CN114058554B - Composting pseudomonas strain and application thereof - Google Patents

Abstract

The invention relates to a composting Pseudomonas strain (Pseudomonas composti), a microbial agent containing the same and application of the composting Pseudomonas strain in the field of water purification, wherein the strain is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.20850, the strain can have an ammonia nitrogen removal effect of more than 95% at an application temperature as low as 8 ℃, and the inoculation amount can be as low as 50 ppm.

Description

Composting pseudomonas strain and application thereof

Technical Field

The invention relates to a composting pseudomonas strain and application thereof, in particular to a composting pseudomonas strain capable of efficiently degrading nitrogenous substances in water under a low-temperature condition, and belongs to the technical field of environmental microorganisms.

Background

The situation of water resources in China is very severe at present, and along with the rapid development of the economic society and the growth of population, the problems of aggravation of water pollution, deterioration of water ecological environment and the like are increasingly prominent, so that the water resource is a main bottleneck for restricting the sustainable development of the economic society. Environmental events, public safety events and even major social events caused by water pollution seriously affect the physical health of people and the harmony and stability of the society, and directly threaten the living space of human beings.

In the sewage treatment process, organic matters, nitrogen, phosphorus and other nutrient substances in the sewage can be degraded and utilized by microorganisms through metabolic action. In this process, temperature is one of the important factors affecting the function and treatment effect of microorganisms in the biological treatment process of sewage. Temperature can affect biochemical reactions in many ways, such as reaction rate, reaction pathways, microbial yield and mortality, among others. Due to the difference of geographical areas and seasonal changes, in a plurality of areas, including cold areas such as Canada, North America, northern Europe, Russia and northern China, the temperature of the sewage can be reduced to 8-15 ℃ or even lower than 5 ℃, and at low temperature, the physiological characteristics of cells, the growth rate of microorganisms, the activity of the microorganisms, the structure of microbial communities and the sedimentation property of sludge can be changed, so that the sewage treatment process effect is poor and the system performance is deteriorated. Therefore, how to maintain and improve the performance of the sewage biological treatment technology under low temperature conditions has been the hot point of research.

The method is characterized by enhancing the removal of pollutants in the sewage under the low-temperature condition, and mainly comprises two strategies of adjusting and optimizing operation parameters and adding a low-temperature microbial agent.

(1) Optimization of the regulation of operating parameters

Operational parameter regulation is a common strategy for enhancing biological treatment systems for wastewater under low temperature conditions. When seasonal cooling or low-temperature impact occurs, the sewage treatment plant generally improves the Sludge load resistance of the reactor by reducing the Sludge load rate, increasing the HRT, the Sludge Retention Time (SRT), the Dissolved Oxygen (DO), and other measures so as to maintain the stability of the system. By adjusting and controlling these process parameters, the adverse effects of low temperatures on the wastewater treatment system can be mitigated.

(2) Adding low-temperature microbial agent

The addition of the low-temperature microbial agent is one of effective ways for improving the low-temperature sewage treatment performance. The quantity and activity of low-temperature functional bacteria can be enhanced by adding the low-temperature microbial agent into the bioreactor, so that the removal rate of pollutants is obviously improved at severe environmental temperature. In recent years, adding low-temperature microbial agents is widely applied to sewage or low-concentration domestic sewage treatment so as to meet stricter sewage discharge requirements in cold regions.

Since the middle of the 70's of the 20 th century, the low-temperature microbial inoculum has been developed and generally applied in sewage treatment worldwide and has achieved good treatment effect, but the following disadvantages exist in research and application:

1. most studies stay in the research stage, and mature products are not formed;

2. in addition, the inoculation amount of the strains in most researches is too large, so that the cost is high in practical application;

3. the most of the minimum effective temperature is above 10 ℃, and the application cost is high.

Disclosure of Invention

Aiming at the defects of the existing low-temperature microbial strains and microbial agents in the field of water purification, the invention provides a composting pseudomonas strain which can still have excellent ability of degrading ammonia nitrogen at 8 ℃, a microbial agent containing the composting pseudomonas strain, a preparation method of the microbial agent and an application of the microbial agent.

The invention claims a compost Pseudomonas sp.AOB001, the 16S rDNA sequence of which is shown as SEQ ID No. 1, and the compost Pseudomonas sp.AOB is preserved in the China general microbiological culture Collection center with the addresses as follows: the No. 3 Xilu Beijing, Chaoyang, has a preservation number of CGMCC No.20850 and a preservation date of 2020, 10 months and 12 days.

