CN113801828B - High-efficiency nitrobenzene degrading bacterium and preparation and application thereof - Google Patents
High-efficiency nitrobenzene degrading bacterium and preparation and application thereof Download PDFInfo
- Publication number
- CN113801828B CN113801828B CN202111310312.9A CN202111310312A CN113801828B CN 113801828 B CN113801828 B CN 113801828B CN 202111310312 A CN202111310312 A CN 202111310312A CN 113801828 B CN113801828 B CN 113801828B
- Authority
- CN
- China
- Prior art keywords
- nitrobenzene
- degrading
- degrading bacteria
- bacteria
- application
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/348—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the way or the form in which the microorganisms are added or dosed
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Water Supply & Treatment (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Environmental & Geological Engineering (AREA)
- Hydrology & Water Resources (AREA)
- Biodiversity & Conservation Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of microorganisms, in particular to a nitrobenzene efficient degradation bacterium and a preparation method and application thereof. The degrading bacteria belong to gram-negative bacteria, the molecular level of the degrading bacteria is identified as pseudomonas stutzeri, the preservation number of the degrading bacteria is CGMCC22758, the nitrobenzene degrading bacteria can degrade nitrobenzene by over 99 percent in 24 hours, nitrobenzene by over 900mg/L can degrade by over 99 percent in 32 hours, the optimal action temperature is 25-35 ℃, the optimal action pH is 6.5-8.0, the degrading bacteria can be continuously cultured in the application of nitrobenzene sewage treatment, nitrobenzene by 500mg/L can be stably degraded in 24 hours, the strain tolerance capability is strong, nitrobenzene with high concentration can be tolerated, the application range of conditions such as temperature, pH is wide, the nitrobenzene is not easy to be impacted by external environment in the application process, and the degrading bacteria have good application value in practical production.
Description
Technical Field
The invention relates to the field of microbial agents, in particular to a nitrobenzene efficient degradation bacterium and a preparation method and application thereof.
Background
Nitrobenzene is a widely used raw material for organic synthesis in industrial production, and is widely used in industrial production, so that a large amount of residue exists in industrial wastewater such as pharmacy, 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 as the front of the environment to preferentially control toxic organic pollutants. With the development of chemical industry, a great deal of nitrobenzene wastewater is produced to cause serious pollution to the environment, so in order to eliminate or reduce the pollution caused by nitrobenzene, the harm caused by nitrobenzene pollution needs to be reduced by artificial measures.
At present, the nitrobenzene treatment technology mainly comprises a physical method, a chemical method and a biological method, and the physicochemical method has high treatment efficiency and relatively simple process, but the reaction conditions generally have special requirements, the treatment cost is also higher, and 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 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 to actual wastewater treatment engineering.
Biological treatment methods of nitrobenzene wastewater can be classified into an anaerobic method and an aerobic method. Nitrobenzene is easily reduced to aniline under anaerobic conditions, and aniline can be further thoroughly mineralized. In comparison with the anaerobic method, nitrobenzene can be mineralized directly under aerobic conditions, so that a large number of aerobic nitrobenzene degrading bacteria including prokaryotes and eukaryotes have been isolated in recent years, wherein prokaryotes are mainly bacteria and eukaryotes are mainly white rot fungi. However, nitrobenzene has a toxic effect on microorganisms due to its poor biodegradability, thereby inhibiting the growth of microorganisms and even killing microorganisms.
