CN113234626A

CN113234626A - Strain with heterotrophic nitrification-aerobic denitrification function and application thereof

Info

Publication number: CN113234626A
Application number: CN202110522226.8A
Authority: CN
Inventors: 高娜; 梁阳阳; 杨坤; 李静; 方婷; 赵秀侠; 卢文轩; 李书杨
Original assignee: Fisheries Research Institute of Anhui AAS
Current assignee: Fisheries Research Institute of Anhui AAS
Priority date: 2021-05-13
Filing date: 2021-05-13
Publication date: 2021-08-10

Abstract

The invention provides a strain with heterotrophic nitrification aerobic denitrification function and application thereof, wherein the strain is Achromobacter strain (Achromobacter xylosidans) pf1 which is preserved in China center for type culture collection with the preservation number of CCTCC NO: m2021273. The strain can synchronously carry out nitrification and denitrification by taking organic carbon as a carbon source under aerobic conditions, and is suitable for removing organic wastewater containing ammonia nitrogen, nitrate and nitrite. In the ammonia nitrogen wastewater with the initial concentration of 175mg/L, the total nitrogen removal rate can reach 100%, and the intermediate product is less in accumulation, so that no secondary pollution is caused; in the nitrate or nitrite wastewater with the initial concentration of 100mg/L, the total nitrogen removal rate can reach 87.1 percent or 65.1 percent. After the industrial turtle raising wastewater is treated, the nitrogen and phosphorus in the wastewater can be obviously removed.

Description

Strain with heterotrophic nitrification-aerobic denitrification function and application thereof

Technical Field

The invention belongs to the field of environmental microorganisms, and particularly relates to a colorless bacillus strain with heterotrophic nitrification-aerobic denitrification functions and application thereof in treatment of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen wastewater.

Background

At present, the great problem of the environmental protection of water bodies in China is that the concentration of ammonia nitrogen in the water bodies is far beyond the self-purification capacity of polluted water bodies. The environmental quality condition of surface water in the whole country in the first half of 2016 shows that the poor V class accounts for 11.3% in ten large watersheds in China, wherein COD, ammonia nitrogen and total phosphorus are main pollution indexes, and the ammonia nitrogen serving as an oxygen-consuming pollutant is a primary index influencing the environmental quality of the surface water body in China. The severe situation of ammonia nitrogen pollution of water bodies becomes a limiting factor of the environmental protection industry of China. When the nitrogen content in the water body exceeds the environmental capacity of the receiving water body, the water ecological balance is broken to cause the water quality deterioration and produce toxic action on aquatic organisms. The removal of nitrogen from water has become a research focus in recent years, and conventional nitrogen removal methods include physical, chemical and biological methods, wherein the biological methods have the advantages of environmental friendliness, low cost, few harmful byproducts and the like. The traditional biological denitrification is completed by the aerobic autotrophic nitrification and the anaerobic heterotrophic denitrification, and the discovery of the heterotrophic nitrification-aerobic denitrification microbial metabolic pathway breaks through the understanding of the traditional biological denitrification field. The new metabolic approach can lead the nitrification and the denitrification to be completed in the same reactor, thereby accelerating the reaction process, reducing the volume of the reactor, shortening the hydraulic retention time, reducing the operation cost and treating the high-concentration ammonia nitrogen wastewater. Therefore, reports of screening, functional identification and water treatment applications of heterotrophic nitrification-aerobic denitrification microorganisms have been occasionally made in recent years.

Disclosure of Invention

The invention provides an achromobacter strain for enriching a denitrogenation strain resource library, wherein the strain is a heterotrophic nitrification-aerobic denitrification strain from a turtle culture pond, and has great application value in the aspect of aquaculture tail water treatment.

The technical scheme of the invention is as follows:

a strain with heterotrophic nitrification-aerobic denitrification function, wherein the strain is Achromobacter strain (Achromobacter xylosoxidans) pf1, which is preserved in China Center for Type Culture Collection (CCTCC) at 3 and 25 months in 2021, with the preservation number being CCTCC NO: m2021273. The preservation address is eight Wuhan university school of 299 # in Wuchang district of Wuhan city, Hubei province.

In a further embodiment, the effective sequence length of 16SrDNA of the Achromobacter strain (Achromobacter xylosoxidans) pf1 is 1453bp, and the sequence is shown as SEQ ID NO: 1 is shown.

