CN109517770B - Aerobic facultative autotrophic denitrifying bacterium and application thereof - Google Patents
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Abstract
The invention discloses an aerobic facultative autotrophic denitrifying bacterium and application thereof, and relates to the aerobic facultative autotrophic denitrifying bacterium and application thereof. The aerobic facultative autotrophic denitrifying bacterium is Pseudomonas (Pseudomonas koreensis) Y5-11, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation address of No. 3 Xilu No.1 Beijing, Chaoyang district, the preservation date of 2018, 10 months and 29 days, and the preservation number of 16651. The strain of the invention can not only utilize organic carbon source for growth, but also can use CO for growth2The invention is used for reducing nitrate by a unique carbon source and is applied to the technical field of environmental microorganisms.
Description
Technical Field
The invention relates to an aerobic facultative autotrophic denitrifying bacterium and application thereof.
Background
Denitrification by microorganisms refers to the process by which microorganisms reduce nitrate to nitrogen. This phenomenon is common in nature (land, fresh water and marine systems). Denitrification is generally considered to be divided into anoxic-heterotrophic denitrification and anaerobic-autotrophic denitrification. The anoxic-heterotrophic denitrification is a process of reducing nitrate to nitrogen under an anoxic condition (dissolved oxygen is about 0.5 mg/L) by using organic matters (organic carbon) as electron donors. The characteristic of anoxic-heterotrophic denitrification is that a large amount of organic carbon source is required to sustain growth (C/N)>10) In the denitrification treatment process of sewage or slightly polluted underground water, the condition of insufficient carbon source often occurs, and organic matters such as methanol and the like are generally required to be additionally added, so that the water production cost is increased. The anaerobic-autotrophic denitrifying bacteria do not need organic carbon source and use CO2、HCO3 -Or CO3 2-The bacteria can be divided into hydrogen autotrophic denitrifying bacteria and sulfur autotrophic denitrifying bacteria according to different electron donors. Since the anaerobic-autotrophic denitrifying bacteria are obligate anaerobes and require additional supply of electron donors (i.e., hydrogen or sulfide), these characteristics increase the difficulty and cost of application of the anaerobic-autotrophic denitrifying bacteria. In recent years, a great deal of research and discovery shows that heterotrophic nitrification-aerobic denitrification bacteria exist, so that denitrification reaction can be carried out under aerobic conditions, however, the bacteria still need high organic carbon sources (usually, the C/N is 10-20).
Disclosure of Invention
The invention provides an aerobic facultative autotrophic denitrifying bacterium and application thereof.
The aerobic facultative autotrophic denitrifying bacterium is Pseudomonas koreensis Y5-11, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation address of No. 3 Xilu No.1 Beijing, Chaoyang district, the preservation date of 2018, 10 and 29 days, and the preservation number of 16651.
The aerobic facultative autotrophic denitrifying bacterium is applied to removing pollutants in underground water.
The screening method of the aerobic facultative autotrophic denitrifying bacteria Y5-11 comprises the following steps: putting 100mL of low-temperature (about 4 ℃) underground water sample into a 4 ℃ incubator for enrichment culture, and periodically adding 0.1g/L NaNO3(ii) a After 7-10 days, the water sample is obviously turbid, the growth of microorganisms is indicated, and the water sample is transferred into a separation and purification solid culture medium; the separation and purification temperature is 6-10 ℃, the culture time of the separation and purification is 3-4 d, after 3 times of purification, the thalli staining observation is carried out, and when no mixed bacteria exist in bacterial colonies, the bacterial colonies are pure bacterial strains and are used for carrying out screening test; and carrying out liquid culture on the separated and purified strains, collecting thalli, inoculating the thalli into a 100mg/L nitrate solution, measuring the concentration of the residual nitrate and calculating the denitrification rate after 30min under the conditions of 6-10 ℃ and the rotating speed of a shaking table of 180rpm/min, and selecting the strains capable of carrying out denitrification, namely the aerobic facultative autotrophic denitrifying bacteria.
Extracting genome DNA of the aerobic facultative autotrophic denitrifying bacteria, performing 16S rDNA sequence amplification, using the obtained fragments for sequencing, identifying the fragments into Pseudomonas koreensis according to gene characteristics of the aerobic facultative autotrophic denitrifying bacteria and Bergey bacteria identification handbook, and finally naming the fragments as Pseudomonas koreensis Y5-11.
