A method for degrading copper ion concentration in water by ligand-binding Thalassiosira wiseri

Abstract

The present invention relates to the technical field of biological ligand binding, and in particular to a method for degrading copper ion concentration based on the binding of a ligand to Thalassiosira wissenii. The method comprises the following steps: preparing chitosan, L-methionine, D-glucuronic acid, and EDTA stock solutions, and preparing concentrated nitric acid and hydrogen peroxide stock solutions; preparing a copper ion solution, and preparing a Thalassiosira wissenii algal culture solution; filtering the stock solution, and diluting the filtrate to obtain a ligand aqueous solution; taking equal amounts of the Thalassiosira wissenii algal culture solution and the copper ion solution into five groups of containers, sequentially adding chitosan, L-methionine, D-glucuronic acid, and EDTA, and culturing to obtain a culture solution; measuring the fluorescence value of the Thalassiosira wissenii algal body to determine photosynthetic activity; determining the chlorophyll content; digesting the sample and measuring the copper ion concentration; and determining the algal growth, photosynthetic activity, and copper ion contamination based on the measured fluorescence value, chlorophyll content, and copper ion concentration of the Thalassiosira wissenii algal body, and determining the optimal type of ligand to be added.

Description

Technical Field

[0001] The present invention relates to the technical field of biological ligand binding, and in particular to a method for degrading copper ion concentration in water by binding to Thalassiosira wiseri using a ligand. Background Art

[0002] Copper sulfate is commonly used in aquaculture to treat parasitic diseases such as Ichthyophthirius, Trichodina, saccharinids, water molds, and some harmful algae. Due to its low cost, effectiveness, availability, and ease of use, copper sulfate is widely used by aquaculture operators. However, failure to precisely control copper sulfate dosage can lead to irreparable economic losses. Crustaceans are particularly sensitive to heavy metals in water, and even the slightest inadvertent use can cause serious damage.

[0003] Chemical precipitation and biological methods can partially remove copper ions from water. Chemical precipitation involves adding chemical reagents to contaminated water, causing heavy metal ions to react with them and form a precipitate. Commonly used chemical reagents include caustic soda, lime, sodium sulfide, and ferric chloride. While chemical precipitation offers the advantages of rapid reaction and immediate results, it also has significant disadvantages. Inappropriate use of chemical reagents can cause secondary contamination of aquaculture waters and lead to the death of farmed animals. Biological methods involve adding organisms such as fungi and algae to water or planting plants with adsorption properties. Biological methods offer the advantages of preventing secondary pollution and providing higher economic value. Therefore, cultivating Thalassiosira wieldingii in water and adding different types of ligands to enhance copper ion adsorption has practical significance and promising application prospects. Summary of the Invention

[0004] The present invention aims to overcome copper ion pollution and prevent copper ion accumulation in the environment and amplification through the food chain, ultimately posing a potential hazard to human health. The present invention seeks a method that has a low inhibitory effect on the growth and photosynthesis of Thalassiosira wiseri and can bind to Thalassiosira wiseri to degrade copper ion concentration. The preparation conditions of the method are easily controllable, the operation method is simple, and the ligand type can be adjusted as needed to inhibit the growth of Thalassiosira wiseri.

[0005] The Thalassiosira wissenii used in the experiment was obtained from the State Key Laboratory of Coastal Marine Environmental Sciences, Xiamen University. Thalassiosira wissenii was stored in f / 2 culture medium (Na2SiO3·H2O was added to the culture medium to maintain the Si concentration at 21.1mmol / L). Algal cells in the exponential growth phase were selected and re-filtered and transferred to new culture medium every 1-2 days to ensure that the cells adapted to the culture conditions. During the experiment, attention was paid to adding nutrients at appropriate concentrations, controlling the pH to around 7.5, and the light intensity to 140μmolphotons·m -2 ·s -1The light-dark ratio was 14h:10h and the temperature was 19°C to ensure the normal growth of Thalassiosira wissenii.

