CN117384781B

CN117384781B - Pseudomonas curvatus LH-HF0019 and its low-temperature-resistant detoxifying agent and use

Info

Publication number: CN117384781B
Application number: CN202311061023.9A
Authority: CN
Inventors: 李慧芬; 王开春; 田凤蓉; 范贤培; 王强; 徐军; 孙文妮; 曲昂; 吴丹
Original assignee: Shanghai Zhonghao Potassium Salt Engineering Technology Center; Bluestar Lehigh Engineering Institute
Current assignee: Shanghai Zhonghao Potassium Salt Engineering Technology Center; Bluestar Lehigh Engineering Institute
Priority date: 2023-08-22
Filing date: 2023-08-22
Publication date: 2025-10-14
Anticipated expiration: 2043-08-22
Also published as: CN117384781A

Abstract

The present invention discloses a Pseudomonas curvatus LH‑HF0019 for degrading acrylonitrile, a low-temperature resistant detoxifying agent and its use. The deposit number of the Pseudomonas curvatus LH‑HF0019 is CGMCC No. 27554. The Pseudomonas curvatus LH‑HF0019 of the present invention has a strong acrylonitrile degradation ability, can produce a variety of enzymes such as amylase, protease, cellulase and lipase, and can degrade and eliminate the toxicity of acrylonitrile using acrylonitrile as a carbon source. At the same time, it can remove the COD _Cr of the system through the organic substrate of the enzymatic system. When the acrylonitrile concentration in the system reaches 600ppm, the bacteria can still achieve an acrylonitrile degradation rate of ≥95.82% at a low temperature of 10°C, and strengthen the activated sludge to achieve a COD _Cr removal rate of ≥83.13%. This strain has strong low-temperature tolerance and acrylonitrile detoxification ability, can improve the ability of the biochemical system to tolerate acrylonitrile toxicity, improve the biodegradability of the system, and has broad application prospects for the treatment of COD _Cr standards for acrylonitrile-rich POP and other wastewaters.

Description

Pseudomonas curvatus LH-HF0019, low temperature resistant detoxication microbial inoculum and application thereof

Technical Field

The invention belongs to the technical field of environment-friendly microorganisms, and particularly relates to pseudomonas curvata LH-HF0019 for degrading acrylonitrile and application thereof in low-temperature detoxification strengthening treatment of wastewater rich in acrylonitrile.

Background

Acrylonitrile (Acrylonitrile) is widely used as a very important chemical raw material for the production of synthetic materials such as rubber, fiber, and plastic. As is well known, acrylonitrile wastewater is considered as refractory high-concentration organic wastewater, has high toxicity, belongs to one of 58 types of preferential control and 114 types of preferential control toxic chemicals specified by United states EPA determined in China, belongs to refractory organic pollutants, and can cause serious pollution to the environment and harm to human health if the refractory organic pollutants are not treated effectively. At present, the methods for treating the acrylonitrile wastewater comprise an adsorption method, an extraction method, a combustion method, a catalytic oxidation method, a biochemical method and the like, and compared with other methods, the biochemical method is greatly related due to low treatment cost. The biological treatment method adopts an activated sludge method and a biological membrane method, but because the components of the acrylonitrile wastewater are complex and most pollutants have biotoxicity, large impact is often generated on microorganisms, and the treatment effect is poor.

The acrylonitrile has a chemical formula of C3H3N, a molecular weight of 53.06g/mol, a density of 0.806g/mLat ℃ and a boiling point of 77 ℃. The acrylonitrile has active chemical property, contains cyano, carbon-carbon double bond and the like in the molecule, and can participate in various reactions. Colorless volatile transparent liquid, sweet and slightly odorous. Can be dissolved in organic solvents such as acetone, benzene, carbon tetrachloride, diethyl ether, ethanol and the like. Slightly soluble in water. Acrylonitrile can be carcinogenic, highly flammable, inhaled, skin contact and toxic to swallow. It also stimulates the respiratory system and skin, causing serious injury to the eyes. Acrylonitrile is toxic to aquatic organisms and can have long-term adverse effects on the water environment. After the impact of the acrylonitrile wastewater, the normal metabolism of microorganisms in a conventional biological treatment system is blocked, such as the blocking of the activity of metabolic enzymes, the poisoning of sludge, the disintegration, the floating and the like, and then the degradation of indexes such as TOC, COD _Cr and the like is affected, so that the common biochemical treatment is difficult to stably operate the acrylonitrile wastewater and is always the bottleneck of the sewage treatment. Some enterprises dilute the clean water to reduce the toxicity of the acrylonitrile and then carry out biochemical treatment, so that the waste of water resources is caused, and meanwhile, the total amount of treatment facilities and sewage is increased. Therefore, how to effectively and economically realize the detoxification of high acrylonitrile wastewater and the degradation of the system COD _Cr becomes a scientific and engineering difficult problem to be solved urgently, and the key to realize the effective and economical detoxification of the acrylonitrile wastewater and the degradation of the COD _Cr is to find microorganisms which can resist acrylonitrile.

The existence of the acrylonitrile tolerant microorganism provides a new thought for biological denitrification treatment of high-salinity wastewater, which is an extreme microorganism in a living high-acrylonitrile exposure environment, widely exists in high-acrylonitrile toxicity environments such as soil, activated sludge and the like, and the acrylonitrile degradation functional bacteria form unique viability under acrylonitrile stress in a long-term evolution process, have extremely special physiological structures and metabolic mechanisms, have specificity in the aspects of enzyme series properties, metabolic pathways, protein nucleic acid components, conformation and the like, adapt to the high-acrylonitrile toxicity environments so as to ensure that the acrylonitrile degradation functional bacteria can continue to grow and reproduce in the acrylonitrile toxicity environments, and degrade acrylonitrile toxic substances in the system.