The Pseudomonas koreensis Y5-11 of the invention can not only utilize organic carbon source for growth, but also CO2Nitrate is reduced for the sole carbon source and no additional electron donor is required. The nitrification rate of Y5-11 under the condition of no carbon source is measured, and when the nitrate concentration reaches 150.0mg/L, the denitrification rate is the highest and is 135.12 mg/L/h.
The Pseudomonas koreensis Y5-11 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of West Lu No.1 Hospital, Chaojing, Chaoyang, the preservation date is 2018, 10 months and 29 days, and the preservation number is 16651.
Drawings
FIG. 1 is a schematic diagram showing the gram stain result of aerobic facultative autotrophic denitrifying bacteria Y5-11 according to the present invention;
FIG. 2 is a schematic diagram showing the colony of aerobic facultative autotrophic denitrifying bacterium Y5-11 according to the present invention.
Detailed Description
The first embodiment is as follows: the aerobic facultative autotrophic denitrifying bacterium is Pseudomonas koreensis Y5-11, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation address of No. 3 Hosieboldo No.1 of Beijing Kogyo Chen, the preservation date of 2018, 10 and 29 days, and the preservation number of 16651.
The screening method of the aerobic facultative autotrophic denitrifying bacterium Pseudomonas koreensis Y5-11 in the embodiment comprises the following steps: putting 100mL of low-temperature (about 4 ℃) underground water sample into a 4 ℃ incubator for enrichment culture, and periodically adding 0.1g/L NaNO3(ii) a After 7-10 days, the water sample is obviously turbid, the growth of microorganisms is indicated, and the water sample is transferred into a separation and purification solid culture medium; the main components of the culture medium are 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，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, (1.8 g/L of agar is added into a solid culture medium); the separation and purification temperature is 6-10 ℃, the culture time of the separation and purification is 3-4 d, after 3 times of purification, the thalli staining observation is carried out, and when no mixed bacteria exist in bacterial colonies, the bacterial colonies are pure bacterial strains and are used for carrying out screening test; and carrying out liquid culture on the separated and purified strains, collecting thalli, inoculating the thalli into a 100mg/L nitrate solution, measuring the concentration of the residual nitrate and calculating the denitrification rate after 30min under the conditions of 6-10 ℃ and the rotating speed of a shaking table of 180rpm/min, and selecting the strains capable of carrying out denitrification, namely the aerobic facultative autotrophic denitrifying bacteria.
Extracting genome DNA of the aerobic facultative autotrophic denitrifying bacteria, performing 16S rDNA sequence amplification, using the obtained fragments for sequencing, identifying the fragments into Pseudomonas koreensis according to gene characteristics of the aerobic facultative autotrophic denitrifying bacteria and Bergey bacteria identification handbook, and finally naming the fragments as Pseudomonas koreensis Y5-11.
The Pseudomonas koreensis Y5-11 of the invention can not only utilize organic carbon source for growth, but also CO2Nitrate is reduced for the sole carbon source and no additional electron donor is required. The denitrification rate of Y5-11 was determined, and when the nitrate concentration reached 150.0mg/L, the denitrification rate was the highest, 135.12 mg/L/h.
The second embodiment is as follows: the first difference between the present embodiment and the specific embodiment is: the aerobic facultative autotrophic denitrifying bacterium Pseudomonas koreensis Y5-11 is gram-negative, bacillus, 2-4 mu m in length and 0.5-0.9 mu m in width, has no spores and flagella, moves by virtue of the flagella, has capsules, forms milky colonies on an LB culture medium, and is irregular and circular, and the surface of the colonies is convex. The rest is the same as the first embodiment.
The third concrete implementation mode: the aerobic facultative autotrophic denitrifying bacteria are applied to removing pollutants in underground water.
The fourth concrete implementation mode: the third difference between the present embodiment and the specific embodiment is that: the contaminant is nitrate. The rest is the same as the third embodiment.
