CN114908016B

CN114908016B - Pseudomonas denitrificans and application thereof in field of sewage and wastewater purification

Info

Publication number: CN114908016B
Application number: CN202210608388.8A
Authority: CN
Inventors: 赵琬玫; 刘圣鹏
Original assignee: Qingdao Weilan Saide Biotechnology Co ltd
Current assignee: Qingdao Weilan Saide Biotechnology Co ltd
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2023-05-19
Anticipated expiration: 2042-05-31
Also published as: CN114908016A

Abstract

The invention relates to a pseudomonas denitrificans (Pseudomonas denitrificans) XA-1 which is preserved in the China general microbiological culture Collection center with the address of: the collection number of the national institute of microbiology, national academy of sciences, no. 3, north Chen West Lu 1, chao yang, beijing, is: CGMCC No.24446, the strain has high degradation efficiency on benzene series, and the benzene degradation rate of 72 hours reaches more than 96 percent aiming at the benzene content below 50mg/L in an evaluation culture medium at 30 ℃; and moderately salt-tolerant, the number of viable bacteria is hardly affected when growing under the salinity condition of less than 5%.

Description

Pseudomonas denitrificans and application thereof in field of sewage and wastewater purification

Technical Field

The invention relates to pseudomonas denitrificans and application thereof in degrading benzene and ammonia nitrogen and reducing COD in the purification process of sewage and wastewater, belonging to the technical field of environmental microorganisms.

Background

Benzene is one of typical aromatic hydrocarbon compounds, belongs to organic pollutants difficult to degrade, has cancerogenic action, and has great toxic action on human bodies and microorganisms. Benzene pollution mainly comes from industries such as pesticides, petrifaction, printing and dyeing, pharmacy, plastic rubber and the like, and if the benzene pollution cannot be properly treated, the benzene pollution can cause adverse effects on human health, economic development and sustainable development of environment.

The industrial wastewater containing benzene mainly comprises coking wastewater, oil refining wastewater, printing and dyeing wastewater, ABS resin production wastewater and the like, and a great deal of researches show that most of the industrial wastewater containing benzene has complex components and is often accompanied with the phenomenon of exceeding standard of inorganic elements such as ammonia nitrogen and the like, so that heterotrophic nitrification-aerobic denitrification degradation strain taking benzene as a carbon source has important theoretical guidance and practical application significance for researching novel water treatment technology.

Currently, benzene is an urgent substance to be treated in wastewater, and treatment methods are mainly classified into physical methods, chemical methods and biological methods. The biological treatment technology has the advantages of small secondary pollution, high removal efficiency, thorough pollutant removal, high economic performance and the like, so that the biological treatment technology becomes a research hot spot of the modern wastewater treatment technology and is receiving more and more attention.

Currently, scientists have screened various strains with benzene degradation function, such as Pseudomonas sp, flavobacterium sp, alcaligenes eutrophus sp, rhodococcus sp, etc., but these strains have not been sufficiently prominent in benzene degradation effect, and thus it is necessary to screen or construct strains having more prominent benzene degradation effect.

Disclosure of Invention

Aiming at the current situation that the effect of the existing benzene series degrading strain is not outstanding enough, the invention provides the pseudomonas denitrificans and the application thereof in the field of sewage and wastewater purification, and the strain can utilize the benzene series as a carbon source to degrade ammonia nitrogen in a water body, thereby achieving the effect of reducing the ammonia nitrogen and COD content while efficiently degrading the benzene series in the water body.

Pseudomonas denitrificans (Pseudomonas denitrificans) XA-1, deposited in China general microbiological culture Collection center (China Committee) for culture Collection, has the following addresses: the collection number of the national institute of microbiology, national academy of sciences, no. 3, north Chen West Lu 1, chao yang, beijing, is: CGMCC No.24446, the preservation date is: in 2022, 28 days of 2 months, the 16S rDNA sequence is shown as SEQ ID No. 1, and the Pseudomonas denitrificans in the invention refers to Pseudomonas denitrificans strain XA-1 unless otherwise specified.

