CN118126871A - Pseudomonas and application thereof in degradation of terpineol and restoration of polluted soil - Google Patents

Pseudomonas and application thereof in degradation of terpineol and restoration of polluted soil Download PDF

Info

Publication number
CN118126871A
CN118126871A CN202410122942.0A CN202410122942A CN118126871A CN 118126871 A CN118126871 A CN 118126871A CN 202410122942 A CN202410122942 A CN 202410122942A CN 118126871 A CN118126871 A CN 118126871A
Authority
CN
China
Prior art keywords
terpineol
pseudomonas
degradation
soil
biochar
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.)
Pending
Application number
CN202410122942.0A
Other languages
Chinese (zh)
Inventor
刘白林
赵海明
莫测辉
叶锦成
向垒
蔡全英
李彦文
李慧
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jinan University
Original Assignee
Jinan University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Jinan University filed Critical Jinan University
Priority to CN202410122942.0A priority Critical patent/CN118126871A/en
Publication of CN118126871A publication Critical patent/CN118126871A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • C12N1/205—Bacterial isolates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • 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
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Inorganic Chemistry (AREA)
  • Environmental & Geological Engineering (AREA)
  • Soil Sciences (AREA)
  • Mycology (AREA)
  • Molecular Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a Pseudomonas strain and application thereof in degradation of terpineol and restoration of polluted soil, wherein the preservation number of the Pseudomonas sp 4 is GDMCC No:63948, which was deposited at the cantonese collection of microorganism strains on the 10 th month 31 of 2023. The invention provides Pseudomonas sp (Pseudomonas sp.) S4 with a high-efficiency terpineol degradation function, which has a degradation rate of up to 95% for 350mg/L terpineol after being cultured under laboratory conditions, generates a composite surfactant containing monose rhamnolipid and bisrhamnolipid, can be used for repairing terpineol-polluted soil, increases the diversity of microorganism species in the soil, can exert synergistic effect when being combined with rhamnolipid or biochar, has more remarkable repairing effect, provides a new technical choice for treating terpineol pollution, and has wide application prospect.

Description

Pseudomonas and application thereof in degradation of terpineol and restoration of polluted soil
Technical Field
The invention relates to the technical field of microorganisms, in particular to pseudomonas and application thereof in degradation of terpineol and restoration of polluted soil.
Background
Flotation agents are drugs used for mineral separation, which are performed by varying the physicochemical properties and hydrophobicity of the surface of different minerals. With the transition of the times, as the demands of people for mineral resources are larger and larger, the scale and exploitation strength of mines are continuously enlarged, so that a large amount of organic flotation agents are widely applied to the mining process. The mine flotation agents are of a wide variety and commonly used include collectors (xanthates, black agents, white agents, fatty acids, mineral oils, etc.), foaming agents (terpineols, creosotes, alcohols, etc.), and conditioning agents (lime, copper sulphate, sodium sulphide, sodium carbonate, sulphur dioxide). In the mining process, the flotation reagent can permeate into the ground to pollute the underground water and farmland.
Terpineol (Terpineol) is commonly called terpineol, and is one of the most widely used foaming agents in flotation because of the simple preparation method, low cost, strong foam stability formed in mining and obvious collecting effect. The terpineol is prepared by adding an emulsifier into turpentine serving as a raw material, performing hydration reaction at normal temperature, and dehydrating. Terpineol has a cyclic structure, has three isomers (alpha-terpineol, beta-terpineol and gamma-terpineol), is usually light yellow oily transparent liquid, has pungent smell, is flammable, is slightly soluble in water and glycerol, is easily soluble in ethanol, and can be oxidized in air.
Terpineol has moderate to strong toxicity, can stably exist in the environment due to the ring structure and the branched chain, is difficult to degrade, and causes serious soil pollution and seriously threatens ecological safety when being used in a large amount in mining and beneficiation. How to economically and effectively remove these harmful substances from the environment is a current challenge for those skilled in the art. At present, terpineol in soil is removed mainly by a physical method (ozonolysis, ultraviolet irradiation, activated carbon adsorption), a chemical method (lime sedimentation, sodium hypochlorite oxidation), a biological method (plant enrichment) and other methods in the prior art, but risks and problems that the treatment cost is high, the difficulty in realizing industrialized mass treatment is high, other pollutants are introduced and the like generally exist. The microbial method has the advantages of simple operation, economy, applicability, no secondary pollution and the like, however, terpineol has broad-spectrum antifungal and antibacterial capabilities, and has great challenges in screening microorganisms capable of degrading the terpineol, and the biological degradation route and mechanism of the terpineol are reported recently.