The fifth concrete implementation mode: the screening method of the aerobic facultative autotrophic denitrifying bacterium Pseudomonas koreensis Y5-11 in the embodiment comprises the following steps: putting 100mL of low-temperature (about 4 ℃) underground water sample into a 4 ℃ incubator for enrichment culture, and periodically adding 0.1g/L NaNO3(ii) a After 7-10 days, the water sample is obviously turbid, the growth of microorganisms is indicated, and the water sample is transferred into a separation and purification solid culture medium; the main components of the culture medium are as follows: NaNO3 0.1～0.5g/L，MnSO40.01～0.05g/L，(NH4)2Fe(SO4)2·6H2O 0.01～0.10g/L，CaCl2 0.01～0.05g/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, (1.8 g/L of agar is added into a solid culture medium); the temperature for separation and purification is 6-10 ℃, and the culture time for separation and purification is3-4 d, after 3 times of purification, carrying out thallus staining observation, and when no mixed bacteria exist in the bacterial colony, obtaining a pure bacterial strain for carrying out a screening test; and carrying out liquid culture on the separated and purified strains, collecting thalli, inoculating the thalli into a 100mg/L nitrate solution, measuring the concentration of the residual nitrate and calculating the denitrification rate after 30min under the conditions of 6-10 ℃ and the rotating speed of a shaking table of 180rpm/min, and selecting the strains capable of carrying out denitrification, namely the aerobic facultative autotrophic denitrifying bacteria.
The aerobic facultative autotrophic denitrifying bacterium Pseudomonas koreensis Y5-11 is gram-negative, bacillus, 2-4 mu m in length and 0.5-0.9 mu m in width, has no spores and flagella, moves by virtue of the flagella, has capsules, forms milky colonies on an LB culture medium, and is irregular and circular, and the surface of the colonies is convex. The gram stain results for Y5-11 are shown in FIG. 1; the colonies of Y5-11 are shown in FIG. 2.
A reference method for measuring the physiological and biochemical characteristics of aerobic facultative autotrophic denitrifying bacteria Pseudomonas koreensis Y5-11 (Dongxu bead, Chuia Miaoying. general bacteria system identification manual. Beijing: scientific Press, 2001.) is provided. The physiological and biochemical characteristics of the aerobic facultative autotrophic denitrifying bacteria in the embodiment are as follows: catalase and oxidase are positive, Tween 80 and arginine can be hydrolyzed, gelatin liquefaction can be carried out, and nitrate can be reduced to generate nitrogen; the glucose can not be hydrolyzed to produce acid, and the starch can not be hydrolyzed; d-arabitol, D-fructose, D-galactose, alpha-D-glucose, D-mannitol, methylpropionic acid, methylsuccinic acid, acetate, propionate, butyrate, citrate, ethanol, benzoate, malate, and lactate may be utilized. : catalase and oxidase are positive, Tween 80 and arginine can be hydrolyzed, gelatin liquefaction can be carried out, and nitrate can be reduced to generate nitrogen; the glucose can not be hydrolyzed to produce acid, and the starch can not be hydrolyzed; d-arabitol, D-fructose, D-galactose, alpha-D-glucose, D-mannitol, methylpropionic acid, methylsuccinic acid, acetate, propionate, butyrate, citrate, ethanol, benzoate, malate, and lactate may be utilized.
The sixth specific implementation mode: identification of Pseudomonas koreensis Y5-11 of the present embodiment:
extracting genome DNA of aerobic facultative autotrophic denitrifying bacterium Pseudomonas koreensis Y5-11, and then carrying out 16S rDNA sequence amplification: the PCR primers used for amplification are universal 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. mu.L Taq enzyme, 2.5. mu.L 10 XBuffer (containing Mg2+), 1. mu.L dNTP (2.5 mmol/L each), 0.5. mu.L forward primer, 0.5. mu.L reverse primer, and 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 registration number is MH 817853. According to the gene characteristics of aerobic facultative autotrophic denitrifying bacteria and Bergey's Manual of identification of bacteria, the strain is identified as Pseudomonas koreensis of the genus Pseudomonas, and finally named as Pseudomonas koreensis Y5-11.