In a further scheme, the Achromobacter strain (Achromobacter xylosoxidans) pf1 is a gram-negative bacillus and is obtained by enrichment culture, separation and purification from a water body of a terrapin aquaculture pond.

In a further embodiment, the Achromobacter strain (Achromobacter xylosoxidans) pf1 is grown using ammonium sulfate, nitrate or nitrite as the sole nitrogen source.

Another object of the present invention is to provide the use of the above-mentioned strain of Achromobacter (Achromobacter xylosidases) pf1 for denitrification in nitrogen-containing water.

In a further scheme, the nitrogen-containing water body refers to organic wastewater containing ammonia nitrogen, nitrate and nitrite.

In a further scheme, the Achromobacter strain (Achromobacter xylosoxidans) pf1 is inoculated in a nitrogen-containing water body for aerobic continuous culture after amplification culture.

In a further scheme, the temperature of the aerobic continuous culture is 30-35 ℃; the Achromobacter strain (Achromobacter xylosoxidans) pf1 was inoculated in an amount of 1% by volume of the nitrogen-containing water body.

The strain pf1 of the invention is gram-negative bacillus, has small bacterial colony, regular edge, milky white color and moist and smooth surface.

The effective sequence length of the 16SrDNA of the Achromobacter (Achromobacter xylosoxidans) pf1 is 1453bp, which is shown as SEQ ID NO.1 in the sequence table. The strain was identified as belonging to Achromobacter (Achromobacter xylosoxidans) designated as Achromobacter (Achromobacter xylosoxidans) pf1 by BLAST sequence homology alignment.

In the application, Achromobacter (Achromobacter xylosoxidans) pf1 is subjected to amplification culture by using R2A culture medium until logarithmic phase, the inoculum sizes are respectively inoculated into conical flasks (250ml) filled with 100ml of heterotrophic Nitrification (NM) and aerobic denitrification (DMA and DMB) culture medium according to 1 percent (volume ratio), the culture is carried out at 30 ℃ and 220R/min, and the cell concentration (OD600), TN and NH are detected by timing sampling₄ ⁺-N，NO₃ ^--N，NO₂ ^-The change in-N reflects the denitrification effect of Achromobacter pf 1. Tests show that the composite material has a removing effect on nitrogen and phosphorus in wastewater.

The technical scheme of the invention has the following advantages:

1. the achromobacter pf1 is a heterotrophic nitrification-aerobic denitrification strain, namely, can synchronously carry out nitrification and denitrification by taking organic carbon as a carbon source under aerobic conditions, and is suitable for removing organic wastewater containing ammonia nitrogen, nitrate and nitrite.

2. Has obvious ammonia nitrogen removing effect. In ammonia nitrogen wastewater with the initial concentration of 175mg/L, the total nitrogen removal rate can reach 100 percent under the conditions of the temperature of 30 ℃, the rotating speed of 220rpm and the pH value of 7, and the NH content of the Achromobacter pf1 is reduced₄ ⁺The removal rate of-N can reach 92.9 percent, the accumulation of intermediate products is less, and the ammonia nitrogen removal effect is good.

3. The strain pf1 has aerobic denitrification energyForce. Under the conditions of initial concentration of 100mg/L nitrate wastewater, temperature of 30 ℃, rotating speed of 220rpm and pH of 7, the total nitrogen removal rate can reach 87.1 percent, and NO is₃The removal rate of-N can reach 97.5 percent.

In the nitrite waste water with the initial concentration of 100mg/L, under the conditions of the temperature of 30 ℃, the rotating speed of 220rpm and the pH value of 7, the total nitrogen removal rate is 65.1 percent, and NO is₂ ^-The removal rate of-N is 76%, and the aerobic denitrification capability is obvious.

4. When the strain pf1 is used for treating the industrial turtle culture wastewater, the removal rate of total nitrogen reaches 90.4 percent after 24 hours of treatment, the removal effect of COD is 76.3 percent, and the removal rate of phosphorus by the strain also reaches 52.6 percent. Therefore, the strain pf1 can be practically applied to nitrogen and phosphorus removal of culture wastewater.

Drawings

Biological denitrification of Achromobacter pf1 is described in detail with reference to the accompanying drawings.

