Pseudomonas ly2 and uses thereof
By screening Pseudomonas LY2, the problem of insufficient nitrate dissimilatory reduction and arsenic oxidation functions in existing technologies was solved, realizing effective arsenic pollution remediation and nitrogen retention in high arsenic and high nitrite nitrogen environments, and providing a theoretical basis for novel arsenic pollution remediation technologies.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HUAZHONG AGRI UNIV
- Filing Date
- 2024-06-28
- Publication Date
- 2026-08-04
AI Technical Summary
Currently, no microbial strains have been found capable of simultaneously reducing nitrate to ammonium and oxidizing arsenic, which would lead to arsenic pollution and nitrogen loss. Furthermore, existing strains have poor survival rates in environments with high arsenic and high nitrite nitrogen, which could potentially cause secondary pollution.
Pseudomonas sp. LY2 was screened out. This strain can survive stably in high arsenic and high nitrite nitrogen environments and has the functions of dissimilatory reduction of nitrate to ammonium and arsenic oxidation, thus avoiding nitrite accumulation. Remediation agents with concentrations of 10⁸ CFU mL⁻¹ to 10¹² CFU mL⁻¹ were prepared for water and soil remediation.
Pseudomonas LY2 can effectively reduce arsenic toxicity and promote nitrogen retention in high arsenic and high nitrite nitrogen environments. It has the highest arsenic oxidation efficiency of 35.90% and the highest nitrate dissimilatory reduction efficiency to ammonium of 59.31%, without producing secondary pollution, thus providing a theoretical basis for arsenic pollution remediation.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of water and soil pollution remediation, specifically to a Pseudomonas sp. LY2 and its applications. Background Technology
[0002] Arsenic pollution is a global environmental problem. Arsenic can be transferred through the food chain and affect human health. During rice cultivation, frequent flooding and drying in paddy soil can promote arsenic activation, potentially exacerbating environmental risks. Simultaneously, the application of nitrogen fertilizers to paddy soil may also pose environmental problems. Ammonium nitrogen in fertilizers easily converts to nitrate nitrogen under flooded conditions, and then releases nitrous oxide through denitrification, leading to nitrogen loss and exacerbating the greenhouse effect. Furthermore, excessive nitrate accumulation in water bodies causes eutrophication, damaging aquatic ecosystems.
[0003] Microorganisms can transform pollutants in the environment through specific metabolic pathways. Functional microorganisms that perform dissimilatory reduction of nitrates to ammonium convert nitrates into ammonium nitrogen, thereby reducing the direct reduction of nitrates to nitrogen gas or nitrites. This process helps retain nitrogen and reduces nitrogen fertilizer loss. The oxidation of arsenite to arsenate is one of the effective measures to reduce arsenic toxicity. Functional microorganisms that oxidize arsenic can oxidize arsenic(III) to arsenic(V) through specific enzyme systems. The latter has lower solubility in water and relatively lower toxicity. Therefore, utilizing functional microorganisms for arsenic oxidation and the dissimilatory reduction of nitrates to ammonium is a promising remediation technology.
[0004] Currently, single-function strains can only play a partial role. Some microbial strains have been reported to be able to perform dissimilatory reduction of nitrate to ammonium or arsenic oxidation, but no strains that can perform these multiple functions simultaneously have been found. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Pseudomonas sp. LY2 strain and its application. This Pseudomonas sp. strain is a bacterium screened from neutral arsenic-contaminated paddy soil that exhibits nitrate dissimilatory reduction to ammonium coupled with arsenic oxidation. It can mediate the denitrification coupled with arsenic oxidation process, thereby reducing arsenic toxicity; and it is effective even in high nitrite nitrogen (200 mg NL) conditions. -1 ) and high arsenic (500mg As L) -1 It can survive in the environment without accumulating nitrite and causing secondary pollution. Therefore, it can not only reduce the toxicity of arsenic, but also promote nitrogen retention.
[0006] To achieve the above objectives, the technical solution designed by the present invention is as follows:
[0007] This invention provides a Pseudomonas sp. LY2, with the accession number: CCTCC NO: M20241108.
[0008] The aforementioned Pseudomonas sp. LY2, after identification, was determined to be a member of the genus Pseudomonas and named Pseudomonas sp. LY2. This bacterium was deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20241108 and deposit date of May 30, 2024.
[0009] This strain is stable at pH 5-10, 30°C, and in high nitrite nitrogen (200 mg NL) conditions. -1 ) and high arsenic (500mg As L) -1 It can survive in the environment without accumulating nitrite and causing secondary pollution.
[0010] The present invention also provides an application of the above-mentioned Pseudomonas LY2 in the dissimilatory reduction of nitrate to ammonium coupled with arsenic oxidation.
[0011] The present invention also provides an application of the above-mentioned Pseudomonas LY2 in the remediation of arsenic pollution in water or soil.