In the prior art, wei Chaohai and the like describe three aerobic degradation bacteria separated in the 'co-matrix and biological synergistic effect of nitrobenzene aerobic degradation', wherein nitrobenzene can be used as the only carbon, nitrogen and energy, and the effect is best that one bacillus subtilis strain degrades 200mg/L for 145 h; wang Jing et al, in the article "Pseudomonas JX165 and aerobic degradation of the whole cell paranitrobenzene thereof", indicate that the maximum tolerance concentration of the separated Pseudomonas paranitrobenzene is 800mg/L, and the separated Pseudomonas paranitrobenzene can be completely degraded within 60 hours when the initial concentration of the nitrobenzene is 200mg/L; lu Wenxiang in the study of separation and degradation characteristics of nitrobenzene degrading bacteria Pseudomonas sp.XY-1, the degradation rate of the separated Pseudomonas to nitrobenzene with the initial concentration of 200mg/L is 97% in 24 hours; zheng Chunli and the like can degrade nitrobenzene with the concentration of 200mg/L in 60 hours; cai Bangcheng in the screening of a nitrobenzene efficient degrading bacterium and the degradation characteristic thereof, the maximum degradation capacity of Acinetobacter for 24 hours separated from the sewage sludge of a chemical plant is 400mg/L. In addition, the pseudomonas 24h provided in the application CN1570079A is a nitrobenzene pollutant high-efficiency degrading bacterium, can degrade 433mg/L, and can degrade 98% for 796mg/L nitrobenzene 100 h. However, the important information such as the preservation of the strain is not disclosed, and the kind of the Pseudomonas is not known.
From the current report, the degradation efficiency of nitrobenzene degrading bacteria is generally low, and the nitrobenzene degrading bacteria have some influence on the application of nitrobenzene degrading bacteria in actual production. Therefore, the separation and breeding of strains with high tolerance and degradation capability are particularly important to be applied to the bio-enhancement technology.
Disclosure of Invention
The invention aims to provide nitrobenzene degrading bacteria 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 nitrobenzene high-efficiency degradation bacteria of the invention collect water samples from sewage treatment systems of petrochemical and leather sewage plants, enrich the water samples, and further enrich the water samples in an inorganic salt culture medium with nitrobenzene as unique carbon, nitrogen and energy sources in a gradient way, and separate and purify the water samples.
According to detection, the nitrobenzene degrading bacteria are gram-negative bacteria, the bacterial colony is circular light yellow, the edge is irregular, the convex umbilicus is shaped like a navel, the surface is dry, the nitrobenzene degrading bacteria are identified as pseudomonas stutzeri (Pseudomonas stutzeri) by 16S rDNA, and the nitrobenzene degrading bacteria are preserved in China general microbiological culture collection center (CGMCC) 22758).
As a further scheme, the invention provides a microbial inoculum of the nitrobenzene degrading bacteria, which is prepared by fermentation:
the fermentation medium comprises the following components in percentage by mass: corn flour 1.0-2.0%, glucose 0.5-1.1%, corn steep liquor dry powder 1.8-2.4%, urea 0.2-0.6%, magnesium sulfate 0.02-0.06%, sodium chloride 0.1-0.2%, potassium dihydrogen phosphate 0.1-0.3%, manganese sulfate 0.001-0.003%, and water for the rest; the pH value is 7-7.5, and the preparation is used after being autoclaved at 121 ℃ for 20min.
The nitrobenzene degrading bacteria are subjected to high-density fermentation culture for 20-30h at 32-37 ℃ to obtain liquid microbial inoculum, wherein the effective viable count of the microbial inoculum is 0.5X10 8 ～1.5×10 10 cfu/mL. The preparation method can be further prepared into solid microbial inoculum and the like, and the preparation method adopts conventional technology.
Further, the fermentation temperature is 35+/-1 ℃, the dissolved oxygen is about 30 percent, the culture is carried out for 20-30 hours, the dissolved oxygen is increased, the pH value is reduced, 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 the ethanol concentration of about 6.5% at most.
The nitrobenzene degrading bacteria can degrade nitrobenzene into acetone and ammonia nitrogen, has very strong degradation capacity, can degrade 900mg/L nitrobenzene by more than 99% in 24 hours, and degrade 1000mg/L nitrobenzene by more than 87% in 24 hours and more than 99% in 32 hours. The strain has higher ethanol tolerance and has certain advantage effect when being impacted by the outside, which indicates that the strain can resist the impact.
When the nitrobenzene degrading bacteria or the microbial inoculum prepared by the nitrobenzene degrading bacteria are used, the strain is directly put into nitrobenzene-containing sewage or a treatment system thereof, the temperature is 25-35 ℃, the pH is 6.5-8.0, and the aeration treatment is carried out to dissolve oxygenAbove 2 mg/L. As a further implementation, the initial viable count of nitrobenzene degrading bacteria in nitrobenzene sewage is 0.5X10 5 ～7.5×10 7 cfu/mL, and the pH of sewage is 6.0-7.0.