In recent years, different species of microorganism strains, such as Arthrobacter, thiobacillus, rhodococcus and azotobacter, are screened out, and certain results are achieved in the aspect of acrylonitrile wastewater treatment. For example, a strain for producing nitrile hydratase and a method for producing p-hydroxyphenylacetamide, disclosed in China patent publication No. CN107586750A, belongs to the field of biotechnology, and the strain is classified and named as bacillus aryabhattai Jn-102, can produce nitrile hydratase, has good genetic stability, low culture cost, high activity of the produced nitrile hydratase and wide applicable substrate spectrum, and can be directly used for converting p-hydroxyphenylacetonitrile to synthesize the p-hydroxyphenylacetamide. The literature 'optimizing the acrylonitrile degradation condition of a high-efficiency acrylonitrile degrading bacterium Rhodococus rhodochrous BX' uses acrylonitrile as a target pollutant, and utilizes a high-efficiency acrylonitrile degrading bacterium Rhodococus rhodochrous BX obtained by screening in a laboratory to research the degradation characteristic of the high-efficiency acrylonitrile degrading bacterium on acrylonitrile, and optimizes the degradation condition to improve the degradation capability of the strain on the acrylonitrile. The degradation rate of acrylonitrile under the conditions that the substrate concentration is 403.51mg/L, p H7.44.44 and the temperature is 34.46 ℃ is 95.1 percent. The added carbon source is glucose or the added nitrogen source is ammonium chloride, which has obvious promotion effect on the growth of the strain and the degradation of acrylonitrile. The strain Rhodococus rhodochrous BX2 can efficiently degrade the acrylonitrile in the synthetic wastewater, and the degradation rate of the acrylonitrile can reach 89.4% at 30 h.

However, when these strains degrade acrylonitrile, the reaction temperature exceeds 30℃and is a conventional treatment temperature. When the actual wastewater is applied, the temperature range of the polluted wastewater is more lower than 15 ℃, and the conventional microbial inoculum is difficult to play a role, so that the low-temperature resistant strain with strong acrylonitrile degradation capability is urgently needed to be screened to expand the application range.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the low-temperature-resistant functional bacterium Pseudomonas curvata LH-HF0019, which can effectively remove the toxicity of acrylonitrile in acrylonitrile wastewater under the low temperature condition and biologically strengthen and promote the degradation capacity of activated sludge COD _Cr.

The invention aims to provide the low-temperature-resistant detoxification microbial inoculum prepared from the low-temperature-resistant degradation pseudomonas acrylonitrile-bending LH-HF 0019.

The invention aims to provide the pseudomonas acrylonitrile-bending (LH-HF 0019) with low temperature resistance and the application of the low temperature resistance detoxification microbial inoculum prepared by the pseudomonas acrylonitrile-bending (LH-HF 0019).

In order to achieve the aim, the invention is realized by the following technical scheme:

The invention provides Pseudomonas curvata (Pseudomonas geniculata) LH-HF0019, which is classified and named as Pseudomonas curvata Pseudomonas geniculata, and the preservation number is CGMCC No.27554. The strain LH-HF0019 is preserved in China general microbiological culture Collection center (CGMCC), the address of North Star Xila No. 1, 3, china academy of sciences microbiological study, and the preservation date of 2023, 6 months and 5 days.

The bacterial strain of the pseudomonas curvata LH-HF0019 is cultured on an Lb culture medium for 24 hours, and the bacterial strain is white, opaque, round, neat in edge, moist and glossy on the surface, has the diameter of 0.2mm, shows that the bacterial strain is rod-shaped under the condition of 20000 times of a G-scanning electron microscope, and has the width of 0.3-0.5 mu m and the length of 1.6-2.3 mu m.

The nucleotide sequence of the pseudomonas curvata LH-HF0019 is shown as SEQ ID No. 1.

The growth temperature range of the pseudomonas curvata LH-HF0019 is 10-50 ℃. Preferably, the Pseudomonas campylobacter LH-HF0019 has an optimum growth temperature of 20 ℃.

The growth NaCl range of the pseudomonas curvata LH-HF0019 is 0-8%. Further, the growth NaCl optimal concentration of the pseudomonas curvata LH-HF0019 is 5%.

The invention also discloses a method for culturing the pseudomonas curvata LH-HF0019, which comprises the steps of culturing the pseudomonas curvata LH-HF0019 in an MSM inorganic salt culture medium with acrylonitrile as a sole carbon source at 10-50 ℃ and 180rpm for 96 hours;

The Pseudomonas curvata LH-HF0019 has the capability of realizing self proliferation by taking acrylonitrile as a sole carbon source and degrading and utilizing the acrylonitrile, is cultured for 96 hours in an MSM inorganic salt culture medium taking the acrylonitrile as the sole carbon source, preferably at 30 ℃ and 180rpm, and has the degradation rate of not less than 96.61 percent for the acrylonitrile with the concentration below 600 ppm. The MSM inorganic salt culture medium consists of ：KH₂PO₄ 1.7g,Na₂HPO₄9.8g,(NH₄)₂SO₄ 5.0g,MgSO₄ 0.1g,NaCl 1％L, trace element mother liquor 1.0ml, trace element mother liquor ：MgO 10.75g,FeSO₄·7H₂O 0.9g,ZnSO4·7H2O 1.44g,CuSO₄·5H₂O 0.25g,H₃BO₃ 0.06g,, distilled water to 1000ml and pH 7.2.

The pseudomonas curvata LH-HF0019 has the capability of producing amylase, protease, cellulase and lipase.

The invention also discloses an enzyme production method of pseudomonas curvata LH-HF0019, which comprises the following steps:

The enzyme-producing culture mediums are respectively as follows:

(1) The amylase production medium comprises 10g of peptone, 5g of beef extract, 5g of NaCl, 2g of soluble starch, 7.1-7.3 of pH, 1L of distilled water and 20g of agar;

(2) The protease production culture medium comprises 10g of casein, 1g of yeast extract, pH 6.9-7.1, 1L of distilled water and 20g of agar;

(3) The cellulase production medium comprises 20g of sodium carboxymethyl cellulose, 0.5g of yeast powder, 2.5g of disodium hydrogen phosphate, 1.5g of potassium dihydrogen phosphate, 2.5g of peptone, 7.1-7.3 of pH, 1L of distilled water and 20g of agar;

(4) The lipase production culture medium comprises 10g of peptone, 5g of yeast powder, 10g of NaCl, 2mL of tributyrin, 7.4-7.6 pH, 1L of distilled water and 20g of agar;

The enzyme production condition is that pseudomonas curvata LH-HF0019 is respectively inoculated on 4 solid culture mediums for producing amylase, protease, cellulase and lipase, and the culture is carried out for 48 hours at the constant temperature of 10-50 ℃, preferably 20 ℃.

