CN113801828A

CN113801828A - Efficient nitrobenzene degrading bacterium and preparation and application thereof

Info

Publication number: CN113801828A
Application number: CN202111310312.9A
Authority: CN
Inventors: 蔡倩倩; 蔡颖辉; 张心青; 杨传伦; 冉新新; 郭南南; 韩立霞; 傅英旬; 张萧萧; 杨丹丹
Original assignee: Chambroad Chemical Industry Research Institute Co Ltd
Current assignee: Chambroad Chemical Industry Research Institute Co Ltd
Priority date: 2021-11-05
Filing date: 2021-11-05
Publication date: 2021-12-17
Anticipated expiration: 2041-11-05
Also published as: CN113801828B

Abstract

The invention relates to the technical field of microorganisms, and particularly relates to a nitrobenzene efficient degrading bacterium and preparation and application thereof. The degrading bacteria belong to gram-negative bacteria, are identified as pseudomonas stutzeri by molecular level, have the preservation number of CGMCC22758, can efficiently degrade nitrobenzene, can degrade more than 99% of 900mg/L nitrobenzene within 24 hours, can degrade more than 99% of 1000mg/L nitrobenzene within 32 hours, have the optimal action temperature of 25-35 ℃ and the optimal action pH of 6.5-8.0, can be continuously cultured in the application of nitrobenzene sewage treatment, can stably degrade 500mg/L nitrobenzene within 24 hours, have strong strain tolerance capability, can tolerate high-concentration nitrobenzene, have wide application range on conditions such as temperature and pH, are not easy to be impacted by external environment in the application process, and have good application value in actual production.

Description

Efficient nitrobenzene degrading bacterium and preparation and application thereof

Technical Field

The invention relates to the field of microbial agents, and particularly relates to a nitrobenzene efficient degrading bacterium and a preparation application thereof.

Background

Nitrobenzene is an organic synthetic raw material widely used in industrial production, and is widely used in industrial production, so that a large amount of nitrobenzene remains in industrial wastewater such as pharmaceuticals, aniline, pesticides, dyes, and the like. Meanwhile, nitrobenzene has the characteristics of stable chemical property, high toxicity, easy biological enrichment, long-term existence in the environment and the like, and is listed in the front row of the environment for preferentially controlling toxic organic pollutants. With the development of chemical industry, the large amount of nitrobenzene wastewater causes serious pollution to the environment, so in order to eliminate or reduce the pollution caused by nitrobenzene, measures are needed to reduce the harm caused by nitrobenzene pollution.

At present, the nitrobenzene treatment technology mainly comprises a physical method, a chemical method and a biological method, and although the physical and chemical method has high treatment efficiency and relatively simple process, the reaction conditions generally have special requirements and higher treatment cost, so that the nitrobenzene treatment technology is difficult to be applied to actual wastewater treatment engineering on a large scale. Compared with the prior art, the biological method for treating the nitrobenzene wastewater has the advantages of low treatment cost, simple operation management, no secondary pollution, strong variability and domestication adaptability of microorganisms, suitability for wastewater of various types and conditions, and easy large-scale application in practical wastewater treatment engineering.

The biological treatment method of nitrobenzene wastewater can be divided into an anaerobic method and an aerobic method. Under the anaerobic condition, nitrobenzene is easy to be reduced into aniline, and the aniline can be further thoroughly mineralized. Compared with the anaerobic method, nitrobenzene can be directly mineralized under aerobic conditions, so a large number of aerobic nitrobenzene degrading bacteria including prokaryotes and eukaryotes have been separated in recent years, wherein the prokaryotes are mainly bacteria, and the eukaryotes are mainly white rot fungi. But because the biodegradability of nitrobenzene is poor and nitrobenzene has toxic effect on microorganisms, the growth of the microorganisms is inhibited and even the microorganisms are poisoned.