Disclosure of Invention
In order to solve the problems of the prior art that microorganisms for efficiently repairing terpineol polluted soil are lack and related repairing methods, the invention provides pseudomonas and application thereof in degradation of terpineol and repair of polluted soil.
It is a first object of the present invention to provide a Pseudomonas sp.s4.
A second object of the invention is to provide the use of Pseudomonas sp for degrading terpineol.
A third object of the invention is to provide the use of Pseudomonas sp for repairing terpineol contaminated soil.
A fourth object of the present invention is to provide the use of Pseudomonas sp in combination with a biosurfactant for repairing terpineol contaminated soil.
A fifth object of the present invention is to provide the use of Pseudomonas sp in combination with biochar for repairing terpineol contaminated soil.
A sixth object of the present invention is to provide a formulation for degrading terpineol.
A seventh object of the present invention is to provide a method for remediation of terpineol contaminated soil.
In order to achieve the above object, the present invention is realized by the following means:
According to the research, terpineol is used as a target pollutant, mine soil is used as a source medium of microorganisms, domestication is carried out through concentration gradient, and Pseudomonas sp for producing a surfactant is screened and separated to obtain a strain S4; optimizing degradation conditions of terpineol by a single factor and response surface curve method; obtaining a crude surfactant extract from a fermentation broth of Pseudomonas sp by adopting an acid precipitation and extraction method, and identifying the type of the biosurfactant by using a Fourier infrared spectrum (FTIR) and a high performance liquid chromatography-time of flight mass spectrometer (HPLC-TOF-MS/MS); the application effect of adding rhamnolipid and charcoal immobilized Pseudomonas (sp.) S4 in repairing terpineol polluted soil is explored, and the structural change of microbial communities in the polluted soil under the repairing effect is revealed by using a high-throughput sequencing technology.
The invention claims the following:
A Pseudomonas sp.) S4, said Pseudomonas sp 4 having the accession number GDMCC No:63948, which was deposited at the cantonese collection of microorganism strains on the 10 th month 31 of 2023.
The application of Pseudomonas sp in degrading terpineol, the application of Pseudomonas sp in treating terpineol pollution and the application of Pseudomonas sp in repairing terpineol polluted soil are within the protection scope of the invention.
Preferably, the Pseudomonas sp is Pseudomonas sp.s4, accession number GDMCC No:63948, which was deposited at the cantonese collection of microorganism strains on the 10 th month 31 of 2023.
More preferably, the conditions under which Pseudomonas sp.s4 degrades terpineol include: the pH is 5-10.
Further preferably, the conditions under which Pseudomonas sp.s4 degrades terpineol include: the pH is 7-9.
Still further preferably, the conditions under which Pseudomonas sp.s4 degrades terpineol include: the pH is 7-8.
Still further preferably, the conditions under which Pseudomonas sp.s4 degrades terpineol include: the pH was 7.
More preferably, the conditions under which Pseudomonas sp.s4 degrades terpineol include: the temperature is 20-40 ℃.
Further preferably, the conditions under which Pseudomonas sp.s4 degrades terpineol include: the temperature is 25-40 ℃.
Still further preferably, the conditions under which Pseudomonas sp.s4 degrades terpineol include: the temperature was 30 ℃.
More preferably, the conditions under which Pseudomonas sp.s4 degrades terpineol include: the inoculation amount is OD 600nm =0.2 to 1.
Further preferably, the conditions under which Pseudomonas sp.s4 degrades terpineol include: the inoculation amount is OD 600nm =0.4-1.
Still further preferably, the conditions under which Pseudomonas sp.s4 degrades terpineol include: the inoculum size was OD 600nm =0.7.
Most preferably, the conditions under which Pseudomonas sp.s4 degrades terpineol include: the pH was 7, the temperature was 30℃and the inoculum size OD 600nm = 0.7.
Preferably, the soil comprises paddy soil and/or mine soil.
The use of a combination of Pseudomonas sp with a biosurfactant for degrading terpineol, the use of a combination of Pseudomonas sp with a biosurfactant for remediating terpineol pollution, and the use of a combination of Pseudomonas sp with a biosurfactant for remediating terpineol-contaminated soil are within the scope of the present invention.
Preferably, the Pseudomonas sp is Pseudomonas sp.s4, accession number GDMCC No:63948, which was deposited at the cantonese collection of microorganism strains on the 10 th month 31 of 2023.
Preferably, the biosurfactant comprises rhamnolipids. The rhamnolipids include monose rhamnolipids and bisrhamnolipids.