FIG. 1 is the heterotrophic nitrification-aerobic denitrification growth characteristics of said Achromobacter;

FIG. 2 shows the denitrification capacity of Achromobacter pf1 in ammonia nitrogen wastewater with an initial concentration of 175 mg/L;

FIG. 3 is a denitrification performance analysis of Achromobacter pf1 using nitrite and nitrate as nitrogen sources.

Detailed Description

The present invention will be described in further detail with reference to the following examples and the accompanying drawings.

The methods for measuring total nitrogen, ammonia nitrogen, nitrate nitrogen and nitrite nitrogen referred to in the following examples are as follows:

the total nitrogen is measured by alkaline potassium persulfate digestion ultraviolet spectrophotometry (GB 11894-89);

measuring ammonia nitrogen by adopting a water quality ammonia nitrogen determination Nashiner reagent spectrophotometry (HJ 535-2009);

nitrate Nitrogen (NO)₃ ^--N) respectively adopting a method (HZ-HJ-SZ-0138) for measuring nitrate nitrogen by adopting ultraviolet spectrophotometry water quality;

nitrous acid Nitrogen (NO)₂ ^-The measurement of-N) was carried out by using naphthyl ethylenediamine hydrochloride spectrophotometry.

Example 1:

a bacterial strain with heterotrophic nitrification and aerobic denitrification functions is Achromobacter strain (Achromobacter xylosoxidans) pf1, the collection site is located in the Special terrapin of the abrupt ditch town Anhui Lantian of the Wuhu city, Anhui province, which is preserved in the China center for type culture Collection in 2021, 3, 25 days, the preservation number is CCTCC NO: m2021273; the preservation address is eight Wuhan university school of 299 # in Wuchang district of Wuhan city, Hubei province.

1. Isolation and purification of the strains

Inoculating a water sample collected from a terrapin aquaculture pond into a heterotrophic nitrification enrichment medium according to the volume ratio of 10% for enrichment and domestication, and performing shake culture at 30 ℃ and 150 r/min; replacing fresh heterotrophic nitrification culture medium according to the volume ratio of 10% every 48h, and continuously culturing for 20 d.

Wherein, the formula of the heterotrophic nitrification enrichment medium is as follows: (NH)₄)₂SO₄0.472g, 4.052g sodium succinate and 50ml Vickers salt solution, adding water for dissolution, supplementing distilled water to 1L, and adjusting the pH to 7.0. Wherein the formula of solute in Vickers salt solution (g/L) is as follows: k₂HPO₄ 5.0g，FeSO₄·7H₂O 0.05g，NaCl 2.5g，MgSO₄·7H₂O 2.5g，MnSO₄·4H₂O 0.05g。

Inoculating the domesticated microorganisms into an aerobic denitrification culture medium for further screening, wherein the aerobic denitrification culture medium takes potassium nitrate as a unique nitrogen source.

Aerobic denitrification culture medium: 0.36g/L KNO₃，10.55g/L Na₂HPO₄·12H₂O，1.5g/L KH₂PO₄，0.1g/L MgSO₄·7H₂O, 4.0g/L sodium citrate and 0.2 percent (volume ratio) of trace element solution. Solution of trace elements: 50g/L EDTA-Na2, 2.2g/L ZnSO₄，5.5g/L CaCl₂，5.06g/L MnCl₂·4H₂O，5.0g/LFeSO₄·7H₂O，1.57g/L CuSO₄·5H₂O，1.61g/L CoCl₂·6H₂O。

1mL of sample subjected to primary screening by the aerobic denitrification culture medium is uniformly coated on a solid GN chromogenic culture medium, and then the sample is placed in a constant temperature incubator at 30 ℃ for culture to obtain bacterial colonies.

To further verify that the single colony picked has the property of changing the GN chromogenic medium to blue, the single colony which is blue in the solid chromogenic medium was picked and inoculated into 3mL of sterilized liquid GN chromogenic medium, shaking-cultured at 30 ℃ and 150r/min, the color change was observed, and a strain which can change the liquid GN chromogenic medium from green to blue was selected as a rescreen.

Wherein liquid GN chromogenic medium: 1.0g/L KNO₃8.5g/L sodium citrate, 1.0g/L L-asparagine, 1.0g/L KH₂PO₄，1.0g/L MgSO₄·7H₂O，0.2g/L CaCl₂·6H₂O，0.05g/L FeCl₃·6H₂O, 0.1% by volume of bromothymol blue (BTB), and the pH was adjusted to 7.0. Adding agar 2% to the liquid chromogenic medium to prepare solid chromogenic medium, and sterilizing at 121 deg.C for 20 min.

