Method and application of Spartina alterniflora extract in inhibiting cyanobacteria bloom

Abstract

The invention relates to the technical field of cyanobacteria treatment, and in particular to a method and application of a Spartina alterniflora extract for inhibiting cyanobacteria blooms. The method comprises the steps of preparing a Spartina alterniflora extract, adding the Spartina alterniflora extract into a cyanobacteria growth environment, wherein the concentration of the Spartina alterniflora extract is 0.5-10 g / L. The invention uses the Spartina alterniflora extract to inhibit cyanobacteria blooms, thereby promoting resource utilization of the invasive plant Spartina alterniflora and treating cyanobacteria blooms. Meanwhile, the treatment process of cyanobacteria blooms is environmentally friendly and will not cause secondary pollution to water bodies, thereby providing better help for sewage treatment in China, reducing the environmental burden, and improving the high efficiency and greenness in the environmental treatment process.

Description

Technical Field

[0001] The invention relates to the technical field of cyanobacteria control, and in particular to a method and application of a Spartina alterniflora extract to inhibit cyanobacteria blooms. Background Art

[0002] Due to the influence of human activities, the eutrophication of water bodies caused by excessive content of nutrients such as N and P in rivers, lakes and bay areas has become increasingly serious, making cyanobacteria blooms an important water environment problem facing the world. The massive reproduction of cyanobacteria changes the physical and chemical properties of water, causing pollution. In addition, after the accumulation and decomposition of cyanobacteria blooms, a large amount of nutrients will be released, and a large amount of derivative substances will be decomposed, including toxins, odorous substances, etc., which seriously affect the water quality and the structure and function of aquatic ecosystems. At the same time, the toxins and carcinogens released by cyanobacteria can be enriched through the food chain / web, endangering human health.

[0003] At present, algae removal technologies mainly include three categories: physical, chemical, and biological methods. Physical methods are used to remove algae from water bodies, and are often used in drinking water areas such as lakes and reservoirs. Common physical algae removal technologies include mechanical algae removal, ultraviolet algae removal, ultrasonic algae removal, clay algae removal, flotation algae removal, and photocatalytic algae removal. Chemical algae removal technology is the most commonly used method at present. Chemical algae removal methods are chemical coagulation and algae removal by algaecides. Biological algae removal technology is a method that uses the principles of ecological balance and the life activities of cultivated or inoculated organisms to transform, degrade, and transfer pollutants in the water, thereby restoring the health of the water environment. Common biological algae removal technologies include aquatic plant and animal algae control technology, biofilm technology, microbial algae removal technology, and artificial wetland algae removal.

[0004] However, the above algae removal methods also have many disadvantages and shortcomings. Most physical algae removal technologies are time-consuming, labor-intensive and expensive. Chemical algae removal technologies are prone to secondary pollution of water bodies. Although biological algae removal technology is simple and safe, it is subject to many restrictive factors, such as time, water quality, and water body size.

[0005] Spartina alterniflora is native to the Atlantic coast of North and South America and is a typical halophyte. From fresh water to sea water, it has a wide adaptability to salinity, strong tolerance to salt stress, strong adaptability and tolerance to climate and environment, and no special requirements for substrate conditions. Because it can prevent waves and sand from eroding coastal embankments and increase the area of ​​mudflat wetlands. However, with the passage of time, the harm of Spartina alterniflora has gradually been exposed. It not only blocked waterways, induced red tides, squeezed out native plants, and invaded bird habitats, but also severely inhibited the growth and habitats of large benthic organisms such as snails, shellfish, and crabs, causing serious economic losses. In addition, after the invasion of Spartina alterniflora, soil nutrients decreased significantly and degradation was obvious.