Preferably, the soil comprises paddy soil and/or mine soil.
The use of Pseudomonas sp in combination with biochar for repairing terpineol contaminated soil should be within the scope of the present invention.
Preferably, the Pseudomonas sp is Pseudomonas sp.s4, accession number GDMCC No:63948, which was deposited at the cantonese collection of microorganism strains on the 10 th month 31 of 2023.
Preferably, the biochar is loaded with the Pseudomonas sp.s4.
More preferably, the Pseudomonas sp 4 is adsorbed on the surface of the biochar, i.e. the formulation is a biochar immobilized microbial agent comprising Pseudomonas sp 4.
Further preferably, the preparation method of the biochar immobilized microbial inoculum comprises the following steps: and (3) fully reacting the bacterial suspension of Pseudomonas sp 4 with the concentration of OD 600nm = 0.5-1.5 with biochar to obtain the microbial preparation.
Still more preferably, the concentration of the bacterial suspension of Pseudomonas sp.s4 is OD 600nm =0.8 to 1.2.
Still further preferably, the concentration of the bacterial suspension of Pseudomonas sp.s4 is OD 600nm =1.
Further preferably, the ratio of the bacterial suspension of Pseudomonas sp.s4 to the amount of biochar is (4-6) mL: (20-30 g).
Still further preferably, the bacterial suspension to biochar ratio of Pseudomonas sp.s4 is 5mL:25g.
Further preferably, the preparation method of the biochar immobilized microbial agent further comprises the following steps: after the reaction is completed, the obtained reaction product is subjected to solid-liquid separation.
Still further preferably, the solid-liquid separation method includes: centrifuging and/or drying.
Specifically, the preparation method of the biochar immobilized microbial inoculum comprises the following steps: and (3) fully reacting 5mL of bacterial suspension of Pseudomonas S4 with the concentration of OD 600nm = 1 with 25g of biochar, centrifuging the obtained reaction product for 10min at 1000r/min, collecting the precipitate, and drying in a drying oven at 30 ℃ to obtain the product.
The application of the biological carbon immobilized microbial inoculum containing Pseudomonas sp in degrading terpineol, the application of the biological carbon immobilized microbial inoculum containing Pseudomonas sp in treating terpineol pollution and the application of the biological carbon immobilized microbial inoculum containing Pseudomonas sp in repairing terpineol polluted soil are within the protection scope of the invention.
Preferably, the Pseudomonas sp is Pseudomonas sp.s4, accession number GDMCC No:63948, which was deposited at the cantonese collection of microorganism strains on the 10 th month 31 of 2023.
Preferably, the preparation method of the biochar immobilized microbial inoculum comprises the following steps: and (3) fully reacting the bacterial suspension of Pseudomonas sp 4 with the concentration of OD 600nm = 0.5-1.5 with biochar to obtain the microbial preparation.
More preferably, the concentration of the bacterial suspension of Pseudomonas sp.s4 is OD 600nm =0.8 to 1.2.
Further preferably, the concentration of the bacterial suspension of Pseudomonas sp.s4 is OD 600nm =1.
More preferably, the ratio of the bacterial suspension of Pseudomonas sp.s4 to the amount of biochar is (4-6) mL: (20-30 g).
Further preferably, the ratio of the bacterial suspension of Pseudomonas sp.) S4 to the amount of biochar is 5mL:25g.
More preferably, the preparation method of the biochar immobilized microbial agent further comprises the following steps: after the reaction is completed, the obtained reaction product is subjected to solid-liquid separation.
Further preferably, the solid-liquid separation method comprises: centrifuging and/or drying.
Specifically, the preparation method of the biochar immobilized microbial inoculum comprises the following steps: and (3) fully reacting 5mL of bacterial suspension of Pseudomonas S4 with the concentration of OD 600nm = 1 with 25g of biochar, centrifuging the obtained reaction product for 10min at 1000r/min, collecting the precipitate, and drying in a drying oven at 30 ℃ to obtain the product.
A formulation for degrading terpineol, the formulation comprising Pseudomonas sp 4, accession number GDMCC No:63948, which was deposited at the cantonese collection of microorganism strains on the 10 th month 31 of 2023.
Preferably, the concentration of Pseudomonas sp.s4 is OD 600nm =0.5 to 1.5.
More preferably, the concentration of Pseudomonas sp.s4 is OD 600nm =0.8 to 1.2.
Further preferably, the concentration of Pseudomonas sp.s4 is OD 600nm =1.
Preferably, the formulation further comprises a biosurfactant or biochar.
