CN109722394B - A strain of arsenic-oxidizing Pseudomonas and its application - Google Patents

Abstract

An arsenic-oxidizing Pseudomonas strain and its application, the invention relates to an arsenic-oxidizing Pseudomonas strain and its application. A strain of Pseudomonas arsenic oxidizing in the present invention is Pseudomonas arsenicoxydans Y24-2, which has been deposited in the General Microbiology Center of the China Microorganism Culture Collection and Administration Commission with the preservation number of CGMCC No. 16655, and the preservation date is October 29, 2018. Under the condition of low temperature and poor nutrition, when the nitrate concentration of the influent is 20-100 mg/L, the nitrite in the groundwater can be efficiently removed, and the nitrate removal rate is 12.94-93.96 mg/L/h. The invention is applied to the technical field of environmental microorganisms.

Description

A strain of arsenic-oxidizing Pseudomonas and its application

technical field

The present invention relates to a strain of arsenic-oxidizing pseudomonas and its application.

Background technique

Due to the application of agricultural fertilizers, nitrate enters the groundwater environment through soil infiltration and affects groundwater quality. Because agricultural non-point source pollution is relatively difficult to control, the phenomenon of excessive nitric acid in groundwater in my country is relatively common. If the drinking water contains excessive nitrates, it is easy to cause methemoglobinemia, and even induce cancer, causing harm to the human body. In my country, especially in the northern region, about 60% of people use groundwater as their water source. Effectively removing nitrates from groundwater is crucial to ensuring the safety of water supply for residents. my country's drinking water hygiene standards (GB5749-2006) implemented since July 2007 also limit the maximum allowable concentration of nitrate nitrogen to 10mg/L.

At present, the biological treatment methods of nitrate in groundwater can be divided into two categories:

One is to use heterotrophic denitrifying bacteria to remove nitrate from groundwater. The principle is that under anoxic conditions (dissolved oxygen is about 0.5 mg/L), organic matter (organic carbon) is used as an electron donor to reduce nitrate to generate nitrogen. Because the organic matter content in groundwater is low and mainly exists in the form of humic acid that is difficult to be bioavailable, the C/N ratio in raw water is usually less than 1, so the carbon source is often insufficient. The currently commonly used solution is to add slow-release carbon sources such as rice straw and corncob to improve the C/N ratio of the treatment process.

The other is to use autotrophic denitrifying bacteria to remove nitrate from groundwater. The principle is that under obligate anaerobic conditions, CO ₂ , HCO ₃ ^- or CO ₃ ^2- is used as carbon source, and H ₂ , S or sulfide is used as electron donor to reduce nitrate to generate nitrogen, such as denitrification sulfur Bacillus (Thiobacillus denitrificans), Thiobacillus ferrooxidans (Thiobacillus ferrooxidans) and the like. Although this type of autotrophic denitrifying bacteria does not require an additional organic carbon source during nitrate removal, a strict anaerobic environment is required and an additional electron donor ₍ H or sulfide) is required, not only during application The process is difficult to achieve, and the cost of water production is also increased accordingly.

In recent years, some related studies have attempted to solve the problems of low groundwater temperature, low C/N ratio and difficult biological treatment. Zhou Shilei (Research on denitrification characteristics and technical application of oligotrophic aerobic denitrifying bacteria in water source reservoirs with mixed oxygenation. Xi'an University of Architecture and Technology. Doctoral dissertation 2017) During the research process, 13 strains that can survive in oligotrophic conditions were isolated and obtained. The bacteria that carry out aerobic denitrification under the condition are Acinetobacter sp., Novosphingobium sp., Aquabacterium sp., Sphingomonas sp. ), Zoogloea sp. and Delftia sp.. Liu Yongbo et al. (Liu Yongbo, Qu Dan, He Jun. Aerobic denitrification performance of a low-temperature aniline-degrading bacteria. Henan Water Conservancy and South-to-North Water Transfer. 2018, 5:78-79) also isolated and obtained aerobic denitrification suitable for cold Bacteria AN-1. However, when the above strains are used for denitrification, the nitrate concentration does not exceed 5 mg/L, while the nitrate concentration in the slightly polluted groundwater is usually 20-150 mg/L.

Pseudomonas arsenicoxydans was originally reported in 2010 to oxidize arsenite (Victor L. Campos et al. Pseudomonas arsenicoxydans sp nov., an arsenite-oxidizing strain isolated from the Atacama desert. Systematic and Applied Microbiology. 2010.33:193-197.). In recent years, there have also been studies on the use of arsenic-oxidizing Pseudomonas for the treatment of arsenite, but there is no report that arsenic-oxidizing Pseudomonas can be used for the treatment of nitrate in groundwater.