The invention also claims a microbial agent containing the composting pseudomonas strain.

The technical scheme of the invention has the beneficial effects that:

(1) the pseudomonas compost strain screened by the method disclosed by the invention is low-temperature resistant, can have an excellent ammonia nitrogen removal effect of more than 95% at an application temperature as low as 8 ℃, and can reduce the cost required by water body temperature rise after being applied to sewage treatment;

(2) the strain or the microbial inoculum can still keep excellent ammonia nitrogen removal effect under the condition that the inoculation amount is as low as 50ppm, and the cost for purchasing the strain or the microbial inoculum can be reduced after the strain or the microbial inoculum is applied to sewage treatment, so the strain or the microbial inoculum has wide application prospect.

The preparation method of the microbial agent comprises the following steps:

(1) seed culture: inoculating the Pseudomonas sp AOB001 to a seed culture medium, and culturing at 25-35 deg.C and 150-;

(2) fermentation: inoculating the seed culture solution obtained in the step (1) into a fermentation culture medium in a fermentation tank according to the inoculation amount of 1-3%, controlling the temperature to be 25-35 ℃, fermenting under the condition of aeration ratio of 1:1-2:1 and 150-300rpm, and stopping fermenting when dissolved oxygen begins to rise to obtain fermentation liquor;

(3) preparing a microbial agent: diluting and filling the fermentation liquor obtained in the step (2) to obtain the microbial agent.

Further, the composition of the seed culture medium is as follows: 3-8g/L of carbon source, 0.1-1.5g/L of nitrogen source, 3-8 vol% of Vickers salt solution, the balance of water and the pH value of 6.5-8;

preferably, the composition of the seed culture medium is as follows: 5-6g/L of carbon source, 0.4-0.6g/L of nitrogen source, 4-6 vol% of Vickers' salt solution and the balance of water, wherein the pH value is 6.5-7.5;

further, the composition of the fermentation medium is as follows: carbon source 15-30g/L, nitrogen source 5-15g/L, K ⁺ 0.2-0.4g/L，Mg ²⁺ 0.05-0.1g/L，Na ⁺ 0.05-0.1g/L，Mn ²⁺ (1.5-3.5)*10 ^-3 g/L，Fe ³⁺ Or Fe ²⁺ (1-2)*10 ^-3 g/L, the balance of water, and the pH value of 6.5-8;

preferably, the composition of the fermentation medium is as follows: carbon source 20-25g/L, nitrogen source 8-12g/L, K ⁺ 0.2-0.4g/L，Mg ²⁺ 0.05-0.08g/L，Na ⁺ 0.05-0.08g/L，Mn ²⁺ (2.0-3.0)*10 ^-3 g/L，Fe ³⁺ Or Fe ²⁺ (1-1.5)*10 ^-3 g/L, the balance of water, and the pH value of 6.5-7.5;

further, the carbon source is selected from one or more of glucose, starch, sucrose, dextrin, sodium succinate and sodium citrate;

further, the nitrogen source is selected from one or more of yeast extract powder, peptone, ammonium salt, urea and soybean meal powder, and the ammonium salt is preferably ammonium sulfate;

further, K is ⁺ The source of the magnesium is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium sulfate, potassium chloride and potassium nitrate, and the Mg ²⁺ The source of (A) is one or more of magnesium sulfate, magnesium nitrate and magnesium chloride, and the Na is ⁺ The source of the Mn-Mn composite material is one or more of sodium chloride, sodium sulfate, sodium nitrate, sodium carbonate, sodium acetate and sodium succinate ²⁺ The source of (A) is one or more of manganese sulfate monohydrate, manganese nitrate and manganese chloride, and the Fe is ³⁺ Is derived from chlorineOne or more of ferric oxide, ferric nitrate and ferric sulfate, wherein the Fe is ²⁺ The source of the (B) is one or more of ferrous sulfate, ferrous chloride and ferrous sulfate.

The aeration ratio in the preparation method of the microbial agent refers to the ratio of the volume of air introduced into the fermentation tank per minute to the total volume of the fermentation liquid.