More preferably, the biosurfactant comprises rhamnolipids. The rhamnolipids include monose rhamnolipids and bisrhamnolipids.
Still more preferably, the concentration of rhamnolipid in the formulation is 50mg/L to 200mg/L.
Still further preferably, the concentration of said rhamnolipid in said formulation is 100mg/L.
More preferably, the biochar is loaded with the Pseudomonas sp.s4.
Further preferably, the Pseudomonas sp 4 is adsorbed on the surface of the biochar, i.e. the formulation is a biochar immobilized microbial agent comprising Pseudomonas sp 4.
Still further preferably, the preparation method of the charcoal immobilized microbial agent comprises the following steps: and (3) fully reacting the bacterial suspension of Pseudomonas sp 4 with the concentration of OD 600nm = 0.5-1.5 with biochar to obtain the microbial preparation.
Still further preferably, the concentration of the bacterial suspension of Pseudomonas sp.s4 is OD 600nm =0.8 to 1.2.
Still further preferably, the concentration of the bacterial suspension of Pseudomonas sp.s4 is OD 600nm =1.
Still further preferably, the ratio of the bacterial suspension of Pseudomonas sp.S4 to biochar is (4-6) mL: (20-30 g).
Still further preferably, the bacterial suspension of Pseudomonas sp.) S4 is used in an amount ratio of 5mL to charcoal: 25g.
Still further preferably, the preparation method of the biochar immobilized microbial agent further comprises: after the reaction is completed, the obtained reaction product is subjected to solid-liquid separation.
Still further preferably, the solid-liquid separation method includes: centrifuging and/or drying.
Specifically, the preparation method of the biochar immobilized microbial inoculum comprises the following steps: and (3) fully reacting 5mL of bacterial suspension of Pseudomonas S4 with the concentration of OD 600nm = 1 with 25g of biochar, centrifuging the obtained reaction product for 10min at 1000r/min, collecting the precipitate, and drying in a drying oven at 30 ℃ to obtain the product.
A method of remediating terpineol contaminated soil, the terpineol contaminated soil being treated with a formulation comprising Pseudomonas sp.) S4 having a deposit number GDMCC No:63948, which was deposited at the cantonese collection of microorganism strains on the 10 th month 31 of 2023.
Preferably, the concentration of Pseudomonas sp.s4 is OD 600nm =0.5 to 1.5.
More preferably, the concentration of Pseudomonas sp.s4 is OD 600nm =0.8 to 1.2.
Further preferably, the concentration of Pseudomonas sp.s4 is OD 600nm =1.
Preferably, the volume to mass ratio of the bacterial suspension of Pseudomonas sp.) S4 to the terpineol contaminated soil is 1mL: (20-30 g).
More preferably, the volume to mass ratio of the bacterial suspension of Pseudomonas sp.) S4 to the terpineol contaminated soil is 1mL:25g.
Further preferably, the volume to mass ratio of the bacterial suspension of Pseudomonas sp 4 with a concentration of OD 600nm = 0.5-1.5 to the terpineol contaminated soil is 1mL: (20-30 g).
Still further preferably, the volume to mass ratio of the bacterial suspension of Pseudomonas sp 4 with a concentration of OD 600nm = 0.8-1.2 to the terpineol contaminated soil is 1mL: (20-30 g).
Still further preferably, the volume to mass ratio of the bacterial suspension of Pseudomonas sp 4 at a concentration of OD 600nm = 1 to the terpineol contaminated soil is 1mL: (20-30 g).
Further preferably, the volume to mass ratio of the bacterial suspension of Pseudomonas sp 4 with a concentration of OD 600nm = 0.5-1.5 to the terpineol contaminated soil is 1mL:25g.
Still further preferably, the volume to mass ratio of the bacterial suspension of Pseudomonas sp 4 with a concentration of OD 600nm = 0.8-1.2 to the terpineol contaminated soil is 1mL:25g.
Still further preferably, the volume to mass ratio of the bacterial suspension of Pseudomonas sp 4 at a concentration of OD 600nm = 1 to the terpineol contaminated soil is 1mL:25g.
Preferably, the formulation further comprises a biosurfactant or biochar.
More preferably, the biosurfactant comprises rhamnolipids. The rhamnolipids include monose rhamnolipids and bisrhamnolipids.
Further preferably, the biosurfactant is rhamnolipid.
Still more preferably, the concentration of rhamnolipid in the formulation is 50mg/L to 200mg/L.
Still further preferably, the concentration of said rhamnolipid in said formulation is 100mg/L.
Still further preferably, the mass ratio of rhamnolipid to terpineol contaminated soil is 1: (1X 10 5～1.5×105).
Still further preferably, the mass ratio of rhamnolipid to terpineol contaminated soil is 1: (1.25X10 5).
More preferably, the biochar is loaded with the Pseudomonas sp.s4.
Further preferably, the Pseudomonas sp 4 is adsorbed on the surface of the biochar, i.e. the formulation is a biochar immobilized microbial agent comprising Pseudomonas sp 4.
Still further preferably, the preparation method of the charcoal immobilized microbial agent comprises the following steps: and (3) fully reacting the bacterial suspension of Pseudomonas sp 4 with the concentration of OD 600nm = 0.5-1.5 with biochar to obtain the microbial preparation.
Still further preferably, the concentration of the bacterial suspension of Pseudomonas sp.s4 is OD 600nm =0.8 to 1.2.
Still further preferably, the concentration of the bacterial suspension of Pseudomonas sp.s4 is OD 600nm =1.
Still further preferably, the ratio of the bacterial suspension of Pseudomonas sp.S4 to biochar is (4-6) mL: (20-30 g).
Still further preferably, the bacterial suspension of Pseudomonas sp.) S4 is used in an amount ratio of 5mL to charcoal: 25g.
Still further preferably, the preparation method of the biochar immobilized microbial agent further comprises: after the reaction is completed, the obtained reaction product is subjected to solid-liquid separation.
Still further preferably, the solid-liquid separation method includes: centrifuging and/or drying.
Specifically, the preparation method of the biochar immobilized microbial inoculum comprises the following steps: and (3) fully reacting 5mL of bacterial suspension of Pseudomonas S4 with the concentration of OD 600nm = 1 with 25g of biochar, centrifuging the obtained reaction product for 10min at 1000r/min, collecting the precipitate, and drying in a drying oven at 30 ℃ to obtain the product.
Still further preferably, the mass ratio of the biochar immobilized microbial inoculum to the terpineol contaminated soil is 1: (20-30).
Still further preferably, the mass ratio of the biochar immobilized microbial inoculum to the terpineol-contaminated soil is 1:25.
Preferably, the terpineol contaminated soil comprises terpineol contaminated rice soil and/or terpineol contaminated mine soil.
Compared with the prior art, the invention has the following beneficial effects:
The invention provides Pseudomonas sp S4 with a function of efficiently degrading terpineol, which has a degradation rate of up to 95% for 350mg/L terpineol after being cultured under laboratory conditions, and can also produce a composite surfactant containing monose rhamnolipid and bisrhamnolipid. Pseudomonas sp 4 can be used for repairing terpineol-polluted soil, the diversity of microorganism species in the soil is increased, and the synergistic effect can be exerted by combining with rhamnolipid or biochar, so that the repairing effect is more remarkable, a new technical choice is provided for treating terpineol pollution, and the method has a wide application prospect.
Drawings
FIG. 1 shows colony morphology characteristics of strain S4 in nutrient agar medium.
FIG. 2 shows morphological features of strain S4 under scanning electron microscope (5000 times).
FIG. 3 is a 16S rDNA phylogenetic tree of strain S4.
FIG. 4 shows the effect of different factors on the degradation rate of P.sp.S.4 on terpineol, A is the degradation rate of terpineol at different pH values, B is the degradation rate of terpineol at different temperatures, and C is the degradation rate of terpineol at different inoculation amounts.
FIG. 5 shows the response surface test results, wherein A is the response analysis curved surface of the degradation rate of terpineol and the temperature and the pH, and B is the contour line of the degradation rate of terpineol and the temperature and the pH.
FIG. 6 is a graph of the degradation kinetics of Pseudomonas sp.S 4 for various initial concentrations of terpineol.
FIG. 7A is a graph showing the results of blue gel plates, and B is a graph showing the emulsification effect of Pseudomonas sp.S4
FIG. 8 is a chart showing the infrared absorption spectrum of the Pseudomonas sp.S4 metabolite.
FIG. 9 is a mass spectrum of the surfactant of Pseudomonas sp.S 4 analyzed by HPLC-TOF-MS/MS, with A-C being C 14H26O7、C14H22O7 and C 20H36O11 in this order.
FIG. 10 shows the results of quantitative analysis of rhamnolipid production by Pseudomonas sp.S 4, A is a standard curve of rhamnose content and absorbance, and B is the case of rhamnolipid production by Pseudomonas sp.S 4 during 24h of cultivation.
