CN109722394B - Pseudomonas arsenic oxide strain and application thereof - Google Patents
Pseudomonas arsenic oxide strain and application thereof Download PDFInfo
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Abstract
The invention discloses a pseudomonas arsenic oxide and application thereof, and relates to a pseudomonas arsenic oxide and application thereof. The Pseudomonas arsenic oxide strain is Pseudomonas arsenicoxydans Y24-2, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.16655 and the preservation date of 2018, 10 months and 29 days. Under the condition of low-temperature poor nutrition of the strain, nitrite in underground water can be efficiently removed when the concentration of nitrate in the inlet water is 20-100 mg/L, and the removal rate of the nitrate is 12.94-93.96 mg/L/h. The invention is applied to the technical field of environmental microorganisms.
Description
Technical Field
The invention relates to pseudomonas arsenic oxide and application thereof.
Background
Due to the application of the agricultural fertilizer, nitrate enters the underground water environment through the soil percolation effect, and the underground water quality is influenced. As the agricultural non-point source pollution is relatively difficult to control, the phenomenon that the nitric acid in the underground water exceeds the standard in China is common. If the drinking water contains excessive nitrate, the hyperhemoglobin is easily caused, even cancer is induced, and the harm to human bodies is caused. In China, particularly in northern areas, about 60 percent of people use underground water as a water source, and the nitrate in the underground water is effectively removed, so that the water supply safety of residents is guaranteed to be critical. The sanitary standard for drinking water in life (GB5749-2006) implemented in China from 7 months in 2007 also limits the maximum allowable concentration of nitrate nitrogen to 10 mg/L.
The existing biological treatment methods for nitrate in underground water can be divided into two types:
one is to remove nitrate in the ground water by using heterotrophic denitrifying bacteria. The principle is that under the condition of oxygen deficiency (dissolved oxygen is about 0.5 mg/L), organic matters (organic carbon) are used as electron donors, and nitrate is reduced to generate nitrogen. Since the organic matter content in the ground water is low and mainly exists in the form of humic acid which is difficult to be utilized biologically, the C/N ratio in the raw water is usually less than 1, and thus the carbon source is often insufficient. The currently generally adopted solution is to add slow-release carbon sources such as rice straws, corncobs and the like so as to improve the C/N ratio of the treatment process.
The other method is to remove nitrate in the groundwater by using autotrophic denitrifying bacteria. The principle is that CO is used under the condition of obligate anaerobism2、HCO3 -Or CO3 2-As a carbon source, with H2S or sulfide is an electron donor to reduce nitrate to generate nitrogen, such as Thiobacillus denitrificans (Thiobacillus densiticus), Thiobacillus ferrooxidans (Thiobacillus ferrooxidans) and the like. Although such autotrophic denitrifying bacteria do not need additional organic carbon source during nitrate removal, strict anaerobic environment is required, and additional electron donor (H) supply is required2Or sulfides) are difficult to realize in the application process, and the water production cost is correspondingly increased.
In recent years, there have been related studies to solve the problems of low groundwater temperature, low C/N ratio, and difficulty in biological treatment. In the research process of perilithic epiphyte (mixed oxygenation reinforced water source reservoir poor nutrition aerobic denitrifying bacteria, research on denitrification characteristics and technical application, western's university of construction and science and technology, doctor's academic paper 2017), 13 strains of bacteria capable of performing aerobic denitrification under poor nutrition conditions are obtained through separation, and the bacteria are respectively Acinetobacter (Acinetobacter sp.), neosphingolipid (Novosphingobium sp.), Aquibacterium (Aquibacterium sp.), Sphingomonas sp.), Acetobacter (Zoogloea sp.) and Delftia (Delftia sp.). Liu Yongbo et al (Liu Yongbo, Qudan, any army. the aerobic denitrification performance of a low-temperature aniline degrading bacterium, Henan water conservancy and south water north of the river, 2018,5:78-79) also separate and obtain the aerobic denitrification bacterium AN-1 with coldness. However, when the strains are subjected to denitrification, the nitrate concentration does not exceed 5mg/L, and the nitrate concentration in the slightly polluted underground water is usually 20-150 mg/L.
Pseudomonas arsenioxide (Pseudomonas arsenicoxidins) originally reported in 2010 to be capable of oxidizing arsenite (Victor L. Campos et al. Pseudomonas arsenicoxidins sp. nov., an arsenite-oxidizing strain isolated from the Atacama receiver. systematic and Applied microbiology.2010.33: 193-197.). There have also been recent studies on the use of pseudomonas arsenic oxide for the treatment of arsenite, but there has been no report on the use of pseudomonas arsenic oxide for the treatment of nitrate in ground water.
Disclosure of Invention
The invention provides pseudomonas arsenic oxide and application thereof.
The Pseudomonas arsenioxidans is Pseudomonas arsenicoxydans Y24-2, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.16655 and the preservation date of 2018, 10 months and 29 days.
The Pseudomonas arsenic oxide is Pseudomonas arsenicoxydans Y24-2 used for removing pollutants in water.
The screening method of Pseudomonas arsenicum arsenicoxydans Y24-2 comprises the following steps: enriching 100ml of low-temperature underground water (water temperature 4 ℃) polluted by nitrate (nitrate concentration 140mg/L) for 12-20 days at 2-6 ℃ and 150rpm/min,continuously replenishing NaNO during the process3And C2H5OH, OD of bacterial liquid600After the value reaches above 0.5, performing gradient dilution and inoculating on a solid culture medium, culturing at 2-6 ℃ for 5-7 days, and selecting a typical single colony for purification culture. And (3) carrying out a nitrate reduction test on the finally obtained pure culture strain, and selecting the strain with the nitrate reduction rate reaching more than 50% within 30 min.
Extracting strain genome DNA, performing 16S rDNA sequence amplification, sequencing the obtained fragment, and performing PCR with model strain Pseudomonas arsenicoxydans CECT 7543TThe similarity of the two groups was 99.21%, and the Pseudomonas arsenoxide was identified as Pseudomonas arsenicoxydans and finally named as Pseudomonas arsenicoxydans Y24-2.
According to the invention, under the low-temperature poor nutrition condition that the concentration of the nitrate in the inlet water is 20-100 mg/L and the temperature of the Pseudomonas arsenicoxoidans Y24-2 is 6-10 ℃ and the C/N ratio is 0-0.5, the nitrate in the underground water can be efficiently removed by Pseudomonas arsenicoxoidans Y24-2, and the removal rate of the nitrate is 12.94-93.96 mg/L/h.
The Pseudomonas arsenicoxaydans Y24-2 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.16655 and the preservation date of 2018, 10 months and 29 days.
Drawings
FIG. 1 is a schematic diagram showing the gram staining result of Pseudomonas arsenicoxydans Y24-2 according to the present invention;
FIG. 2 is a schematic diagram of a colony of Pseudomonas arsenicoxydans Y24-2 of the present invention.
Detailed Description
The first embodiment is as follows: the Pseudomonas arsenic oxide strain is Pseudomonas arsenicoxydans Y24-2, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.16655 and the preservation date of 2018, 10 months and 29 days.
The screening method of Pseudomonas arsenicum of the embodiment as Pseudomonas arsenicoxydans Y24-2 comprises the following steps: low temperature groundwater (water temperature 4 deg.C) contaminated with nitrates (nitrate concentration 140mg/L))100ml, enriching for 12-20 days at 2-6 ℃ and 150rpm/min, and continuously supplementing NaNO during the enrichment3And C2H5OH, OD of bacterial liquid600After the value reaches above 0.5, performing gradient dilution and inoculating on a solid culture medium, culturing at 2-6 ℃ for 5-7 days, and selecting a typical single colony for purification culture. And (3) carrying out a nitrate reduction test on the finally obtained pure culture strain, and selecting the strain with the nitrate reduction rate reaching more than 50% within 30 min.
Extracting strain genome DNA, amplifying 16S rDNA sequence, sequencing the obtained fragment, and performing sequencing with model strain Pseudomonas arsenicoxydans CECT 7543TThe similarity of the two groups was 99.21%, and the Pseudomonas arsenoxide was identified as Pseudomonas arsenicoxydans and finally named as Pseudomonas arsenicoxydans Y24-2.
In the embodiment, the concentration of nitrate in inlet water is 20-100 mg/L under the low-temperature poor nutrition condition that the temperature of the Pseudomonas arsenicoxoidans Y24-2 is 6-10 ℃ and the C/N ratio is 0-0.5, the Pseudomonas arsenicum arsenicoxoidans Y24-2 can effectively remove nitrite in underground water, and the removal rate of nitrate is 12.94-93.96 mg/L/h.
The second embodiment is as follows: the culture medium formula used in the screening process of pseudomonas arsenic oxide of the embodiment is as follows: NaNO3 0.1～0.5g/L，MnSO4 0.01～0.05g/L，(NH4)2Fe(SO4)2·6H2O 0.01～0.10g/L，CaCl2 0.01～0.05g/L，C2H5OH 0.1～2.0mL/L，Na2HPO4 0.3～0.9g/L，MgSO4·7H20.01-0.05 g/L of O, 0.3-0.9 g/L of NaCl and 7.0-7.4 of pH value. The rest is the same as the first embodiment.
In the case of solid culture medium, 1.8g/L agar is added to the above formulation.
The third concrete implementation mode: the Pseudomonas arsenic oxide of the embodiment is Pseudomonas arsenicoxydans Y24-2 used for removing pollutants in water.
The fourth concrete implementation mode: the third difference between the present embodiment and the specific embodiment is that: the water is micro-polluted low-temperature underground water. The rest is the same as the third embodiment.
The fifth concrete implementation mode: this embodiment is different from the third or fourth embodiment in that: the temperature of the low-temperature underground water is 4-10 ℃. The other is the same as the third or fourth embodiment.
The sixth specific implementation mode: the difference between this embodiment and one of the third to fifth embodiments is: the contaminant is nitrate. The rest is the same as one of the third to fifth embodiments.
The seventh concrete implementation mode: the screening method of Pseudomonas arsenicum of the embodiment as Pseudomonas arsenicoxydans Y24-2 comprises the following steps: low temperature underground water (water temperature 4 ℃) polluted by nitrate (nitrate concentration 140mg/L) is 100ml, and is enriched for 12-20 days at 2-6 ℃ and 150rpm/min, and NaNO is continuously supplemented in the period3And C2H5OH, OD of bacterial liquid600After the value reaches above 0.5, performing gradient dilution and inoculating on a solid culture medium, culturing at 2-6 ℃ for 5-7 days, and selecting a typical single colony for purification culture. And (3) carrying out a nitrate reduction test on the finally obtained pure culture strain, and selecting the strain with the nitrate reduction rate reaching more than 50% within 30 min.
The culture medium used in the above process is: NaNO3 0.1～0.5g/L，MnSO4 0.01～0.05g/L，(NH4)2Fe(SO4)2·6H2O 0.01～0.10g/L，CaCl2 0.01～0.05g/L，C2H5OH 0.1～2.0mL/L，Na2HPO40.3～0.9g/L，MgSO4·7H20.01-0.05 g/L of O, 0.3-0.9 g/L of NaCl and 7.0-7.4 of pH value (1.8 g/L of agar is added into a solid culture medium).
The strain Pseudomonas arsenicoxoxydans Y24-2 is obligate aerobic bacteria, gram staining is negative (shown in figure 1), bacterial colony is convex, viscous and milky (shown in figure 2); the oxidase and the catalase are positive, the growth temperature is 4-37 ℃, and the optimal pH value is 7.5-8.0; can not generate hydrogen sulfide and indole, can hydrolyze urea, and can reduce nitrate; ethanol, sodium acetate, sodium butyrate, sodium benzoate and glucose can be used as carbon sources for growth; citric acid and maltose cannot be used.
The sixth specific implementation mode: identification of Pseudomonas arsenicoxydans Y24-2 in this embodiment:
extracting bacterial genome DNA, and then carrying out 16S rDNA sequence amplification, wherein PCR reaction primers for amplification are general primers: a forward primer 5'-CAGAGTTTGATCCTGGCT-3'; the reverse primer 5'-AGGAGGTGATCCAGCCGCA-3'. The PCR reaction system is as follows: 20-50 ng template DNA, 0.2 uL Taq enzyme, 2.5 uL 10 Xbuffer (containing Mg)2+) mu.L dNTP (2.5 mmol/L each), 0.5. mu.L forward primer, 0.5. mu.L reverse primer, sterile deionized water was added to 25. mu.L. PCR amplification conditions: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 45s, amplification at 55 ℃ for 45s, extension at 72 ℃ for 1min, 30 cycles, extension at 72 ℃ for 10min, and termination of reaction at 4 ℃, wherein the obtained fragment is used for sequencing, and the gene sequence is shown as SEQ ID NO: 1, GenBank accession number MH817850, with a similarity of 99.21% to the model strain Pseudomonas arsenicoxydans CECT 7543T, was identified as Pseudomonas arsenicoxydans, and was finally named as Pseudomonas arsenicoxydans Y24-2.
The denitrification function of Pseudomonas arsenicoxydans Y24-2 under the low-temperature and poor-nutrition condition is verified:
test 1: nitrate solutions with the concentrations of 10.0, 20.0, 40.0, 60.0, 100.0, 150.0 and 200.0mg/L are prepared, and 7-10 mg/L of MnSO is added into the nitrate solutions to simulate the water quality characteristics of underground water4And 5-8 mg/L of FeSO4Obtaining a degradation solution, and then culturing the Pseudomonas arsenicoxydans Y24-2 strain to 107～109Taking 50ml, centrifuging at 6000g for 10min to obtain thallus precipitate, cleaning the thallus precipitate for 2-3 times by using sterile deionized water, transferring the thallus into 50ml of degradation solution, using ethanol as a carbon source, determining the reduction rate of nitrate after degrading for 30min under the low-temperature conditions of pH value of 7.0 and temperature of 6 ℃, wherein the C/N ratio is 0.5.
The test finds that the nitrate reduction rate is higher as the nitric acid concentration is increased, and the nitrate reduction rate is highest and is 89.72mg/L/h when the nitrate concentration reaches 100.0 mg/L.
Test 2: the preparation concentration is 100.0mg/L nitrate solution, in order to simulate the water quality characteristics of underground water, 7-10 mg/L MnSO is added into the solution4And 5-8 mg/L of FeSO4Obtaining a degradation solution, and then culturing the Pseudomonas arsenicoxydans Y24-2 strain to 107～109Taking 50ml, centrifuging at 6000g for 10min to obtain thallus sediment, cleaning the thallus sediment for 2-3 times by using sterile deionized water, transferring the thallus into 50ml of degradation solution, using sodium acetate as a carbon source, adjusting the C/N ratio to be 0.5, 1, 2, 4, 8, 10, 15 and 20 respectively, reducing nitrate for 30min under the conditions of 4-10 ℃, pH value of 6-8 and 80-180 rpm (dissolved oxygen is more than 2-10 mg/L), and then measuring the reduction rate of nitrate.
The results showed that the nitrate reduction rate was highest at 91.24 mg/L.h at a C/N ratio of 0.5, a temperature of 6 ℃, a pH of 7.2, and 180rpm (dissolved oxygen of about 9.0 mg/L).
Test 3: 2 actual underground water samples are collected from different underground water sampling plots, the water quality is shown in table 1, and the C/N ratio of the 2 water samples is lower than 0.1 according to the water quality condition, so that the underground water sampling plots belong to a poor nutrition environment.
TABLE 1 Water quality profiles of groundwater samples
Is cultured to 10 degrees by using the strain Pseudomonas arsenicoxydans Y24-27～109And (2) centrifuging 50mL of bacterial liquid at 6000g for 10min to obtain thallus precipitates, cleaning the thallus precipitates for 2-3 times by using sterile deionized water, collecting the thallus, transferring the thallus into 2 water samples respectively, treating the thallus in the water samples at 4-5 ℃ for 30min and 8h, determining the concentration of the residual nitrate in the water, and calculating the removal rate of the nitrate, wherein the results are shown in Table 2. The result shows that Y24-2 can remove about 80% of nitrate in the groundwater after 8h treatment, and the nitrate can meet the requirements of sanitary Standard for Drinking Water (GB5749-2006) in China (when groundwater is used as water source, the nitrate is less than or equal to 20mg/L)
TABLE 2Pseudomonas arsenicoxydans Y24-2 effect on nitrate removal from groundwater after 30min and 8h
As shown in the test 1-3, the strain Pseudomonas arsenicoxydans Y24-2 can efficiently remove nitrite in underground water under the condition of low temperature and poor nutrition and when the concentration of nitrate in inlet water is 20-100 mg/L, the removal rate of nitrate is 12.94-93.96 mg/L/h; after 8h of treatment, about 80% of nitrate in the groundwater can be removed by Y24-2.
Sequence listing
<110> university of Heilongjiang
<120> Pseudomonas arsenic oxide strain and application thereof
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<213> Pseudomonas arsenoxide (Pseudomonas arsenicoxydans)
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tcctacggga gaaagcaggg gaccttcggg ccttgcgcta tcagatgagc ctaggtcgga 180
ttagctagtt ggtgaggtaa tggctcacca aggcgacgat ccgtaactgg tctgagagga 240
tgatcagtca cactggaact gagacacggt ccagactcct acgggaggca gcagtgggga 300
atattggaca atgggcgaaa gcctgatcca gccatgccgc gtgtgtgaag aaggtcttcg 360
gattgtaaag cactttaagt tgggaggaag ggcattaacc taatacgtta gtgttttgac 420
gttaccgaca gaataagcac cggctaactc tgtgccagca gccgcggtaa tacagagggt 480
gcaagcgtta atcggaatta ctgggcgtaa agcgcgcgta ggtggttcgt taagttggat 540
gtgaaatccc cgggctcaac ctgggaactg cattcaaaac tgtcgagcta gagtatggta 600
gagggtggtg gaatttcctg tgtagcggtg aaatgcgtag atataggaag gaacaccagt 660
ggcgaaggcg accacctgga ctgatactga cactgaggtg cgaaagcgtg gggagcaaac 720
aggattagat accctggtag tccacgccgt aaacgatgtc aactagccgt tgggagcctt 780
gagctcttag tggcgcagct aacgcattaa gttgaccgcc tggggagtac ggccgcaagg 840
ttaaaactca aatgaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg 900
aagcaacgcg aagaacctta ccaggccttg acatccaatg aactttccag agatggattg 960
gtgccttcgg gaacattgag acaggtgctg catggctgtc gtcagctcgt gtcgtgagat 1020
gttgggttaa gtcccgtaac gagcgcaacc cttgtcctta gttaccagca cgtaatggtg 1080
ggcactctaa ggagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaagtcat 1140
catggccctt acggcctggg ctacacacgt gctacaatgg tcggtacaga gggttgccaa 1200
gccgcgaggt ggagctaatc ccagaaaacc gatcgtagtc cggatcgcag tctgcaactc 1260
gactgcgtga agtcggaatc gctagtaatc gcgaatcaga atgtcgcggt gaatacgttc 1320
ccgggccttg tacacaccgc ccgtcacacc atgggagtgg gttgctccag aagtagctag 1380
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<213> Artificial sequence
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CAGAGTTTGATCCTGGCT 18
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<220>
<223> PCR reverse primer nucleotide sequence.
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AGGAGGTGATCCAGCCGCA 19
Claims (2)
1. Pseudomonas arsenic oxide (A)Pseudomonas arsenicoxydans) Y24-2, which has been preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.16655 and the preservation date of 2018, 10 months and 29 days.
2. The use of Pseudomonas arsenic oxide as claimed in claim 1 for removing nitrate from water, wherein the water is groundwater at 4-10 ℃.
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