The invention also discloses a pseudomonas curvata low temperature resistant detoxification microbial inoculum which is prepared by the following steps that after the pseudomonas curvata LH-HF0019 is activated by an LB culture medium, the pseudomonas curvata LH-HF0019 is inoculated into a functional bacteria fermentation culture medium with the concentration of 0.3-0.8g/L inducer cobalt chloride respectively in a volume ratio of 5%, and the pseudomonas curvata LH-HF0019 is cultured under the conditions of 10-50 ℃ and 180rpm until the bacterial content of fermentation broth is not less than 450 multiplied by 10 ⁸ CFU/ml;

The formula of the functional bacteria fermentation medium comprises 20g of glucose, 5g of peptone, 3g of yeast extract, 3g of maltose, 0.6g of cobalt chloride, 0.2g of K ₂HPO₄ 1g、KH₂PO₄ 1g、MgSO₄·7H₂ O and regulating the pH value to 7.0.

The low temperature resistant Jie Dujun agent of the pseudomonas curvata is preferably inoculated into a functional bacteria fermentation culture medium with the concentration of 0.6g/L inducer cobalt chloride respectively in a volume ratio, and is cultured at 20 ℃ and 180rpm until the bacteria content of fermentation liquor is not lower than 450 multiplied by 10 ⁸ CFU/ml.

The invention also discloses application of the pseudomonas curvata LH-HF0019 in preparing a low temperature resistant Jie Dujun agent of pseudomonas curvata for degrading acrylonitrile by using the pseudomonas curvata LH-HF 0019.

The invention also discloses application of the pseudomonas curvata low-temperature-resistant detoxication microbial inoculum in treating acrylonitrile-containing wastewater. In the application, the fermentation broth with the bacterial content not lower than 450 multiplied by 10 ⁸ CFU/mL is prepared into diluted bacterial liquid with the bacterial content of 1.125 multiplied by 10 ⁸～1.5×10⁸ CFU/mL, and the diluted bacterial liquid is added in the proportion of 5% -6% for application.

In the application of the invention, when the wastewater containing acrylonitrile is treated, the acrylonitrile is degraded and detoxified by taking the acrylonitrile as the sole carbon source, and the COD of the system is removed by the organic substrate of the enzymolysis system.

In the application of the invention, when the acrylonitrile wastewater is treated, the treatment temperature is 10-50 ℃.

Compared with the prior art, the invention has the following advantages and technical effects:

1. The pseudomonas curvata LH-HF0019 has the capability of realizing self proliferation by taking acrylonitrile as a sole carbon source and degrading and utilizing the acrylonitrile, is cultured for 96 hours in an MSM inorganic salt culture medium taking the acrylonitrile as the sole carbon source under the conditions of 20 ℃ and 180rpm, and has the degradation rate of more than or equal to 96.61 percent for the acrylonitrile with the concentration of less than 600ppm, and can be well adapted to and relieve the toxicity of acrylonitrile wastewater.

2. The growth temperature range of the pseudomonas curvata LH-HF0019 is 10-50 ℃, the optimal temperature is 20 ℃, the concentration range of the tolerant NaCl is 0-8%, and the optimal NaCl concentration is 5%. When the environmental temperature for treating the acrylonitrile wastewater is 10 ℃, the acrylonitrile degradation rate of more than or equal to 95.82 percent can be still realized at the low temperature of 10 ℃ when the acrylonitrile concentration of the system reaches 600ppm, and the COD _Cr removal rate of more than or equal to 83.13 percent can be realized by strengthening the activated sludge.

3. The Pseudomonas curvata LH-HF0019 can produce various enzymes such as amylase, protease, cellulase, lipase and the like, and can degrade and remove the toxicity of acrylonitrile by taking the acrylonitrile as a carbon source and remove the COD _Cr of an organic substrate removal system of an enzymolysis system.

4. The Pseudomonas curvata LH-HF0019 is separated from the activated sludge of the acrylonitrile wastewater, and is applied to the detoxification and the biologically enhanced COD _Cr reduction treatment of the acrylonitrile wastewater, so that the Pseudomonas curvata LH-HF0019 has a stronger in-situ adaptation effect.

5. The preparation method of the pseudomonas crooked fermentation broth is simple and reasonable, is rapid to operate, can obviously improve the COD _Cr removing effect of a biochemical system on the premise of not changing a structure, has stronger acrylonitrile detoxification capacity and low-temperature COD _Cr removing effect, and has good industrial implementation prospect.

Drawings

FIG. 1 is a graph showing comparison of acrylonitrile-degrading ability of a selected strain;

FIG. 2 is a photograph of a colony of Pseudomonas curvatus LH-HF 0019;

FIG. 3 is a scanning electron micrograph of Pseudomonas curvata LH-HF 0019;

FIG. 4 is a Neighbor-joining phylogenetic tree of Pseudomonas curvatus LH-HF 0019;

FIG. 5 shows the strain growth-acrylonitrile degradation curve of Pseudomonas campaigns LH-HF 0019;

FIG. 6 is a graph comparing FPLC of 0h versus 120h of Pseudomonas curvatus LH-HF0019 treatment of acrylonitrile;

FIG. 7 is a graph showing the effect of initial acrylonitrile concentration and temperature on LH-HF0019 acrylonitrile degradation rate;

FIG. 8 is a graph showing the effect of inducer concentration on the ability of Pseudomonas curvatus LH-HF0019 to degrade acrylonitrile in acrylonitrile wastewater.

Detailed Description

The technical scheme of the invention is further described in detail by combining the following specific examples.

In the following examples, unless otherwise specified, the test methods used are conventional and materials, reagents, etc. used are available from biological or chemical reagent companies.

The media or test reagent formulations required in the examples are as follows:

1. The first-generation enrichment culture medium comprises 2.465g of glucose, 2.50g of molasses, 2.0g of beef extract powder and 0.5g of KH ₂PO₄ 0.50g,K₂HPO₄0.50g,MgSO₄·7H₂ O, 4.75ml of Shandong Zibo POP wastewater is inoculated to adjust the concentration of acrylonitrile in a system to 8ppm, the pH is adjusted to 7, and the volume is fixed to 1000ml.

2. The second-generation enrichment culture medium comprises 2.465g of glucose, 2.50g of molasses, 2.0g of beef extract powder, 0.5g of KH ₂PO₄ 0.50g,K₂HPO₄0.50g,MgSO₄·7H₂ O, 11.88ml of Shandong Zibo POP wastewater, regulating the concentration of acrylonitrile in the system to 20ppm, regulating the pH to 7, and fixing the volume to 1000ml.

