CN109161513B

CN109161513B - Sphingobacterium and application thereof

Info

Publication number: CN109161513B
Application number: CN201811346777.8A
Authority: CN
Inventors: 邓敬轩; 阮文权; 黄振兴; 施万胜; 单晓红
Original assignee: Jiangnan University
Current assignee: Hengzhen Wuxi Biotechnology Co ltd
Priority date: 2018-11-13
Filing date: 2018-11-13
Publication date: 2020-09-04
Anticipated expiration: 2038-11-13
Also published as: WO2020097994A1; CN109161513A

Abstract

The invention discloses a sphingosine bacillus and application thereof, belonging to the technical field of biological engineering and environmental engineering. The sphingosine bacillus is preserved in China general microbiological culture Collection center (CGMCC) No.16596 in 2018, 10 and 17 months, has heterotrophic nitrification and aerobic denitrification characteristics, can be applied to the fields of nitrogenous sewage treatment and river treatment, has an ammonia nitrogen removal rate of more than 90 percent, has no pollution to the environment, achieves the effects of rapid biological denitrification and river self-cleaning capacity recovery, and has great significance in the field of environmental water treatment.

Description

Sphingobacterium and application thereof

Technical Field

The invention relates to a sphingosine bacillus and application thereof, belonging to the technical field of bioengineering and environmental engineering.

Background

The over-standard ammonia nitrogen content in rivers and lakes often causes eutrophication of water bodies and reduction of dissolved oxygen in the water, which becomes one of the main causes of water body pollution in China. The ammonia nitrogen in the water mainly comes from the use of chemical fertilizers in farmlands, animal excrement sewage generated in the breeding industry and the like, and the pollutants enter lakes and rivers through surface runoff to bring in a large amount of nitrate nitrogen and ammonia nitrogen, so that the quality of the water is deteriorated and the structure of the water ecological environment is damaged.

In the traditional biological denitrification theory, ammonia nitrogen is gradually converted into nitrite nitrogen and nitrate nitrogen through autotrophic nitrification and nitrite bacteria, and then is converted into nitrogen through the action of denitrifying bacteria to be removed from the environment. Due to the complex real environment, the two parts are difficult to realize in a real ecosystem.

The facultative heterotrophic nitrification and aerobic denitrification bacteria can not only make the nitrification process and the denitrification process be synchronously performed, but also greatly shorten the growth period because the growth rate is far higher than that of the autotrophic bacteria. The product of the nitration process can directly become a substrate of the denitrification process, thereby avoiding the inhibition effect of the accumulation of the nitration product on the nitration reaction and greatly improving the biological denitrification efficiency. The relative balance of acid and alkali can be kept in the whole process, and the products of denitrification can supplement the environmental alkalinity, thereby keeping the pH within a certain range. Most facultative heterotrophic nitrification and aerobic denitrification bacteria have strong environmental adaptability and are suitable for treating large-area polluted water areas.

At present, researchers have made few studies on facultative heterotrophic nitrification and aerobic denitrification bacteria, and the only few studies have focused on their degradation mechanisms. Therefore, the deep research on the physiological and biochemical characteristics and denitrification efficiency of the facultative heterotrophic nitrification and aerobic denitrification bacteria is necessary, and has important theoretical and practical application values.

Disclosure of Invention

The first purpose of the invention is to provide a sphingosine bacillus (Sphingobacterium sp.) which is preserved in China general microbiological culture Collection center (CGMCC) within 10 and 17 months in 2018, wherein the preservation number is CGMCC No.16596, and the preservation address is No. 3 of the West Lu No.1 of the North Chen of the sunward area in Beijing, China academy of sciences microbial research institute.

It is a second object of the present invention to provide a microbial preparation containing Sphingobacterium.

In one embodiment of the invention, the viable bacteria content of the Sphingobacterium in the microbial preparation is more than or equal to 1 × 10⁹cfu/g。

In one embodiment of the invention, the microbial agent is a biological microcapsule.