In the prior art, three aerobic degradation bacteria separated from Wachaohai et al introduced in 'co-matrix and biological synergistic effect of nitrobenzene aerobic degradation' can take nitrobenzene as the only carbon, nitrogen and energy, and the best effect is that one strain of bacillus subtilis degrades 200mg/L in 145 h; the Wangxi et al in the article 'aerobic degradation of pseudomonas JX165 and intact cell p-nitrobenzene' states that the maximum tolerant concentration of the pseudomonas p-nitrobenzene obtained by separation is 800mg/L, and the pseudomonas p-nitrobenzene can be completely degraded within 60h when the initial concentration of the nitrobenzene is 200 mg/L; luwenxiang reported in the research on the separation _ identification and degradation characteristics of nitrobenzene degrading bacteria Pseudomonas sp.XY-1, wherein the degradation rate of the separated pseudomonas to nitrobenzene with the initial concentration of 200mg/L within 24h is 97 percent; the nitrobenzene degrading bacteria separated from Zhengchunli and the like can degrade nitrobenzene with the concentration of 200mg/L for 60 hours; in Chua bang Cheng et al screening and degradation characteristics of a strain of efficient nitrobenzene degradation bacteria, the maximum 24-hour degradation capacity of acinetobacter separated from bottom mud of a sewer of a chemical plant is 400 mg/L. In addition, the patent application CN1570079A is a nitrobenzene pollutant efficient degrading bacterium and the pseudomonas 24h provided by the using method can degrade 433mg/L, and can degrade 98% of nitrobenzene 100h with 796 mg/L. However, important information such as the preservation of the strain is not disclosed, and the species of the Pseudomonas bacterium cannot be known.

From the current reports, the degrading efficiency of the nitrobenzene degrading bacteria is generally low, and the nitrobenzene degrading bacteria have some influence on the application of the nitrobenzene degrading bacteria in actual production. Therefore, the isolation and breeding of strains with high tolerance and degradation capability are especially important for the application of the strains in the bioaugmentation technology.

Disclosure of Invention

The invention aims to provide a nitrobenzene degrading bacterium with strong environmental adaptability so as to solve the problems of poor biodegradability and high toxicity of nitrobenzene and difficult degradation, and realize one-step mineralization of nitrobenzene.

Firstly, the efficient nitrobenzene degrading bacteria are obtained by collecting water samples from sewage treatment systems of petrochemical and leather sewage plants, enriching the water samples, and further performing gradient enrichment, separation and purification on the water samples in an inorganic salt culture medium which takes nitrobenzene as unique carbon, nitrogen and energy.

The nitrobenzene degrading bacteria is a gram-negative bacteria, the bacterial colony is circular faint yellow, the edge is irregular, the shape of a convex navel is realized, the surface is dry, the nitrobenzene degrading bacteria is identified as Pseudomonas stutzeri by 16S rDNA, the Pseudomonas stutzeri is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC 22758.

As a further scheme of the invention, the invention provides a microbial inoculum of the nitrobenzene degradation bacteria, which is prepared by fermentation:

the fermentation medium comprises the following components in percentage by mass: 1.0-2.0% of corn flour, 0.5-1.1% of glucose, 1.8-2.4% of corn steep liquor dry powder, 0.2-0.6% of urea, 0.02-0.06% of magnesium sulfate, 0.1-0.2% of sodium chloride, 0.1-0.3% of monopotassium phosphate, 0.001-0.003% of manganese sulfate and the balance of water; the pH is 7-7.5, and the product is sterilized by autoclaving at 121 deg.C for 20 min.

The nitrobenzene degrading bacteria are subjected to high-density fermentation culture for 20-30h at the temperature of 32-37 ℃, and after the fermentation is finished, a liquid microbial inoculum is obtained, wherein the effective viable count of the microbial inoculum is 0.5 multiplied by 10⁸～1.5×10¹⁰cfu/mL. Can be further prepared into solid microbial inoculum and the like, and the preparation method adopts the conventional technology.

Furthermore, the fermentation temperature is 35 +/-1 ℃, the dissolved oxygen is about 30 percent, the culture is carried out for 20-30h, the dissolved oxygen rises, the pH value drops, and the fermentation is stopped.

Furthermore, the nitrobenzene degrading bacteria can be continuously cultured and applied in nitrobenzene sewage, and the strain has good ethanol tolerance and can tolerate about 6.5 percent of ethanol concentration at most.

The nitrobenzene degrading bacteria can degrade nitrobenzene into acetone and ammonia nitrogen, has very strong degradation capability, can degrade 900mg/L of nitrobenzene by more than 99% within 24h, can degrade 1000mg/L of nitrobenzene by 87% within 24h, and can degrade by more than 99% within 32 h. The strain has high ethanol tolerance and has a certain advantage effect when being impacted by the outside, which shows that the strain can resist the impact.