The invention also claims a method for purifying a water body using the Pseudomonas composting strain Pseudomonas sp.aob001 or a microbial agent comprising the Pseudomonas composting strain Pseudomonas sp.aob001, comprising the step of inoculating the Pseudomonas composting strain AOB001 or applying a microbial agent comprising the Pseudomonas composting strain AOB001 to a water body, preferably wherein the inoculum size of the strain or microbial agent is above 50ppm, and wherein the applicable temperature for purifying a water body is above 8 ℃, preferably 8-30 ℃, more preferably 8-15 ℃, most preferably 8-10 ℃.

The invention also claims the use of the composting Pseudomonas strain Pseudomonas sp aob001 or a microbial agent comprising the composting Pseudomonas strain Pseudomonas sp aob001 in the field of water purification, preferably the composting Pseudomonas strain Pseudomonas sp aob001 or a microbial agent comprising the composting Pseudomonas strain Pseudomonas sp aob001 is used for degrading nitrogen-containing substances, preferably ammonia nitrogen, in water, and the inoculum size of the composting Pseudomonas strain or microbial agent is above 50ppm, the applicable temperature of the use is above 8 ℃, preferably 8-30 ℃, more preferably 8-15 ℃, most preferably 8-10 ℃.

Detailed Description

The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

Example 1 screening and Performance testing of strains

1. Enrichment culture

Collecting sewage of aeration section of sewage treatment station of certain industrial enterprise in Weifang City, sucking 10mL of sewage, and transferring to a medium containing 100mL of enrichment medium (the composition of the enrichment medium is (NH) ₄ ) ₂ SO ₄ 0.5g, sodium succinate 5.62g, vickers' salt solution 50mL, deionized water 1L, pH 7) 250mL threeThe first enrichment was performed in a corner flask by culturing at 10 ℃ and 120rpm for 72 hours. Then, 10mL of the primary enrichment solution is sucked and added into a fresh enrichment medium, and the mixture is cultured for 48 hours at the temperature of 15 ℃ and the rpm of 120 so as to carry out secondary enrichment. And performing the third enrichment according to the method of the second enrichment.

2. Separation of

Adopting a gradient dilution method to carry out gradient dilution on the third enrichment solution to 10 ^-6 Respectively suck 10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 200 μ L of each of the gradient dilutions was added to a separation medium (the composition of the separation medium was: (NH) ₄ ) ₂ SO ₄ 0.5g, 5.62g of sodium succinate, 50mL of Vickers' salt solution, 1L of deionized water, pH 7 and 20.0g of agar powder), and after being uniformly coated, the mixture is poured and cultured for about 72 hours at 30 ℃ until a single colony grows. Selecting single colonies with different forms, transferring to a test tube slant separation medium, culturing at 30 ℃ for about 48h, and transferring to a refrigerator at 4 ℃ for storage.

6 strains are obtained by the separation method, and are respectively numbered as follows: AOB001, AOB002, AOB003, AOB004, AOB005 and AOB 006.

3. Preliminary evaluation

In an aseptic environment, 1 ring of each of the 6 strains obtained by primary screening is selected and inoculated into a 250mL triangular flask containing 100mL of enrichment medium, and the mixture is cultured for 48h at 30 ℃ and 120rpm for activation.

Respectively absorbing 5 mu L of each activation solution and inoculating the activation solutions into a fresh ammonia nitrogen culture medium containing 100mL of ammonia nitrogen culture medium (the ammonia nitrogen culture medium comprises (NH) ₄ ) ₂ SO ₄ 1g, 5.62g of sodium succinate, 50mL of vickers' salt solution, 1L of deionized water, pH 7) in a 250mL Erlenmeyer flask, and culturing at 15 ℃ and 120 rpm. Sterile water was used as a blank instead of the activation solution and 3 replicates were set up for each experimental group. The ammonia nitrogen content in the culture medium was periodically measured, and the results are shown in table 1.

The ammonia nitrogen detection method is executed according to a nano reagent spectrophotometry for measuring ammonia nitrogen in HJ 535 + 2009 water quality.

TABLE 1.15 ℃ Ammonia nitrogen degradation capability test results of each strain

According to the detection results in the table 1, in 6 strains of preliminarily screened strains, each strain does not show obvious ammonia nitrogen removal capability due to small inoculation amount, AOB001 shows stronger ammonia nitrogen removal capability than other strains at 48h, the ammonia nitrogen degradation rate reaches 51.1%, and the ammonia nitrogen degradation rate reaches 66.2% after 72 h.