Compared with the prior art, the invention has the beneficial effects that: the nitrobenzene high-efficiency degradation bacterium has the characteristics of simplicity and high efficiency in application, meanwhile, the strain has strong tolerance capability, can tolerate high-concentration nitrobenzene, has wide application range to 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: 2021, 6, 23
Preservation unit name: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
Preservation number: CGMCC No.22758
Deposit unit address: beijing city, chaoyang area, north Chenxi Lu No. 1 and 3
Classification naming Pseudomonas stutzeri (Pseudomonas stutzeri)
Drawings
FIG. 1 is a graph showing the degradation ability of 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 detection of the system before and after the nitrobenzene degrading bacteria treatment in example 5.
FIG. 5 is a graph of the trace degradation of the simulated application of the nitrobenzene degrading bacterial agent of example 6;
FIG. 6 is a graph of the trace degradation of the simulated application of the nitrobenzene degrading bacterial agent of example 7;
FIG. 7 is a graph of the trace degradation of the simulated application of the nitrobenzene degrading bacterial agent of example 8;
Detailed Description
The above-described aspects of the present invention will be described in further detail with reference to the following detailed description, 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 techniques realized based on the above description of the present invention are within the scope of the present invention, and the following examples are accomplished by conventional techniques unless otherwise specified.
The term "dissolved oxygen" in the microbial inoculum fermentation process in the following examples refers to air saturation (%), and is a general expression method of 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 separation of Nitrobenzene degrading bacteria
And (3) taking activated sludge of a petrochemical sewage treatment plant and a leather sewage treatment plant for enrichment and screening treatment, and enriching high-efficiency nitrobenzene degrading bacteria by adopting a method for gradually increasing nitrobenzene concentration.
The specific operation method comprises the following steps: taking 10mL of activated sludge sample, inoculating the activated sludge sample into 100mL of enrichment culture medium (initial concentration of nitrobenzene is 10 mg/L), performing shake culture at 30 ℃ for 2-5d by using a shaking table at 160r/min to perform enrichment culture, taking out 10mL of bacterial liquid when the content of nitrobenzene in the sampling detection system is degraded to be lower than 2mg/L, and inoculating the bacterial liquid into fresh inorganic salt culture medium (concentration of nitrobenzene is 20 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.
The final enrichment culture solution is coated on an inorganic salt culture medium flat plate which takes 100mg/L nitrobenzene as the only carbon, nitrogen and energy source after gradient dilution, and is cultured for 2-3d in a 30 ℃ incubator. Single colonies with different forms which are grown on the flat plate are respectively streaked on an LB flat plate for purification for at least 3 times, the single colonies are picked up after the purification 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 of nitrobenzene with the inoculum size of 2% (V/V) in the next day, the single colonies are cultured for 72 hours at 30 ℃ and 160r/min, and then the sample is sampled and the content of the nitrobenzene is detected by adopting a reduction-azo photometry. 30 strains of bacteria are separated by the method, wherein 50mg/L nitrobenzene can be completely degraded within 72h by the bacteria with the number of N12, and the bacteria are determined to be nitrobenzene degrading bacteria and are stored.
The inorganic salt culture medium is prepared from Na 2 HPO 4 ·12H 2 O3.8 g/L; KH2PO4 1g/L; naCl 1g/L; mgSO4 0.2g/L; the pH is natural, the preparation is carried out by using distilled water, the sterilization is carried out for 20min at 121 ℃, the agar is added into the solid culture medium for 20g, and nitrobenzene is added before use.
Nitrobenzene is added in the culture medium, and nitrobenzene absolute ethanol solution is prepared according to 1/150 (V/V) for dissolution assistance and then is added.
The enrichment medium is prepared by adding 10.0g of peptone, 5.0g of yeast powder and 10.0g of sodium chloride into 1.0L of water, adjusting the pH value to 7.0, adding 20.0g of agar into the solid medium, and sterilizing for 20min at 121 ℃.