3. 1.0Ml of modified MSM inorganic salt domestication culture medium ：KH₂PO₄ 1.7g,Na₂HPO₄9.8g,(NH₄)₂SO₄ 5.0g,MgSO₄ 0.1g,NaCl 1％L, microelement mother liquor containing gradient acrylonitrile, adding distilled water into the microelement mother liquor ：MgO 10.75g,FeSO₄·7H₂O 0.9g,ZnSO4·7H2O 1.44g,CuSO₄·5H₂O 0.25g,H₃BO₃ 0.06g, to fix volume to 1000ml;pH 7.2,116 ℃ for sterilization for 30min, cooling, adding acrylonitrile as unique carbon source, and the gradient acrylonitrile concentration is 30-50ppm respectively.

4. The improved nutrient agar separating and screening culture medium comprises peptone 10.0g, beef extract powder 5.0g, naCl 5.0g, agar 18.0g, distilled water and water to 1000ml, pH 7.3+ -0.1. Sterilizing at 116 deg.C for 30min, and adding acrylonitrile to a concentration of 50ppm before cooling.

5. The culture medium for optimizing the fermentation of the functional bacteria comprises 20g of glucose, 5g of peptone, 3g of yeast extract, 3g of maltose, 0.6g of cobalt chloride, 0.2g of K ₂HPO₄ 1g、KH₂PO₄ 1g、MgSO₄·7H₂ O and regulating the pH to 7.0.

6. The amylase production analysis culture medium comprises 10g of peptone, 5g of beef extract, 5g of NaCl, 2g of soluble starch, 7.1-7.3 of pH, 1L of distilled water and 20g of agar.

7. The protease production analysis culture medium comprises 10g of casein, 1g of yeast extract, pH 6.9-7.1, 1L of distilled water and 20g of agar.

8. The cellulase production analysis culture medium comprises 20g of sodium carboxymethyl cellulose, 0.5g of yeast powder, 2.5g of disodium hydrogen phosphate, 1.5g of potassium dihydrogen phosphate, 2.5g of peptone, 7.1-7.3 of pH, 1L of distilled water and 20g of agar.

9. The lipase production analysis culture medium comprises 10g of peptone, 5g of yeast powder, 10g of NaCl, 2mL of tributyrin, 7.4-7.6 of pH, 1L of distilled water and 20g of agar.

10. Iodine solution, i.e. 5g of iodine, 10g of KI and 1000mL of distilled water;

11. The acidic mercury reagent comprises HgCl ₂ g, concentrated hydrochloric acid 200mL and distilled water 1000mL;

12. Congo red dye liquor, namely, congo red 1g and distilled water 1000ml;

13.1mol/L NaCl solution, 58.5g NaCl, 1000ml distilled water;

the above culture medium is sterilized at 116 deg.C for 30min before use.

EXAMPLE 1 isolation screening and identification of Pseudomonas curvatus LH-HF0019

1. Isolation and screening of Pseudomonas curvatus LH-HF0019

The POP wastewater of Shandong Zibo is rich in 1685ppm acrylonitrile, 1732ppm TOC and 7000ppm COD,pH 7.96, and the corresponding activated sludge concentration is 5758ppm. The method comprises the steps of taking a sludge sample, inoculating the sludge sample into 200ml of enrichment culture medium according to the inoculation proportion of 10%, storing the sludge sample in a sealed blue mouth bottle, wherein the concentration of acrylonitrile in a first-generation enrichment culture medium is 100ppm, enrichment culture conditions are 37 ℃ and 150rpm, enrichment culture is carried out for 3d, the first-generation enrichment culture medium is filtered by 4 layers of sterile gauze, centrifuging for 10min at 4000rpm, collecting bacterial sludge, washing with sterile water, centrifuging again, re-suspending the bacterial sludge, inoculating the bacterial sludge into a fresh second-generation enrichment culture medium, and carrying out enrichment culture for 3d at 37 ℃ and 150rpm, thereby obtaining the second-generation enrichment culture solution of acrylonitrile.

Centrifuging the acrylonitrile 2 generation enrichment culture solution at 4000rpm for 10min, collecting bacterial sludge, washing with sterile water, centrifuging again, re-suspending bacterial sludge 10ml MSM, inoculating into 100ml MSM inorganic salt domestication culture medium containing 300ppm acrylonitrile at 37deg.C and 150rpm, domesticating for 2-3d, sequentially increasing concentration domestication according to turbidity of bacterial solution, and sequentially selecting 300ppm, 400 ppm and 500ppm acrylonitrile gradient in domestication culture medium.

The domestication screening culture solution is diluted in a gradient way, uniformly coated on an improved nutrient agar separation screening culture medium (the concentration of the acrylonitrile is 300 ppm), and placed in a biochemical incubator for culture at 37 ℃ for 2 days to obtain single colony of the acrylonitrile degradation functional bacteria.

Single colony is selected from a flat plate and inoculated into 3mL LB test tube for culture until the exponential phase, then 1mL is taken and inoculated into 100mL of MSM culture medium with 1% LB, acrylonitrile with the final concentration of 300ppm is added, the culture is carried out in a serum bottle at 30 ℃ and 180rpm until the exponential phase, centrifugation is carried out for 15min at 10000rpm, the supernatant is removed, bacterial cells are collected, the aseptic MSM culture medium is added for bacterial washing twice, and finally 2mL of MSM culture medium is added for re-suspension, thus obtaining seed liquid.

The seed solution was inoculated into 100mL of MSM medium, the initial OD600 = 0.1 of the cells was adjusted, and acrylonitrile was added to a final concentration of 425ppm, and the cells were cultured at 30℃and 180rpm for 3d, whereby the effect of degrading acrylonitrile was examined.

20 Strains of acrylonitrile degradation functional bacteria are obtained on NA acrylonitrile selection plates (containing 300ppm of acrylonitrile), wherein LH-HF0019 has stronger acrylonitrile degradation effect, and the degradation rate reaches 80.13 percent, which is shown in a figure 1.

2. Morphology and scanning electron microscope of pseudomonas curvata LH-HF0019

The bacterial colony of the strain LH-HF0019 cultured for 24 hours on the Lb culture medium is white, opaque, round, neat in edge, moist and glossy on the surface, and has the diameter of 0.2mm, and the bacterial colony is G ^- as shown in figure 2.