Example 2 detection and identification of Strain AOB001

2.1 Experimental methods

2.1.1 extraction of bacterial genomic DNA

(1) Collecting 1.0X 10 with 2ml centrifuge tube ⁹ (1ml of bacterial suspension OD600 1-1.5), and centrifuged at 12,000 Xg for 30s, and the supernatant was discarded. The pellet was suspended with 150. mu.l Buffer S to which RNase A had been added.

(2) Add 20. mu.l lysozyme stock solution, mix well, and let stand at room temperature for 5 min.

(3) Add 30. mu.l of 0.25mol/L EDTA (pH 8.0), mix well and ice-wash for 5 min.

(4) Add 450. mu.l Buffer G-A, vortex for 15s, water bath at 65 ℃ for 10 min.

(5) Mu.l of Buffer G-B and 1ml of Buffer DV (precooled at 4 ℃) were added, mixed vigorously and centrifuged at 12,000 Xg for 2 min.

(6) The upper phase was discarded as much as possible, the interphase precipitate and the lower phase were retained, 1ml of 4 ℃ precooler DV was added, mixed vigorously, and centrifuged at 12,000 Xg for 2 min.

(7) The upper phase was discarded and the lower phase was transferred to a filter (filter placed in a 2ml centrifuge tube) and centrifuged at 12,000 Xg for 1 min.

(8) Discard the filter, add 400. mu.l Buffer BV to the filtrate and mix well.

(9) The preparation tube was placed in a 2ml centrifuge tube, the mixture from step 8 was transferred to the preparation tube and centrifuged at 12,000 Xg for 1 min.

(10) The filtrate was discarded, and the preparation tube was returned to the original 2ml centrifuge tube, 500. mu.l of Buffer W1 was added, and centrifugation was carried out at 12,000 Xg for 1 min.

(11) The filtrate was discarded, and the preparation tube was returned to the original 2ml centrifuge tube, and 700. mu.l of Buffer W2 was added and centrifuged at 12,000 Xg for 1 min.

(12) In the same manner, 700. mu.l of Buffer W2 was washed once more.

(13) The filtrate was discarded, and the preparation tube was returned to the original 2ml centrifuge tube and centrifuged at 12,000 Xg for 1 min.

(14) The preparation tube was placed in another clean 1.5ml centrifuge tube, and 200. mu.l of Eluent or deionized water was added to the center of the silica membrane and allowed to stand at room temperature for 1 min. The DNA was eluted by centrifugation at 12,000 Xg for 1 min.

2.1.2 PCR amplification of bacterial genomes

TABLE 2 PCR amplification primer design

Primer name	Sequence of
		27F	5-AGAGTTTGATCCTGGCTCAG-3
1492R	5-CTACGGCTACCTTGTTACGA-3

PCR amplification reaction system

The following ingredients were added to a 0.2ml centrifuge tube:

TABLE 3PCR amplification reaction System

Flicking and uniformly mixing, performing instantaneous centrifugation to collect liquid drops on the tube wall to the tube bottom, and performing PCR reaction on a PCR amplification instrument, wherein the reaction parameters are as follows:

TABLE 4 PCR amplification reaction procedure

Pre-denaturation	Denaturation of the material	Annealing	Extension	Final extension	Number of cycles
						95℃，5min	95℃，30s	58℃，30s	72℃，1min30s	72℃，7min	35

After the reaction was completed, 3. mu.l of the PCR product was subjected to 1% agarose gel electrophoresis to confirm the PCR-amplified fragment.

2.1.3 recovery of PCR products

The PCR product is recovered by using an AxyPrep DNA gel recovery kit, the specific operation is carried out according to the kit instruction, and the steps are as follows:

(1) the agarose gel containing the desired DNA was cut under an ultraviolet lamp and placed in a clean centrifuge tube and the weight was weighed.

(2) Add 3 gel volumes of Buffer DE-A, mix well and heat at 75 ℃ until the gel mass is completely melted.

(3) Adding 0.5 Buffer DE-B with the volume of the Buffer DE-A, and uniformly mixing; when the isolated DNA fragment was less than 400bp, 1 gel volume of isopropanol was added.

(4) The mixture was transferred to a DNA preparation tube and centrifuged at 12,000 Xg for 1 min. The filtrate was discarded.

(5) The preparation tube was put back into a 2ml centrifuge tube, 500. mu.l of Buffer W1 was added, and the mixture was centrifuged at 12,000 Xg for 30 seconds, and the filtrate was discarded.