2. Molecular biological identification

Single colonies of the rescreened bacteria were picked for 16S rRNA gene amplification and PCR amplification was performed using bacterial 16S rRNA gene universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5 '-TACGGYTACCTTGTTACGACTT-3'). Wherein the 50 mu L PCR reaction system comprises: 2 XTaq PCR Master Mix 25. mu.L, 27F (10. mu.M) 1. mu.L, 1492R (10. mu.M) 1. mu.L, DNA template as single colony picked, ddH₂O24. mu.L. Setting a PCR program: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 45s, and extension at 72 ℃ for 40s, and amplification for 30 cycles; final extension at 72 ℃ for 10 min. The sequencing result is shown in SEQ ID NO: 1, and then submitting the 16SrDNA Sequence of the bacteria obtained by sequencing to a Genbank database (https:// blast.ncbi.nlm.nih.gov/blast.cgi) for Sequence alignment, wherein the database is '16 Sribosome RNA databases', and the most similar species to the target strain is determined according to the highest Sequence coverage rate (Sequence coverage) and similarity (Identity).

The effective sequence length of the sequenced 16SrDNA is 1453bp, and is shown as the sequence SEQ ID NO: 1 is shown. The 16SrDNA sequence of the bacterium is confirmed to have homology of more than 99 percent with Achromobacter xylosoxidans (Achromobacter xylosoxidans) published in NCBI database through Blastn analysis, so that the bacterium is confirmed to be the Achromobacter and named as Achromobacter pf 1.

The Achromobacter pf1 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation address of eight 299 Wuhan university schools in the Wuhan district of the city of Wuhan, Hubei province and the preservation number of CCTCC NO: m2021273.

Example 2 cultivation and analysis of Denitrification Performance of Achromobacter pf1

The bacterial colony of the rescreened strain separated in example 1 is picked up to an R2A liquid culture medium, cultured for 18h at 30 ℃ under the condition of 200rpm of a gas bath shaker, centrifuged for 2min at 4000R/min, the strain is collected, and the strain is resuspended by using the same amount of sterile water and is respectively inoculated into NM (heterotrophic nitrification culture medium), DMA (nitrate is used as a nitrogen source) and DMB (nitrite is used as a nitrogen source) liquid culture media according to 1 percent (volume ratio). Shaking-flask culture at 30 deg.C and 200rpm, and periodically sampling to determine optical density (OD600), Total Nitrogen (TN), and ammonia Nitrogen (NH)₄ ⁺-N), Nitrate (NO)₃ ^--N) and Nitrite (NO)₂ ^--N), the growth curve of the bacterial cells is drawn according to the OD600 value, and the denitrification performance of the bacterial strain is judged by analyzing the total nitrogen removal rate.

Wherein the formula of the R2A liquid culture medium is as follows: 0.5g/L of tryptone, 0.5g/L of yeast extract, 0.5g/L of starch, 0.5g/L of enzymatic hydrolysis casein, 0.5g/L of glucose, 0.3g/L of sodium pyruvate, 0.3g/L of dipotassium hydrogen phosphate and 0.024g/L of magnesium sulfate.

The formula of NM culture medium is: (NH4)₂SO₄0.945g/L, 16.34g/L sodium citrate and MgSO₄·7H₂O 1g/L，KH₂PO₄ 0.25g/L，Na₂HPO₄ 0.3g/L。

The formula of the DMA culture medium is as follows: KNO₃ 0.722g/L(NaNO₂0.5g/L), sodium citrate 5g/L, MgSO₄·7H₂O 1g/L，KH₂PO₄ 0.25g/L，Na₂HPO₄ 0.3g/L。

The formula of the DMB culture medium is as follows: NaNO₂0.5g/L, 5g/L sodium citrate，MgSO₄·7H₂O 1g/L，KH₂PO₄0.25g/L，Na₂HPO₄ 0.3g/L。

As can be seen from figures 1 and 2, the growth adaptation period of the achromobacter strain pf1 is longer, when ammonia nitrogen is used as a unique nitrogen source, the strain is cultured for 16h and then grows in a logarithmic phase, and after 48h, the strain enters a plateau phase; the removal rate of the total nitrogen by the strain after 40 hours as shown in b in FIG. 2 reaches 100%; fig. 2a shows that nitrate nitrogen and nitrite nitrogen are scarcely accumulated as intermediate products at this time.