FIG. 11 is a scanning electron microscope image before and after the bacteria are immobilized on the charcoal, wherein A is before the bacteria are immobilized on the charcoal, and B is after the bacteria are immobilized on the charcoal.
FIG. 12 shows the results of terpineol contaminated soil remediation test, wherein A is the statistics of degradation rate of terpineol in contaminated rice soil of CK group, A group, B group, C group and D group; b is the degradation rate statistics of terpineol in the contaminated paddy soil of the CK group, the contaminated mine soil of the E group and the contaminated mine soil of the F group.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The reagents used in the embodiment of the invention comprise: terpineol (Terpineol), purchased from Shanghai Aladin chemical industry Co., ltd., CAS number: 8000-41-7, purity >95%; dichloromethane was purchased from Sigma-Aldrich, usa, CAS number: 75-09-2, purity >98%; rhamnose, available from Shanghai Meilin Biochemical technologies Co., ltd., CAS number: 6155-35-7; biochar, available from Henan Lize environmental protection technologies Co., ltd., product number: LZ-04-05; rhamnolipids, available from Shanghai Meilin Biochemical technologies Co., ltd., CAS number: 869062-42-0.
The culture medium used in the embodiment of the invention comprises the following components: liquid basal salt (MSM) medium (K2HPO4,5.8g/L;KH2PO4,4.5g/L;(NH4)2SO4,2.0g/L;MgCl2,0.15g/L;CaCl2,0.03g/L;Na2MoO4·2H2O,0.0025g/L;FeCl3,0.0016g/L;MnCl2·2H2O,0.0014g/L; double distilled water, 1000mL; pH 7); LB medium (peptone, 10g/L; yeast extract, 5g/L; sodium chloride, 5g/L; double distilled water, 1000mL; pH 7); nutrient agar (LB solid) medium (peptone, 10g/L; yeast extract, 5g/L; sodium chloride, 5g/L; agar, 20g/L; double distilled water, 1000mL; pH 7).
EXAMPLE 1 screening, isolation and identification of Pseudomonas
1. Gradient domestication
And (3) taking a soil sample of a polluted mining area in Guangxi nan Dan county as a target pollutant, and performing strain domestication by adopting a concentration gradient method.
Firstly, 10g of mine soil sample is inoculated into 100mL of MSM culture medium containing 1% (v/v) terpineol, and placed into a shaking table at 25 ℃ and at 150rpm for 5d culture; subsequently, inoculating the strain with 10% (v/v) of terpineol to MSM medium containing 2% of terpineol, and culturing for 5d under the same condition; inoculating 10% (v/v) of the strain into MSM culture medium containing 3% terpineol, and culturing under the same condition for 5d; inoculating 10% (v/v) of the strain into MSM culture medium containing 4% terpineol, and culturing under the same condition for 5d; inoculating 10% (v/v) of the strain into MSM culture medium containing 5% terpineol, and culturing under the same condition for 5d to obtain culture bacterial liquid. Each concentration was repeated three times.
The obtained culture bacterial liquid is separated and purified by an LB solid culture medium to obtain a single colony, the corresponding strain number is S4, the strain S4 is inoculated into a test tube slant culture medium, and the culture bacterial liquid is preserved in a refrigerator at the temperature of 4 ℃ for standby.
2. Identification of strains
1. Morphological identification
(1) Colony characterization
The strain S4 was inoculated onto LB plate, placed in a constant temperature incubator for culturing for 24 hours, and the shape, size, surface, transparency, edge, color, etc. of the strain S4 were observed during the culturing.
As shown in FIG. 1, after the strain S4 was cultured on LB solid medium for 24 hours, the colony had a round transparent convex shape and was brown yellow in color.
(2) Observation of morphological characteristics of the strain by scanning electron microscope
Selecting single colony of strain S4 from LB solid medium by inoculating loop, adding into LB liquid medium, and culturing at 25deg.C with shaking table of 160rpm for 24 hr; sucking 1mL of bacterial liquid from the triangular flask, putting the bacterial liquid into a 2.5mL centrifuge tube, centrifuging at 6000rpm for 6min, discarding the supernatant, and collecting bacterial bodies; adding 1mL of sterilized 0.9% NaCl solution, mixing well, and centrifuging to collect thalli (repeated 3 times); then 1mL of 2.5% glutaraldehyde is added, stirred uniformly and then placed in a refrigerator at 4 ℃ for fixing for 4 hours; after the fixation is finished, adding ethanol for dehydration; the cells were collected by centrifugation, freeze-dried, and then observed for morphological characteristics of the strain S4 by using a scanning electron microscope.