In one embodiment of the invention, the biological microcapsule is prepared by (1) weighing and dissolving 10-50g/L of activated carbon, 5-50g/L of chitosan and 10-50g/L of sodium alginate, and mixing uniformly to obtain a gel solution, and (2) inoculating 50-100g of solid thallus into the gel solution to obtain a thallus concentration of 1 × 10⁸-1×10¹¹cfu/ml of microbial gel solution; (3) dripping the microorganism gel solution into CaCl with mass concentration of 1-50g/L at a speed of 15-25 drops/min₂Obtaining microbial active particles in the solution; (4) cross-linking the microbially active particles in a 5% glutaraldehyde solution at room temperature for 24 hours; (5) soaking the microbial active granules cross-linked in the step (4) in sterile water for 24-48 hours, and addingObtaining the sphingosine bacillus BT1 biological microcapsule.

The second purpose of the invention is to provide the application of the sphingosine bacillus.

In one embodiment of the invention, the application comprises degrading river, lake pollutants or sewage treatment and the like.

In one embodiment of the invention, the application is the treatment of nitrogen-containing wastewater.

In one embodiment of the invention, the nitrogen-containing sewage is treated by adding the sphingosine bacillus BT1 into the nitrogen-containing sewage at a final concentration of 10-30 mg thallus/L.

In one embodiment of the invention, the sphingobacterium BT1 is administered to a body of water in the form of a biological microcapsule.

Has the advantages that: the invention provides a sphingosine bacillus strain, which has the characteristics of heterotrophic nitrification and aerobic denitrification, can be applied to the fields of nitrogenous sewage treatment and river treatment, has the ammonia nitrogen removal rate of more than 90 percent, has no pollution to the environment, achieves the effects of rapid biological denitrification and river self-cleaning capability recovery, and has great significance in the field of environmental water treatment.

Biological material preservation

A strain of sphingosine bacillus (Sphingobacterium sp.) is preserved in China general microbiological culture Collection center (CGMCC) at 17.10.2018 with the preservation number of CGMCC No.16596 and the preservation address of Beijing university Hokko No.1 of the sunward area, China academy of sciences, and the microbial research institute.

Drawings

FIG. 1 is a graph showing the growth and ammonia nitrogen degradation curves of Sphingobacterium BT 1;

FIG. 2 is a graph showing the change of degradation of nitrate nitrogen by Sphingobacterium BT 1;

FIG. 3 is a graph showing the change of degradation of nitrite nitrogen by Sphingobacterium BT 1;

FIG. 4 is a gel electrophoresis image of the amoA gene validation; wherein, Marker: DL2000(100 bp-2000 bp); 1: gene fragment: (ii) an amoA;

FIG. 5 is a gel electrophoresis image of nirK gene validation; wherein, Marker: DL2000(100 bp-2000 bp); 1: the gene fragment is nirS; 2: the gene fragment is nirK.

Detailed Description

Heterotrophic nitrification medium (g/L): (NH)₄)₂SO₄0.47,C₄H₄Na₂O₄5.62, vickers' saline solution 50ml, C/N10, pH 7.0, agar 2%.

Vickers salt solution (g/L): k₂HPO₄5.0，MgSO₄·7H₂O 2.5，NaCl 2.5，MnSO₄·4H₂O 0.05，FeSO₄·7H₂O 0.05。

Aerobic denitrification culture medium: nitrate medium: KNO₃2g, 5g sodium citrate, K₂HPO₄1g，KH₂PO₄1g，MgSO₄·7H₂0.2g of O, 2mL of trace element solution, and distilled water to 1L, pH7.2-7.5

Nitrite culture medium: NaNO₂2g, 5g sodium citrate, K₂HPO₄1g，KH₂PO₄1g，MgSO₄·7H₂O0.2g, 2mL of trace element solution, and distilled water to 1L, pH7.2-7.5

Solution of trace elements: EDTA 50.0g, ZnSO₄2.2g，CaCl₂·2H₂O 5.5g，MnCl₂·4H₂O 5.06g，FeSO₄·7H₂O 5.0g，(NH₄)6Mo₇O₂·4H₂O 1.1g，CuSO₄·5H₂O 1.57g，CoCl₂·6H₂O1.61 g, adding distilled water to 1L, pH 6.0

Example 1

The activated sludge of the landfill leachate aerobic fermentation tank in a certain period with stable operation is used as the separation sludge. Separating on heterotrophic nitrification culture medium by dilution mixing plate method to obtain pure Sphingobacterium BT1 strain. The genome of the strain sphingobacterium BT1 was extracted and subjected to 16S rDNA sequencing. The sequencing result is compared with the similarity of known sequences in a Genbank database, and the result shows that the strain sphingosine BT1 has the highest sequence homology with sphingosine (Sphingobacterium) and is identified as the sphingosine.