(6) The preparation tube was put back into a 2ml centrifuge tube, 700. mu.l of Buffer W2 was added, and the mixture was centrifuged at 12,000 Xg for 30 seconds, and the filtrate was discarded. The cells were centrifuged again at 700. mu.l Buffer W2, 12,000 Xg, for 1min in the same manner.

(7) The prepared tube was placed back in a 2ml centrifuge tube and centrifuged at 12,000 Xg for 1 min.

(8) The preparation tube was placed in a clean 1.5ml centrifuge tube (provided in the kit), 25-30. mu.l deionized water was added to the center of the preparation membrane, and the membrane was allowed to stand at room temperature for 1 min. The DNA was eluted by centrifugation at 12,000 Xg for 1 min.

2.2 sequencing and analysis

Taking the PCR products after each strain purification, using a sequencer ABI3730-XL to carry out DNA sequencing, wherein the 16S rDNA sequence of the pseudomonas composting AOB001 is shown as SEQ ID No. 1.

2.3 sequence analysis

The spliced sequence file is compared with data in an NCBI 16S (https:// blast.ncbi.nlm.nih.gov/blast.cgi PROGRAM ═ blastn & PAGE _ TYPE ═ blastSearch & LINK _ LOC ═ blastthom) database by using an NCBI Blast program, a sequence with the maximum similarity is selected as a species identification result (see the analysis and comparison result in the table 2 in detail), the sequence is identified to have more than 99% of homology with pseudomonas composta, and the initial pseudomonas composta is judged as pseudomonas composta.

The AOB001 strain slant was subjected to 16S rDNA gene sequence sequencing, and the sequences obtained by the sequencing were compared in NCBI, and the results are shown in Table 5.

TABLE 5 comparison of NCBI on AOB001 samples of strains

ID No.	DNA identification results	Identities
			Low temperature AOB-001	Pseudomonas composti	100％

Example 3 evaluation of Ammonia Nitrogen degradation Capacity of Strain AOB001 in simulated Sewage

3.1 activation of the bacterial species

Enrichment culture medium: (NH) ₄ ) ₂ SO ₄ 0.5g, 5.62g of sodium succinate, 50mL of vickers' salt solution, 1L of deionized water at pH 7, and the composition of the medium was evaluated as that of the enrichment medium.

A1-ring AOB001 strain is selected in an aseptic environment and inoculated into a 250mL triangular flask containing 100mL enrichment medium and cultured for 24h under the conditions of 30 ℃ and 120rpm to obtain activated bacterial liquid. Diluting the bacteria content to 50 hundred million CFU/ml for later use.

3.2 evaluation experiment of Ammonia Nitrogen degradation ability of Strain AOB001

To a 250mL Erlenmeyer flask containing 100mL of the evaluation medium was added 5. mu.l of the activated bacteria solution, and the mixture was incubated at 8 ℃ and 120rpm, and the ammonia nitrogen content in the medium was measured every 24 hours. In total, 3 parallel experimental groups and 1 blank control group in which the activated bacteria solution was replaced with sterile water were set.

3.3 results of the experiment

The results of the evaluation experiments are shown in Table 6.

Table 68 ℃ simulation of Ammonia Nitrogen degradation capability of Strain AOB001 in Sewage

Evaluation time/h	Ammonia nitrogen content/ppm
		0	211.12
72	217.5
		96	31.2
120	9

As can be seen from Table 6, the ammonia nitrogen removal rate of 120 hours after inoculation of the strain at 8 ℃ can reach more than 95%, so that the strain has good ammonia nitrogen removal capability under low temperature conditions.

Example 4 evaluation of Ammonia Nitrogen degradation ability of Strain AOB001 in Sewage

4.1 Experimental materials

LT-AOB low-temperature ammonia nitrogen removal agent and biochemical water of a certain chemical industry park in Tengzhou;

the preparation method of the LT-AOB low-temperature ammonia nitrogen removal microbial inoculum comprises the following steps:

(1) seed culture: taking 1 ring pilePseudomonas putida sp. AOB001 was inoculated into 1L of a seed medium (wherein the composition of the seed medium was 0.5g (NH) ₄ ) ₂ SO ₄ 5.62g of sodium succinate, 50ml of Vickers salt solution and the balance of water, wherein the pH value is 7), and the mixture is shake-cultured for 6h at the temperature of 25-30 ℃ and the speed of 220rpm to obtain a seed culture solution;