The strain LH-HF0019 is cultured in a sterile LB liquid medium at 30 ℃ and 150rpm for 24 hours, bacterial liquid is centrifuged at 5000rpm for 10 minutes to enrich the precipitate, the precipitate is immersed in PBS (0.1M without NaCI), cells or tissues are rinsed for several times, supernatant is removed by centrifugation, 2.5% glutaraldehyde precooled at 4 ℃ is added, the mixture is fixed at 4 ℃ overnight, the fixative is sucked out, and PBS (0.1M without NaCI) is used for immersing for 3-5 times, and 15 minutes each time. Dehydrating with serial gradient alcohol (30%, 50%, 70%, 80%, 90%, 95%, 100%), dehydrating with alcohol of each concentration for 1 time, thoroughly dehydrating with alcohol of 100% for 15min each time for 1 time, and replacing with isoamyl acetate for 2 times each time for 20min. And (5) naturally drying. Vacuum spraying method is used. Spraying is uniform, and the zeiss GEMINI SEM model 300 scanning electron microscope is adopted for observation under the scanning electron microscope after finishing. The scanning electron microscope 20000 times results show that the bacterial strain LH-HF0019 has a rod shape, 0.3-0.5 μm and a length of 1.6-2.3 μm, and is shown in FIG. 3.

3. 16S rRNA sequencing of Pseudomonas curvatus LH-HF0019

The DNA of the strain LH-HF0019 is used as a template, and the sequence is determined after the amplification by using a 16S rDNA universal primer, wherein the sequence is shown as SEQ ID No. 1. The 16S universal gene primer is 27F, AGAGTTTGATCCCTGGCTCAG, 1492 2R, TACGGCTACCTTGTTACGACGACTT. Inputting the 16S rDNA sequencing result of the strain LH-HF0019 into NCBI database for BLAST comparison to obtain related mode strain, performing preliminary treatment on the target sequence and the mode strain sequence by ClustalX1.81, and constructing a phylogenetic tree by adopting MEGA4.0 according to a Neighbor-joining method. As a result, as shown in FIG. 4, the homology of strain LH-HF0019 with Pseudomonas geniculata was the highest, and thus strain LH-HF0019 was determined as Pseudomonas curvata.

4. Bacterial preservation of Pseudomonas curvatus LH-HF0019

The screened strain LH-HF0019 is subjected to strain preservation, wherein the preservation unit of the pseudomonas curvata LH-HF0019 is China general microbiological culture Collection center (CGMCC), the address is North Star XU No. 1, 3 of the Kao-yang area of Beijing, the institute of microbiological study, the preservation date is 2023, 6 months and 5 days, and the preservation number of the pseudomonas curvata Pseudomonas geniculata is CGMCC No.27554.

Example 2 Strain growth-Acrylonitrile degradation Curve and physiological Biochemical Properties of Pseudomonas Curve LH-HF0019

1. Bacterial strain growth-acrylonitrile degradation curve of Pseudomonas curvatus LH-HF0019

The Pseudomonas curvata LH-HF0019 single colony is picked from a flat plate, inoculated into 100mL 10% LB culture, cultured to an exponential phase at 180rpm based on 30 ℃, centrifuged at 6000rpm for 5min, collected, washed twice by adding 15mL of sterile MSM culture medium, and finally resuspended by adding 5mL of MSM culture medium to obtain seed liquid. The seed solution was inoculated into 35mL of MSM medium, the initial OD600 = 0.1 of the cells was adjusted, and acrylonitrile was added at a final concentration of 425ppm, and the cells were cultured at 20℃and 180rpm for 120 hours. Sampling at regular time and every 12h, detecting acrylonitrile concentration (HJ 806-2016 water quality acrylonitrile and acrolein measurement purge and trap meteorological chromatography) and microorganism growth condition (detection OD 600), and drawing strain growth-acrylonitrile degradation curve after acrylonitrile degradation is completed, as shown in figure 5

The pseudomonas curvata LH-HF0019 can tolerate 425ppm of acrylonitrile, degrade 80.13% of acrylonitrile in 72 hours, degrade 92.4% of acrylonitrile in 96 hours and degrade 99.96% of acrylonitrile in 120 hours, the pseudomonas curvata LH-HF0019 grows slowly in the first 12 hours, the bacterial strain grows into a logarithmic proliferation period in 12 hours to 60 hours, the highest OD value reaches 0.82, and the bacterial strain can well utilize the acrylonitrile as a carbon source and an energy source to carry out self bacterial proliferation and realize degradation of a toxic substrate acrylonitrile. FIG. 6 and Table 1 show a comparison of acrylonitrile-FPLC at different times from 0h to 120h, and comparison data, showing that Pseudomonas curvatus LH-HF0019 treatment at 120h degraded 99.96% acrylonitrile.

TABLE 1 FPLC contrast data for 0h vs 120h of Pseudomonas curvatus LH-HF0019 treatment of acrylonitrile

2. Physiological and biochemical characteristics of Pseudomonas curvatus LH-HF0019

After Pseudomonas curvata LH-HF0019 is cultured in an improved nutrient agar medium, the physiological and biochemical characteristics of the Pseudomonas curvata LH-HF0019 are measured according to physiological and biochemical detection methods in the "Burger's bacteria identification Manual" and the "common bacteria System identification Manual", and the results are shown in Table 2:

TABLE 2 physiological and biochemical Properties of Pseudomonas curvatus LH-HF0019

Note that "+" indicates positive for biochemical reaction or gram staining and "one" indicates negative for biochemical reaction or gram staining.

Pseudomonas curvatus LH-HF0019 can grow and reproduce within the temperature range of 10-50 ℃, but is most suitable for growing at 20 ℃, which shows that the strain can resist low temperature and has wider low temperature tolerance. Pseudomonas curvatus LH-HF0019 can normally grow in the NaCl range of 0% -8%, and the optimal growth salinity NaCl is 5%, which indicates that the strain has a certain salinity tolerance. Pseudomonas curvatus LH-HF0019 can produce contact enzyme, ornithine decarboxylase and lysine decarboxylase, can liquefy gelatin, and can produce acid by utilizing various carbohydrates such as glucose, sucrose, inositol, lactic acid, sodium acetate, sodium succinate, sodium malonate, sodium succinate, beta-galactoside and the like.