As shown in FIG. 2, the strain S4 was observed under a scanning electron microscope to be in the form of a straight or slightly curved bacillus, and was arranged singly or in pairs, with a length of about 1. Mu.m.
2. Identification of physiological and biochemical characteristics
The identification is carried out with reference to Bergey's Manual of Determinative Bacteriology (Lippincott Williams & Wilkins, 1994).
(1) Gram staining
Placing a loop of distilled water on a slide by using an inoculating loop; the inoculating loop is burnt and sterilized by an alcohol lamp flame, the strain S4 is picked from LB solid medium and evenly mixed with distilled water on a slide, and then the smear is placed beside the alcohol lamp flame for fixation. Dropping a drop of crystal violet staining solution, waiting for 1min, and washing with water; adding gram iodine solution, standing for 1min, and washing with clear water; then a large amount of 95% ethanol is dripped, decolorized for 10s and washed with water; finally adding the counterstain liquid, and washing after 1 min; and (5) performing microscopic examination after drying.
(2) Contact enzyme assay
Bacterial strain S4 is picked from LB solid medium by an inoculating loop, placed into a beaker containing 3% hydrogen peroxide, observed whether bubbles are generated or not, and the situation that no bubbles are generated is negative, otherwise, the situation is positive.
(3) Methyl Red experiment
Strain S4 was inoculated into a universal medium, after 3 days of culture, 3 drops of methyl red indicator were added, and the color change was observed, with bright red positive and yellow negative.
(4) Oxidase test
Bacterial strain S4 is selected from LB solid medium by an inoculating loop, and put into 1% hydrochloric acid dimethyl p-phenylene diamine solution, and the color change is observed, and purple is positive.
(5) Indole experiments
The strain S4 is inoculated into peptone culture solution containing tryptophan, cultured for 48 hours, indole reagent is added, and the contact position of two liquid surfaces is changed to be positive when the color is not changed to be negative when the observation result is that the contact position of two liquid surfaces is changed to be positive.
(6) Urease experiment
Strain S4 was inoculated into a urea-containing medium and cultured for 48 hours, and the color change was observed, with positive red and negative no change in color.
(7) Hydrogen sulfide experiments
And (3) selecting the strain S4 from the LB solid medium by using an inoculating loop, inoculating the strain S4 onto a trisaccharide iron agar medium, culturing for 24 hours, and observing the color change condition of the medium, wherein the black generation is positive, and the negative is negative.
(8) Citrate experiments
The strain S4 is selected from LB solid medium by an inoculating loop and inoculated on citrate LB medium, the culture is carried out for 3d, the color change of the medium is observed, the blue generation is positive, and the negative is negative.
(9) Experimental results
TABLE 1 physiological and biochemical characterization results of strain S4
Note that: "+" is positive and "-" is negative.
As shown in Table 1, strain S4 belongs to gram-positive bacteria, and can grow by using citrate; the experimental results of the strain contact enzyme, urease, hydrogen sulfide and oxidase are positive. The experimental results of methyl red and indole are negative.
3. 16S rDNA sequence homology analysis
The 16S rDNA fragment of the strain S4 is amplified by using a universal primer pair (27F and 1492R), and the PCR reaction system is as follows: taq mix 25. Mu.L; primer 27F 1 μl; primer 1499r1μl; 1 μl of DNA template; ddH 2 O22 μL; the total volume was 50. Mu.L. The PCR reaction conditions were: initial preheating at 95 ℃ for 5min; fusing at 94 ℃ for 45s, annealing at 55 ℃ for 45s, extending at 72 ℃ for 1min for 15s, circulating for 32 times, and finally maintaining at 72 ℃ for 10min. After the PCR amplification was completed, the size and specificity of the amplified fragment were detected by 1.5% agarose gel electrophoresis and photography in a gel imaging system.
The PCR product was recovered and submitted to the Biotechnology company for sequencing to obtain the 16S rDNA sequence of strain S4 as follows:
CGGGGGAGCTACACATGCAGTCGAGCGGATGACGGGAGCTTGCTCCTTGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCGAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAATCCTTGAGATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCACGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCACCCTTGTCTAGTACAGCACGTTATGGTGGGCACTCTAGAGACTGCGTGACAACGAGATGTGGATGACGTCAGTCATCATGCCT(SEQ ID NO.1).