When the nitrobenzene degrading bacteria or the microbial inoculum prepared by the nitrobenzene degrading bacteria are applied, the bacterial strain is directly put into nitrobenzene-containing sewage or a treatment system thereof, the temperature is 25-35 ℃, the pH value is 6.5-8.0, and the dissolved oxygen is more than 2mg/L through aeration treatment. As a further implementation, the initial viable count of the nitrobenzene-degrading bacteria in the nitrobenzene sewage is 0.5X 10⁵～7.5×10⁷cfu/mL, and the pH of the sewage is 6.0-7.0.

Compared with the prior art, the invention has the beneficial effects that: the invention provides a nitrobenzene high-efficiency degrading bacterium which has the characteristics of simplicity and high efficiency in application, has strong strain tolerance capability, can tolerate nitrobenzene with high concentration, has wide application range on conditions such as temperature, pH and the like, is not easy to be impacted by external environment in the application process, and has good application value in actual production.

Preservation information

Preservation time: 23/6/2021

The name of the depository: china general microbiological culture Collection center

The preservation number is: CGMCC No.22758

The address of the depository: xilu No. 1 Hospital No. 3 of Beijing market facing Yang district

Classification nomenclature Pseudomonas stutzeri

Drawings

FIG. 1 is a graph showing the comparative degradation ability of the nitrobenzene degrading bacteria in example 5.

FIG. 2 is a graph showing the effect of temperature on the degradation rate of nitrobenzene degrading bacteria in example 5.

FIG. 3 is a graph showing the effect of pH on the degradation rate of nitrobenzene degrading bacteria in example 5.

FIG. 4 shows the results of gas quality measurements of the system before and after the treatment with the nitrobenzene degrading bacteria in example 5.

FIG. 5 is a graph showing the simulated application of nitrobenzene degrading bacteria in example 6;

FIG. 6 is a graph showing the simulated application of the nitrobenzene degrading microbial inoculum of example 7;

FIG. 7 is a graph showing the simulated application of nitrobenzene degrading bacteria in example 8;

Detailed Description

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention, and the following embodiments are all completed by adopting the conventional prior art except for the specific description.

The "dissolved oxygen" of the microbial inoculum fermentation process referred to in the following examples refers to air saturation (%), which is a common method for indicating the dissolved oxygen concentration in the fermentation industry.

The "dissolved oxygen" in the aeration process of sewage treatment in the following examples refers to the absolute concentration of dissolved oxygen in water, expressed in mg/L, and is a common dissolved oxygen expression method for sewage treatment in the environmental protection industry.

Example 1 enrichment, screening and isolation of Nitrobenzene-degrading bacteria

Activated sludge of certain petrochemical sewage treatment plants and leather sewage treatment plants is enriched and screened, and the high-efficiency nitrobenzene degrading bacteria are enriched by adopting a method for gradually increasing the concentration of nitrobenzene.

The specific operation method comprises the following steps: taking 10mL of an activated sludge sample, inoculating the activated sludge sample into 100mL of enrichment medium (the initial concentration of nitrobenzene is 10mg/L), carrying out enrichment culture by shaking and culturing for 2-5d at 30 ℃ in a shaking table at 160r/min, sampling and detecting the content of nitrobenzene in a system, and taking 10mL of bacterial liquid out and inoculating the bacterial liquid into a fresh inorganic salt medium (the concentration of nitrobenzene is 20mg/L) when the content of nitrobenzene is degraded to be lower than 2 mg/L. Repeating the steps, and gradually increasing the concentration of nitrobenzene in the culture medium to 200mg/L by taking 50, 75, 100, 125, 150 and 200mg/L as gradients.

And (3) after the final enrichment culture solution is diluted in a gradient manner, coating the final enrichment culture solution on an inorganic salt culture medium plate which takes 100mg/L nitrobenzene as unique carbon, nitrogen and energy, and culturing for 2-3d in an incubator at 30 ℃. The single colonies with different forms growing on the plate are respectively streaked on an LB plate for purification for at least 3 times, the single colonies after purification are selected and inoculated in 10mL of LB liquid culture medium, the single colonies are respectively inoculated in 100mL of inorganic salt liquid culture medium containing 50mg/L nitrobenzene in an inoculum size of 2% (V/V) the next day, the single colonies are cultured for 72h at 30 ℃ at 160r/min, and then the single colonies are sampled and detected by adopting a reduction-azo photometry method to detect the content of the nitrobenzene. 30 strains of bacteria are separated by the method, wherein the bacteria with the number of N12 can completely degrade 50mg/L nitrobenzene in 72 hours, and the bacteria are determined to be the nitrobenzene degradation bacteria and stored.