Example 3 comparison of the ability of Pseudomonas curvatus LH-HF0019 to degrade acrylonitrile under different conditions

1. Effect of initial acrylonitrile concentration on LH-HF0019 acrylonitrile degradation Rate

Acrylonitrile standard was purchased from Shanghai Meilin Biochemical technology Co., ltd, and had a molecular weight of 53.06g/moL and a chemical formula of C ₃H₃ N. Preparing an MSM inorganic salt culture medium with initial gradient acrylonitrile concentration, inoculating a single colony of pseudomonas curvata LH-HF0019 to 100mL 10% LB culture, culturing to an exponential phase at 180rpm based on 20 ℃, centrifuging for 5min at 6000rpm, collecting thalli, adding 15mL of sterile MSM culture medium to wash the bacteria twice, and finally adding 5mL of MSM culture medium to resuspension to obtain seed liquid. Inoculating the seed solution into 35mL MSM culture medium, regulating the initial OD600 = 0.1 of the bacterial cells, adding acrylonitrile with the final concentration range of 100-1000ppm, culturing for 96 hours at 20 ℃ and 180rpm, measuring the final concentration of the acrylonitrile by adopting a measurement purge and trap meteorological chromatography of HJ 806-2016 water quality acrylonitrile and acrolein, examining the influence of the initial acrylonitrile concentration on the degradation rate of LH-HF0019 acrylonitrile, and as shown in a result of a graph (A) of fig. 7, gradually reducing the degradation rate of the pseudomonas curvata LH-HF0019 on the acrylonitrile along with the increase of the initial concentration of the acrylonitrile, wherein the degradation rate is more than or equal to 96.61% for the acrylonitrile with the concentration below 600ppm, which indicates that the bacterial has stronger acrylonitrile tolerance.

2. Effect of temperature on LH-HF0019 acrylonitrile degradation Rate

The Pseudomonas curvatus LH-HF0019 single colony is inoculated to 100mL 10% LB culture, the culture is carried out to an exponential phase under the conditions of 20 ℃ and 180rpm, the centrifugation is carried out for 5min at 6000rpm, the thalli are collected, 15mL of sterile MSM culture medium is added for bacterial washing twice, and finally 5mL of MSM culture medium is added for resuspension, so that seed liquid is obtained. Inoculating the seed solution into 35mL MSM culture medium, regulating the initial OD600 = 0.1 of the bacterial cells, adding acrylonitrile with the final concentration of 50ppm, culturing for 96 hours under the conditions of gradient temperature of 10 ℃,20, 30, 40 and 50 ℃ and 180rpm, measuring the final concentration of the acrylonitrile by adopting HJ 806-2016, examining the influence of the temperature on the degradation rate of LH-HF0019 acrylonitrile, and as shown in a result of a graph (B) in fig. 7, the degradation rate of the pseudomonas curvata LH-HF0019 on the acrylonitrile is more than or equal to 92.21 percent in the range of 10-40 ℃, and the degradation rate of the bacterial cells against the acrylonitrile is more than or equal to 85.22 percent even when the bacterial cells grow slowly at 50 ℃, thereby indicating that the bacterial cells have stronger low-temperature tolerance.

Example 4 comparison of the enzyme Productivity of Pseudomonas curvatus LH-HF0019

Pseudomonas curvatus LH-HF0019 is inoculated to 4 analysis solid culture mediums of amylase production, protease production, cellulase production and lipase production, and cultured for 48 hours at a constant temperature of 20 ℃, and the diameter of a hydrolysis circle is measured after the respective treatments. And (3) dropwise adding a proper amount of iodine solution on the amylase production analysis culture medium, shaking uniformly, determining the amylase production capacity of the strain according to the diameter of the amylase enzymolysis ring, dropwise adding a proper amount of acidic mercury on the protease production analysis culture medium, shaking uniformly, and determining the protease production capacity of the strain according to the diameter of the protease enzymolysis ring. And (3) dropwise adding a proper amount of Congo red dye liquor on a cellulase production analysis culture medium, shaking uniformly, dropwise adding a 1mol/L NaCl solution for washing, determining the cellulase production capacity of the strain according to the diameter of a cellulase enzymolysis ring, and if the strain produces lipase, producing transparent lipase enzymolysis rings around the specific strain. The enzyme production capacity of Pseudomonas curvatus LH-HF0019 is positively correlated with the diameter of the hydrolytic circle and negatively correlated with the diameter of the thallus, and specifically, the enzyme production capacity=the diameter of the hydrolytic circle/the diameter of the thallus. The ability of Pseudomonas curvatus LH-HF0019 to produce 4 enzymes is shown in Table 3:

TABLE 3 enzyme Productivity of Pseudomonas curvatus LH-HF0019

The ratio of the amylase produced by LH-HF0019 is 4.67, the ratio of the protease produced is 3.75, the ratio of the cellulase produced is 3.67, and the ratio of the lipase produced is 2.50, which indicates that the strain has stronger low-temperature enzyme producing capability, which is beneficial to the biological enzymolysis of organic pollutants such as system amylase, proteins, hemicellulose, cellulose, fat and the like when the strain is applied to environmental liquid or solid waste, thereby reducing the index of the system COD _Cr.

EXAMPLE 5 preparation of Pseudomonas curvatus LH-HF0019 microbial inoculum

After the Pseudomonas curvata LH-HF0019 is activated by an improved NB culture medium, respectively inoculating the strain into functional bacteria fermentation culture mediums with different inductants and cobalt chloride concentrations in a volume ratio of 5% to 0.3-0.8g/L, and culturing the strain to an exponential phase at 20 ℃ and 180rpm, wherein the bacterial content of a fermentation broth is not lower than 450 multiplied by 10 ⁸ CFU/ml. Diluting Pseudomonas curvatus LH-HF0019 bacteria 300-400 times with a blank functional bacteria fermentation medium to obtain diluted bacteria liquid with the bacterial load of 1.125-1.5X10 ⁸ CFU/ml. The diluted Pseudomonas curvata LH-HF0019 bacterial liquid is inoculated into a closed blue mouth bottle containing 50ml of 30 times dilution of Shandong catalpa waste water (containing 56.17ppm of acrylonitrile and 233.34ppm COD,pH 7.96) and reacted for 96 hours at 150rpm/min and 20 ℃, the final concentration of the acrylonitrile is measured by adopting HJ 806-2016, and the influence of the concentration of an inducer on the acrylonitrile degradation capability of the Pseudomonas curvata LH-HF0019 in the acrylonitrile waste water is examined, and the result is shown in figure 8. As the concentration of the inducer increases, the degradation rate of the acrylonitrile of LH-HF0019 in the POP diluted wastewater gradually increases, and finally, 0.6g/L is determined to be the concentration of cobalt chloride which is a proper inducer.