Example 2 determination of Ammonia Nitrogen degrading ability of Sphingobacterium (Sphingobacterium) Sphingobacterium BT1

After activated enrichment culture of Sphingobacterium BT1, 1ml of the suspension was transferred to 100ml of agar-free heterotrophic nitrification medium and cultured at 30 ℃ and 160rpm/min for 48 hours. The culture broth was taken every 8 hours. Measuring the growth curve of Sphingobacterium BT1 at 600nm of part of culture solution, filtering the rest culture solution with 0.22 μm microporous membrane, and detecting the indexes of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, COD, etc. The results are shown in table 1 and fig. 1.

TABLE 1 changes in degradation rates of Ammonia Nitrogen by Sphingobacterium BT1

From table 1OD₆₀₀The indicator can obtain the growth curve of the strain. The strain can enter an exponential phase only after 8 hours of delay phase, the OD value reaches 1.349 from 0.01 within 24 hours, and the strain enters a stationary phase after 24 hours, so that the growth period is shortened by 24-48 hours compared with the growth period of common nitrobacteria.

In 40h, the ammonia nitrogen is reduced from 90.21mg/L to 4.49mg/L, the degradation rate of the ammonia nitrogen reaches 95.01%, the total nitrogen is reduced from 93.77mg/L to 5.11mg/L, and the degradation rate of the total nitrogen is 94.65%.

It is noted that, within 48 hours, nitrite nitrogen content was accumulated from 0mg/L to 0.3mg/L and nitrate nitrogen was reduced from 0.6mg/L to 0.05mg/L, indicating that Sphingobacterium BT1 has the ability to accumulate nitrite nitrogen and degrade nitrate nitrogen, and we speculate that Sphingobacterium BT1 may have the ability to aerobically denitrify.

Compared with common nitrifying bacteria or anaerobic denitrifying bacteria, the sphingosine bacillus BT1 has the functions of heterotrophic nitrification and aerobic denitrification, the nitrification process and the denitrification process can be carried out simultaneously, and the nitrified product can be directly used as a substrate of denitrification. As can be seen from the table 1, the growth cycle of the sphingosine bacillus BT1 is short, the platform stage can be reached within 24 hours, and the growth rate is far greater than that of autotrophic nitrifying bacteria; strong environmental adaptability and strong tolerance to high-concentration ammonia nitrogen.

Example 3 determination of aerobic denitrifying capability of Sphingobacterium BT1

(1) The denitrification performance of the sphingosine bacillus BT1 is screened and characterized by adopting a nitrate culture medium.

Activating and enriching Sphingobacterium BT1, transferring 1ml of suspension into 100ml of agar-free nitrate culture medium or nitrite culture medium, culturing at 30 deg.C and 160rpm/min, and measuring bacterial concentration (OD) of culture solution every 8 hr₆₀₀) Nitro nitrogen, nitrous nitrogen.

TABLE 2 changes in degradation of nitrate nitrogen by Sphingobacterium BT1

As can be seen from Table 2 and FIG. 2, Sphingobacterium BT1 began to grow at 46 hours and peaked at 80 hours. In 100 hours, the nitrate nitrogen is reduced from 240.42mg/L to 133.32mg/L, the degradation rate of the nitrate nitrogen is 44.5%, and the nitrite is accumulated from 0 to 40.59mg/L, which shows that the sphingosine bacillus BT1 has better removal rate of the nitrate nitrogen and stronger denitrification function.

(2) The denitrifying performance of the sphingosine bacillus BT1 is screened and characterized by adopting a nitrite culture medium. On the basis of a nitrate culture medium, potassium nitrate in the medium is replaced by sodium nitrite.

TABLE 3 changes in degradation of nitrite nitrogen by Sphingobacterium BT1

As can be seen from Table 3 and FIG. 3, the seed strain started to grow at 27 hours after inoculation and reached a peak at 68 hours. In 100 hours, the nitrite nitrogen content is reduced from 439.86mg/L to 375.97mg/L, the nitrite content is reduced by 14.5%, the nitrate nitrogen content is reduced from 80.27mg/L to 53.13mg/L, and the content is reduced by 33.8%.