[0012] The present invention also provides the application of the above-mentioned Pseudomonas LY2 in the preparation of arsenic contamination remediation agents.
[0013] The present invention also provides an arsenic contamination remediation agent comprising Pseudomonas LY2.
[0014] Furthermore, the concentration of Pseudomonas LY2 in the repair agent is 10. 8 CFU mL -1 ~10 12 CFU mL -1 .
[0015] The beneficial effects of this invention are:
[0016] (1) The Pseudomonas LY2 strain of the present invention has two functions. In the present invention, Pseudomonas LY2 can simultaneously undergo arsenic oxidation and nitrate dissimilatory reduction to ammonium, with the highest arsenic oxidation efficiency being 35.90% and the highest nitrate dissimilatory reduction to ammonium efficiency being 59.31%.
[0017] (2) The *Pseudomonas LY2* strain of this invention exhibits high arsenic resistance. In this invention, *Pseudomonas LY2* can withstand 500 mg AsL... -1 Survival in the environment.
[0018] (3) The Pseudomonas LY2 strain of the present invention will not cause secondary pollution. In the present invention, Pseudomonas LY2 will not accumulate nitrite nitrogen after reducing nitrate nitrogen.
[0019] In summary, the *Pseudomonas* LY2 strain of this invention can mediate the denitrification-coupled arsenic oxidation process, thereby reducing arsenic toxicity. This provides an important theoretical basis for the development of novel arsenic pollution remediation technologies. Attached Figure Description
[0020] Figure 1 Transmission electron microscopy image of Pseudomonas LY2;
[0021] Figure 2 This is a phylogenetic tree of Pseudomonas LY2.
[0022] Figure 3 The graph shows the effect of Pseudomonas LY2 on nitrogen and arsenic over time.
[0023] In the figure, a represents the change in nitrate nitrogen content, b represents the change in nitrite nitrogen content, c represents the change in ammonium nitrogen content, and d represents the growth OD. 600 The graph shows the change in the content of trivalent arsenic oxide; e represents the change in the content of trivalent arsenic oxide.
[0024] Figure 4 A schematic diagram showing the growth of Pseudomonas LY2 under different arsenic concentrations;
[0025] Figure 5 A schematic diagram showing the growth of Pseudomonas LY2 under different pH conditions;
[0026] Figure 6 This is a schematic diagram illustrating the utilization of different carbon and nitrogen sources by Pseudomonas LY2.
[0027] Figure 7 The effect of the remediation agent on arsenic pollution in water;
[0028] In the figure, a represents the nitrate nitrogen content, b represents the nitrite nitrogen content, c represents the ammonium nitrogen content, and d represents the growth OD. 600 Figure e shows the content of trivalent arsenic oxidation. Detailed Implementation
[0029] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.
[0030] The culture medium formulations used in the following examples are as follows:
[0031] Liquid denitrification medium: sodium citrate 5.0 g / L -1 KNO3 1.0g L -1 K2HPO4 1.0g L -10.2 g L of CaCl2·H2O -1 MgSO4·H2O 1.0g L -1 FeSO4·7H2O 0.006g L -1 .
[0032] Liquid denitrification arsenic-containing medium: sodium citrate 5.0 g / L -1 KNO3 1.0g L -1 K2HPO4 1.0g L -1 0.2 g L of CaCl2·H2O -1 MgSO4·H2O 1.0g L -1 FeSO4·7H2O 0.006g L -1 NaAsO2 50mg As L -1 .
[0033] Liquid LB medium: 10 g / L tryptone -1 5g / L yeast powder -1 10g L of NaCl -1 .
[0034] To prepare solid plates, add 20 g / L agar to each of the above liquid culture media. -1 This refers to the corresponding solid culture medium.
[0035] All the above culture media were sterilized at 121℃ for 30 minutes.
[0036] The carbon and nitrogen sources were determined using a biolog-ECO plate containing 32 carbon and nitrogen sources.
[0037] The bacterial suspension concentration used in the following examples is 10. 8 CFU mL -1 (OD 600 =0.8).
[0038] Actual soil samples: Soil samples were collected from farmland soil in Zhongxiang City, Hubei Province. Fresh samples were stored in resealable bags and transported to the laboratory at low temperature.
[0039] Example 1: Screening of nitrate dissimilatory reduction to ammonium coupled with arsenic oxidizing bacteria
[0040] 1. Isolation and purification of bacteria
[0041] Soil samples collected from farmland in Zhongxiang City, Hubei Province, were inoculated into sterile water at a volume ratio of 1%. After shaking for 30 minutes, 1 mL of the suspension sample was added to 9 mL of sterile physiological saline and serially diluted to 10⁻⁶. -9100 μL of the diluted solution was spread onto solid denitrification arsenic-containing medium plates and inverted in a 28°C constant temperature incubator. The growth of bacteria on the plates was then observed. When a large number of single colonies grew on the plates, the single colonies were picked off and streaked to obtain pure cultures. The pure cultures were then stored at -80°C using glycerol tubes.