SUMMARY OF THE INVENTION

The invention provides a strain of arsenic oxidizing pseudomonas and its application.

A strain of Pseudomonas arsenic oxidizing in the present invention is Pseudomonas arsenicoxydans Y24-2, which has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration Committee, with the preservation number of CGMCC No. 16655, and the preservation date is October 29, 2018.

The Pseudomonas arsenic oxidizing bacteria of the present invention is Pseudomonas arsenicoxydans Y24-2, which is used for removing pollutants in water.

The screening method for Pseudomonas arsenicoxydans Y24-2 in the present invention is: 100ml of low-temperature groundwater (water temperature 4°C) contaminated by nitrate (nitrate concentration 140mg/L), at 2-6°C, 150rpm/min After enrichment for 12-20 days, NaNO ₃ and C ₂ H ₅ OH were continuously supplemented during the period. After the OD ₆₀₀ value of the bacterial solution reached above 0.5, gradient dilution was performed and inoculated on solid medium, and cultured at 2-6 °C for 5- 7d, select a typical single colony for purification and culture. The finally obtained pure cultured strains were subjected to nitrate reduction test, and the strains whose nitrate reduction rate reached more than 50% within 30 minutes were selected.

After extracting the genomic DNA of the strain, the 16S rDNA sequence was amplified, and the obtained fragment was used for sequencing. The similarity with the type strain Pseudomonas arsenicoxydans CECT 7543 ^T was 99.21%. It was identified as Pseudomonas arsenicoxydans, and finally named Pseudomonas arsenicoxydans Y24-2.

The Pseudomonas arsenicoxydans Y24-2 of the present invention is 6-10 DEG C, the C/N ratio is 0-0.5 under the low-temperature nutrient-poor condition, when the influent nitrate concentration is 20-100 mg/L, the arsenic-oxidizing Pseudomonas (Pseudomonas arsenicoxydans) Y24-2 can efficiently remove nitrate in groundwater, and the removal rate of nitrate is 12.94～93.96mg/L/h.

The present invention, Pseudomonas arsenicoxydans Y24-2, has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee, the deposit number is CGMCC No. 16655, and the deposit date is October 29, 2018.

Description of drawings

Fig. 1 is the schematic diagram of the Gram staining result of Pseudomonas arsenicoxydans Y24-2 of the present invention;

Figure 2 is a schematic diagram of the colony of Pseudomonas arsenicoxydans Y24-2 of the present invention.

Detailed ways

Specific embodiment 1: A strain of Pseudomonas arsenic oxidizing in this embodiment is Pseudomonasarsenicoxydans Y24-2, which has been deposited in the General Microbiology Center of the China Microbial Culture Collection and Management Committee, with the preservation number of CGMCC No. 16655, and the preservation date is October 2018. 29th.

In this embodiment, the screening method for Pseudomonas arsenicoxydans Y24-2 is as follows: 100ml of low-temperature groundwater (water temperature 4°C) contaminated by nitrate (nitrate concentration 140mg/L), at 2-6°C, 150rpm/L Min to enrich for 12 ~ 20d, during which NaNO ₃ and C ₂ H ₅ OH were continuously supplemented. After the OD ₆₀₀ value of the bacterial solution reached above 0.5, the gradient was diluted and inoculated on solid medium, and cultured at 2 ~ 6 ℃ for 5 ~7d, select a typical single colony for purification and culture. The finally obtained pure cultured strains were subjected to nitrate reduction test, and the strains whose nitrate reduction rate reached more than 50% within 30 minutes were selected.

After extracting the genomic DNA of the strain, the 16S rDNA sequence was amplified, and the obtained fragment was used for sequencing. The similarity with the type strain Pseudomonas arsenicoxydans CECT 7543 ^T was 99.21%. It was identified as Pseudomonas arsenicoxydans, and finally named Pseudomonas arsenicoxydans Y24-2.

In the present embodiment, Pseudomonas arsenicoxydans Y24-2 is at 6-10° C., C/N ratio of 0-0.5 under low temperature and nutrient-poor conditions, and when the nitrate concentration of the influent is 20-100 mg/L, Pseudomonas arsenicoxydans (Pseudomonas arsenicoxydans )Y24-2 can efficiently remove nitrite in groundwater, and the removal rate of nitrate is 12.94～93.96mg/L/h.