The mechanism of the heterotrophic nitrification-aerobic denitrification reaction of the pseudomonas is more complex, a more authoritative explanation mechanism does not exist at present, the more popular theory at present is mainly a reaction mechanism (Thiosphaera pantotropha) of HN-AD action proposed by Robertson and the like, electrons can be received by a denitrification enzyme system and oxygen in the nitrogen metabolism process, denitrification can be realized under the aerobic condition, the joint points of the heterotrophic nitrification and the aerobic denitrification are nitrite nitrogen, and the main ways are as follows: ammonia nitrogen-hydroxylamine-nitrous nitrogen-nitrate nitrogen-nitrous nitrogen-nitric oxide-nitrous oxide-nitrogen.

Benzene is used as the only carbon source and energy source under the aerobic condition, and enters the cells through passive or active transportation of the cells. All aromatic hydrocarbons must be converted to either ortho or para dihydroxybenzene before ring cleavage can occur. Catechol (catechol), 3, 4-dihydroxybenzoic acid (protocatechuic acid), 2, 5-dihydroxyphenylacetic acid (homogentisate), and 2, 5-dihydroxybenzoic acid (plateau catechol) are the predominant few dihydroxybenzenes. Under the catalysis of dioxygenase, dihydroxybenzene is cracked into small molecular organic acid to enter TCA circulation, so as to produce energy for biological utilization and synthesize self-material.

The pseudomonas denitrificans provided by the invention has the beneficial effects that:

1) The strain has high degradation efficiency on benzene series, and the benzene degradation rate of 72 hours reaches more than 96 percent aiming at the benzene content below 50mg/L in an evaluation culture medium at the temperature of 30 ℃;

2) Moderately salt tolerant, when grown under a salinity condition below 5%, the viable count is hardly affected, but cannot tolerate a salinity above 6%;

3) The strain can degrade benzene series and simultaneously reduce ammonia nitrogen content in water to a certain extent, thereby achieving two purposes;

4) The strain has the advantages of simple culture method, high growth speed, strong environmental adaptability, high safety, no damage to the original environment and no secondary pollution.

Benzene-based compounds described in the present invention include, but are not limited to, benzene, toluene, ethylbenzene, ortho-xylene, meta-xylene, and para-xylene.

The invention also claims a microbial agent comprising the Pseudomonas denitrificans.

Preferably, the fermentation method of the pseudomonas denitrificans comprises the following steps:

(1) Primary seed culture: inoculating Pseudomonas denitrificans into liquid LB culture medium under aseptic condition, and culturing at 25-35deg.C and 150-300rpm for 12-48 hr to obtain primary seed culture solution of Pseudomonas denitrificans;

(2) Secondary seed culture: inoculating the primary seed culture solution of the pseudomonas denitrificans into a liquid LB culture medium according to an inoculum size of 1-10vol% under a sterile condition, and culturing for 12-48h at 25-35 ℃ and 150-300rpm to obtain a secondary seed culture solution of the pseudomonas denitrificans;

(3) Fermentation: after the fermentation medium in the fermentation tank is disinfected, inoculating the second-level seed culture solution of the pseudomonas denitrificans obtained in the step (2) into the fermentation medium according to the inoculum size of 5-10vol%, controlling the temperature to be 25-35 ℃ and the rotating speed to be 150-300rpm, fermenting under the condition that the aeration ratio is 1 (1-2), and stopping fermenting when dissolved oxygen starts to rise to obtain the fermentation solution of the pseudomonas denitrificans.

The aeration ratio in the present invention means the ratio of the volume of air introduced into the fermenter per minute to the total volume of the fermentation liquid.

Wherein the composition of the liquid LB culture medium is as follows: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and water as a solvent, wherein the pH=7-7.5.

Further, the fermentation medium comprises the following components: 10-30g/L of carbon source and 20-40g/L, PO of nitrogen source ₄ ^3- 0.8-1.5g/L、K ⁺ 0.5-1.0g/L、Mg ²⁺ 0.05-0.2g/L、Na ⁺ 0.1-0.3g/L、Mn ²⁺ 0.03-0.1g/L, water as solvent, pH=6-8.