Example 2 identification of species and colony characteristics of Nitrophenyl degrading bacteria
And (3) strain identification: and (3) carrying out 16S rDNA identification on the obtained nitrobenzene degrading bacteria: using primer 27F:5 '-AGAGTTTGATCCTGGCTCAG-3' and 1492R:5 '-TACCTTGTTACGACTT-3' amplifying 16S rDNA of the strain, connecting the strain to a cloning vector pMD19T in a T/A cloning mode, constructing a recombinant cloning vector pMD19T-16S, transforming the recombinant cloning vector pMD19T-16S into cloning host bacteria Escherich coli DH alpha to obtain recombinant microorganism Escherich coli DH5 alpha (pMD 19T-16S), sequencing the obtained exogenous fragment of the recombinant microorganism, comparing the 16S rDNA sequences by NCBI database, identifying that the strain belongs to Pseudomonas stutzeri at a molecular level, and naming the strain 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 bacterium pseudomonas stutzeri N12 is gram negative bacteria, bacterial colony is round faint yellow, irregular in edge, convex umbilical and dry in surface.
EXAMPLE 3 preparation of Nitrobenzene degrading microbial inoculant
The nitrobenzene degrading bacterial agent is prepared by utilizing the bacterial strain, and the specific preparation method is as follows:
inoculating the purified pseudomonas stutzeri on a test tube slant culture medium, placing the test tube slant culture medium in a constant temperature incubator, and culturing until a colony grows on the culture medium to obtain the pseudomonas stutzeri;
secondly, culturing the single colony obtained in the first step in LB liquid medium (100 ml) at the temperature of 35 ℃ and 185r/min overnight to obtain primary seed liquid, inoculating the obtained primary seed liquid into LB medium according to 10% (V/V), culturing for 16 hours under the same conditions to obtain secondary seed liquid, and storing at the temperature of 4 ℃;
thirdly, inoculating the second-level seed solution of pseudomonas stutzeri into a fermentation culture medium according to 5% (V/V) for fermentation culture, controlling the fermentation temperature to be 35+/-1 ℃ and the dissolved oxygen to be about 30%, culturing for 20-30 hours, raising the dissolved oxygen, lowering the pH value, and stopping fermentation to obtain the nitrobenzene degradation microbial inoculum.
The viable count of the basic degrading microbial inoculum obtained in the embodiment is 0.8X10 9 cfu/mL。
The fermentation medium comprises the following components in percentage by mass: corn flour 1.0-2.0%, glucose 0.5-1.1%, corn steep liquor dry powder 1.8-2.4%, urea 0.2-0.6%, magnesium sulfate 0.02-0.06%, sodium chloride 0.1-0.2%, potassium dihydrogen phosphate 0.1-0.3%, manganese sulfate 0.001-0.003%, water balance, pH7-7.5, and sterilizing under high pressure at 121deg.C for 20min.
The LB culture medium is prepared from 10.0g of peptone, 5.0g of yeast powder and 10.0g of sodium chloride by adding water to 1.0L, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 20min.
Example 4 detection of degradation Properties of Nitrobenzene
The nitrobenzene degrading bacterial agent 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, and the degradation capacity is detected.
Wherein nitrobenzene is added in the culture medium, nitrobenzene absolute ethanol solution is prepared according to 1/150 (V/V) for dissolution assistance, and then the nitrobenzene absolute ethanol solution is added.
Under the conditions, 7 groups are respectively arranged, the adding amount of nitrobenzene in the inorganic salt culture medium is 80, 180, 390, 525, 590, 770 and 970mg/L, shake culture is carried out at 30 ℃ and 160r/min after inoculation, sampling is carried out after 24 hours, the sample is centrifuged for 3-5 min at 4000-8000 r/min, the supernatant is taken for detecting the nitrobenzene content, the degradation rate can reach more than 99% when the concentration is 400mg/L (ethanol content is 5%), the 24-hour degradation rate is 50% when the degradation rate is 525mg/L (ethanol content is 6.5%), and the degradation effect is reduced when the degradation rate is 48 hours is 99% and 600 mg/L. The bacterium can tolerate 6.5% ethanol and degrade 500mg/L nitrobenzene.
EXAMPLE 5 detection of degradation Properties of Nitrobenzene degrading bacteria
The nitrobenzene degrading bacterial agent 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, and degradation performance and detection are carried out.