The inorganic salt culture medium is composed of Na₂HPO₄·12H₂O3.8 g/L; KH2PO 41 g/L; NaCl 1 g/L; MgSO40.2g/L; the pH is natural, distilled water is used for preparation, sterilization is carried out at the temperature of 121 ℃ for 20min, agar 20g is added into a solid culture medium, and nitrobenzene is added before use.

Nitrobenzene in the culture medium is added after an anhydrous ethanol solution of nitrobenzene is prepared according to 1/150(V/V) for assisting dissolution.

The enrichment medium is prepared from peptone 10.0g, yeast powder 5.0g, sodium chloride 10.0g, water to 1.0L, adjusting pH to 7.0, adding agar 20.0g into solid medium, and sterilizing at 121 deg.C for 20 min.

Example 2 identification of the species of Nitrobenzene-degrading bacteria and the characteristics of the colonies

And (3) strain identification: 16S rDNA identification is carried out on the obtained nitrobenzene degradation bacteria: using primer 27F: 5 '-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5 '-TACCTTGTTACGACTT-3' amplifying the 16S rDNA of the strain, connecting to a cloning vector pMD19T by a T/A cloning mode, constructing a recombinant cloning vector pMD19T-16S, transforming the recombinant cloning vector pMD19T-16S into a cloning host strain Escherichia coli DH5 alpha to obtain a recombinant microorganism Escherichia coli DH5 alpha (p MD19T-16S), sequencing the obtained recombinant microorganism exogenous fragment, comparing the 16S rDNA sequence with an NCBI database, identifying the strain to Pseudomonas stutzeri on a molecular level, and naming the strain to Pseudomonas stutzeri as Pseudomonas stutzeri N12, wherein the nucleotide sequence of the 16S r DNA is shown as SEQ ID in a sequence table.

The nitrobenzene degrading bacteria pseudomonas stutzeri N12 is gram-negative bacteria, and the bacterial colony is circular yellowish, irregular in edge, convex navel-shaped and dry in surface.

EXAMPLE 3 Nitrobenzene degradation microbial inoculum preparation

The nitrobenzene degrading microbial inoculum is prepared by the strain, and the specific preparation method comprises the following steps:

the first step, inoculating the purified pseudomonas stutzeri on a test tube slant culture medium, placing the culture medium in a constant temperature incubator, and culturing until a bacterial colony grows on the culture medium to obtain the pseudomonas stutzeri;

secondly, putting the single colony obtained in the first step into an LB liquid culture medium (100ml), culturing overnight at 35 ℃ and 185r/min to obtain a primary seed solution, inoculating the obtained primary seed solution into the LB liquid culture medium according to 10% (V/V), culturing for 16h under the same condition to obtain a secondary seed solution, and storing at 4 ℃;

and thirdly, inoculating the secondary seed solution of the pseudomonas stutzeri to a fermentation culture medium according to the concentration of 5% (V/V) for fermentation culture, controlling the fermentation temperature to be 35 +/-1 ℃, controlling the dissolved oxygen content to be about 30%, culturing for 20-30h, increasing the dissolved oxygen content, reducing the pH value, and stopping fermentation to obtain the nitrobenzene degradation microbial inoculum.

The number of viable bacteria of the basic degradation microbial inoculum obtained in the embodiment is 0.8 multiplied by 10⁹cfu/mL。

The fermentation medium comprises the following components in percentage by mass: 1.0-2.0% of corn flour, 0.5-1.1% of glucose, 1.8-2.4% of corn steep liquor dry powder, 0.2-0.6% of urea, 0.02-0.06% of magnesium sulfate, 0.1-0.2% of sodium chloride, 0.1-0.3% of monopotassium phosphate, 0.001-0.003% of manganese sulfate, the balance of water, the pH value of 7-7.5, and the corn steep liquor is used after being sterilized under high pressure at 121 ℃ for 20 min.