Preferably, the K ⁺ The source of the catalyst is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium sulfate, potassium chloride and potassium nitrate, and the Mg ²⁺ Is one or more of magnesium sulfate and magnesium chloride, and the Na is ⁺ The source of the Mn is one or more of sodium chloride, sodium nitrate or sodium sulfate ²⁺ The source of the catalyst is one or more of manganese sulfate monohydrate, manganese sulfate tetrahydrate, manganese nitrate or manganese chloride.

Further, the carbon source is selected from one or more of glucose, sucrose, starch, sodium acetate or sodium citrate.

Further, the nitrogen source is selected from one or more of yeast extract powder, peptone, corn steep liquor dry powder, ammonium sulfate or potassium nitrate.

In the practical application process, the form of the final pseudomonas denitrificans product can be determined according to the practical use and storage requirements, when the liquid product is required to be used, the fermentation liquor can be diluted to the required concentration for direct use, when the solid product is required to be used, the fermentation liquor can be centrifuged to obtain bacterial mud, and then the solid bacterial powder is prepared by adopting a spray drying or freeze drying process.

The invention also claims a method for purifying sewage and wastewater by using the activated liquid or microbial agent of the pseudomonas denitrificans, which comprises the step of applying the activated liquid of the pseudomonas denitrificans or the microbial agent containing the pseudomonas denitrificans to the sewage and wastewater.

Further, the benzene series concentration in the sewage/wastewater is 50mg/L or less, preferably 30mg/L or less.

Further, the salinity of the sewage and wastewater is 5% or less, preferably 3% or less, and most preferably 2% or less.

Further, the inoculation amount of the activated liquid of the Pseudomonas denitrificans or the microbial agent is more than 1000ppm, preferably 1000-20000ppm, and most preferably 1000-10000ppm.

The invention also claims the application of the pseudomonas denitrificans and the microbial agent in the field of sewage and wastewater purification.

Preferably, the pseudomonas denitrificans and the microbial agent are used for degrading benzene compounds in sewage and wastewater; more preferably, the Pseudomonas denitrificans and microbial agents are used for degrading benzene in wastewater.

Drawings

FIG. 1 is a photograph of a colony of Pseudomonas denitrificans;

FIG. 2 is a photograph of a Pseudomonas denitrificans colony magnified by a 100-fold microscope.

Detailed Description

The principles and features of the present invention are described below in connection with examples, which are set forth only to illustrate the present invention and not to limit the scope of the invention.

The respective media used in the examples were composed as follows:

enrichment medium: 1g of dipotassium hydrogen phosphate, 1g of monopotassium phosphate, 1g of ammonium sulfate, 0.2g of magnesium sulfate, 1g of potassium nitrate, 0.01g of calcium chloride, 1ml of microelement solution, and fixing the volume to 1L, wherein pH=7;

basal medium: 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 1000ml of water and pH=7;

evaluation of the medium: 1g of dipotassium hydrogen phosphate, 1g of monopotassium phosphate, 1g of ammonium sulfate, 0.2g of magnesium sulfate, 1g of potassium nitrate, 0.01g of calcium chloride and 1ml of microelement solution, and adjusting the pH to 1L and 7, and adjusting different benzene concentrations according to requirements;

trace element solution: 1.5g of ferric chloride, 0.1g of manganese sulfate, 70mg of zinc chloride, 2mg of copper chloride, 30mg of nickel chloride, 200mg of cobalt chloride, 5mg of sodium molybdate and the volume to 1L, wherein pH=7.

EXAMPLE 1 screening separation and purification of Pseudomonas denitrificans

1. Water sample enrichment

Taking 100mL of a water sample from an aerobic tank of a certain chemical plant in Shandong province, inoculating 10mL of the water sample into a triangular flask filled with 100mL of enrichment medium, adding 10mg/L of benzene into the enrichment medium, performing shake culture at 30 ℃ for 5 days under the condition of 200r/min, measuring the content of benzene every other day, and adjusting the pH; when the benzene concentration is reduced to below 50%, transferring the next stage of enrichment, namely taking 10mL of culture solution after 5 days of culture, inoculating the culture solution into 100mL of enrichment medium again, increasing the benzene concentration by 10mg/L each time, and repeatedly culturing for 5 times until the benzene concentration of the enrichment medium is 50mg/L.