(2) fermentation: inoculating the seed culture solution into a fermentation medium in a 50L fermentation tank according to the inoculation amount of 1-3 vol% (wherein the fermentation medium comprises 21.68g of a carbon source, 10g of a nitrogen source, 0.67g of potassium dihydrogen phosphate, 0.33g of magnesium sulfate, 0.17g of sodium chloride, 0.0067g of manganese sulfate monohydrate and 0.0067g of ferrous sulfate heptahydrate in every 1L of the fermentation medium, the carbon source can be any one of glucose, starch, sucrose, dextrin, sodium succinate and sodium citrate, the nitrogen source can be any one of yeast extract powder, peptone, ammonium sulfate, ammonium chloride, urea and soybean meal powder), the initial pH is adjusted to 7.2, the temperature is controlled to be 30 ℃, the aeration ratio is about 1:1, and the aeration ratio is increased to about 2 in the middle and later stages according to the condition of dissolved oxygen reduction: 1, in the process, microscopic examination of thallus growth condition, monitoring of wet weight change, liquid supplement (pH adjustment and carbon source supplement) by using sterilized dilute acetic acid, controlling the temperature at 35 ℃ in the middle and later stages, and stopping fermentation when dissolved oxygen begins to rise to obtain fermentation liquid;

(3) preparing a microbial agent: diluting the fermentation liquor obtained in the step (2) by 6 times to obtain the fermented liquid

LT-AOB low-temperature ammonia nitrogen removal agent.

4.2 Experimental methods

To be produced

LT-AOB low-temperature ammonia nitrogen removal microbial inoculum (microbial inoculum for short) is inoculated into biochemical water in a chemical industrial park according to the inoculation amount of 50ppm, different carbon sources or alkalis are respectively added according to the table 7, the addition amount of the carbon source is 0.4g/L, the addition amount of the alkali is 0.02g, and the ammonia nitrogen content is measured at the shaking culture interval of 120rpm at 10 ℃.

TABLE 7 evaluation results of ammonia nitrogen reduction ability in biochemical water of Tengzhou chemical industry park

As shown in Table 7, when methanol was used as a carbon source in the wastewater containing 12.42ppm of ammonia nitrogen,

the LT-AOB has higher ammonia nitrogen removal efficiency, can reduce the ammonia nitrogen content to 1.29ppm within 24 hours, has a degradation rate of 92.51 percent, further proves that the bacterium is heterotrophic nitrobacteria, can greatly improve the ammonia nitrogen removal efficiency and shorten the retention time under the condition of providing enough carbon source.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Qingdao Ulish blue Saide Biotechnology Co., Ltd