[0042] After culturing a single colony in liquid LB medium for 12 hours, a 1% bacterial suspension was inoculated into liquid denitrifying arsenic-containing medium. The medium was then incubated at 30°C and 150 rpm for 48 hours using a shaker. The contents of nitrate nitrogen, nitrite nitrogen, ammonium nitrogen, trivalent arsenic, and pentavalent arsenic in the medium were measured. The selected strain was determined based on the final measured ammonium nitrogen production and arsenic oxidation levels. Figure 1 ).
[0043] 2. Molecular biological identification
[0044] Single colonies of LY2 were selected for 16S rDNA gene amplification. PCR amplification was performed using the universal primers 27F / 1492R for the 16S rDNA gene.
[0045] 27F: 5'-AGTTTGATCMTGGCTCAG-3',
[0046] 1492R: 5'-GGTTACCTTGTTACGACTT-3';
[0047] The 30 μL PCR reaction system includes: 15 μL of 2×Taq PCR Master Mix, 2 μL of 27F (10 μM), 2 μL of 1492R (10 μM), 1 μL of DNA template (a single picked colony), and 10 μL of ddH2O.
[0048] PCR program settings: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 45 s, 72℃ extension for 40 s, 30 cycles of amplification; 72℃ final extension for 5 min.
[0049] To ensure the specificity of PCR amplification, after PCR amplification, 2 μL of PCR product was subjected to agarose gel electrophoresis (1% concentration). The banding pattern of the PCR product was used to determine the specificity of each sample's amplified product. PCR products with bands matching the theoretical target size were sent for Sanger sequencing using an ABI 3730xL DNA Analyzer sequencer. Finally, the obtained bacterial 16S rDNA sequence was submitted to the NCBI nt / nr database for sequence alignment. The species most similar to the target strain were determined based on the highest sequence completeness and similarity. The effective sequence length of the sequenced 16S rDNA was 1387 bp, as shown in SEQ ID NO.1. After BLAST comparison analysis, a phylogenetic tree of the strain was constructed. Figure 2 ),
[0050] In summary, the nitrate dissimilatory reduction to ammonium coupled with arsenic oxidation bacteria belongs to the genus *Pseudomonas*, named *Pseudomonas sp. LY2* (i.e., *Pseudomonas sp.* LY2). This bacterium was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, China, on May 30, 2024, with accession number CCTCCNO: M 20241108.
[0051] Example 2: Functional identification of Pseudomonas LY2
[0052] 1. Identification of the function of dissimilatory reduction of nitrate to ammonium coupled with arsenic oxidation
[0053] Pseudomonas LY2 was activated and cultured. The prepared bacterial suspension was inoculated into liquid denitrification arsenic-containing medium at a volume ratio of 1%, and incubated at 30℃ and 150 rpm for 2 days. Samples were taken at 0, 12, 24, 36, and 48 hours to determine nitrate nitrogen, nitrite nitrogen, ammonium nitrogen, trivalent arsenic, pentavalent arsenic, and solution OD. 600 absorbance ( Figure 3 ).
[0054] The results showed that Pseudomonas LY2 could dissimilate nitrate to ammonium and oxidize trivalent arsenic to pentavalent arsenic without nitrite accumulation.
[0055] 2. Arsenic resistance identification of strains
[0056] First, NaAsO2 concentrations of 50, 100, 300, 500, and 1000 mg As L were prepared. -1Solid LB arsenic-containing medium was used, with a pH of 7. The content and concentration of other substances, except for arsenic, were the same as those in LB medium. After high-temperature sterilization, 100 μL of the prepared bacterial solution was spread evenly on solid denitrification arsenic-containing medium with different arsenic concentrations. Then, it was placed in a constant temperature incubator at 28°C and incubated in the dark for 1 day. The growth was then photographed and recorded.
[0057] The results showed that Pseudomonas LY2 could survive at a NaAsO2 concentration of 500 mg As L. -1 It can survive in environments within a certain range and has high arsenic resistance. Figure 4 ).
[0058] Example 3: Selection of Culture Conditions for Pseudomonas LY2
[0059] 1. Detection of pH for bacterial growth
[0060] First, liquid denitrification media with pH values of 4, 5, 6, 7, 8, 9, and 10 were prepared. After high-temperature sterilization, the prepared bacterial solutions were inoculated into the liquid denitrification media at a 1% inoculation rate. The media were then incubated at 30°C and 150 rpm on a shaker for 1 day, and the OD of the solution was measured. 600 Absorbance.
[0061] The results showed that Pseudomonas LY2 could survive in environments with pH values ranging from 5 to 10. Figure 5 ).
