CN113373086A - Denitrifying bacterium pseudomonas strain JNB12 and application thereof - Google Patents

Abstract

The invention provides Pseudomonas JNB12(Pseudomonas sp. JNB12) with the preservation number of CGMCC No.21605 and application thereof. The strain can effectively degrade nitrogen in the environment, almost has no accumulation of ammonium nitrogen and nitrite, and does not produce N₂O。

Description

Denitrifying bacterium pseudomonas strain JNB12 and application thereof

Technical Field

The present invention relates to the field of environmental microbiology. Specifically, the invention relates to a strain material for assimilating and reducing nitrate, namely a Pseudomonas strain JNB12(Pseudomonas sp. JNBB 12) which is preserved by China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 21605. The invention also relates to the use thereof.

Background

China is a big agricultural country, and in order to increase the yield, a large amount of chemical nitrogen fertilizers are applied in the agricultural production process, wherein a considerable part of the chemical nitrogen fertilizers are not absorbed and utilized by crops and accumulate and remain in soil in the form of nitrates. On one hand, nitrate remained in the soil enters the water body in the form of surface runoff, and the ecological balance of rivers and lakes is changed, so that algal bloom and eutrophication of the water body are caused; on the other hand, the nitrate in the drinking water is over-high and harmful to human health because of entering the underground water in a leaching way. Data indicate that large amounts of leaching of farmland nitrate caused by high-strength fertilizer application leads to an increase in groundwater pollution level year by year. In order to solve the problem of nitrate leaching, researchers have provided various solutions based on agricultural measures such as controlled release fertilizer, nitrification inhibitor and water fertilizer regulation. Different schemes are different in economic benefit, feasibility and the like.

Bacteria in the soil are an integral part of the soil and have a significant effect on the function of the soil. The denitrification process mediated by the microorganisms is taken as a low-cost, high-efficiency and sustainable biological nitrogen removal measure, is widely applied to wastewater treatment, and has the potential of preventing and controlling the nitrate leaching of farmland soil.

The denitrification process is mediated by microorganisms, and NO in high valence state is converted into₃ ^--N or NO₂ ^-The process of reducing N to gaseous nitrogen is one of the important pathways of the natural nitrogen cycle. Although denitrification as an important active nitrogen sink can be used to help offset global artificial nitrogen imbalance, there is still an increase in N while enhancing soil denitrification activity₂Possibility of emission of greenhouse gases. Traditional theories suggest that bacterial denitrification is a strictly anaerobic process. However, in recent years, aerobic denitrification has been carried outThe bacteria are found to overcome the defect that the traditional denitrifying bacteria can only carry out denitrification under the anoxic condition. In recent years, more and more denitrifying bacteria at home and abroad are separated from soil or sludge and are well applied. The aerobic denitrifying bacteria are used for biological denitrification, so that the investment cost and the system operation cost can be greatly reduced, and the method is the most economic denitrification method. Therefore, the development of the research on the biological characteristics of the aerobic denitrifying bacteria has important theoretical value and practical significance for the application of the aerobic denitrifying bacteria.

Disclosure of Invention

The invention aims to solve the technical problem of providing a denitrifying strain for assimilating and reducing nitrate, and the invention also comprises application of the strain.

In one aspect of the present invention, the present invention provides a Pseudomonas strain (Pseudomonas sp) JNB12, which is deposited by the common microorganism center of the china committee for culture collection of microorganisms (CGMCC) at the institute of microbiology, china academy of sciences, No. 3, western road-1, north chen, township, beijing; the product is preserved in 2021, 1, 8 days with preservation number of CGMCC No. 21605.

In one aspect of the invention, the strain provided by the invention can be used for treating excess nitrate in soil, and specifically comprises: the microorganisms are used for converting nitrate nitrogen in soil or water into inorganic nitrogen or nitrogen of organisms through a microorganism denitrification process by taking the nitrate nitrogen in the soil or water as a nitrogen source for life activities, so that the deep soil nitrate leaching loss and underground water nitrate pollution can be reduced, and nitrogen-containing sewage can be purified and treated.