Embodiment 2: The formulation of the medium used in the screening process of Pseudomonas arsenides in this embodiment is: NaNO ₃ 0.1-0.5g/L, MnSO ₄ 0.01-0.05g/L, (NH ₄ ) ₂ Fe ( SO ₄ ) ₂ ·6H ₂ O 0.01～0.10g/L, CaCl ₂ 0.01～0.05g/L, C ₂ H ₅ OH 0.1～2.0mL/L, Na ₂ HPO ₄ 0.3～0.9g/L, MgSO ₄ · 7H ₂ O 0.01～0.05g/L, NaCl 0.3～0.9g/L, pH value 7.0～7.4. Others are the same as the first embodiment.

If it is a solid medium, add 1.8g/L agar to the above formula.

Embodiment 3: In this embodiment, Pseudomonas arsenicoxydans is Pseudomonas arsenicoxydansY24-2, which is used to remove pollutants in water.

Embodiment 4: The difference between this embodiment and Embodiment 3 is that the water is slightly polluted low-temperature groundwater. Others are the same as the third embodiment.

Embodiment 5: The difference between this embodiment and Embodiment 3 or 4 is that the temperature of the low-temperature groundwater is 4-10°C. Others are the same as the third or fourth embodiment.

Embodiment 6: The difference between this embodiment and one of Embodiments 3 to 5 is that the pollutants are nitrates. Others are the same as one of the third to fifth embodiments.

Embodiment 7: The screening method for Pseudomonas arsenicoxydansY24-2 in this embodiment is: 100ml of low-temperature groundwater (water temperature 4°C) polluted by nitrate (nitrate concentration 140mg/L), at 2-6 ℃, 150rpm/min enrichment for 12 ~ 20d, during which NaNO ₃ and C ₂ H ₅ OH were continuously supplemented. After the OD ₆₀₀ value of the bacterial solution reached above 0.5, gradient dilution and inoculation on solid medium were carried out. After 2 ~ 6 Cultivate at ℃ for 5-7 days, and select a typical single colony for purification and culture. The finally obtained pure cultured strains were subjected to nitrate reduction test, and the strains whose nitrate reduction rate reached more than 50% within 30 minutes were selected.

The culture medium used in the above process is: NaNO ₃ 0.1～0.5g/L, MnSO ₄ 0.01～0.05g/L, (NH ₄ ) ₂ Fe(SO ₄ ) ₂ ·6H ₂ O 0.01～0.10g/L, CaCl ₂ 0.01～0.05g/L, C ₂ H ₅ OH 0.1～2.0mL/L, Na ₂ HPO ₄ 0.3～0.9g/L, MgSO ₄ ·7H ₂ O 0.01～0.05g/L, NaCl 0.3～0.9g/ L, pH value 7.0～7.4, (add 1.8g/L agar to solid medium).

Strain Pseudomonas arsenicoxydans Y24-2 obligate aerobic bacteria, negative for Gram staining (as shown in Figure 1), colonies raised, viscous, and milky white (as shown in Figure 2); positive for oxidase and contactase, growth The temperature is 4～37℃, the optimum pH value is 7.5～8.0; hydrogen sulfide and indole cannot be produced, urea can be hydrolyzed, and nitrate can be reduced; ethanol, sodium acetate, sodium butyrate, sodium benzoate, and glucose can be used as carbon sources For growth; can not use citric acid, maltose.

Embodiment 6: Identification of Pseudomonas arsenicoxydans Y24-2 in this embodiment:

Bacterial genomic DNA was extracted for 16S rDNA sequence amplification. The PCR primers used for amplification were universal primers: forward primer 5'-CAGAGTTTGATCCTGGCT-3'; reverse primer 5'-AGGAGGTGATCCAGCCGCA-3'. The PCR reaction system is as follows: 20～50ng template DNA, 0.2μL Taq enzyme, 2.5μL 10×Buffer (containing Mg ²⁺ ), 1μL dNTP (2.5mmol/L each), 0.5μL forward primer, 0.5μL reverse primer, Add sterile deionized water to 25 μL. PCR amplification conditions: pre-denaturation at 94°C for 4 min, followed by denaturation at 94°C for 45s, amplification at 55°C for 45s, extension at 72°C for 1min, after 30 cycles, extension at 72°C for 10min, and then termination of the reaction at 4°C, the obtained fragment was used for Sequencing, the gene sequence is shown in SEQ ID NO: 1, the GenBank accession number is MH817850, the similarity with the type strain Pseudomonas arsenicoxydans CECT 7543T is 99.21%, it is identified as Pseudomonas arsenicoxydans, and finally named as Pseudomonas arsenicoxydansY24-2.

The denitrification function of Pseudomonas arsenicoxydans Y24-2 of the present embodiment is verified under low-temperature oligotrophic conditions:

Experiment 1: Nitrate solutions with concentrations of 10.0, 20.0, 40.0, 60.0, 100.0, 150.0, and 200.0 mg/L were prepared. In order to simulate the characteristics of groundwater quality, 7-10 mg/L of MnSO ₄ and MnSO were added to the nitrate solution. 5 ~ 8mg/L FeSO ₄ to obtain a degradation solution, and then the Pseudomonas arsenicoxydans Y24-2 strain was cultured to 10 ⁷ ~ 10 ⁹ /ml, and 50ml was centrifuged at 6000g for 10min to obtain the cell precipitate, and the bacteria were precipitated with sterile deionized water. The bacteria were precipitated and washed for 2 to 3 times, and the bacteria were transferred to 50ml of degradation solution, using ethanol as the carbon source, the C/N ratio was 0.5, and the pH was 7.0 and the temperature was 6 ℃. Nitrate reduction rate.

This experiment found that with the increase of nitric acid concentration, the nitrate reduction rate was higher. When the nitrate concentration reached 100.0 mg/L, the nitrate reduction rate was the highest, which was 89.72 mg/L/h.

Experiment 2: A nitrate solution with a concentration of 100.0 mg/L was prepared. In order to simulate the water quality characteristics of groundwater, 7-10 mg/L MnSO ₄ and 5-8 mg/L FeSO ₄ were added to the solution to obtain a degradation solution. Pseudomonasarsenicoxydans Y24-2 strain was cultured to 10 ⁷ to 10 ⁹ cells/ml, 50 ml was centrifuged at 6000g for 10 min to obtain the cell pellet, washed with sterile deionized water for 2 to 3 times, and the cell was transferred to 50 ml for degradation The solution, using sodium acetate as the carbon source, adjusted the C/N ratio to 0.5, 1, 2, 4, 8, 10, 15, and 20, respectively, at 4 to 10 ° C, pH value of 6 to 8, and 80 to 180 rpm (dissolved). Under the condition of oxygen greater than 2～10mg/L), the nitrate reduction rate was measured after 30min reduction of nitrate.

The results show that when the C/N ratio is 0.5, the temperature is 6℃, the pH value is 7.2, and 180rpm (dissolved oxygen is about 9.0mg/L), the nitrate reduction rate is the highest, which is 91.24mg/L·h.

Experiment 3: Take two actual groundwater samples from different groundwater sampling sites. The water quality is shown in Table 1. From the water quality, it can be seen that the C/N ratio of the two water samples is lower than 0.1, which is a nutrient-poor environment.

Table 1 General situation of water quality of groundwater samples

The strain Pseudomonas arsenicoxydans Y24-2 was cultured to 10 ⁷ to 10 ⁹ cells/mL, and 50 mL of bacterial liquid was centrifuged at 6000 g for 10 min to obtain the bacterial precipitation, and the bacterial precipitate was washed 2 to 3 times with sterile deionized water. The samples were collected and transferred to 2 water samples respectively. After treatment at 4-5 °C for 30 min and 8 h, the residual nitrate concentration in the water was measured, and the nitrate removal rate was calculated. The results are shown in Table 2. The results show that after 8h treatment, Y24-2 can remove about 80% of the nitrate in groundwater. After treatment, the nitrate can meet the requirements of my country's "Drinking Water Sanitation Standard" (GB5749-2006) (when groundwater is used as the water source). , nitrate≤20mg/L)

Table 2 The removal effect of Pseudomonas arsenicoxydans Y24-2 on nitrate in groundwater after 30min and 8h

It can be seen from test 1-3 that the strain Pseudomonas arsenicoxydans Y24-2 of the present invention can efficiently remove nitrite in groundwater under low-temperature and nutrient-poor conditions and when the influent nitrate concentration is 20-100 mg/L, and the removal rate of nitrate is high. It is 12.94～93.96mg/L/h; after 8h treatment, Y24-2 can remove about 80% of nitrate in groundwater.

sequence listing

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Claims (2)

1. A strain of Pseudomonas arsenicoxydans Y24-2 has been deposited in the General Microbiology Center of the China Microbial Culture Collection and Management Committee, the deposit number is CGMCC No. 16655, and the deposit date is October 29, 2018. 2 . The application of the arsenic-oxidizing Pseudomonas as claimed in claim 1 in removing nitrate in water, wherein the water is groundwater at 4-10° C. 3 .

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