(1) Detection of maximum degradation capability: under the above conditions, 12 groups are respectively arranged, 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, shake culture is carried out under the condition of 30 ℃ and 160r/min after inoculation, sampling is carried out after 24 hours, the sample is centrifuged for 3-5 minutes at 4000-8000 r/min, the supernatant is taken for detecting the nitrobenzene content, when the concentration is 850mg/L, the degradation rate of 24 hours can reach 99%, when the concentration is 1000mg/L, the degradation rate of 24 hours is 87%, the degradation rate of 30 hours can reach more than 99%, and when the concentration is above 1000mg/L, the degradation capacity is reduced, as shown in figure 1.
(2) Influence of the culture temperature: setting 7 groups, adding 800mg/L nitrobenzene into 3 repeated inorganic salt culture mediums of each group, naturally adjusting pH, respectively setting different culture temperatures after inoculation, setting experiment temperatures to 20-40 ℃ (20 ℃,25 ℃,28 ℃,30 ℃,33 ℃,35 ℃ and 40 ℃ respectively), placing the culture mediums in a shaking table for 160r/min for shake culture, sampling and centrifuging after 24 hours, and detecting to obtain the optimal acting temperature of the culture mediums to be 25-35 ℃, as shown in figure 2;
(3) Influence of medium pH: setting 11 groups, adding 800mg/L nitrobenzene into 3 repeated inorganic salt culture mediums of each group, respectively setting different pH values, setting the experimental range of the pH values to be 5-10 (5,5.5,6,6.5,7,7.5,8,8.5,9,9.5,10 respectively), carrying out shake culture at 30 ℃ in a shaking table at 160r/min after inoculation, sampling and centrifuging after 24 hours, and detecting to obtain the pH value for the best adaptation to be 6.5-8.0, wherein the application range is wider, and shown in figure 3 in detail.
The nitrobenzene mother liquor needs to be prepared firstly for adding nitrobenzene in the culture medium: accurately sucking 1333 mu L nitrobenzene sterilized by a 0.22 mu m filter membrane into 1000mL inorganic salt culture medium by a pipette, uniformly mixing, standing for 2-3d to obtain the nitrobenzene, and then diluting according to proportion to perform experiments.
And in the experimental group with the nitrobenzene addition amount of 700mg/L in the detection of the maximum degradation capacity, the detection is carried out by adopting gas chromatography-mass spectrometry, the system before and after the nitrobenzene degradation bacteria are treated is sampled and centrifuged for 16 hours, the supernatant is taken for detection, the detection result is shown in figure 4, and from the detection result, after the nitrobenzene is metabolized, the nitrogen source is converted into ammonia nitrogen, the carbon source is converted into acetone and carbon dioxide, so that the thorough mineralization of the nitrobenzene is realized.
Example 6 application of nitrobenzene degrading bacteria in nitrobenzene wastewater treatment 1
A nitrobenzene degrading bacterial preparation was prepared in the same manner as in example 3, the cultivation temperature was 35.+ -. 1 ℃ and the dissolved oxygen was controlled to 30%, and the viable count of the bacterial preparation obtained was 1X 10 9 cfu/mL。
Simulation application: the source of the treated water is the water entering an aerobic section of a leather sewage treatment plant, the treatment capacity is 5L, the adding amount of nitrobenzene degrading bacteria is 0.1% (V/V), the aeration treatment uses dissolved oxygen above 2mg/L, the pH of raw sewage is 6.0-8.0, COD is about 3000mg/L, ammonia nitrogen is about 200mg/L, 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 as shown in figure 5.