The LB culture medium is prepared from peptone 10.0g, yeast powder 5.0g, sodium chloride 10.0g, water to 1.0L, adjusting pH to 7.0, and sterilizing at 121 deg.C for 20 min.

EXAMPLE 4 examination of Nitrobenzene degradation characteristics

The nitrobenzene degradation microbial inoculum obtained in the example 3 is inoculated into 5mL to 100mL of inorganic salt culture medium, wherein nitrobenzene is used as the only carbon, nitrogen and energy source for the detection of degradation capability.

Wherein, the nitrobenzene in the culture medium is added after the nitrobenzene absolute ethyl alcohol solution is prepared according to 1/150(V/V) for assisting the dissolution.

Under the above conditions, 7 groups are respectively set, the addition amount of nitrobenzene in the inorganic salt culture medium is 80, 180, 390, 525, 590, 770 and 970mg/L, after inoculation, shaking culture is carried out at 30 ℃ and 160r/min, sampling is carried out after 24h, a sample is centrifuged for 3-5 min at 4000-8000 r/min, and the supernatant is taken for carrying out detection on the content of nitrobenzene, wherein the degradation rate can reach more than 99% when the concentration is 400mg/L (5% of ethanol), the degradation rate is 50% when the concentration is 525mg/L (6.5% of ethanol) in 24h, the degradation rate is 99% when the concentration is 48 mg/L, and the degradation effect is reduced when the concentration is more than 600 mg/L. The bacteria can tolerate 6.5% of ethanol and degrade 500mg/L of nitrobenzene at the same time.

Example 5 detection of degradation characteristics of Nitrobenzene-degrading bacteria

And (3) inoculating the nitrobenzene degrading microbial inoculum obtained in the example 3 into 5mL to 100mL of inorganic salt culture medium, wherein nitrobenzene is used as the only carbon, nitrogen and energy source for degradation performance and detection.

(1) Detection of maximum degradation capacity: under the above conditions, 12 groups are respectively set, the concentration adding amount of nitrobenzene in the inorganic salt culture medium is respectively 90, 180, 300, 400, 480, 560, 700, 780, 850, 1000, 1200 and 1350mg/L, 3 groups are repeated, after inoculation, shaking culture is carried out at 30 ℃ and 160r/min, sampling is carried out after 24h, the sample is centrifuged for 3-5 min at 4000-8000 r/min, and the supernatant is taken for detecting the content of nitrobenzene, wherein when the concentration is 850mg/L, the degradation rate of 24h can reach 99%, when the concentration is 1000mg/L, the degradation rate of 24h is 87%, the degradation rate of 30h can reach more than 99%, and when the concentration is more than 1000mg/L, the degradation capability is reduced, as shown in figure 1.

(2) Influence of the incubation temperature: setting 7 groups, adding 800mg/L nitrobenzene into 3 repeated inorganic salt culture media in each group, setting the pH to be natural, setting different culture temperatures after inoculation, setting the experiment temperature to be 20-40 ℃ (setting the experiment temperature to be 20 ℃, 25 ℃, 28 ℃, 30 ℃, 33 ℃, 35 ℃ and 40 ℃) respectively, placing the mixture in a shaking table for 160r/min for shaking culture, sampling and centrifuging after 24 hours, and detecting to obtain the optimal action temperature of 25-35 ℃, wherein the detailed action temperature is shown in figure 2;

(3) influence of the pH of the medium: the method comprises the steps of setting 11 groups, adding 800mg/L nitrobenzene into 3 repeated inorganic salt culture media in each group, setting different pH values respectively, setting the experimental range of the pH values to be 5-10 (setting the experimental range to be 5,5.5,6,6.5,7,7.5,8,8.5,9,9.5 and 10 respectively), carrying out shake culture in a shaking table at 30 ℃ at 160r/min after inoculation, sampling and centrifuging after 24 hours, detecting to obtain the optimum application pH value of 6.5-8.0, and obtaining the optimum application pH value with a wider adaptable range, wherein the detailed range is shown in figure 3.

The addition of nitrobenzene in the culture medium requires the preparation of nitrobenzene mother liquor: accurately sucking 1333 mu L of nitrobenzene sterilized by a 0.22 mu m filter membrane into 1000mL of inorganic salt culture medium by using a liquid transfer gun, uniformly mixing and standing for 2-3d to obtain the product, and then diluting according to the proportion to perform the experiment.