The technical scheme of the invention mainly comprises the following steps:

a. isolated culture of bacteria

b. Screening of denitrifying strains: carrying out color reaction on the purified strains by BTB (bromothymol blue), selecting the strains with positive reaction, carrying out denitrification test, and screening the strains with higher denitrification capability.

c. Scanning electron microscope for observing strain morphology

d. And (3) strain identification: morphological features were performed in conjunction with 16S rDNA sequence analysis;

e. and (4) identifying a denitrification functional gene.

Specifically, the invention relates to the following technical scheme:

1. pseudomonas JNB12(Pseudomonas sp. JNB12) with the collection number of CGMCC No. 21605.

2. A Pseudomonas preparation comprising Pseudomonas JNB12 according to item 1.

3. The Pseudomonas preparation according to item 2, which is a solid or liquid preparation.

4. The Pseudomonas formulation of item 3, wherein when the formulation is a liquid formulation, the viable count in the formulation>1×10¹⁰CFU ml^-1。

5. The Pseudomonas formulation of clause 3, wherein the Pseudomonas F1 is adsorbed on a carrier (e.g., ground straw) when the formulation is a solid formulation.

6. A method for denitrogenating an environmental sample, the method comprising adding Pseudomonas bacteria according to item 1 or a Pseudomonas preparation according to item 2 to the environmental sample, and incubating the mixture to degrade the nitrogen content of the environmental sample.

7. The method of clause 6, wherein the environmental sample is soil or a body of water.

8. The method of item 7, wherein when the environmental sample is a water body, the carbon-nitrogen ratio in the water body is 6-8, and the saturated dissolved oxygen is 50-98%.

9. The method of clause 6, wherein the nitrogen present in the environmental sample is ammonium nitrogen or nitrate nitrogen, such as nitrate or nitrite.

10. Use of the pseudomonas of item 1 or the pseudomonas preparation of item 2 in environmental denitrification.

ADVANTAGEOUS EFFECTS OF INVENTION

The inventor obtains two strains showing positive by purifying the separated colonies and carrying out BTB color reaction, and the two strains are treated at 100mg/kg NO₃ ^-Further screening was carried out in a liquid medium of-N, and as a result, it was found that the strain JNB12 had the ability to rapidly remove nitrate. By JNB12 and the model bacterium Paracoccus dentificans strain PD1222 at 100mg/kg NO₃ ^-The culture result of the-N denitrification culture medium shows that the removal of nitrate nitrogen of the JNB12 strain occurs within 24-48 h of the culture time at the fastest speed, the strain has a good effect on the removal capability of nitrate, and the removal rate of nitrate reaches more than 89% in 48h, and the effect is stable (see figure 2).

And strain identification results are as follows: the JNB12 colony was milky white, and in the denitrification broth, the culture process was a pale white liquid, with cell rods. The strains with the homology of more than 99 percent with the strain JNB12 in Genbank are mainly Pseudomonas (Pseudomonas), the sequence similarity of the Pseudomonas (Pseudomonas sp.HYS, Pseudomonas sp.ID2, Pseudomonas sp.A3) and the strain JNB12 reaches 100 percent, so the strain is determined to be Pseudomonas, and the temporary name is Pseudomonas JNB 12.

Drawings

FIG. 1 shows the morphology of Pseudomonas strain JNB12 cells under Scanning Electron Microscopy (SEM);

FIG. 2 shows the nitrate nitrogen removal (a) and N by Pseudomonas JNB12 and model bacterium PD1222 (positive control)₂Influence of O emission (b).

FIG. 3 shows ammonium nitrogen accumulation in Pseudomonas JNB12 during nitrate nitrogen removal.

Detailed Description

In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.

The methods used in the following examples are conventional methods unless otherwise specified, and the reagents used are commercially available reagents unless otherwise specified.

Example 1, material preparation:

btb isolation medium:

B. denitrification Medium (DM):

wherein, the microelements are: (ultrapure water 1L)

CuSO₄·5H₂O 0.2g ZnSO₄·7H₂O 0.2g

FeSO₄·7H₂O 0.2g

C. Beef extract peptone culture medium

Beef extract 3g peptone 10g

NaCl 5g pH：7.0±0.2

Agar: 1.5% (m/v) ultrapure water 1L

After the preparation of the culture medium, adjusting the pH value to 7.0 by NaOH, and performing moist heat sterilization at 115 ℃ for 30min to prepare a flat plate. The components are prepared into the corresponding liquid culture medium without adding agar.