[0006] Since the prevention and control of Spartina alterniflora is difficult and costly, in the comprehensive management of Spartina alterniflora, on the one hand, we should actively explore economical and effective control measures to curb its spread rate and minimize its harm to the ecosystem; on the other hand, we should change our way of thinking and make full use of the characteristics of Spartina alterniflora such as rapid growth and strong stress resistance, and use it as a resource to achieve the goal of "turning harm into benefit and waste into treasure" through resource development. Combining prevention and control with utilization may reap both ecological and economic benefits.

[0007] There are various forms of utilization of Spartina alterniflora in my country, but it is still in the research stage. The utilization of Spartina alterniflora is mainly the low-value utilization of straw, such as fertilizer, raw material, feed and fuel. It also includes research on the medicinal value of Spartina alterniflora and high-tech utilization such as salt-tolerant genes. Among the publicly published research papers on the utilization of Spartina alterniflora, the utilization of straw as fuel accounts for the largest proportion, which is 43%, while the utilization of straw as fertilizer and other utilizations such as salt-tolerant genes account for the smallest proportion, which are 3% and 5% respectively. The resource utilization of Spartina alterniflora is still in the laboratory research stage, and it is still a long way from scale, marketization and industrialization. Summary of the invention

[0008] In view of the above-mentioned deficiencies in the prior art, the present invention aims to provide a method and application of an extract of Spartina alterniflora to inhibit cyanobacterial blooms, so as to provide an application of Spartina alterniflora in the treatment of cyanobacteria.

[0009] In order to solve the above problems, the present invention adopts the following technical solutions:

[0010] A method for inhibiting blue algae blooms with a Spartina alterniflora extract comprises preparing the Spartina alterniflora extract and adding the Spartina alterniflora extract into a blue algae growth environment.

[0011] Further, the preparation of the Spartina alterniflora extract comprises:

[0012] During the heading period of Spartina alterniflora, the aboveground parts of Spartina alterniflora are collected, the stems, leaves and ears of Spartina alterniflora are separated after washing, and then dried;

[0013] The water extraction method was used to prepare the extract of Spartina alterniflora. The separated stems, leaves and panicles of Spartina alterniflora were cut into sections. Different tissues of Spartina alterniflora and deionized water were added into a beaker at a mass-to-volume ratio of 1:10 for soaking. The mixture was stirred 3-5 times a day. After soaking at room temperature for 48 hours, the extracts of different tissues of Spartina alterniflora were obtained by filtering.

[0014] Furthermore, the Spartina alterniflora extract is a Spartina alterniflora spike extract.

[0015] Furthermore, after the Spartina alterniflora extract is added into the blue algae growth environment, the concentration of the Spartina alterniflora extract is 0.5-10 g / L.

[0016] Furthermore, after the Spartina alterniflora extract is added into the blue algae growth environment, the concentration of the Spartina alterniflora extract is 5g / L-10g / L.

[0017] Application of Spartina alterniflora extract in the preparation of cyanobacteria bloom inhibitors.

[0018] Furthermore, the concentration of the Spartina alterniflora extract after being added into the blue algae growth environment is 0.5-10 g / L; the Spartina alterniflora extract is the Spartina alterniflora spike extract.

[0019] Furthermore, the Spartina alterniflora extract inhibits the content of chlorophyll a in cyanobacteria.

[0020] Furthermore, the Spartina alterniflora extract increases the activities of SOD and CAT and the content of MDA in cyanobacteria.

[0021] Furthermore, the Spartina alterniflora extract increases the GSH content and GST activity in cyanobacteria.

[0022] The beneficial effect of the present invention is that the present invention uses the extract of Spartina alterniflora to inhibit cyanobacterial blooms, which not only promotes the resource utilization of the invasive plant Spartina alterniflora, but also can control the cyanobacterial blooms. At the same time, the control process of the cyanobacterial blooms is environmentally friendly and will not cause secondary pollution to the water body, thereby providing better help for China's sewage treatment, thereby reducing the environmental burden and improving the high efficiency and greenness of the environmental treatment process. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Graph showing the inhibition rate of different tissues and extracts of different concentrations on Microcystis aeruginosa in the examples of the present invention.