Example 6 experiments with Pseudomonas curvatus LH-HF0019 to enhance sludge degradation of acrylonitrile and COD _Cr at different temperatures

After the Pseudomonas curvata LH-HF0019 is activated by the modified NB culture medium, the modified NB culture medium is inoculated into a functional bacteria fermentation culture medium with the cobalt chloride concentration of 0.6g/L in a volume ratio, and is cultured to an exponential phase at 20 ℃ and 180rpm, wherein the bacteria content of a fermentation broth is not lower than 450 multiplied by 10 ⁸ CFU/ml. Diluting Pseudomonas curvatus LH-HF0019 bacteria 300-400 times with a blank functional bacteria fermentation medium to obtain diluted bacteria liquid with the bacterial load of 1.125-1.5X10 ⁸ CFU/ml. 30-fold dilution of Shandong Zibo certain POP wastewater (containing 56.17ppm acrylonitrile, 233.34ppm COD _Cr, pH 7.96) is used as a research object, and a 250ml sealed blue-mouth bottle is used as an experimental system. The Shandong Zibo sludge with the concentration of 5758ppm is washed by distilled water for 2 times and then resuspended by the POP wastewater diluent in equal quantity for standby. The control series group only adds a certain amount of Shandong-Bo sludge with the sludge amount of 5 ml/group, the experimental series group adds a pseudomonas curvata LH-HF0019 microbial inoculum with the proportion of 5% -6% on the basis of adding an equal amount of Shandong-Bo sludge, and three reaction temperatures of 10 ℃,20 ℃ and 30 ℃ are respectively set by taking 150rpm/min reaction for 96 hours as the basis reaction conditions. The final concentration of acrylonitrile was measured by HJ 806-2016, the COD _Cr content of the system was measured by dichromate method (HJ 828-2017) of water quality chemical oxygen demand, and the capability of Pseudomonas curvatus LH-HF0019 to enhance the degradation of acrylonitrile and COD _Cr by sludge at different temperatures was examined, and the results are shown in Table 4:

TABLE 4 Pseudomonas curvatus LH-HF0019 enhanced sludge degradation of acrylonitrile and COD at different temperatures _Cr

As can be seen from table 3, when the treatment temperature is gradually reduced from 30 ℃ to 10 ℃, only the control group of the sludge has the acrylonitrile content of the effluent gradually increased, and the removal rate of COD _Cr is gradually reduced from 68.09% to 55.61%, and even 60% of the satisfactory treatment effect is hardly achieved, which means that the capacity of removing COD _Cr is poorer when the activated sludge is subjected to the toxicity stress of acrylonitrile at low temperature. In contrast, pseudomonas curvata LH-HF0019 provided by the invention can degrade acrylonitrile, has strong low-temperature tolerance, and has the acrylonitrile degradation rate of more than or equal to 95.83% for POP diluted wastewater when the treatment temperature is gradually reduced from 30 ℃ to 10 ℃, the COD _Cr removal rate of more than or equal to 83.64%, and compared with a control group only using activated sludge, the improvement amplitude of the COD _Cr degradation rate after biological reinforcement is 17.04% -32.37%, wherein the biological reinforcement effect at 20 ℃ is most obvious, and the COD _Cr is increased by 32.37%. The pseudomonas curvata LH-HF0019 has stronger low temperature resistance, acrylonitrile toxicity decomposition and biochemical strengthening effect of promoting the degradation of the COD of the system, and the technical effect is better than the prior art.

Example 7 experiments on enhanced sludge degradation of acrylonitrile and COD _Cr by Pseudomonas curvatus LH-HF0019 at different acrylonitrile concentrations at 10 ℃

After the Pseudomonas curvata LH-HF0019 is activated by the modified NB culture medium, the modified NB culture medium is inoculated into a functional bacteria fermentation culture medium with the cobalt chloride concentration of 0.6g/L in a volume ratio, and is cultured to an exponential phase at 30 ℃ and 180rpm, wherein the bacteria content of a fermentation broth is not lower than 450 multiplied by 10 ⁸ CFU/ml. Diluting Pseudomonas curvatus LH-HF0019 bacteria 300-400 times with a blank functional bacteria fermentation medium to obtain diluted bacteria liquid with the bacterial load of 1.125-1.5X10 ⁸ CFU/ml. The POP diluted wastewater with gradient concentration acrylonitrile and COD _Cr concentration is prepared by utilizing different dilution multiples of POP wastewater of Shandong Zibo, and is used as an experimental study object of the experiment, and a 250ml closed blue-mouth bottle is used as an experimental system. The Shandong Zibo sludge with the concentration of 5758ppm is washed by distilled water for 2 times and then resuspended by the POP wastewater diluent in equal quantity for standby. The control series group only adds a certain Shandong-Bobo sludge with the sludge amount of 5 ml/group, the experimental series group adds a pseudomonas curvata LH-HF0019 microbial inoculum with the proportion of 5% -6% on the basis of adding an equivalent Shandong-Bobo sludge, adopts HJ 806-2016 to measure the final concentration of acrylonitrile under the reaction conditions of 150rpm/min, 10 ℃ and 48 hours, adopts a water quality chemical oxygen demand measurement dichromate method (HJ 828-2017) to measure the COD _Cr content of the system, and examines the capability of strengthening sludge degradation acrylonitrile and COD _Cr of the pseudomonas curvata LH-HF0019 under different acrylonitrile concentrations at the temperature of 10 ℃, and the results are shown in Table 5:

TABLE 5 enhanced sludge degradation of acrylonitrile and COD _Cr by Pseudomonas curvatus LH-HF0019 at different acrylonitrile concentrations at 10 ℃

As can be seen from Table 4, when the acrylonitrile concentration is gradually increased from 400ppm to more than 1000ppm, the gradually increased acrylonitrile toxicity stress and the environmental condition of 10 ℃ severely limit the exertion of the biochemical COD _Cr reducing effect of the activated sludge, and only the control group of the sludge has the acrylonitrile degradation rate gradually reduced from 13.45% to 6.38%, the COD _Cr removing rate gradually reduced from 58.28% to 33.15%, which means that the gradually increased acrylonitrile concentration brings more toxicity stress to the activated sludge, obvious sludge poisoning phenomenon occurs, the COD _Cr treatment effect is less than 60%, and the effluent reaching standard is seriously affected. In contrast, the pseudomonas curvata LH-HF0019 can play a role in low-temperature detoxification of functional strains at the treatment temperature of 10 ℃, even if the acrylonitrile concentration is gradually increased from 400ppm to more than 1000ppm, the acrylonitrile degradation rate of an experimental group is still generally more than 71.87%, at 405.2ppm, the acrylonitrile degradation rate reaches 96.06%, the COD _Cr removal rate of the corresponding POP diluted wastewater is also between 80.96% and 86.74%, and compared with a control group only using activated sludge, the improvement amplitude of the COD _Cr degradation rate after biological reinforcement is between 28.46% and 47.81%, and the biological reinforcement effect is obvious when the treated acrylonitrile concentration is higher. The pseudomonas curvata LH-HF0019 can assist the activated sludge system to contact with gradually increased acrylonitrile toxicity at a lower temperature of 10 ℃ and restore the degradation capability of organic substrates of a biochemical system, so that the COD _Cr removal rate of the system is improved, and the low-temperature-resistant detoxification microbial inoculum for acrylonitrile toxic wastewater with a stronger biological strengthening technical effect is provided.