As shown in figures 1 and 3, when nitrate is used as a nitrogen source, the strain is cultured for 24 hours and grows in a logarithmic phase, the strain grows for 40 hours and grows in a plateau phase, and the removal rate (DMA-TN) of the strain to total nitrogen reaches 87.1 percent at the maximum. When nitrite is used as a nitrogen source, the strain is cultured for 40 hours and grows in a logarithmic phase, the strain grows in a plateau phase after 56 hours, and the removal rate (DMB-TN) of the strain to the total nitrogen reaches 65.1 percent at the maximum.

Example 3: achromobacter pf1 practical denitrification application

Adding 1000ml of turtle industrial aquaculture wastewater with initial COD of 760mg/L into a 3L beaker, adding 15g of sodium citrate, adding a carbon source, and increasing COD of the turtle industrial aquaculture wastewater to 8160 mg/L; then, 0.5g of pf1 wet cells which had been extensively cultured in the R2A medium of example 2 to logarithmic phase were added thereto, and the mixture was left at room temperature (18 to 25 ℃ C.) and dissolved oxygen was maintained at 2ppm or more by an aerator. Meanwhile, turtle breeding wastewater without pf1 inoculation is used as a control group after the same treatment. And sampling after 24h, centrifuging to remove precipitates, and detecting the total nitrogen, total phosphorus and COD content in the supernatant. The details are shown in the following table:

TABLE 1 Effect of Achromobacter pf1 on removing nitrogen and phosphorus from wastewater

Table 1 shows that the industrial turtle breeding wastewater contains high-concentration nitrogen, phosphorus and COD, and the achromobacter pf1 is cultured in the turtle wastewater supplemented with carbon source for 24 hours, so that the total nitrogen removal rate is over 90%, the COD removal efficiency is 76%, and the total phosphorus removal rate by the strain is 52.6% compared with the turtle industrial breeding wastewater before final treatment. Compared with a control group, the removal rate of total nitrogen reaches over 84%, the removal efficiency of COD is 71.6%, and the removal rate of total phosphorus by the strain also reaches 40%. Therefore, the achromobacter pf1 is a microorganism with good application prospect in the culture wastewater treatment.

The above-described embodiments are merely preferred embodiments of the present invention, and the embodiments of the present invention are not limited to the above-described embodiments, and it should be understood that many other modifications and embodiments can be devised by those skilled in the art, which will fall within the spirit and scope of the principles of this disclosure.

Sequence listing

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Claims

1. A bacterial strain with heterotrophic nitrification-aerobic denitrification function is characterized in that: the strain is Achromobacter strain (Achromobacter xylosoxidans) pf1, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2021273.

2. The strain of claim 1, wherein: the effective sequence length of the 16SrDNA of the Achromobacter strain (Achromobacter xylosoxidans) pf1 is 1453bp, and the sequence is shown as SEQ ID NO: 1 is shown.

3. The strain of claim 1, wherein: the Achromobacter strain (Achromobacter xylosoxidans) pf1 is a gram-negative bacillus and is obtained by enrichment culture, separation and purification from a water body of a terrapin aquaculture pond.

4. The strain of claim 1, wherein: the Achromobacter strain (Achromobacter xylosoxidans) pf1 was grown using ammonium sulfate, nitrate and nitrite as sole nitrogen sources.

5. Use of a strain according to any one of claims 1 to 4, characterized in that: the Achromobacter strain (Achromobacter sp.) pf1 is used for denitrification in nitrogen-containing water bodies.

6. Use according to claim 5, characterized in that: the nitrogen-containing water body refers to organic wastewater containing ammonia nitrogen, nitrate and nitrite.

7. Use according to claim 5, characterized in that: the Achromobacter strain (Achromobacter sp.) pf1 is inoculated in a nitrogen-containing water body for aerobic continuous culture after amplification culture.

8. Use according to claim 7, characterized in that: the temperature of the aerobic continuous culture is 30-35 ℃; the Achromobacter strain (Achromobacter xylosoxidans) pf1 was inoculated in an amount of 1% by volume of the nitrogen-containing water body.