[0062] 2. Selection of carbon and nitrogen sources for strain growth
[0063] Take 200 μL of the prepared bacterial culture into an ECO plate and incubate in the dark at 28℃ for 5 days. Place a damp cotton cloth under the plate to reduce sample evaporation. During this period, measure the absorbance at 590 nm wavelength on a microplate reader at 0, 4, 8, 12, 16, 20, 24, 36, 48, 60, 72, 96 and 120 h.
[0064] The results showed that *Pseudomonas LY2* could utilize a wide range of carbon and nitrogen sources, most of which were complex compounds, including methyl pyruvate, Tween 40, Tween 80, D-galacturonic acid, 4-hydroxybenzoic acid, γ-hydroxybutyric acid, L-arginine, L-asparagine, L-serine, and putrescine. Among these, LY2 showed the strongest utilization of L-asparagine. Figure 6 ).
[0065] Based on the above results: the culture medium for Pseudomonas LY2 is 5.0 g / L L-asparagine. -1 K2HPO4 1.0g L -1 0.2 g L of CaCl2·H2O -1 MgSO4·H2O 1.0g L -1FeSO4·7H2O 0.006g L -1 Its pH is 7.0.
[0066] Example 4: Preparation of arsenic contamination remediation agent
[0067] After activating Pseudomonas LY2 in LB medium for 24 hours, the bacterial suspension was collected and the viable count was adjusted to 102. 8 CFU mL -1 Subsequently, an inoculum of 1% by volume was added to the sterilized *Pseudomonas* LY2 from Example 3, and the mixture was incubated at 30°C and 150 rpm on a shaker for 10 hours. The bacterial suspension was then collected and the viable count was adjusted to 102. 10 CFU mL -1 Bacterial agents are also known as repair agents.
[0068] Example 5: Application of Arsenic Pollution Remediation Agents in Water Remediation
[0069] An environment simulating nitrate enrichment and arsenic contamination was simulated by artificially setting up different gradients. First, two nitrogen sources (KNO3 and KNO2) were prepared at concentrations of 50, 100, and 200 mg NL, respectively. -1 The aquatic environment was analyzed in six treatments, with the pH adjusted to 7. The content and concentration of substances other than the nitrogen source were as follows: sodium citrate 5.0 g / L. -1 KNO3 1.0g L -1 K2HPO4 1.0g L -1 0.2 g L of CaCl2·H2O -1 MgSO4·H2O 1.0g L -1 FeSO4·7H2O 0.006g L -1 After high-temperature sterilization, the above-mentioned repair agent was inoculated into different treatments at a volume ratio of 1%, and incubated at 30℃ and 150rpm in a shaker for 2 days. Then, nitrate nitrogen, nitrite nitrogen, ammonium nitrogen, trivalent arsenic, pentavalent arsenic, and the OD of the solution were measured. 600 Absorbance.
[0070] The results showed that, in the remedy, *Pseudomonas LY2* was able to revive at concentrations of 50, 100, and 200 mg NL for two nitrogen sources (KNO3 and KNO2). -1 It can survive in environments with high nitrite toxicity. LY2 can survive in environments with a NaAsO2 concentration of 50 mg As L. -1 The arsenic oxidation efficiency was greater than or equal to 20.62% at nitrogen source concentrations of 100 and 200 mg NL. -1 Under these conditions, the dissimilatory reduction efficiency of nitrate to ammonium ranged from 22.03% to 59.31%, with the highest efficiency at 200 mg / L KNO2. -191.61 mg NL was converted. -1 Ammonium nitrogen and 78.17 mg NL -1 Only 10.40 mg NL of nitrate nitrogen remained. -1 The treatment with KNO3 as the nitrogen source resulted in almost no accumulation of nitrite nitrogen, effectively reducing arsenic content in the water and providing important technical support for the remediation and protection of soil and water bodies. Figure 7 ).
[0071] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A type of Pseudomonas ( Pseudomonas sp.)LY2, its accession number is: CCTCC NO: M 20241108.
2. The application of the Pseudomonas LY2 of claim 1 in the dissimilatory reduction of nitrate to ammonium coupled with arsenic oxidation.
3. The application of the Pseudomonas LY2 of claim 1 in the remediation of arsenic pollution in water bodies.
4. The use of the Pseudomonas LY2 of claim 1 in the preparation of an arsenic contamination remediation agent.
5. A remediation agent for arsenic contamination, characterized in that: The repair agent includes the Pseudomonas LY2 of claim 1, and the concentration of the Pseudomonas LY2 in the repair agent is 10 8 CFU / mL -1 ~10 12 CFU / mL -1 .
6. The repair agent according to claim 5, characterized in that: The concentration of Pseudomonas LY2 in the repair agent is 10. 10 CFU mL -1 .