2. Screening and isolation of strains

Taking 0.5mL of the culture solution of the last stage, and carrying out gradient dilution by using sterile water to respectively dilute to 10 ^-4 ，10 ^-5 ，10 ^-6 ，10 ^-7 And 10 ^-8 The diluted culture solution is coated on a nutrient solid culture medium flat plate, and then the culture solution is placed in a 30 ℃ incubator for culture, after the colony grows to a proper size, the morphological characteristics of the colony are observed, single colony is selected for streak purification, after three generations of purification, inclined-plane preservation at 4 ℃ is carried out, 6 strains are totally separated, and the strains are respectively named as XA-1-XA-6.

3. Double screen

The 6 strains obtained by primary screening are respectively inoculated into a basic culture medium, shake culture is carried out for 24 hours at 30 ℃, 10mL of culture solution is respectively taken and centrifuged for 10 minutes at 5000rpm, supernatant is discarded, each strain is respectively inoculated into 100mL of evaluation culture medium (benzene content is 50 mg/L), shake culture is carried out at 30 ℃, three groups of strains are arranged in parallel, sterile water is used as a blank control, and the concentration change of benzene in the culture medium is periodically detected, and the results are shown in table 1.

Table 1 evaluation results of benzene by the strain obtained by preliminary screening

As can be seen from the data in Table 1, strain XA-1 has the strongest benzene-removing effect and significantly better benzene-degrading ability than the other 5 strains.

Example 2 identification of species

1. Morphological observation

Strain XA-1 was inoculated on a nutrient solid medium plate, and morphological characteristics of colonies were observed. The colony of XA-1 strain is shown in FIG. 1, and the 100 times microscopic photograph is shown in FIG. 2, and the colony is punctiform, semitransparent, milky white, neat in edge, clear and smooth, and wet in surface.

2. Molecular biological identification

Genomic DNA of strain XA-1 was extracted and 16S rDNA was amplified using this as a template.

Primer: 16S rDNA-27F 5'-TTGCGGAGGCTACCATGCAG-3',

1492r:5’-GACGGTATACTCCGTGGTAC-3’。

the PCR products were then extracted and DNA sequenced using a sequencer ABI 3730-XL. The spliced sequence file is compared with data in an NCBI 16S database by using an NCBI Blast program to obtain species information with the maximum sequence similarity with a species to be detected, the species information is found to belong to the genus Pseudomonas, the species information is identified as Pseudomonas denitrificans (Pseudomonas denitrificans), and the species information is named as Pseudomonas denitrificans XA-1.

EXAMPLE 3 salinity tolerance test of Pseudomonas denitrificans XA-1

Under aseptic condition, the Pseudomonas denitrificans XA-1 is inoculated into a 250mL conical flask containing 100mL of liquid LB culture medium, and is subjected to shaking culture for 24 hours at 30 ℃ in a shaking table of 200rpm to perform strain activation, so as to obtain an activated liquid of Pseudomonas denitrificans, and the number of viable bacteria of the activated liquid is 75 hundred million CFU/mL through test.

With the liquid LB medium as a basal medium (salinity: 1%), 1g,2g,3g,4g,5g,6g,7g,8g,9g of NaCl was additionally added to a flask containing 100mL of the liquid LB medium, and then salinity gradients of 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10% were set. Respectively taking 5mL of each activating solution of Pseudomonas denitrificans, respectively inoculating into culture mediums with different salinity gradients, shake culturing in shaking table at 30deg.C, and periodically measuring OD ₆₀₀ Values and viable count to determine the extent of strain growth, the results are shown in Table 2.

TABLE 2 OD of Pseudomonas denitrificans cultured under different salinity conditions ₆₀₀ Value and viable count

As can be seen from the data in Table 2, strain XA-1 can normally grow at a salinity of 1-5%, 1% is the optimum salinity, and the growth condition of the strain is significantly better than that at a salinity of more than 5% when the salinity is less than 5%.