Example 7 application of nitrobenzene degrading bacteria in nitrobenzene wastewater treatment 2
A nitrobenzene degrading bacterial preparation was prepared in the same manner as in example 3, the cultivation temperature was 33.+ -. 1 ℃ and the dissolved oxygen was 30% to give a bacterial preparation viable count of 0.5X10 8 cfu/mL。
Simulation application: the source of the treated water is the inflow water of an aerobic section of a pesticide sewage treatment plant, the treatment capacity is 5L, the addition amount of nitrobenzene degrading bacteria is 0.3% (V/V), dissolved oxygen is used for aeration treatment and is above 2mg/L, the pH value of sewage is 6.0-8.0, the COD of raw sewage is about 4500mg/L, the ammonia nitrogen is about 480mg/L, the concentration of organic amine is about 300mg/L, the concentration of nitrobenzene is below 100mg/L, the concentration of nitrobenzene is artificially added to 600mg/L, the nitrobenzene content of sewage is less than 1mg/L after 24h, the degradation rate reaches above 99.9%, and the degradation process of the sewage is timely sampled, tracked and detected in the process, which is shown in figure 6.
Example 8 application of nitrobenzene degrading bacteria in nitrobenzene wastewater treatment 3
A nitrobenzene degrading bacterial preparation was prepared in the same manner as in example 3, the cultivation temperature was 36.+ -. 1 ℃ and the dissolved oxygen was controlled to 30%, and the viable count of the bacterial preparation obtained was 1.5X10 10 cfu/mL。
The source of the treated water is the inflow water of an aerobic tank of a petrochemical sewage treatment plant, the treatment capacity is 5L, the addition amount of nitrobenzene degrading bacteria is 0.3% (V/V), dissolved oxygen is used for aeration treatment and is above 2mg/L, the pH of sewage is 6.0-8.0, the concentration of nitrobenzene is below 5mg/L, COD is about 2500mg/L, volatile phenol is about 280mg/L, ammonia nitrogen is about 100mg/L, sulfide is about 10mg/L, the concentration of nitrobenzene is artificially added to 1000mg/L, the nitrobenzene content of sewage is 136mg/L when the nitrobenzene content is reduced to less than 1mg/L for 24 hours, the degradation rate is above 99.9%, and the degradation process is shown in figure 7 after the timed sampling tracking detection.
Example 9 application of nitrobenzene degrading bacteria in nitrobenzene wastewater treatment 4
A nitrobenzene degrading bacterial preparation was prepared in the same manner as in example 3, the cultivation temperature was 36.+ -. 1 ℃ and the dissolved oxygen was controlled to 30%, and the viable count of the bacterial preparation obtained was 1.5X10 10 cfu/mL。
The source of the treated water is the inflow water of an aerobic tank of a petrochemical sewage treatment plant, the treatment capacity is 10L, the addition amount of nitrobenzene degrading bacteria is 0.35% (V/V), the dissolved oxygen is more than 2mg/L by aeration treatment, the pH value of sewage is 6.0-8.0, the concentration of nitrobenzene is less than 5mg/L, the COD is about 2500mg/L, the volatile phenol is about 280mg/L, the ammonia nitrogen is about 100mg/L, the sulfide is about 10mg/L, the content of nitrobenzene which is inflow water is regulated at any time in the treatment process, the inflow water is continuously inflow and outflow water for 24 hours, the inflow water speed is about 17mL/min, the retention time is about 10 hours, the nitrobenzene content of water is sampled and detected, and the detailed process and the detection result are shown in the following table:
the experiment and the simulation results of each application show that the nitrobenzene degrading bacteria have remarkable effect in the sewage treatment process, and have higher tolerance to ethanol in a nitrobenzene system based on ethanol.