The denitrification function of the Pseudomonas koreensis Y5-11 of the embodiment is verified under the conditions of low temperature and no addition of organic carbon source:
test 1: measuring the denitrification rate of aerobic facultative autotrophic denitrifying bacterium Pseudomonas koreensis Y5-11 at 6-10 ℃ under the condition of no organic carbon source: simulating the water quality of underground water: respectively preparing nitrate solutions with the concentrations of 10.0, 20.0, 40.0, 60.0, 100.0, 150.0 and 200.0mg/L, and then adding MnSO4And FeSO4Mixed solution of MnSO4The concentration is 7-10 mg/L, FeSO4The concentration is 5-8 mg/L; the strain Pseudomonas koreensis Y5-11 was then cultured to 10%7～109And (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, transferring the thallus into 50mL of simulated underground water, measuring the concentration of the residual nitrate after 30min, and calculating the denitrification rate, wherein when the concentration of the nitrate reaches 150.0mg/L, the denitrification rate is the highest and is 135.12 mg/L/h. When the nitrate concentration exceeds 150mg/L, the denitrification rate tends to decrease.
As can be seen from the tests, Pseudomonas koreensis Y5-11 can be expressed as CO2The nitrate is reduced by the single carbon source without additionally providing an electron donor, and the denitrification rate is 135.12mg/L/h under the conditions of 6-10 ℃ and no carbon source and when the concentration of the nitrate reaches 150.0 mg/L.
Test 2: 50mL of 4 samples of actual groundwater were collected from different groundwater samples, and the water quality was as shown in Table 1.
TABLE 1 groundwater quality
Is to culture the strain Pseudomonas koreensis Y5-11 to 107～109And (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 4 water samples respectively, determining the concentration of the residual nitrate in the water after treating for 30min and 8h, and calculating the removal rate of the nitrate, wherein the results are shown in Table 2. The result shows that Y5-11 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 koreensis Y5-11 effect on nitrate removal from groundwater after 30min and 8h
As can be seen from tests 1 and 2, Pseudomonas koreensis Y5-11 can be expressed as CO2The nitrate is reduced by the single carbon source without additionally providing an electron donor, and the denitrification rate is 135.12mg/L/h under the conditions of 6-10 ℃ and no carbon source and when the concentration of the nitrate reaches 150.0 mg/L. Y5-11 can remove about 80% of nitrate in groundwater after 8h of treatment without adding extra carbon source and electron donor.
Sequence listing
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<120> aerobic facultative autotrophic denitrifying bacterium and application thereof
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gtgaaatccc cgggctcaac ctgggaactg catccaaaac tggcaagcta gagtatggta 600
gagggtggtg gaatttcctg tgtagcggtg aaatgcgtag atataggaag gaacaccagt 660
ggcgaaggcg accacctgga ctgatactga cactgaggtg cgaaagcgtg gggagcaaac 720
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gtgccttcgg gagcattgag acaggtgctg catggctgtc gtcagctcgt gtcgtgagat 1020
gttgggttaa gtcccgtaac gagcgcaacc cttgtcctta gttaccagca cgttatggtg 1080
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Claims (2)
1. An aerobic facultative autotrophic denitrifying bacterium, characterized in that it is a Korean pseudomonas (A) (B)Pseudomonas koreensis) Y5-11, deposited in China general microbiological culture Collection center, with the deposit address being No. 3 of Xilu No.1 of Beijing, Chaoyang, the date of deposit being 2018, 10 months and 29 days, and the deposit number being CGMCC No. 16651.
2. The use of an aerobic facultative autotrophic denitrifying bacterium according to claim 1, wherein said bacterium is Pseudomonas aeruginosa (Pseudomonas aeruginosa)Pseudomonas koreensis) Y5-11 was used to remove nitrate from groundwater.
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| CN110951658B (en) * | 2020-01-15 | 2022-11-18 | 广州市微生物研究所有限公司 | Pseudomonas and application thereof |
| CN112760268B (en) * | 2021-02-09 | 2022-11-18 | 黑龙江大学 | Ultraviolet-resistant bacillus and application thereof |
| CN112813004B (en) * | 2021-02-09 | 2022-05-10 | 黑龙江大学 | Ultraviolet-resistant and antioxidant immobile bacterium and application thereof |
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| CN113005062B (en) * | 2021-03-24 | 2022-05-13 | 中国科学院成都生物研究所 | Facultative ammonia oxidizing bacteria and application thereof |
| CN116121148A (en) * | 2023-03-15 | 2023-05-16 | 浙江商达公用环保有限公司 | Synchronous deoiling and denitrifying bacteria, screening, application and preparation method of microbial inoculum |
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