And (3) detecting the experimental group with the nitrobenzene addition amount of 700mg/L in the detection of the maximum degradation capacity by adopting gas chromatography-mass spectrometry, sampling and centrifuging the system before and after the nitrobenzene degrading bacteria are treated for 16 hours, and then taking the supernatant for detection, wherein the detection result is shown in figure 4.

Example 6 application of Nitrobenzene degrading bacteria in Nitrobenzene Sewage treatment 1

The nitrobenzene degradation microbial inoculum was prepared according to the method of example 3, the culture temperature was 35 + -1 deg.C, the dissolved oxygen was controlled to 30%, and the viable count of the obtained microbial inoculum was 1X 10⁹cfu/mL。

Simulation application: the water quality source for treatment is water inlet of an aerobic section of a leather sewage treatment plant, the treatment capacity is 5L, the addition amount of a nitrobenzene degradation microbial inoculum is 0.1% (V/V), dissolved oxygen used in aeration treatment is more than 2mg/L, the pH of raw sewage is 6.0-8.0, COD is about 3000mg/L, ammonia nitrogen is about 200mg/L and total salt is 3000mg/L, sulfide is 50mg/L, heavy metal chromium is about 500mg/L, the detection concentration of added nitrobenzene is about 93mg/L, and the degradation condition is tracked and shown in figure 5.

Example 7 application of Nitrobenzene degrading bacteria in Nitrobenzene Sewage treatment 2

The nitrobenzene degradation microbial inoculum was prepared according to the method of example 3, the culture temperature was 33 + -1 deg.C, the dissolved oxygen was controlled to 30%, and the viable count of the obtained microbial inoculum was 0.5X 10⁸cfu/mL。

Simulation application: the water quality source for treatment is water inlet at an aerobic section of a pesticide sewage treatment plant, the treatment capacity is 5L, the addition amount of a nitrobenzene degradation microbial inoculum is 0.3% (V/V), dissolved oxygen used in aeration treatment is more than 2mg/L, the pH of the sewage is 6.0-8.0, the COD of the raw sewage is 4500mg/L, ammonia nitrogen is 480mg/L, organic amine is 300mg/L, the concentration of nitrobenzene is less than 100mg/L, the concentration of nitrobenzene is artificially added to 600mg/L, the nitrobenzene content of the sewage is less than 1mg/L after 24 hours, the degradation rate is more than 99.9%, and the degradation process is detected by sampling, tracking and detecting at regular time in the process as shown in figure 6.

Example 8 application of Nitrobenzene degrading bacteria in Nitrobenzene Sewage treatment 3

The nitrobenzene degradation microbial inoculum was prepared according to the method of example 3, the culture temperature was 36 + -1 deg.C, the dissolved oxygen was controlled to 30%, and the viable count of the obtained microbial inoculum was 1.5X 10¹⁰cfu/mL。

The water quality source for treatment is aerobic tank inlet water of a petrochemical sewage treatment plant, the treatment capacity is 5L, the addition amount of a nitrobenzene degrading microbial inoculum is 0.3% (V/V), dissolved oxygen used for aeration treatment is more than 2mg/L, the pH of sewage is 6.0-8.0, the concentration of nitrobenzene is less than 5mg/L, COD is about 2500mg/L, volatile phenol is about 280mg/L, ammonia nitrogen is about 100mg/L, sulfide is about 10mg/L, nitrobenzene is artificially added to 1000mg/L, the nitrobenzene content of sewage is 136mg/L after 24h, the nitrobenzene content is reduced to less than 1mg/L after 32h, the degradation rate is more than 99.9%, and the degradation process is detected by timing sampling, tracking and detection in the process shown in figure 7.

Example 9 application of Nitrobenzene degrading bacteria in Nitrobenzene Sewage treatment 4

The water quality source for treatment is water fed into an aerobic tank of a petrochemical sewage treatment plant, the treatment capacity is 10L, the addition amount of a nitrobenzene degrading microbial inoculum is 0.35% (V/V), the dissolved oxygen is more than 2mg/L through aeration treatment, the pH of sewage is 6.0-8.0, the concentration of nitrobenzene is less than 5mg/L, the COD is about 2500mg/L, the concentration of volatile phenol is about 280mg/L, the concentration of ammonia nitrogen is about 100mg/L, and the concentration of sulfide is about 10mg/L, the content of the influent nitrobenzene is adjusted at any time in the treatment process, 24 hours of continuous water inlet and outlet are realized, the water inlet speed is about 17mL/min, the retention time is about 10 hours, and the effluent nitrobenzene content is sampled and detected, and detailed processes and detection results are shown in the following table:

the experiments and various application simulation results show that the nitrobenzene degrading bacteria have obvious effect when being used in the sewage treatment process, and have higher tolerance to ethanol in a nitrobenzene system based on ethanol.

Sequence listing

<110> Jingbo chemical research institute of yellow river delta Ltd

<120> efficient nitrobenzene degradation bacterium and preparation application thereof

<160> 1

<170> SIPOSequenceListing 1.0

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ctgattcacg attactagcg attccgactt cacgcagtcg agttgcagac tgcgatccgg 180

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gcggtcgact taatgcgtta gctgcgccac taagatctca aggatcccaa cggctagtcg 660

acatcgttta cggcgtggac taccagggta tctaatcctg tttgctcccc acgctttcgc 720

acctcagtgt cagtattgtc agtattagcc caggtggtcg ccttcgccac tggtgttcct 780

tcctatatct acgcatttca ccgctacaca ggaaattcca ccaccctctg ccatactcta 840

gcttgccagt tttggatgca gttcccaggt tgagcccggg gctattcaca ttcaacttaa 900

caaaccacct acgcgcgctt tacgccagta attccgatta acgcttgcac ccttcgtatt 960

accgcggctg ctggcacgaa agttagccgg tgcttattct gtcggtaaac gtcaaaacac 1020

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gctcatctga tagcgcaagg ccc 1283

Claims

1. A nitrobenzene efficient degradation bacterium is characterized in that the bacterium is Pseudomonas stutzeri (Pseudomonas stutzeri) and is preserved in China general microbiological culture Collection center (CGMCC 22758), and the 16SrDNA of the bacterium is shown in SEQ ID NO. 1.

2. The bacterium for degrading nitrobenzene efficiently according to claim 1, wherein the strain can degrade nitrobenzene with concentration of 1000mg/L or less and has tolerance to ethanol.

3. The bacteria for degrading nitrobenzene efficiently according to claim 1, wherein the strain can tolerate ethanol concentrations of 6.5% or less.

4. A nitrobenzene degradation microbial inoculum is characterized in that the preparation method comprises the following steps: performing high-density fermentation culture on the nitrobenzene degradation bacteria of claim 1 at 32-37 ℃ for 20-30 h;

wherein the fermentation medium comprises the following components in percentage by mass: 1.0-2.0% of corn flour, 0.5-1.1% of glucose, 1.8-2.4% of corn steep liquor dry powder, 0.2-0.6% of urea, 0.02-0.06% of magnesium sulfate, 0.1-0.2% of sodium chloride, 0.1-0.3% of monopotassium phosphate, 0.001-0.003% of manganese sulfate and the balance of water.

5. The nitrobenzene degradation microbial inoculum according to claim 4, wherein the effective viable count of the microbial inoculum is 0.5 x 10⁸～1.5×10¹⁰cfu/mL。

6. The nitrobenzene degradation microbial inoculum according to claim 4, wherein in the fermentation culture process, the fermentation temperature is 35 plus or minus 1 ℃, the dissolved oxygen is 30 percent, the fermentation is stopped after the culture is carried out for 20 to 30 hours, the dissolved oxygen is increased, the pH is reduced.

7. The use of the bacterium of claim 1 for degrading nitrobenzene.

8. The method for treating nitrobenzene-containing sewage by using the efficient nitrobenzene degradation bacteria of claim 1, wherein the bacterial strain or the microbial inoculum thereof is put into the nitrobenzene-containing sewage or a treatment system thereof at a temperature of 25-35 ℃ and a pH of 6.5-8.0, and the dissolved oxygen is aerated to be more than 2 mg/L.

9. The method according to claim 8, wherein the number of viable nitrobenzene-degrading bacteria in the wastewater is 0.5X 10⁵～7.5×10⁷cfu/mL。