[0024] Figure 2 This is a graph showing the effects of different tissues and different concentrations of extracts on the chlorophyll a content of Microcystis aeruginosa in an embodiment of the present invention.

[0025] Figure 3This is a graph showing the effects of different tissues and different concentrations of extracts on the SOD activity of Microcystis aeruginosa in an example of the present invention.

[0026] Figure 4 This is a graph showing the effects of different tissues and different concentrations of extracts on the CAT activity of Microcystis aeruginosa in the examples of the present invention.

[0027] Figure 5 This is a graph showing the effects of different tissues and different concentrations of extracts on the MDA content of Microcystis aeruginosa in an embodiment of the present invention.

[0028] Figure 6 This is a graph showing the effects of different tissues and different concentrations of extracts on the GSH content of Microcystis aeruginosa in the examples of the present invention.

[0029] Figure 7 This is a graph showing the effects of different tissues and different concentrations of extracts on the GST activity of Microcystis aeruginosa in the examples of the present invention. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments.

[0031] It should be noted that these embodiments are only used to illustrate the present invention rather than to limit the present invention. Simple improvements to the method based on the concept of the present invention all fall within the scope of protection claimed by the present invention.

[0032] The method for inhibiting water bloom by extracts from different tissues of Spartina alterniflora comprises the following steps:

[0033] S1: Collection of Spartina alterniflora: The aboveground parts of Spartina alterniflora were collected during the heading period, and the stems, leaves and ears of Spartina alterniflora were separated after washing, and dried in an oven at (37±1)°C. After complete drying, the stems, leaves and ears were sealed and stored in a cool and dry place.

[0034] S2: Cultivate Microcystis aeruginosa in BG-11 medium. For the subsequent performance test of algaecides, expand the culture of Microcystis aeruginosa two weeks before the experiment. The culture conditions are: light intensity 3000lx, light-dark ratio 12h:12h, temperature (28±1)℃, shaking 3-5 times a day to make the algae cells enter the logarithmic growth phase. The main components of the culture medium are: NaNO3, 1.5g / L; K2HPO4·3H2O, 0.04g / L; MgSO4·7H2O, 0.075g / L; CaCl2·2H2O, 0.036g / L; citric acid, 0.006g / L; ammonium ferric citrate, 0.006g / L; EDTA, 0.001g / L; Na2CO3, 0.02g / L; A5 solution, 0.1mL.

[0035] The formula of A5solution is H3BO3, 2.86g / L; MnCl2·4H2O, 1.86g / L; ZnSO4·7H2O, 0.22g / L; Na2MoO4·2H2O, 0.39g / L; CuSO4·5H2O, 0.08g / L; Co(NO3)2·6H2O, 0.05g / L.

[0036] S3: Prepare the extract of Spartina alterniflora by water extraction: Cut the stems, leaves and spikes in step S1 into 1 cm segments with scissors, add different tissues of Spartina alterniflora and deionized water in a beaker at a mass (g): volume (mL) ratio of 1:10, stir 3-5 times a day, soak at room temperature for 48 hours, and filter through a 0.22 μm filter membrane. Obtain 50 g / L Spartina alterniflora tissue extract for algae inhibition experiment.

[0037] S4: Add different volumes of deionized water to the extract in step S3 to dilute the extract to 2.5, 5, 25, and 50 g / L respectively.

[0038] S5: Test the inhibitory effects of different concentrations of extracts from different tissues of Spartina alterniflora on Microcystis aeruginosa. Add 20 mL of Microcystis aeruginosa solution in the logarithmic growth phase and 140 mL of BG-11 culture medium into a 250 mL conical flask.

[0039] S6: Add 40 mL of the extracts of different concentrations from step S4 to the conical flask in step S5, so that the final volume of the algae solution is 200 mL and the final density of the algae cells in the conical flask is 5×10 5 cells / mL, so that the final concentrations of extracts from different tissues of Spartina alterniflora in the algae solution were 0.5, 1, 5, and 10 g / L, respectively. Three parallels were set up for each group, and each group was cultured under the culture conditions of step S2.

[0040] S7: Count the cells every 48 hours for 7 consecutive days, for a total of 4 times. And calculate the inhibition rate (IR) of each group of extracts on algae according to the inhibition rate formula.

[0041] S8: After counting on the 7th day, the algae liquid was centrifuged, the supernatant was discarded, and the chlorophyll a content in the algae mud was detected by 95% ethanol extraction method. The malondialdehyde (MDA), reduced glutathione (GSH) content and superoxide dismutase (SOD), catalase (CAT) and glutathione-s-transferase (GST) activities in the sediment algae mud were detected using a kit to obtain the inhibitory ability of extracts from different tissues of Spartina alterniflora on algae.

[0042] In step S8, when determining the chlorophyll a content, the algae mud is extracted in 95% ethanol in the dark for 24 hours, and then the absorbance value is measured at wavelengths of 649nm and 665nm; the chlorophyll a calculation formula is: chlorophyll a (mg / L) = 13.7×A665-5.76×A649.

[0043] In step S8, when the MDA content is measured, the absorbance values ​​are measured at wavelengths of 532 nm and 600 nm after the reagent kit is added to the reaction system; the MDA content is calculated by the formula: MDA content (nmol / 10 4 cell)=((A 532 -A 600 )×total volume of reaction system (4×10 -4 L)÷(MDA molar extinction coefficient(155×10 3 L / mol / cm)×light path of cuvette (1cm))×10 9 )÷(algae density of the sample to be tested(10 4 cell / mL)×added sample volume (0.1 mL).

[0044] In step S8, when the GSH content is measured, the absorbance value is measured at a wavelength of 412 nm after the reagent kit is added to the reaction system; the GSH content is calculated by the formula: GSH (nmol / 10 4 cell) = (A test - A blank) ÷ 0.75 × the volume of supernatant added to the reaction system (0.02 mL) ÷ (the volume of supernatant added to the reaction system (0.02 mL) × the algae density of the sample to be tested (10 4 cell / mL)).

[0045] In step S8, when SOD activity is measured, the absorbance value is measured at a wavelength of 450nm after the reagent kit is added to the reaction system; SOD activity unit definition: the enzyme activity corresponding to 50% SOD inhibition rate in this reaction system is one SOD activity unit (U / mL). SOD activity calculation formula: SOD activity (U / 10 4 cell) = (inhibition percentage ÷ (1-inhibition percentage) × total volume of reaction system (0.2 mL)) ÷ (volume of sample added to reaction system (0.01 mL) × algae density of sample to be tested (10 4 cell / mL)).

[0046] In step S8, when measuring CAT activity, after adding the reagent kit to the reaction system, the absorbance value is measured at a wavelength of 405 nm; the definition of CAT activity unit is: per 10 4 The degradation of 1 μmol H2O2 per minute catalyzed by the cell is defined as one unit of enzyme activity. The CAT activity calculation formula is: CAT activity (μmol / min / 10 4cell)=(A 对 -A 测 -0.0013)÷0.1×total volume of reaction system (0.445mL)÷(volume of added sample (0.05mL)×algae density of sample to be tested (10 4 cell / mL))÷reaction time (10min).

[0047] In step S8, when measuring the activity of GST, after adding the reagent kit to the reaction system, the absorbance values ​​at 450 nm for 10 s and 310 s are measured; the unit definition of GST activity is: at 25°C, every 10 4 The cell catalyzes 1 nmol / LCDNB and GSH binding per minute as 1 unit of enzyme activity. GST activity calculation formula: GST activity (nmol / min / 10 4 cell)=(A 310 -A 10 )÷product molar extinction coefficient (9.6×10 3 L / mol / cm)÷light diameter of cuvette (1cm)×10 9 × total volume of reaction system (2.2×10 -4 L)÷(volume of added sample (0.02 mL)×algae density of the sample to be tested (10 4 cell / mL))÷reaction time (5min).

[0048] Depend on Figure 1 It can be seen that high-concentration extracts of different tissues of Spartina alterniflora all show the phenomenon of promotion at low concentrations and inhibition at high concentrations. The inhibition rate of extracts with high concentrations of 5 g / L and above reached more than 50% on the first day, and the inhibition rate of 10 g / L extract of the ear reached more than 90% on the third day (in the figure, L represents leaf, E represents ear, and S represents stem. 1, 2, 3, and 4 represent concentrations of 0.5, 1, 5, and 10 g / L, respectively).

[0049] As the main pigment of algae, chlorophyll a directly reflects the efficiency of photosynthesis and represents the growth and proliferation of algae. Figure 2 The results show that different tissues and concentrations of Spartina alterniflora result in different chlorophyll a contents, among which the 10 g / L extract of spike has the greatest inhibitory effect on chlorophyll a content, with an inhibition rate of 70% (Ct is the blank control group).

[0050] SOD and CAT are important enzymatic antioxidants that can help remove excess reactive oxygen species in the body and protect cells. MDA is a byproduct of lipid peroxidation (LPO), which is often evaluated as a biomarker in the early stages of oxidative stress response.

[0051] Depend on Figure 3 , Figure 4 and Figure 5 It can be seen that the low concentration of Spartina alterniflora tissue extract reduced the SOD, CAT activity and MDA content of Microcystis aeruginosa, while the high concentration of the extract increased the SOD, CAT activity and MDA content, and the SOD, CAT activity and MDA content of E4 were the highest. This shows that the high concentration of the extract of Spartina alterniflora spike caused a stronger oxidative stress response in Microcystis aeruginosa and caused greater damage to lipids.

[0052] GSH is an anti-ROS protective factor responsible for maintaining the redox balance in cells and can directly react with ROS. GST is a multifunctional enzyme widely present in organisms that can catalyze the reaction between GSH and ROS and plays an important role in enhancing biological resistance. Figure 6 and Figure 7 It can be seen that the low concentration (0.5, 1g / L) of the extract reduces the GSH content and GST activity in Microcystis aeruginosa, and the GSH content and GST activity increase at high concentrations. The GSH content and GST activity of the 5g / L extract of the spike are the highest. The GSH content and GST activity of the 10g / L extract of the spike and stem are marked as 0 because the test results are negative. It can be seen that the extract of the spike of Spartina alterniflora has the greatest oxidative stress on Microcystis aeruginosa, and all Microcystis aeruginosa die at a concentration of 10g / L.

[0053] In summary, the 10g / L extract of Spartina alterniflora spike has the strongest inhibitory effect on Microcystis aeruginosa and the best inhibitory effect.

[0054] Using the extract of Spartina alterniflora to inhibit cyanobacterial blooms not only promotes the resource utilization of the invasive plant Spartina alterniflora, but also can control cyanobacterial blooms. At the same time, the control process of cyanobacterial blooms is environmentally friendly and will not cause secondary pollution to water bodies, thus providing better help for China's sewage treatment, thereby reducing the environmental burden and improving the efficiency and greenness of the environmental governance process.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described with reference to the preferred embodiments of the present invention, it should be understood by those skilled in the art that various changes may be made in form and details without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. The use of an extract of Spartina alterniflora in the preparation of a cyanobacteria bloom inhibitor, characterized in that: The concentration of the Spartina alterniflora extract after being added to the blue algae growth environment is 0.5-10g / L; the Spartina alterniflora extract is a Spartina alterniflora spike extract; the Spartina alterniflora extract is prepared by a water extraction method; the Spartina alterniflora extract inhibits the chlorophyll a content in blue algae; the Spartina alterniflora extract increases the activities of SOD and CAT and the content of MDA in blue algae; the Spartina alterniflora extract increases the GSH content and GST activity in blue algae.

Images