By combining the effect of degrading nitrate and nitrite by the sphingosine bacillus BT1, the method can be obtained that when the nitrate or nitrite is used as a nitrogen source by the sphingosine bacillus BT1, a latent period of 25-40 hours exists, the logarithmic growth phase of the strain is 12-22 hours, and the strain begins to enter a stationary phase and a decline phase after 30 hours. The sphingosine bacillus BT1 can simply utilize nitrate or nitrite as a nitrogen source to achieve the denitrification effect, so that the sphingosine bacillus BT1 has a strong denitrification function.

Example 4 determination of aerobic denitrifying ability of Sphingobacterium BT1

(1) PCR was used to verify the presence of the nitrification gene amoA in Sphingobacterium BT 1. The primer sequences are as follows:

amoA1F(5’-GGGGTTTCTACTGGTGGT-3’)；

amoA2R(5’-CCCCTCKGSAAAGCCTTCTTC-3’)

reaction conditions are as follows: 3min at 95 ℃; 1min at 94 ℃, 45s at 54.5 ℃, 1min at 72 ℃ and 35 cycles; 72 ℃ for 10 min; the PCR products were detected by 0.8% agarose gel electrophoresis, and the results are shown in FIG. 4:

the PCR product was a 491bp band, and the sequence was confirmed to be identical to the sequence of the amoA gene, whereby the existence of the amoA gene in Sphingobacterium BT1 and the nitrifying function were confirmed.

(2) Denitrification functional gene Nir: the Nir genes are divided into two groups, nirK and nirS, respectively. The nirK gene encodes the Cu-type nitrite reductase, and the other nirS gene encodes cytochrome reductase. It should be noted that the two coding genes do not exist in the same strain at the same time. PCR was used to verify the presence of nirK and nirS genes in Sphingobacterium BT 1. The primer sequences are as follows:

nirS-cd3aF(5’-GTSAACGTSAAGGARACSGG)

nirS-R3cd(5’-GASTTCGGRTGSGTCTTGA)

nirK-F1aCu(5’-ATCATGGTSCTGCCGCG)

nirK-R3Cu(5’-GCCTCG ATCAGRTTGTGGTT)

reaction conditions are as follows: 10min at 95 ℃; 30s at 95 ℃, 30s at 56 ℃, 30s at 72 ℃ and 35 cycles; 72 ℃ for 10 min; the PCR products were detected by 0.8% agarose gel electrophoresis, and the results are shown in FIG. 5.

As can be seen from the figure, the nirS gene is absent and the nirK gene is present in the strain Sphingobacterium BT 1. Namely, the existence of the Cu-type nitrite reductase in the sphingosine bacillus BT1 is shown, and the genotype is consistent with the aerobic denitrification function shown by the Cu-type nitrite reductase.

Example 5

Culturing the sphingosine bacillus BT1 on a heterotrophic nitrification culture medium for 24 hours, and then putting the cultured sphingosine bacillus BT1 into a DN reactor according to the volume proportion of 1%, wherein the DN reactor is a high-efficiency denitrification reactor, a water distribution system, a three-phase separator and other core structures are arranged in the DN reactor, the height-diameter ratio is set to be larger than 2, the controllable parameter is HRT, and when the HRT is set to be 48 hours, the operation parameters of the reactor are as follows:

the parameters show that the Sphingobacterium BT1 can efficiently remove ammonia nitrogen in practical application, the ammonia nitrogen content is reduced from 2000mg/L to 35mg/L within 48 hours, the removal rate reaches 40.9mg/L x h, meanwhile, the Total Nitrogen (TN) content is reduced from 2500mg/L to 200mg/L within 48 hours, the COD is reduced from 8000mg/L to 700mg/L, and the degradation rates of the total nitrogen and the COD respectively reach 92% and 91.25%. DO is always kept to be less than 1mg/L, which shows that the sphingosine bacillus BT1 has low requirement on the concentration of dissolved oxygen and strong adaptability, and is particularly suitable for treating eutrophic and high ammonia nitrogen water bodies.