[0006] The technical solution adopted to achieve the purpose of the present invention is: a method for degrading copper ion concentration by combining different ligands with Thalassiosira wissenii, comprising the following steps:

[0007] 1) Prepare 0.5mmol·L -1 Chitosan, L-methionine, D-glucuronic acid and EDTA stock solutions were stored in the dark and ready for use; the concentration was 0.5 mg·L -1 Concentrated nitric acid and 0.2 mg·L -1 The stock solution of hydrogen peroxide was stored in the dark for future use; a copper ion solution with a concentration of 20 μmol / L was prepared and stored in the dark for future use; and a culture solution of Thalassiosira wissenii algae was prepared and set aside;

[0008] 2) Filter the stock solution through a microporous membrane, take the filtered solution, dilute it with distilled water, and prepare the ligand aqueous solution;

[0009] 3) Equal amounts of 50 ml of Thalassiosira wissenii culture medium and 5 ml of copper ion solution were respectively taken into five sets of containers, and 10 ml each of chitosan, L-methionine, D-glucuronic acid, and EDTA were added to the above containers in sequence. One container was not added with ligand and served as a blank for culture to obtain culture medium;

[0010] 4) using a phytoplankton classification fluorescence instrument to measure the fluorescence value of the Thalassiosira wiseri algae in each container to determine the photosynthetic activity;

[0011] 5) Determine the chlorophyll content in each container using a phytoplankton classification fluorometer;

[0012] 6) digesting the sample and measuring the copper ion concentration using an inductively coupled plasma mass spectrometer;

[0013] 7) Based on the measured fluorescence value, chlorophyll content, and copper ion concentration of the Thalassiosira wielkii algae, the algae growth, photosynthetic activity, and copper ion contamination were determined, and the optimal ligand type to be added was determined.

[0014] Preferably, in the above method of degrading copper ion concentration by combining different ligands with Thalassiosira welshii, in step 1), the stock solution is stored in the dark at a temperature of 4°C.

[0015] Preferably, in the above method of degrading copper ion concentration by combining different ligands with Thalassiosira wissenii, in step 2), the concentration of the ligand aqueous solution is 0.1 mmol·L -1 .

[0016] Preferably, in the above method of degrading copper ion concentration by combining different ligands with Thalassiosira welshii, in step 3), the culture temperature of the Thalassiosira welshii algal culture solution is 19°C.

[0017] Preferably, in the above-mentioned method of degrading copper ion concentration by combining different ligands with Thalassiosira welshii, in step 4), the fluorescence value of the Thalassiosira welshii algae in each triangular flask is measured using a phytoplankton classification fluorescence instrument, and a dark reaction is required before the measurement.

[0018] Preferably, in the above-mentioned method of degrading copper ion concentration by combining different ligands with Thalassiosira wiseri, in step 5), a dark reaction is required for 15 minutes before each measurement using a phytoplankton classification fluorescence instrument.

[0019] Preferably, in the above-mentioned method of degrading copper ion concentration by combining different ligands with Thalassiosira wissenii, in step 6), the specific step of sample digestion is to add concentrated nitric acid and hydrogen peroxide to the culture solution, shake well, and then digest under microwave conditions.

[0020] Preferably, in the above method of degrading copper ion concentration by combining different ligands with Thalassiosira wiseri, in step 6), the microwave pressure during sample digestion needs to be set to 15 atm. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the effect of the ligand in the method of the present invention on the copper ion concentration in the water degraded by Thalassiosira welchii.

[0022] Figure 2 It is the effect of the ligand in the method of the present invention on the photosynthesis of Thalassiosira welchii.

[0023] Figure 3 This is the effect of the ligand in the method of the present invention on the growth of Thalassiosira wiseri. DETAILED DESCRIPTION

[0024] The present invention will be further described below with reference to specific embodiments.

[0025] Example 1 Effect of Ligand Binding to Thalassiosira wiseri on the Degradation of Copper Ion Concentration in Water

[0026] 1. Thalassiosira wissenii used in this experiment was obtained from the State Key Laboratory of Coastal Marine Environmental Sciences, Xiamen University. Thalassiosira wissenii was maintained in f / 2 culture medium (Na2SiO3·H2O was added to maintain a Si concentration of 21.1 mmol / L). Algal cells were selected during the exponential growth phase and re-filtered and transferred to fresh culture medium every 1-2 days to ensure that the cells adapted to the culture conditions. During the experiment, appropriate nutrient concentrations were added, the pH was maintained at approximately 7.5, and the light intensity was 140 μmol photons2 m -22s -1 ; The light-dark ratio is 14h:10h; the temperature is 19℃ to ensure the normal growth of Thalassiosira wiseri.