Sequence listing
<110> yellow river delta Beijing and Bo chemical industry institute of Co., ltd
<120> a strain of nitrobenzene high-efficiency degradation bacteria and preparation and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1283
<212> DNA
<213> Pseudomonas stutzeri (Pseudomonas stutzeri)
<400> 1
cggacggaat cgtcgaggta ccgtcccccg aaggttagac tagctacttc tggagcaacc 60
cactcccatg gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgtgacatt 120
ctgattcacg attactagcg attccgactt cacgcagtcg agttgcagac tgcgatccgg 180
actacgatcg gttttatggg attagctcca cctcgcggct tggcaaccct ttgtaccgac 240
cattgtagca cgtgtgtagc ccaggccgta agggccatga tgacttgacg tcatccccac 300
cttcctccgg tttgtcaccg gcagtctcct tagagtgccc accttaacgt gctggtaact 360
aaggacaagg gttgcgctcg ttacgggact taacccaaca tctcacgaca cgagctgacg 420
acagccatgc agcacctgtg tcagagttcc cgaaggcacc aatccatctc tggaaagttc 480
tctgcatgtc aaggcctggt aaggttcttc gcgttgcttc gaattaaacc acatgctcca 540
ccgcttgtgc gggcccccgt caattcattt gagttttaac cttgcggccg tactccccag 600
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
taacgtatta ggttaatgcc cttcctccca acttaaagtg ctttacaatc cgaagacttc 1080
ttcacacacg cggcatggct ggatcaggct ttcgcccatt gtccaatatt ccccactgct 1140
gcctcccgta ggagtctgca ccgtgtctca gttccagtgt gactgatcat cctctcagac 1200
cagttacgga tcgtcgcctt ggtgagccgt tacctcacca actagctaat cctgacctag 1260
gctcatctga tagcgcaagg ccc 1283
Claims (9)
1. A nitrobenzene high-efficiency degrading bacterium is characterized in that the strain is pseudomonas stutzeri (Pseudomonas stutzeri), and is preserved in China general microbiological culture Collection center (CGMCC) 22758, and the 16SrDNA is shown as SEQ ID NO. 1.
2. The nitrobenzene high-efficiency degrading bacterium according to claim 1, wherein the strain can degrade nitrobenzene below 1000mg/L and has tolerance to ethanol.
3. The nitrobenzene highly effective degrading bacterium as set forth in claim 1, wherein the strain can tolerate ethanol concentration of 6.5% and below.
4. A nitrobenzene degrading bacterial agent is characterized in that the preparation method comprises the following steps: culturing the nitrobenzene degrading bacteria of claim 1 at 32-37 ℃ for 20-30h by high-density fermentation;
wherein, the fermentation culture medium comprises the following components in percentage by mass: corn flour 1.0-2.0%, glucose 0.5-1.1%, corn steep liquor dry powder 1.8-2.4%, urea 0.2-0.6%, magnesium sulfate 0.02-0.06%, sodium chloride 0.1-0.2%, potassium dihydrogen phosphate 0.1-0.3%, manganese sulfate 0.001-0.003%, and water in balance.
5. The nitrobenzene degrading bacterial agent as set forth in claim 4, wherein the effective viable count of the bacterial agent is 0.5X10 8 ～1.5×10 10 cfu/mL。
6. The nitrobenzene degrading bacterial agent according to claim 4, wherein in the fermentation culture process, the fermentation temperature is 35+/-1 ℃, dissolved oxygen is 30%, the culture is carried out for 20-30 hours, the dissolved oxygen is increased, the pH is reduced, and the fermentation is stopped.
7. The use of the nitrobenzene high-efficiency degrading bacterium in degrading nitrobenzene of claim 1.
8. The method for treating nitrobenzene-containing sewage by using the nitrobenzene high-efficiency degradation bacteria as defined in claim 1, which is characterized in that the bacterial strain or the bacterial agent thereof is put into the nitrobenzene-containing sewage or the treatment system thereof, the temperature is 25-35 ℃, the pH is 6.5-8.0, and the dissolved oxygen is more than 2mg/L by aeration treatment.