Example 6 application of Sphingobacterium BT1 in Nitrogen-containing wastewater treatment

In the nitrogen-containing sewage treatment experiment of a certain sewage treatment plant from Nanjing by using sphingosine bacillus BT1, sphingosine bacillus BT1 bacterial liquid is fed at the front end of an aerobic pool according to the concentration of 20 ppm. Within 48 hours, the ammonia nitrogen content is reduced from 20mg/L to 5mg/L, the ammonia nitrogen removal rate reaches 75 percent, and the daily sewage treatment capacity can reach 55000-³It shows that the sphingosine bacillus BT1 can effectively reduce nitrogen when used for treating large-scale sewageThe concentration of ammonia nitrogen in the sewage.

Example 7 application of the Strain Sphingobacterium BT1 in immobilization of microorganisms

The sphingosine bacillus BT1 strain is combined with a biological carrier to prepare the immobilized microorganism which is suitable for the river water body and can be slowly released.

The preparation method of the immobilized strain comprises the following steps:

fermenting and culturing the strain, performing pure culture of Sphingobacterium BT1 in a mechanically-stirred fermentation tank by adopting a liquid fermentation mode, wherein the culture medium is a heterotrophic nitrification liquid culture medium, performing pure culture at 30 ℃ for 12-48 hours to obtain high-concentration Sphingobacterium BT1 pure culture bacterial liquid, and counting the viable count of the bacterial liquid at 1 × 10⁸-1×10¹¹cfu/ml。

Preparation of solid thallus: and after the fermentation of the strain is finished, centrifuging to remove the fermentation liquor to obtain solid thalli.

Preparation and immobilization of biological carrier (1) weighing and dissolving 10-50g/L of activated carbon, 5-50g/L of chitosan and 10-50g/L of sodium alginate, and mixing uniformly to obtain gel solution, (2) inoculating 50-100g of solid thallus into the gel solution to obtain thallus concentration of 1 × 10⁸-1×10¹¹cfu/ml of microbial gel solution. (3) Dripping the microorganism gel solution into CaCl with mass concentration of 1-50g/L at a speed of 15-25 drops/min₂And (4) obtaining the microbial active particles in the solution. (4) The microbially active particles were crosslinked in a 5% glutaraldehyde solution at 1:50 for 24 hours at room temperature. (5) And (3) soaking the cross-linked microbial active particles in the step (4) in sterile water for 24-48 hours, and proliferating to obtain the sphingosine bacillus BT1 biological microcapsule.

The release amount of the obtained sphingosine bacillus BT1 biological microcapsule reaches more than 85 percent within 30 days, the survival rate of the bacteria is more than 5 percent, and the effective viable count reaches 10 percent after verification⁹cfu/g。

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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Claims

1. A strain of sphingosine bacillus (B)Sphingobacterium sp.) And the strain is preserved in the China general microbiological culture Collection center (CGMCC) at 17 months in 2018, the preservation number is CGMCC No.16596, the preservation address is No. 3 of Xilu No.1 of Beijing university and Chaoyang district, China academy of sciences.

2. A microbial preparation comprising the bacterium Sphingobacterium of claim 1.

3. The microbial preparation according to claim 2, wherein the viable bacteria content of Sphingobacterium is not less than 1 × 10⁹CFU/g。

4. The microbial preparation of claim 2, wherein said microbial preparation is a biological microcapsule.

5. The microbial preparation according to claim 4, wherein the biological microcapsule comprises a shell and a core material in the shell, wherein the shell material comprises but is not limited to sodium alginate gel, the core material in the shell comprises but is not limited to calcium chloride solution, and 1 × 10 is dispersed in the core material in the shell⁸～1×10¹¹CFU/mL of said cells of Sphingobacterium.

6. The use of the sphingobacterium according to claim 1 in the chemical and environmental fields for degrading nitrogen-containing pollutants in rivers and lakes or treating nitrogen-containing sewage.

7. A method for treating nitrogen-containing sewage, characterized by comprising administering the sphingosine bacterium according to claim 1 to nitrogen-containing sewage at a final concentration of 10 to 30 mg/L.

8. The method of claim 7, wherein the Sphingobacterium are delivered to the body of water in the form of biological microcapsules.