Example 8 experiments on enhanced treatment of Acrylonitrile-containing wastewater by Cold-resistant detoxified Pseudomonas curvata LH-HF0019 at Low temperature

After the Pseudomonas curvata LH-HF0019 is activated by the modified NB culture medium, the modified NB culture medium is inoculated into a functional bacteria fermentation culture medium with the cobalt chloride concentration of 0.6g/L in a volume ratio, and is cultured to an exponential phase at 30 ℃ and 180rpm, wherein the bacteria content of a fermentation broth is not lower than 450 multiplied by 10 ⁸ CFU/ml. Diluting Pseudomonas curvatus LH-HF0019 bacteria 300-400 times with a blank functional bacteria fermentation medium to obtain diluted bacteria liquid with the bacterial load of 1.125-1.5X10 ⁸ CFU/ml. The waste water of a POP (POP) is diluted to a system acrylonitrile concentration of 601.85ppm and a COD (chemical oxygen demand) _Cr of 2502.41ppm, and a 250ml sealed blue-mouth bottle is used as an experimental system as an experimental study object. The Shandong Zibo sludge with the concentration of 5758ppm is washed by distilled water for 2 times and then resuspended by the POP wastewater diluent in equal quantity for standby. The control series group only adds a certain Shandong-Bobo sludge with the sludge amount of 5 ml/group, the experimental series group adds a pseudomonas curvata LH-HF0019 microbial inoculum with the proportion of 5% -6% on the basis of adding an equal amount of Shandong-Bobo sludge, adopts HJ 806-2016 as a reaction condition with the speed of 150rpm/min and the temperature of 10 ℃ for 48 hours to measure the final concentration of acrylonitrile, and adopts a dichromate method (HJ 828-2017) to measure the COD _Cr content of the system. The results show that the control group only added with activated sludge has the acrylonitrile degradation rate of 11.24-11.30%, the COD _Cr removal rate of 54.23-54.30%, the sludge is disintegrated and floats, and the like, which proves that the poisoning is serious and the standard effluent can not be achieved. Compared with the method, the activated sludge is additionally added with the pseudomonas curvata LH-HF0019 bacterial agent group which is resistant to low temperature and has the effect of removing the acrylonitrile, the degradation rate of the acrylonitrile is between 95.82 and 95.84 percent, the removal rate of COD _Cr is between 83.13 and 83.23 percent, and the sludge state is good. the comparison test shows that the Pseudomonas curvata LH-HF0019 with low temperature resistance and acrylonitrile detoxification property has stronger technical effects of detoxification and biologically enhanced assisted COD _Cr degradation on low temperature acrylonitrile-containing wastewater, and is superior to the prior art.

In conclusion, the pseudomonas curvata LH-HF0019 is separated from activated sludge of POP wastewater of Zibo in Shandong province, has stronger acrylonitrile detoxification capability, and has stronger acrylonitrile degradation capability when the inducer cobalt chloride is 0.6 g/L. Pseudomonas curvatus LH-HF0019 can grow and reproduce in the temperature range of 10-50 ℃, has wider low-temperature tolerance, can generate contact enzyme, ornithine decarboxylase and lysine decarboxylase, can liquefy gelatin, and can produce acid by utilizing various carbohydrates such as inositol, lactic acid, sodium acetate, sodium succinate, sodium malonate, sodium succinate, beta-galactoside and the like. The strain can also produce various enzymes such as amylase, protease, cellulase and lipase, can assist in acrylonitrile degradation and COD _Cr removal in a low-temperature environment, can still realize the acrylonitrile degradation rate of more than or equal to 95.82% at the low temperature of 10 ℃ when the acrylonitrile concentration of the system reaches more than 600ppm, and can strengthen the activated sludge to realize the COD _Cr removal rate of more than or equal to 83.13%. Multiple experiments prove that the strain has stronger acrylonitrile detoxification capability and can still exert biological strengthening effect at low temperature to 10 ℃.

The above embodiments are only for illustrating the technical solution of the present invention, but not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it will be apparent to those skilled in the art that modifications may be made to the technical solution described in the above embodiments or equivalents may be substituted for some of the technical features thereof, and the modifications or substitutions do not depart from the spirit and scope of the technical solution as claimed in the present invention.

Claims

1. The Pseudomonas curvata (Pseudomonas geniculata) LH-HF0019 is characterized in that the preservation number is CGMCC No. 27554.

2. An enzymatic production method of pseudomonas curvata LH-HF0019 as set forth in claim 1, wherein:

The enzyme-producing culture mediums are respectively as follows:

The amylase production medium comprises 10g of peptone, 5g of beef extract, 5g of NaCl, 2g of soluble starch, 7.1-7.3 of pH, 1L of distilled water and 20g of agar;

The protease production culture medium comprises 10g of casein, 1g of yeast extract, pH 6.9-7.1, 1L of distilled water and 20g of agar;

The cellulase production medium comprises 20g of sodium carboxymethyl cellulose, 0.5g of yeast powder, 2.5g of disodium hydrogen phosphate, 1.5g of potassium dihydrogen phosphate, 2.5g of peptone, 7.1-7.3 of pH, 1L of distilled water and 20g of agar;

the lipase production culture medium comprises 10g of peptone, 5g of yeast powder, 10g of NaCl, 2mL of tributyrin, 7.4-7.6 pH, 1L of distilled water and 20g of agar;

The enzyme production condition is that pseudomonas curvata LH-HF0019 is respectively inoculated to 4 solid culture mediums for producing amylase, protease, cellulase and lipase, and the culture is carried out for 48 hours at the constant temperature of 10-50 ℃.

3. Use of pseudomonas curvata LH-HF0019 as claimed in claim 1 for the preparation of a low temperature Jie Dujun agent for the degradation of acrylonitrile comprising pseudomonas curvata LH-HF 0019.