NFT515 (Neben Farb Teiler515). Channel 1 collects wavelengths of 420-515 nm, representing the nucleotide-DAPI signal. Channel 2 is set to collect wavelengths of 530-550 nm, representing the conversion of the PolyP-DAPI signal.

[0062] 3. Determination of polyphosphate (PolyP) in red coral under different conditions

[0063] Each group has 3 replicates. The experiment uses a recirculating indoor seawater culture system. The aquarium is 0.6 m x 0.4 m x 0.3 m in size, and the water volume is about 60 L. Each group of samples is placed in it for a 3-7 day culture experiment. Before the experiment, the aquarium is soaked with potassium permanganate for 24 hours, then brushed clean and filled with artificial seawater. After 48 hours of oxygenation, the sponges are placed in the aquarium. The temporary cultivation time is 7 days, with 24 hours of oxygenation. If the sponges are found to be whitening or dead during the experiment, they should be recorded and cleaned up in a timely manner. Different single factor variables are set to detect the polyphosphate content of the sponges.

[0064] II. Results and analysis

[0065] 1、Different species of sponges in different environments of the ability to analyze the differences in phosphorus

[0066] Collected along the coast of Hainan, Fujian Dongshan sponges for research, the results showed that, whether DIP eutrophication or oligotrophic sea, sponges can form polyphosphate to gather dissolved inorganic phosphorus (DIP) in seawater, but different sponges species, the situation has a great difference in phosphorus Figure 1 ). And for the eutrophic environment of the aquaculture area, the DIP concentration in seawater up to 4.7 uM, bath angle bone sponges not only grow vigorously show high polyphosphate formation ability, PolyP / sponge dry weight (mg / g) ratio > 3.722, and the enrichment rate of up to 46732 times, showing good ability to enrich phosphorus.

[0067] 2, the ability of four kinds of sponges in the offshore phosphorus and microbial distribution of microscopic observation

[0068] Under the confocal microscope to observe the DAPI stained sponge tissue sections, bath angle bone sponges can be seen obvious yellow-green fluorescent particles Figure 2 ). The same fluorescent particles in the beautiful sponges and mountain sponges are difficult to find. Microscopic observation results provide direct evidence for the results of PolyP detection. At the same time, through the scanning electron microscope, found that the bath angle bone sponges for high abundance of microorganisms, there are much higher than other sponges microbial abundance and diversity Figure 3 ), for the DIP in the enrichment of the environment provides an excellent microbial community.

[0069] 3, the ability of four kinds of sponges in the offshore phosphorus monthly changes

[0070] Investigated the ability of four kinds of sponges in the offshore phosphorus, the results as Figure 4 shown, of the four sponges, bath angle bone sponges and moss sponges show better phosphorus ability, of which bath angle bone sponges show the highest phosphorus. However, with the change of different months of the season, the ability of phosphorus changes, among them 2-6 months of phosphorus increases, in April and June, there is a small peak, in June, the highest value, but after June, the ability of phosphorus gradually decreased, in winter, the minimum value. This is consistent with the growth and reproduction of sponges, sponges 4-9 months for the reproductive and growth period, and in winter, sponges into dormancy and decline period, therefore, in the natural environment, the ability of sponges to change with the seasons.

[0071] 4, the effect of different conditions on the ability of sponges to phosphorus

[0072] By setting different conditions to understand the changes in the ability of sponges to phosphorus and the optimal phosphorus conditions, such as Figure 5The results showed that the content of polyphosphate in the sponge of A. bathylinum increased with the increase of temperature, and the optimal temperature was 30℃. The polyphosphate accumulation in the sponge of L. sp. increased slightly with the increase of temperature. The polyphosphate accumulation in the sponges of H. sp. and P. sp. did not change significantly. The highest content of polyphosphate was detected in the sponge of A. bathylinum at the DO of 3.5 mg / L. The polyphosphate accumulation in the sponge decreased with the increase of DO. Figure 5 b) The sponge of A. bathylinum showed a high level of polyphosphate accumulation at the salinity of 28-29‰. The polyphosphate accumulation in the sponge decreased with the increase of salinity. The other sponges showed a low level of polyphosphate accumulation. Figure 5 c) The polyphosphate accumulation in the sponge of A. bathylinum increased with the increase of DIP concentration in seawater, and reached the highest value at the DIP concentration of 4.0 mM. The polyphosphate accumulation in the sponge decreased with the further increase of DIP concentration. Figure 5 d) Although the polyphosphate accumulation in the sponges changed with the conditions, the sponge of A. bathylinum showed the highest polyphosphate accumulation among the sponges detected.

[0073] The above description is only the preferred embodiment of the present application, and therefore cannot limit the scope of the present application. Any equivalent changes and modifications made according to the scope and content of the present patent should still be within the scope of the present application.

Claims

1. Use of a marine sponge in the enrichment of seawater in dissolved inorganic phosphorus, characterized in that: The offshore sponges are bath angle bone sponges, and the offshore sponges are bred in seawater which needs to be enriched with dissolved inorganic phosphorus, and the breeding conditions are that the temperature is not more than 30 DEG C, the dissolved oxygen is 3-3.5 mg / L, the seawater salinity is 28-29 ‰, and the DIP concentration is 1-4 mM.

2. A method of enriching dissolved inorganic phosphorus in seawater, characterized by: The offshore sponges are bred in seawater which needs to be enriched with dissolved inorganic phosphorus, and the offshore sponges are bath angle bone sponges, and the breeding conditions are that the temperature is not more than 30 DEG C, the dissolved oxygen is 3-3.5 mg / L, the seawater salinity is 28-29 ‰, and the DIP concentration is 1-4 mM.

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