9. The method for treating nitrobenzene-containing sewage as claimed in claim 8, wherein the initial viable count of nitrobenzene degrading bacteria in nitrobenzene sewage is 0.5 x 10 5 ～7.5×10 7 cfu/mL。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111310312.9A CN113801828B (en) | 2021-11-05 | 2021-11-05 | High-efficiency nitrobenzene degrading bacterium and preparation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111310312.9A CN113801828B (en) | 2021-11-05 | 2021-11-05 | High-efficiency nitrobenzene degrading bacterium and preparation and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113801828A CN113801828A (en) | 2021-12-17 |
| CN113801828B true CN113801828B (en) | 2023-05-12 |
Family
ID=78898597
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111310312.9A Active CN113801828B (en) | 2021-11-05 | 2021-11-05 | High-efficiency nitrobenzene degrading bacterium and preparation and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113801828B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101096644A (en) * | 2006-06-26 | 2008-01-02 | 谢明 | Pseudomonas stutzeri JSD-008 and its degradation function for organophosphorus pesticide |
| CN101496820A (en) * | 2009-02-26 | 2009-08-05 | 大连交通大学 | Active extract of facultative anaerobic sea Pseudomonas stuszeri as well as production method and use thereof |
| CN103667161A (en) * | 2013-12-25 | 2014-03-26 | 天津凯英科技发展有限公司 | Pseudomonas stutzeri KY-02 and application thereof |
| CN111607543A (en) * | 2020-06-28 | 2020-09-01 | 黄河三角洲京博化工研究院有限公司 | Pseudomonas stutzeri with aerobic denitrification function and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200318163A1 (en) * | 2019-03-29 | 2020-10-08 | Metabolik Technologies Inc. | Biodegradation of toxic organic compounds in contaminated environments |
-
2021
- 2021-11-05 CN CN202111310312.9A patent/CN113801828B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101096644A (en) * | 2006-06-26 | 2008-01-02 | 谢明 | Pseudomonas stutzeri JSD-008 and its degradation function for organophosphorus pesticide |
| CN101496820A (en) * | 2009-02-26 | 2009-08-05 | 大连交通大学 | Active extract of facultative anaerobic sea Pseudomonas stuszeri as well as production method and use thereof |
| CN103667161A (en) * | 2013-12-25 | 2014-03-26 | 天津凯英科技发展有限公司 | Pseudomonas stutzeri KY-02 and application thereof |
| CN111607543A (en) * | 2020-06-28 | 2020-09-01 | 黄河三角洲京博化工研究院有限公司 | Pseudomonas stutzeri with aerobic denitrification function and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113801828A (en) | 2021-12-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107201325B (en) | Pseudomonas strain and culture method and application thereof | |
| CN110643548B (en) | Microbacterium flavum for degrading aniline and application thereof | |
| CN112725240B (en) | Acinetobacter livelii, and microbial inoculum and application thereof | |
| CN114703095B (en) | Pseudomonas adulthood and application thereof in field of sewage and wastewater purification | |
| CN108300674B (en) | Petroleum degrading bacteria, obtaining method thereof and application of petroleum degrading bacteria in crude oil degradation | |
| CN115786179A (en) | Bacterial strain for degrading o-dichlorobenzene and application thereof | |
| CN104371948A (en) | Microbacterium sp. strain and application thereof | |
| CN114292764B (en) | Achromobacter strain JD417 and application thereof | |
| CN114045239B (en) | Paracoccus ubiquitously YBH-7 with dimethylacetamide degradation capability and application thereof | |
| CN113980851B (en) | Paracoccus YBH-X with dimethylacetamide degradation capability and application thereof | |
| CN113249273B (en) | Salt-tolerant bacillus subtilis and application thereof in high-salt ammonia nitrogen wastewater treatment | |
| CN109706101B (en) | Sporosarcina psychrophilum and application thereof | |
| CN113801828B (en) | High-efficiency nitrobenzene degrading bacterium and preparation and application thereof | |
| CN115125174B (en) | Lead-resistant bacillus pumilus and application thereof | |
| CN114107068B (en) | Fungus capable of degrading petroleum hydrocarbon and application of fungus in petroleum degradation | |
| CN114045238B (en) | Rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide and application thereof | |
| CN116004459A (en) | Rhodococcus YZ-1 and application thereof in degrading organic pollutants | |
| CN116333916A (en) | High-efficiency nitrobenzene degrading microbial inoculum, preparation and application thereof | |
| CN104745515A (en) | Acinetobacter sp. for degrading polycyclic aromatic hydrocarbon and application of acinetobacter sp. | |
| CN115960745A (en) | Salt-tolerant epichlorohydrin degrading bacterium and application thereof in treatment of epichlorohydrin in high-salinity water | |
| CN102021126A (en) | Salt-resistant degrading bacterium for treating high-concentration persistent organic wastewater and application thereof | |
| CN108467842B (en) | Bacterial strain and application thereof | |
| CN114214254B (en) | Rhodococcus ruber and application thereof | |
| CN114107076B (en) | Candida tropicalis strain EP1 and application thereof | |
| CN113862177B (en) | Lysogen-aminoglutaric acid bacillus for synchronously degrading mixed phenol and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |