Application of pyocyanin as a reagent for cadmium and chromium removal from rice seeds

Abstract

This invention discloses the application of pyocyanin as a cadmium and chromium reduction agent in rice grains, belonging to the field of safe utilization technology for contaminated farmland. Adding pyocyanin to cadmium and / or chromium-contaminated farmland soil can effectively reduce the accumulation of cadmium and / or chromium in rice grains, thereby achieving the goal of safe utilization of cadmium and / or chromium-contaminated farmland soil.

Description

Technical Field

[0001] This invention relates to a novel use of pyocyanin, specifically a method for applying pyocyanin as a cadmium and chromium-reducing agent to cadmium and / or chromium-contaminated farmland soil to reduce the cadmium / chromium content in rice grains, belonging to the field of safe utilization technology for contaminated farmland. Background Technology

[0002] Rice is a staple food in many countries worldwide, making food safety crucial. Cadmium (Cd) is currently the main inorganic pollutant affecting the environmental quality of paddy field soil, with an exceedance rate reaching 7%. Chromium (Cr) ranks sixth with an exceedance rate of 1.1%. Heavy metal pollution in arable land is mainly at the light to moderate levels, while heavy metal pollution in paddy field soil threatens human health. Remediating contaminated soil and reducing the accumulation of heavy metals in agricultural products are urgent tasks. Therefore, there is an urgent need to research a green, economical, and efficient method to control the accumulation of cadmium / chromium complex pollution in rice, in order to improve the safe utilization rate of arable land, ensure food security, and reduce human health risks.

[0003] For paddy field soils contaminated with cadmium and chromium, current control technologies mainly include low-accumulation rice varieties, soil pH adjustment, water management, and foliar regulation, i.e., VIP+n technology. Through long-term breeding research in China, the "Xizi No. 3" low-cadmium-accumulation rice variety has been the first to pass the national preliminary approval. In field experiments, the average Cd content in paddy fields was 0.02 mg / kg, but the average yield was only 500-600 kg / mu (approximately 0.067 hectares), indicating a need for further improvement in both quality and yield. In actual field experiments, considering the simplicity of implementation, cost-effectiveness, and potential environmental impact, the aforementioned control technologies are still difficult to effectively promote and apply on a large scale in paddy fields. Currently, small molecule compounds such as ethylenediaminetetraacetic acid (EDTA) have been shown to directly affect changes in plant roots and rhizosphere microorganisms to control the migration of heavy metals. Therefore, researchers have begun to focus on small molecule organic compounds secreted by microorganisms with significant environmental effects, hoping to prepare them through microbial fermentation extraction or chemical synthesis for large-scale application in cadmium / chromium contaminated paddy field soils.

[0004] Pyocyanin is widely used as an antibacterial agent in aquaculture, agriculture, biosensors, and medicine. However, to date, there are no studies or reports on its application in controlling the accumulation of cadmium / chromium complex pollution in rice grains in farmland. Summary of the Invention

[0005] In view of the technical problems of cadmium / chromium co-contamination in rice grains in cadmium / chromium co-contaminated farmland in the prior art, the first objective of this invention is to provide a method for reducing cadmium and chromium in rice grains by adding pyocyanin to cadmium / chromium co-contaminated farmland soil. This method can effectively reduce the accumulation of cadmium / chromium in rice grains, thereby achieving the goal of safe utilization of cadmium / chromium co-contaminated farmland soil.

[0006] The second objective of this invention is to provide a Pseudomonas aeruginosa CdO3 strain that is resistant to heavy metals and produces pyocyanin at a high yield.

[0007] To achieve the above-mentioned technical objectives, the present invention provides the application of pyocyanin as a cadmium and chromium reducing agent in rice seeds.

[0008] The pyocyanin of the present invention has the following chemical structure:

[0009]

[0010] The pyocyanin of this invention can be synthesized by microorganisms: Pseudomonas aeruginosa Cd03 (scientific name: Pseudomonas aeruginosa Cd03, deposit date: December 30, 2019; depositary institution: China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China; deposit number: CCTCC NO: M 20191130) was picked from LB culture dishes and added to GA medium (main components and concentrations of medium: glycerol 2%, alanine 6g, magnesium sulfate 3g, dipotassium hydrogen phosphate 0.1g, ferrous sulfate 0.01g, distilled water was added to make up to 1L, and the pH was adjusted to 7). The medium was placed in a constant temperature air bath shaker at 30℃ and shaken at 150r / min for 2 days. 150mL of the seed culture was taken and centrifuged in a floor centrifuge at 8000r / s for 15min to remove the bacterial cells. The supernatant was collected, extracted with chloroform-HCl, and then rotary evaporated to obtain pyocyanin powder. 10 mg of the extracted pyocyanin powder was weighed and dissolved in 10 mL of acetonitrile. After complete dissolution, the solution was transferred to a 1.5 mL HPLC vial for analysis. The identification instrument used in this invention was a Shimadzu LC-MS / MS-8045. Instrument conditions were as follows: Mobile phase A: water + 0.1% formic acid; Mobile phase B: acetonitrile; Injection volume: 50 μL; Flow rate: 0.5 mL / min; Column temperature: 40℃; UV wavelength: 520 nm, 521 nm; Run time: 35 min; Gradient program: 0-10 min 10-60% acetonitrile; 10-20 min 60-90% acetonitrile; 20-30 min 90% acetonitrile; 30-32 min 90-10% acetonitrile; 32-35 min 10% acetonitrile; MS parameters: Capillary voltage: 4 kV; Gas temperature: 300℃; Gas flow rate: 12 L / min; Nebulizer pressure: 35 psi; Scan range: 100-2000 m / z; Results are as follows.Figure 7 As shown.

[0011] The application of pyocyanin in this invention as a cadmium and chromium-reducing agent in rice seeds involves direct application to cadmium and / or chromium-contaminated soil (rhizosphere of rice). On one hand, pyocyanin chelates with free metal ions in the soil to form pyocyanin-cadmium complexes, which are actively transported and absorbed by the rice rhizosphere, accumulating primarily in the roots and stems. Furthermore, during flooding, pyocyanin can also reduce the cadmium content of highly toxic chromium ions in the rice rhizosphere through redox reactions. 6+ Reduced to Cr 3+ Pyocyanin reduces chromium toxicity, affects chromium transport in the soil-rice system, and reduces chromium accumulation in brown rice. On the other hand, it disrupts the rice's own metabolic processes, primarily stimulating the plant to secrete large amounts of secondary defense substances such as benzoxazines and indole compounds like tryptophan. These compounds further react with cadmium / chromium within the plant, thereby reducing the migration of cadmium / chromium complex pollution into the grains. Pyocyanin application also affects the expression of cadmium transport-related genes (such as OsNRAMP1, OsHMA2, OsHMA3, and OsPCS1), altering the redistribution capacity of cadmium in different rice plant tissues, thus reducing its accumulation in rice grains.

[0012] As a preferred embodiment, the pyocyanin is applied to cadmium and / or chromium-contaminated soil in rice-growing areas. More preferably, the pyocyanin is applied to the rice rhizosphere. Pyocyanin is primarily absorbed by the rice rhizosphere, thereby influencing the translocation and distribution of cadmium / chromium complex pollution in various parts of rice tissues by regulating rice metabolism and the expression of related genes. Therefore, applying it to the rice rhizosphere is more conducive to its effectiveness.

[0013] As a preferred embodiment, the pH value of the cadmium and / or chromium contaminated soil is 4.5–6.5, the cadmium concentration is no higher than 1 mg / kg, and the chromium concentration is no higher than 300 mg / kg. Pyocyanin is adaptable to cadmium-contaminated farmland soil, chromium-contaminated farmland soil, and farmland soil with combined cadmium and chromium contamination, and it adapts to a wide range of cadmium and chromium concentrations.

[0014] As a preferred embodiment, the application rate of pyocyanin in cadmium and / or chromium contaminated soil is 0.1–1.0 g / m³. 2 If too little pyocyanin is added, the desired effect will not be achieved; if too much pyocyanin is added, it will have an adverse effect on the growth and development of rice.

[0015] As a preferred embodiment, the pyocyanin is applied at any or several stages during the jointing, heading, grain-filling, and maturity stages of rice. Pyocyanin can be added almost throughout the entire growth period of rice.

[0016] As a preferred embodiment, the pyocyanin is prepared as a solution with a concentration of 0.3 to 1.0 g / L for use.

[0017] As a preferred embodiment, the pyocyanin is produced by Pseudomonas aeruginosa CdO3; the preservation number of Pseudomonas aeruginosa CdO3 is: CCTCC NO: M 20191130.

[0018] This invention also provides a *Pseudomonas aeruginosa* CdO3, with accession number CCTCC NO: M 20191130. It exhibits characteristics such as resistance to heavy metals and high-yield production of pyocyanin.

[0019] Compared with existing technologies, the beneficial technical effects of the present invention are as follows:

[0020] The technical solution of this invention, by applying pyocyanin to cadmium / chromium contaminated farmland soil, can significantly alleviate the stress of cadmium and / or chromium combined pollution on rice, and reduce the accumulation of cadmium and / or chromium combined pollution in rice grains. Pyocyanin affects the metabolism of rice root exudates, synthesizing benzoxazine and indole substances, thus alleviating and / or affecting the stress on rice, and influencing the absorption of cadmium in the rhizosphere; the addition of pyocyanin can regulate the expression of genes such as OsNRAMP1, OsHMA2, OsHMA3, and OsPCS1, affecting the translocation and distribution of cadmium in various parts of rice tissues. For example, when pyocyanin solution was applied to the rhizosphere soil of rice in farmland with combined pollution, compared with no application of pyocyanin solution, the cadmium content in rice grains decreased by 30.12% to 48.92%, and the concentration decreased from 0.31 mg / kg to 0.18 mg / kg, meeting national safety standards; the chromium content in rice grains decreased by 45.26% to 58.82%, and the concentration decreased from 1.47 mg / kg to 0.86 mg / kg, meeting national safety standards.

[0021] The pyocyanin of this invention is readily available and inexpensive, and has the advantages of being economical, efficient, and easy to use, making it convenient for widespread application. Attached Figure Description

[0022] Figure 1 The figure shows the cadmium content in the rhizosphere soil and different tissues of *Pyrrosia lingua* rice at maturity, with and without the addition of pyocyanin in Example 1. The figure shows that after adding pyocyanin at different growth stages, the cadmium content in the roots and stems of rice increased, while the cadmium content in the jointing and heading stages decreased slightly, and the cadmium content in the mature brown rice decreased. The cadmium content in the brown rice decreased from 0.31 mg / kg to 0.18 mg / kg, which is lower than the limit of 0.2 mg / kg in the National Food Safety Standard - Limits of Contaminants in Food (GB2762-2017).

[0023] Figure 2The translocation coefficients of cadmium in different tissues of rice with and without the addition of pyocyanin in Example 1 are shown in the figure. The figure shows that the addition of pyocyanin leads to the accumulation of cadmium in the roots of rice, but reduces the transport of cadmium to the aboveground parts and reduces the distribution of cadmium from the stem to the leaves, brown rice and other parts.

[0024] Figure 3 The figures show the chromium content in the rhizosphere soil and different tissues of *Pyrrosia lingua* rice during the flooded period and the chromium content in mature brown rice after adding and not adding pyocyanin in Example 1. The figures show that after adding pyocyanin, the chromium concentration in the rhizosphere soil decreased at different growth stages, the chromium concentration in the rice roots increased, the chromium concentration in the leaves decreased, and the chromium content in the brown rice decreased. Specifically, the chromium content in the brown rice decreased from 1.47 mg / kg to 0.86 mg / kg, which is lower than the limit of 1.0 mg / kg in the National Food Safety Standard - Limits of Contaminants in Food (GB2762-2017).

[0025] Figure 4 The translocation coefficients of chromium in different tissues of rice were shown in the figure for Huang Huazhan after adding and not adding pyocyanin in Example 1. The figure shows that adding pyocyanin will enrich chromium in the rice roots. The jointing and heading stages will lead to an increase in the transport of chromium from the roots to the aboveground parts, while the grain-filling and maturity stages will reduce the transport of chromium to the aboveground parts. However, the addition of pyocyanin will reduce the distribution of chromium from the stem to the leaves and brown rice at different growth stages.

[0026] Figure 5 The figures show the changes in the content of benzoxazine and indole in root exudates during the grain-filling stage and the abundance of related genes regulating their synthesis after adding and not adding pyocyanin in Example 2. The figures show that the content of benzoxazine and indole is consistent with the gene expression. Benzoxazine and indole compounds are both secondary metabolites secreted by roots, which can effectively alleviate cadmium stress on plants. After adding pyocyanin, the content of benzoxazine and indole in root exudates increased. On the one hand, it can alleviate the stress of cadmium / chromium on rice, and on the other hand, it can bind to cadmium / chromium and promote the translocation of cadmium from the soil to the plant.

[0027] Figure 6 The figure shows the abundance changes of genes related to maturity after adding and not adding pyocyanin in Example 3. The figure shows that after adding pyocyanin, OsNRAMP1 was upregulated, indicating that the root's ability to absorb cadmium is increased; OsHMA2 was downregulated and OsHMA3 was upregulated, indicating that more cadmium was fixed in the roots and the translocation to the aboveground parts was reduced, which is consistent with the cadmium content in different tissues of rice; the abundance of OsPCS1 increased, indicating that adding pyocyanin can alleviate the toxicity of cadmium / chromium combined pollution to rice.

[0028] Figure 7The LC-MS / MS structure of pyocyanin was identified. The retention time of pyocyanin is 7.37067 seconds. The secondary mass spectrum shows that the relative molecular mass of pyocyanin is 210.08. The mass-to-charge ratio is 197.2 when the -CH3 bond is broken; 168.3 when the -CH3 and -COH bonds are broken; and 181.2 when the -CH3 and -OH bonds are broken. Detailed Implementation

[0029] The present invention will be further described below with reference to specific implementation examples. These examples are specific implementation examples of the present invention and do not represent all implementation methods and applications. Non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are still within the scope of protection of the present invention.

[0030] Example 1

[0031] Soil culture experiment with added pyocyanin:

[0032] The soil used in the experiment was paddy field soil collected from Yangji Village, Xiangtan City, Hunan Province. The concentrations of cadmium and chromium were measured to be 0.56 mg / kg and 261.24 mg / kg, respectively, with a pH of 5.6 ± 0.05. The soil was air-dried and passed through a 20-mesh sieve for later use. Approximately 45 kg of the soil was placed in a square foam box (100cm × 45cm × 72cm), and 27 'Huanghuazhan' rice seedlings were transplanted into it. 2 L of chlorophyll solution (prepared at 0.3 g / L) was added at the jointing, heading, grain-filling, and maturity stages, and the seedlings were continuously cultured until harvest, a period of 110 days. The results are as follows: Figures 1-4 As shown.

[0033] Example 2

[0034] Determination of benzoxazine and indole concentrations in root exudates:

[0035] Transfer all samples to a 2 mL centrifuge tube, add a 6 mm diameter grinding bead, and add 300 μL of extraction buffer (methanol:water = 4:1 (v:v)) containing four internal standards (L-2-chlorophenylalanine (0.02 mg / mL, etc.). Grind the samples using a cryogenic homogenizer for 6 min (-10℃, 50 Hz), followed by low-temperature ultrasonic extraction for 30 min (5℃, 40 kHz). Allow the samples to stand at -20℃ for 30 min, then centrifuge for 15 min (13000 g, 4℃). Transfer the supernatant to a vial with an inner tube for analysis. Chromatographic conditions: Column: ACQUITY UPLC HSS T3 (100 mm × 2.1 mm). (id, 1.8 μm; Waters, Milford, USA); Mobile phase A was 95% water + 5% acetonitrile (containing 0.1% formic acid), mobile phase B was 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid), injection volume was 3 μL, column temperature was 40℃. Mass spectrometry conditions: The sample was electrospray ionized, and mass spectrometry signals were acquired using positive and negative ion scanning modes, respectively; scan range 70–1050 m / z, sheath gas flow rate 50 arb, auxiliary gas flow rate 13 arb, heating temperature 425℃; capillary temperature 325℃, spray voltage ±3500 V. Results are as follows. Figure 5 As shown.

[0036] Example 3

[0037] Rice gene expression:

[0038] Leaf tips from hydroponically grown and soil-grown rice at the grain-filling stage were collected, ground into powder using liquid nitrogen, and then a high-purity rice RNA solution was obtained using a plant total RNA extraction kit (Shanghai Sangon Biotech Co., Ltd.). Rice cDNA solution was then obtained using a reverse transcription kit (Shanghai Yisheng Biotechnology Co., Ltd.) and stored at -20℃. Changes in the relative expression levels of rice genes were measured using quantitative real-time PCR (Lightcycler 96, Roche). Results are shown below. Figure 6 As shown.

[0039] Example 4

[0040] Repair cost calculation:

[0041] The remediation cost mainly includes the production cost and application cost of pyocyanin. Taking the remediation of one acre of land as an example, using industrial-grade reagents and pure water fermentation, the production cost is approximately 55.2 yuan / acre, the electricity and equipment cost is approximately 57.6 yuan / acre, and the labor cost during production and application is 20 yuan / acre, for a total cost of approximately 133.1 yuan / acre.

[0042] The scope of this invention is not limited to the specific embodiments described above. Any modifications or additions made by those skilled in the art to the specific embodiments described above, or any similar approaches adopted, shall be within the scope of protection of this invention.

Claims

1. Pyocyanin as a rice seed cadmium and chromium reducing agent applied to cadmium and / or chromium contaminated soil where rice is planted; the amount of pyocyanin applied to the cadmium and / or chromium contaminated soil is 0.1 to 1.0 g / m 2 .

2. The application according to claim 1, characterized in that: The pyocyanin was applied to the rice rhizosphere.

3. The application according to claim 1, characterized in that: The pH value of the cadmium and / or chromium contaminated soil is 4.5 to 6.5, the cadmium concentration is not higher than 1 mg / kg, and the chromium concentration is not higher than 300 mg / kg.

4. The application according to claim 1, characterized in that: The pyocyanin is applied at any time or at any combination of times during the jointing, heading, grain-filling, and ripening stages of rice.

5. The application according to claim 4, characterized in that: The pyocyanin is prepared into a solution with a concentration of 0.3~1.0 g / L for use.

6. The application according to claim 1, 2, 3, 4 or 5, characterized in that: The pyocyanin is produced by Pseudomonas aeruginosa CdO3; the preservation number of Pseudomonas aeruginosa CdO3 is: CCTCC NO: M 20191130.

7. Pseudomonas aeruginosa Cd03, preservation number: CCTCC NO: M 20191130.

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