Example 2 bacterial isolation

Diluting 20g of soil (such as red soil of Zhaoqing, Guangdong) with a soil-water ratio of 1:10 in sterile distilled water to obtain surface soil of 0-15 cm, mixing, diluting 1ml to 9ml of sterile water by 10 times, spreading 0.2ml of suspension on the denitrification culture plate, and culturing at 30 deg.C for 2 weeks. Selecting a culture dish with the colony number of 30-100, selecting single colonies with different colony characteristics, carrying out streak isolation culture, and carrying out the culture conditions.

Example 3 screening of bacterial strains having Denitrification Activity

Qualitative detection of btb (bromothymol blue):

because the denitrification reaction is an alkali production process (consuming H in the environment)⁺) When denitrification reaction occurs, indicator bromothymol blue of the BTB culture medium is blue, namely colonies are blue or blue halo. The selected single colonies were streaked on a BTB medium plate and incubated at 30 ℃ for 3 days. When denitrification reaction occurs, indicator bromothymol blue of the BTB culture medium is blue, namely colonies can be blue or blue halo. Single colonies with blue halos were selected from BTB medium plates and streaked for isolation and culture under the same conditions as above.

B. Quantitative screening of Denitrification Medium

Scraping single colony of 2 positive strains in A and Paracoccus densificans strain PD1222 in inoculating loop, inoculating into the denitrifying liquid culture medium at 30 deg.C and 160rpm, culturing for 36 hr to obtain seed liquid, inoculating the seed liquid into the denitrifying liquid culture medium at a ratio of 1% (v/v), culturing for 0, 12, 24, 36, and 48 hr, destructively sampling, determining OD value, NO, and collecting₃ ^--N，NO₂ ^--N，NH₄ ⁺The content of-N, the culture conditions were the same as above. The above liquid medium without inoculated culture was used as a blank and 3 replicates were set for each treatment. And selecting the strain with the highest nitrogen removal efficiency.

The pseudomonas JNB12 with high efficiency denitrification can be obtained by screening and is preserved in China center for type culture Collection/China Committee for culture Collection of microorganisms (CGMCC), the preservation date is 2021 year, month and 8 day, and the preservation number is CGMCC No. 21605; the address of the depository is: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

The results show (see FIG. 2) that the removal of nitrate nitrogen from the strain JNB12 occurs most rapidly between 24 and 48 hours of the cultivation time, the nitrate removal rate reaches more than 89% at 48 hours, the nitrate removal capacity of JNB12 is stronger than that of Paracoccus denitificas strain PD1222, and almost NO NO is generated during the whole cultivation process₂ ^--N and NH₄ ⁺The accumulation of-N (see FIG. 3, the accumulation amount of ammonium nitrogen is lower than 1mg/L), and the secondary pollution of the water body in the nitrate removal process is avoided. JNB12 did not have the greenhouse gas N during the denitrification culture process₂O production, so that it does not cause greenhouse gas pollution, N of JNB12 in the same culture as Paracoccus densificans strain PD1222₂O is significantly lower than that of the PD1222 strain, and it can convert N₂Reduction of O to N₂Ensure that N is not generated in the denitrification process₂O causes secondary environmental pollution.

Example 4 morphological Observation of Strain JNB12

The JNB12 colony was milky white, and in the denitrifying liquid medium, the culture process was a pale white liquid. JNB12 cells were rod-shaped and found to have a bacterial size of 0.4X 0.6 μm-0.8 μm by Scanning Electron Microscopy (SEM).