Example 4 degradation efficiency determination of Pseudomonas denitrificans XA-1 at different benzene concentrations

Under aseptic condition, the Pseudomonas denitrificans XA-1 is inoculated into a 250mL conical flask containing 100mL of liquid LB culture medium, and is subjected to shaking culture for 24 hours at 30 ℃ in a shaking table of 200rpm to perform strain activation, so as to obtain an activated liquid of Pseudomonas denitrificans, and the number of viable bacteria of the activated liquid is 85 hundred million CFU/mL through test.

Evaluation media with benzene concentration of 100mg/L, 50mg/L, 40mg/L, 30mg/L, 20mg/L and 10mg/L and ammonia nitrogen concentration of 100mg/L are respectively prepared, the activated liquid of the pseudomonas denitrificans is respectively inoculated into 100mL of each sterilized evaluation medium according to an inoculation amount of 1vol%, and then placed into a shaking table at 30 ℃ and 200rpm for shaking culture, 3 parallel groups are arranged, sterile water is added into the evaluation medium as a blank control, the experimental grouping is arranged as shown in table 3, and then benzene content and ammonia nitrogen content in each medium are detected every 24 hours, and the results are shown in tables 4 and 5.

The benzene concentration detection method utilizes a gas chromatograph-mass spectrometer to detect.

The ammonia nitrogen detection method is implemented according to HJ535-2009 ammonia nitrogen determination-Nashi reagent spectrophotometry.

Table 3 each experimental group evaluates benzene content and ammonia nitrogen content of the culture medium

Table 4 each experimental group and control group evaluated the change in benzene content of the medium

TABLE 5 evaluation of the ammonia-nitrogen content variation of the Medium for each of the experimental and control groups

As can be seen from the data in tables 4 and 5, the Pseudomonas denitrificans XA-1 showed a good benzene degradation effect at a benzene concentration of 50mg/L or less, a benzene degradation rate of 96% or more for 72 hours, and a degradation rate of ammonia nitrogen gradually increased with an increase in benzene concentration of 50mg/L or less, with a maximum degradation rate of 37%. However, when the benzene concentration reaches 100mg/L, the benzene degradation rate is only 11.6% after 72 hours, and the ammonia nitrogen degradation rate is also lower, about 7%, so that the result shows that the Pseudomonas denitrificans can tolerate 50mg/L of benzene but not 100mg/L of benzene.

EXAMPLE 5 fermentation Process of Pseudomonas denitrificans

The fermentation method of the pseudomonas denitrificans comprises the following steps:

1) The slant seeds of the pseudomonas denitrificans are picked by an inoculating loop and inoculated into 100mL of liquid LB culture medium, and are cultured for 18 hours at 30 ℃ and 200rpm to obtain first-stage seed liquid;

2) Inoculating the cultured primary seed liquid into 1L of liquid LB culture medium according to 10vol% of inoculation amount, and culturing at 30 ℃ and 200rpm for 18 hours to obtain secondary seed liquid;

3) Under the aseptic condition, inoculating the secondary seed liquid into a fermentation medium according to an inoculum size of 5-10vol%, wherein the formula of the fermentation medium is as follows: 20g/L of glucose, 30g/L of sodium citrate, 10g/L of corn steep liquor dry powder, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 0.5g/L of sodium chloride and 0.2g/L of manganese sulfate, fermenting under the conditions that the temperature is controlled to be 25-35 ℃ and the rotating speed is 150-300rpm and the ventilation ratio is 1 (1-2), stopping fermenting when dissolved oxygen starts to rise, and obtaining fermentation liquor of pseudomonas denitrificans, wherein the viable count of the fermentation liquor is 120 hundred million CFU/mL through test.

EXAMPLE 6 treatment effect of Pseudomonas denitrificans activated liquid on raw Water of chemical plant

Raw water of a certain dyeing chemical factory in Taian city of Shandong province, wherein the COD content before treatment is 2742mg/L, the benzene content is 328.66 mug/L, the ammonia nitrogen content is 128mg/L, and the pH=8.3.