<120> pseudomonas compost strain and application thereof

<130> 1

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1439

<212> DNA

<213> Pseudomonas composti AOB001

<400> 1

gtaccgtccc cccgaaggtt agactagcta cttctggagc aacccactcc catggtgtga 60

cgggcggtgt gtacaaggcc cgggaacgta ttcaccgtga cattctgatt cacgattact 120

agcgattccg acttcacgca gtcgagttgc agactgcgat ccggactacg atcggtttta 180

tgggattagc tccacctcgc ggcttggcaa ccctttgtac cgaccattgt agcacgtgtg 240

tagccctggc cgtaagggcc atgatgactt gacgtcatcc ccaccttcct ccggtttgtc 300

accggcagtc tccttagagt gcccaccata acgtgctggt aactaaggac aagggttgcg 360

ctcgttacgg gacttaaccc aacatctcac gacacgagct gacgacagcc atgcagcacc 420

tgtgtctgag ttcccgaagg caccaatcca tctctggaaa gttctcagca tgtcaaggcc 480

aggtaaggtt cttcgcgttg cttcgaatta aaccacatgc tccaccgctt gtgcgggccc 540

ccgtcaattc atttgagttt taaccttgcg gccgtactcc ccaggcggtc aacttaatgc 600

gttagctgcg ccactaagtt ctcaaggaac ccaacggcta gttgacatcg tttacggcgt 660

ggactaccag ggtatctaat cctgtttgct ccccacgctt tcgcacctca gtgtcagtat 720

cagtccaggt ggtcgccttc gccactggtg ttccttccta tatctacgca tttcaccgct 780

acacaggaaa ttccaccacc ctctaccgta ctctagctcg ccagttttgg atgcagttcc 840

caggttgagc ccggggcttt cacatccaac ttaacgaacc acctacgcgc gctttacgcc 900

cagtaattcc gattaacgct tgcacccttc gtattaccgc ggctgctggc acgaagttag 960

ccggtgctta ttctgtcggt aacgtcaaaa ttgcagagta ttaatctaca acccttcctc 1020

ccaacttaaa gtgctttaca atccgaagac cttcttcaca cacgcggcat ggctggatca 1080

ggctttcgcc cattgtccaa tattccccac tgctgcctcc cgtaggagtc tggaccgtgt 1140

ctcagttcca gtgtgactga tcatcctctc agaccagtta cggatcgtcg ccttggtgag 1200

ccattacctc accaactagc taatccgacc taggctcatc taatggcgcg aggtccgaag 1260

atcccccgct ttctcccgta ggacgtatgc ggtattagcg tccgtttccg aacgttatcc 1320

cccaccacta ggcagattcc taggcattac tcacccgtcc gccgctctca agagaagcaa 1380

gctcctctct accgctcgac ttgcatgtgt taggcctgcc gccagcgttc aatctgagc 1439

Claims (17)

1. A Pseudomonas sp compost strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 20850.

2. A microbial agent comprising the pseudomonas composting strain of claim 1.

3. The method for preparing a microbial agent according to claim 2, comprising the steps of:

(1) seed culture: inoculating the pseudomonas composting strain into a seed culture medium under an aseptic condition, and culturing for 5-24h at the temperature of 25-35 ℃ and the speed of 150-;

(2) and (3) fermentation: after the fermentation medium in the fermentation tank is disinfected, inoculating the seed culture solution obtained in the step (1) into the fermentation medium according to the inoculation amount of 1-3 vol%, controlling the temperature to be 25-35 ℃, fermenting under the condition of aeration ratio of 1:1-2:1 and 150-plus-one rotation speed of 300rpm, and stopping fermenting when dissolved oxygen begins to rise to obtain fermentation liquid;

(3) preparing a microbial agent: diluting and filling the fermentation liquor obtained in the step (2) to obtain the microbial agent.

4. The method of claim 3, wherein the seed medium has the following composition: 3-8g/L of carbon source, 0.1-1.5g/L of nitrogen source, 3-8 vol% of Vickers' salt solution and the balance of water, wherein the pH value is 6.5-8;

the fermentation medium had the following composition: carbon source 15-30g/L, nitrogen source 5-15g/L, K ⁺ 0.2-0.4g/L，Mg ²⁺ 0.05-0.1g/L，Na ⁺ 0.05-0.1g/L，Mn ²⁺ 1.5×10 ^-3 -3.5×10 ^-3 g/L，Fe ³⁺ Or Fe ²⁺ 1×10 ^-3 -2×10 ^-3 g/L, the balance of water, and the pH value of 6.5-8.

5. The preparation method according to claim 4, wherein the carbon source is selected from one or more of glucose, starch, sucrose, dextrin, sodium succinate, and sodium citrate;

the nitrogen source is selected from one or more of yeast extract powder, peptone, ammonium salt, urea and soybean meal powder.

6. A method of purifying a water body comprising the step of inoculating the strain of claim 1 or applying the microbial agent of claim 2 to the water body.

7. The method of claim 6, wherein the applicable temperature of the water purification process is 8 ℃ or higher.

8. The method of claim 7, wherein the applicable temperature of the water purification process is 8-30 ℃.

9. The method of claim 8, wherein the applicable temperature of the water purification process is 8-15 ℃.

10. The method of claim 9, wherein the applicable temperature for the water purification process is 8-10 ℃.

11. Use of the pseudomonas composting strain of claim 1 or the microbial agent of claim 2 in the field of water purification.

12. The use of claim 11, wherein the pseudomonas composting strain of claim 1 or the microbial inoculant of claim 2 is used to degrade nitrogen-containing materials in water.

13. Use according to claim 12, characterized in that the composting pseudomonas strain according to claim 1 or the microbial inoculum according to claim 2 is used for degrading ammoniacal nitrogen in water.

14. Use according to any of claims 11-13, wherein the temperature for use is 8 ℃ or higher.

15. Use according to claim 14, wherein the temperature suitable for use is 8-30 ℃.

16. Use according to claim 15, wherein the temperature suitable for use is 8-15 ℃.

17. Use according to claim 16, characterised in that the temperature for which the use is applicable is most preferably 8-10 ℃.