Example 5 extraction of bacterial genomic DNA and analysis of 16S rDNA sequence

Bacterial genomic DNA was extracted by QIAamp DNA kit (Qiagen), PCR amplification was performed with bacterial 16SrDNA universal primers (for specific information, see Huo YY, Xu XW, Wang CS, Yang JY, Wu M.bacterial diversity of the fragment from cancer Large Fisng Bay. acta Ecologica Sinica,2008,28(10): 5166-. The determined sequences were subjected to sequence alignment and manual alignment using DNAStar software package and nucleotide homology comparisons in GenBank were performed using NCBI (national Center for Biotechnology information) BLAST program. The results show that the strains with more than 90% homology with the strain JNB12 in Genbank are mainly Pseudomonas (Pseudomonas), the similarity of the 16S rRNA sequence of the Pseudomonas (Pseudomonas sp. FBF102, Pseudomonas sp.2-92) and the strain JNB12 reaches 100%, so the strain is determined to be Pseudomonas, and the temporary name is Pseudomonas JNB 12.

TABLE 1 BLAST results for 16S rDNA of Strain JNB12

Example 6 identification of Denitrification functional Gene

Bacterial genomic DNA was extracted by QIAamp DNA kit (Qiagen), narG gene amplification was performed using primers narG1960f and narG2650r (see Philippot, L., Piutti, S., Martin-Laurent, F., Hallet, S., and Germon, J.C. (2002) Molecular analysis of the nitrate reduction from plated and mail-soil tissues. applied Environ Microbiol 68: 6121-plate 6128), and narSC 1F and nirSC1R (see Wei, W., OBe, K., Nishiz for specific information on the primersawa, T., Zhu, L., Shirati, Y., Ohte, N., et al (2015) Higher diversity and availability of differentiating microorganisms in environmental regulation ISME J9: 1954. the gene amplification was carried out using primers nos Z1F and nos Z1R (for specific information on the primers see HenJN, S., Bru, D., Stres, B., Hal, S., and Philippot, L. (2006) Quantitative detection of the gene, encoding nitrate oxide recovery, and amplification of nitrite in the microorganism of 16S, nag, K. and 5181. the gene amplification was carried out using agarose gel containing nitrate reducing gene, S89, S. sub-gene, S. sub-gel, and agarose gel containing nitrate reducing gene. Wherein the nosZ gene can reduce nitrous oxide into nitrogen, which reduces the increase of greenhouse gas N in the denitrification process₂Risk of O emissions. The method has good utilization value for the prevention and control of the leaching of the nitrate in the farmland and the removal of the nitrate in the water body.

Example 7 microbial inoculum information of denitrifying bacteria

Liquid microbial inoculum: the seed bacteria are obtained by activating the strains on a beef extract peptone culture medium, and are added into a liquid beef extract peptone culture medium according to the proportion of 1 percent, and the culture is carried out for 24 hours under the culture conditions of 30 ℃ and 180 rpm. Will obtain the viable count>1×10¹⁰CFU ml^-1The liquid microbial inoculum can be sterilized by crushed straws at 121 ℃ for 2h, and can be used as a carrier after being dried, and the microbial inoculum is adsorbed to the straws and is sprayed to the required soil. Has good application value.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. Pseudomonas JNB12(Pseudomonas sp. JNB12) with the collection number of CGMCC No. 21605.

2. A Pseudomonas preparation comprising the Pseudomonas JNB12 according to claim 1.

3. The pseudomonas formulation of claim 2 which is a solid or liquid formulation.

4. The pseudomonas formulation of claim 3, wherein the viable count in the formulation is when the formulation is a liquid formulation>1×10¹⁰CFU ml^-1。

5. The pseudomonas formulation of claim 3, wherein the pseudomonas F1 is adsorbed onto a carrier (e.g., ground straw) when the formulation is a solid formulation.

6. A method of denitrogenating an environmental sample comprising adding the pseudomonas of claim 1 or the pseudomonas preparation of claim 2 to the environmental sample and incubating to degrade the nitrogen content of the environmental sample.

7. The method of claim 6, wherein the environmental sample is a soil or a water body.

8. The method according to claim 7, wherein when the environmental sample is a water body, the carbon-nitrogen ratio in the water body is 6-8, and the saturated dissolved oxygen is 50-98%.

9. The method of claim 6, wherein the nitrogen present in the environmental sample is ammonium nitrogen or nitrate nitrogen, such as nitrate or nitrite.

10. Use of the pseudomonas of claim 1 or the pseudomonas formulation of claim 2 in environmental denitrification.

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