[0027] 2. Use distilled water to prepare the concentration of 0.5mmol·L -1 Chitosan, L-methionine, D-glucuronic acid, or EDTA stock solutions were stored at 4°C in the dark until ready for use. A 20 μmol / L copper ion solution was prepared with distilled water and stored in the dark until ready for use.

[0028] 3. Filter the stock solution through a 0.45 μm microporous membrane, take the filtered solution, dilute it with distilled water, and prepare the 0.1 mmol·L -1 Aqueous ligand solution.

[0029] 4. Take equal amounts of 50 ml of Thalassiosira wissenii algal culture medium and 5 ml of copper ion solution and place them in 5 groups of 250 ml Erlenmeyer flasks. Add 10 ml of the ligand aqueous solution (chitosan, L-methionine, D-glucuronic acid and EDTA) used in the experiment to the Erlenmeyer flasks in sequence. One group does not add the ligand and serves as the blank group. Label the group accordingly and culture the algae to obtain the culture medium.

[0030] 5. Use a phytoplankton classification fluorometer to measure algal fluorescence, requiring a dark reaction before measurement, to determine photosynthetic activity. Chlorophyll content is also measured using a phytoplankton classification fluorometer, requiring a dark reaction before measurement, and compared with the data obtained from the blank group. This will analyze the effects of the addition of various ligands on the growth and photosynthetic activity of Thalassiosira wiseri.

[0031] 6. To destroy organic matter, dissolve particulate matter, and reduce the impact of nutrients in the Thalassiosira wissenii culture medium on copper ion measurement, 5 ml of the algae sample with different ligands was taken in sequence. 2.0 ml of concentrated nitric acid and 1.0 ml of hydrogen peroxide were added to each sample. After shaking, the samples were microwave-digested at 15 atm for 10 minutes.

[0032] 7. After sample digestion, measure the copper ion concentration using an inductively coupled plasma mass spectrometer, analyze the degradation of copper ions in the algal culture medium with different ligands added, and determine the optimal type of ligand to be added.

[0033] Figure 3 The effects of the ligands used in the present invention on the growth of Thalassiosira welshii were investigated. Compared to the blank control, the culture medium supplemented with EDTA promoted the growth of Thalassiosira welshii or maintained growth at the same level as the blank control within a certain timeframe. The remaining ligands, namely, those supplemented with L-methionine, D-glucuronic acid, and chitosan, inhibited the growth of Thalassiosira welshii throughout the entire testing period, with the inhibitory effects increasing in descending order.

[0034] Figure 2 The effects of the ligands used in the present invention on the photosynthesis of Thalassiosira welshii were investigated. Compared to the blank control, the culture medium supplemented with EDTA promoted photosynthesis in Thalassiosira welshii during the initial culture period, but subsequently reached the same level as the blank control. The remaining ligands, namely, those supplemented with L-methionine, D-glucuronic acid, and chitosan, inhibited the photosynthetic activity of Thalassiosira welshii throughout the entire testing period, with the inhibitory effect increasing in descending order.

[0035] Figure 1 The effect of the ligand on the degradation of copper ion concentration in water by Thalassiosira wieldii in the method of the present invention is shown in FIG. 2+ The concentration did not decrease significantly. The culture media with the addition of other ligands, namely chitosan, D-glucuronic acid, and L-methionine, all showed a significant decrease in copper ion concentration throughout the detection period. Among them, the culture media with the addition of L-methionine had the best effect in reducing copper ion concentration, and there was no increase in copper ion concentration during the detection period.

[0036] like Figure 1 As shown, among the four groups of culture solutions with added ligands, the culture solution with added EDTA had the highest Cu 2+ The concentration was significantly higher than that of the culture medium with other ligands, and slightly higher than that of the blank group on the sixth day, which was probably due to the interaction between EDTA and Cu 2+ The complexation constant is large, and the photoreaction rate of the formed complex EDTA-Cu is very fast, but its light stability is weak. Therefore, it is easy to release copper ions under light, and it is difficult to effectively reduce the copper ion concentration in the solution. Therefore, the copper ion solution of the algae with EDTA added is significantly higher than that of the other three groups of culture solutions. Although the copper ion concentration in the culture solution with D-glucuronic acid added is lower than that of the blank group, it is higher than that of the culture solution with chitosan added on the third day. This may be because under this condition, the copper ion concentration of the algae is higher than that of the culture solution with chitosan added. 2+ The electron transfer speed is faster and thus exhibits stronger electrochemical activity. 2+ The binary system formed with D-glucuronic acid is relatively unstable, and the free Cu dissociated in the solution 2+ The concentration of the solution with chitosan added was significantly lower than that of the culture medium with EDTA and D-glucuronic acid, and only higher than that of the culture medium with L-methionine. This may be because the chemical stability of polysaccharide molecules is very strong and the metal polysaccharide complex formed is difficult to be destroyed by light. 2+ The solution concentration was the lowest, probably because L-methionine and Cu 2+The complex formed has a considerable coordination bond component, and in the bonding between the central ion and the coordinating atom, not only the in-plane σ bonding but also the in-plane π bonding plays an important role. The d orbital of the central ion and the π orbital of the coordinating atom also have considerable overlap, so the complex formed at this time is quite stable and not easily decomposed by light.

[0037] Therefore, in actual production, if it is necessary to quickly reduce the copper ions in the water body, adding an appropriate amount of L-methionine to the water body will not have an excessive inhibitory effect on the growth and photosynthesis of Thalassiosira wiseri, but can also better promote the degradation of copper ions in the solution by Thalassiosira wiseri.

Claims

1. A method for degrading copper ion concentration by combining different ligands with Thalassiosira wiseri, characterized in that: The steps include: 1) Prepare 0.5mmol·L -1 Chitosan, L-methionine, D-glucuronic acid and EDTA stock solutions were stored in the dark and ready for use; the concentration was 0.5 mg·L -1 Concentrated nitric acid and 0.2 mg·L -1 The stock solution of hydrogen peroxide was stored in the dark for future use; a copper ion solution with a concentration of 20 μmol / L was prepared and stored in the dark for future use; and a culture solution of Thalassiosira wissenii algae was prepared and set aside; 2) Filter the stock solution through a microporous membrane, take the filtered solution, dilute it with distilled water, and prepare the ligand aqueous solution; 3) Equal amounts of 50 ml of Thalassiosira wissenii culture medium and 5 ml of copper ion solution were respectively taken into five sets of containers. 10 ml each of chitosan, L-methionine, D-glucuronic acid, and EDTA were added to the above containers in sequence. One container was not added with ligand and served as a blank for culture to obtain culture medium. 4) Using a phytoplankton classification fluorometer, measure the fluorescence value of the Thalassiosira wiseri algae in each container to determine photosynthetic activity; 5) Determine the chlorophyll content in each container using a phytoplankton classification fluorometer; 6) Sample digestion and measurement of copper ion concentration using inductively coupled plasma mass spectrometry; 7) Based on the measured fluorescence value, chlorophyll content, and copper ion concentration of Thalassiosira wielkii, the algal growth, photosynthetic activity, and copper ion contamination were determined, and the optimal ligand type to be added was determined to be L-methionine.

2. The method of claim 1, wherein: In step 1), the stock solution was stored at 4°C in the dark.

3. The method of claim 1, wherein: In step 2), the concentration of the ligand aqueous solution is 0.1 mmol·L -1 .

4. The method of claim 1, wherein: In step 3), the culture temperature of the Thalassiosira welshii algal culture solution is 19°C.

5. The method of claim 1, wherein: In step 4), the fluorescence value of the Thalassiosira wiseri algae in each triangular flask is measured using a phytoplankton classification fluorescence instrument, and a dark reaction is required before the measurement.

6. The method of claim 1, wherein: In step 5), a dark reaction of 15 minutes is required before each measurement using the phytoplankton classification fluorescence instrument.

7. The method of claim 1, wherein: In step 6), the specific steps of sample digestion are: adding concentrated nitric acid and hydrogen peroxide to the culture solution, shaking and then digesting under microwave conditions.

8. The method of claim 7, wherein: In step 6), the microwave pressure should be set to 15 atm during sample digestion.

Images