Pseudomonas denitrificans XA-1 is inoculated into a 250mL serum bottle containing 100mL liquid LB culture medium under aseptic condition, and is subjected to shaking culture for 24 hours at 30 ℃ in a shaking table at 200rpm to obtain an activated liquid, and the activated liquid has the viable count of 80 hundred million CFU/mL through test.

100mL of the chemical raw water is respectively placed in two identical 250mL serum bottles, an experimental group is inoculated with bacterial strain activating solution according to 1% of the volume of the chemical raw water, a control group is inoculated with sterile water with the same volume, the control condition is that the temperature is 30 ℃, the shaking treatment is carried out in a shaking table at 200rpm, and the pH value is natural. Samples were taken every 24 hours during the reaction and the results are shown in tables 6, 7 and 8.

Wherein, the COD detection method is implemented according to the technical requirement of the rapid tester of HJ 924-2017COD photometry and the detection method. The benzene concentration detection method utilizes a gas chromatograph-mass spectrometer to detect.

TABLE 6 variation of COD content in raw water of chemical plant with time

TABLE 7 variation of benzene content in raw water of chemical plant with time

TABLE 8 Ammonia nitrogen content in raw water of chemical plant over time

From the data in tables 6 to 8, the COD degradation rate is 36%, the benzene degradation rate is 80% and the ammonia nitrogen degradation rate is 24% in five days after the bacterial strain activation solution is added, which indicates that the Pseudomonas denitrificans provided by the invention has high-efficiency benzene degradation capability and a certain COD and ammonia nitrogen degradation capability.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Sequence listing

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Claims

1. Pseudomonas denitrificans strainPseudomonas denitrificans) The method is characterized in that the strain is preserved in China general microbiological culture Collection center (China Committee) with the preservation number: CGMCC No.24446.

2. A microbial agent comprising the Pseudomonas denitrificans according to claim 1 as an active ingredient.

3. The fermentation process of Pseudomonas denitrificans according to claim 1, comprising the steps of:

4. A fermentation process according to claim 3, wherein the fermentation medium composition is: 10-30g/L of carbon source and 20-40g/L, PO of nitrogen source ₄ ^3- 0.8-1.5g/L、K ⁺ 0.5-1.0g/L、Mg ²⁺ 0.05-0.2g/L、Na ⁺ 0.1-0.3g/L、Mn ²⁺ 0.03-0.1g/L, water as solvent, and pH=6-8.

5. The fermentation process of claim 4, wherein the carbon source is selected from one or more of glucose, sucrose, starch, sodium acetate or sodium citrate;

the nitrogen source is selected from one or more of yeast extract powder, peptone, corn steep liquor dry powder, ammonium sulfate or potassium nitrate.

6. A method for purifying benzene in sewage and wastewater, comprising the step of applying the activated liquid of pseudomonas denitrificans according to claim 1 or the microbial agent according to claim 2 to sewage and wastewater.

7. The method of claim 6, wherein the benzene concentration in the wastewater is 50mg/L or less.

8. The method of claim 7, wherein the benzene concentration in the wastewater is 30mg/L or less.

9. The method of any one of claims 6-8, wherein the waste water has a salinity of 5% or less.

10. The method of claim 9, wherein the waste water has a salinity of 3% or less.

11. The method of claim 10, wherein the waste water has a salinity of 2% or less.

12. The method according to any one of claims 6 to 8 and 10 to 11, wherein the amount of the activated liquid of pseudomonas denitrificans or the microbial agent inoculated is 1000ppm or more.

13. The method according to claim 9, wherein the amount of the activated liquid of Pseudomonas denitrificans or the microbial agent inoculated is 1000ppm or more.

14. The method according to claim 12, wherein the amount of inoculation of the activated liquid of pseudomonas denitrificans or the microbial agent is 1000-20000ppm.

15. The method according to claim 13, wherein the amount of inoculation of the activated liquid of pseudomonas denitrificans or the microbial agent is 1000-20000ppm.

16. The method according to claim 14 or 15, wherein the amount of the activated liquid of pseudomonas denitrificans or the microbial agent inoculated is 1000-10000ppm.

17. Use of the pseudomonas denitrificans of claim 1 and the microbial agent of claim 2 for degrading benzene in wastewater.