The 16S rDNA sequence (SEQ ID NO. 1) of strain S4 was aligned with the 16S rDNA of a similar microorganism in the GenBank database, and the results showed that strain S4 was closely related to Pseudomonas putida strain J. Selecting some sequences with higher homology from BLAST comparison results, realizing multi-sequence association by using biological software Clustal W, constructing a phylogenetic tree shown in figure 3 by using an adjacent method (Neighborhood-joining) analysis relation, and determining that the genetic relationship between the strain S4 and pseudomonas Pseudomonas putida strain J (MH 814578.1) is closest by comparing the differences between different species.
4. Degradation of terpineol
(1) Preparation of standard solutions
Quantitative terpineol is accurately measured, dichloromethane is added and mixed uniformly, and 10mg/L of terpineol standard mother liquor is finally prepared. The terpineol mother liquor is diluted by dichloromethane with chromatographic purity to obtain terpineol standard solutions with the concentration of 0.1mg/L, 0.2mg/L, 0.4mg/L, 0.6mg/L, 0.8mg/L and 1.0mg/L respectively.
(2) Culture of strain S4 and preparation of sample to be tested
The strain S4 is inoculated in MSM culture medium containing 350mg/L terpineol, placed in a shaking table at 30 ℃ and 150rpm for continuous culture for 3d, and the culture medium is collected after the culture is finished, so as to obtain a sample A.
MSM medium containing 350mg/L terpineol was placed in a shaker at 30℃and 150rpm for continuous 3d to give sample B as a control medium.
Respectively filling the culture medium A and the culture medium B into triangular flasks, respectively adding 50mL of dichloromethane, oscillating at 150rpm for 10min, and completely and uniformly mixing; then ultrasonic for 10min, repeating twice; standing at room temperature for 10min, collecting lower organic phase dichloromethane, filtering with 0.45 μm microporous membrane to obtain filtrate, respectively obtaining sample A and sample B, respectively placing into 2.5mL sample injection bottle, and measuring terpineol content by gas chromatography-mass spectrometry (GC-MS).
(3) GC-MS detection
Sampling is carried out without diversion, and the sampling amount is 1.0 mu L.
Detection conditions: chromatographic column, DB-5ms; the carrier gas is helium, the temperature of the sample inlet is 230 ℃, and the sample is fed by an automatic sample feeder; the initial pressure was 55kPa and the flow rate was 1.5mL/min.
Heating program: the initial temperature is maintained at 60 ℃ for 1min; after heating to 120 ℃ at 10 ℃/min, the temperature is kept for 2min, and then heating to 200 ℃ at 10 ℃/min, and the temperature is kept for 1min. Qualitative and quantitative mass spectrometry: EI ion source for mass spectrum, temperature of 200deg.C, target ion of terpineol of 59m/z, reference ion of 93m/z and 121m/z, and retention time of 6.393min.
By adopting the detection method established above, the blank sample adding recovery rate of the terpineol compound is measured, and the recovery rate is 96.1% -109.7%.
And (3) measuring terpineol standard solutions with different concentrations, and establishing a standard curve for obtaining the concentration (X) and the peak area (Y) to obtain a regression equation. Measuring the peak area of a sample to be measured, substituting the peak area into a regression equation, calculating to obtain the terpineol content of the sample to be measured, and then calculating according to a formula (1) to obtain the degradation rate of the terpineol:
Formula (1): degradation rate (%) = (1-D 1/D0) ×100%;
Wherein D 1 is the terpineol content of medium A after the end of culture of strain S4, and D 0 is the terpineol content of the control medium (medium B).
(4) Detection result
The condition of degrading terpineol by the strain S4 is measured by GC-MS, and the result shows that the strain S4 can effectively degrade terpineol with the concentration of 350mg/L, and the degradation rate of terpineol by culturing for 3d is as high as 95%. The strain S4 is shown to be capable of efficiently degrading terpineol.
In summary, the strain finally selected in the invention is a Pseudomonas sp S4 strain capable of degrading terpineol and is deposited in the microorganism strain collection (GDMCC) of Guangdong province at 10.31 of 2023, with the deposit number GDMCC No:63948, the deposit is No. 59 building 5 of Guangzhou martyr, guangdong province, china, and the deposit name is Pseudomonas sp.S4.
EXAMPLE 2 degradation Properties of Pseudomonas sp.S4 on terpineol
1. Experimental method
1. Preparation of bacterial suspension
A small amount of Pseudomonas sp.s4 is selected from the test tube of the preserved strain, added into a triangular flask containing LB liquid culture medium, subjected to constant temperature shake culture for 24 hours, centrifuged for 5min to collect bacterial precipitate, and after supernatant removal, the bacterial precipitate is washed three times with 0.9% sterile physiological saline and resuspended to obtain bacterial suspension with OD 600nm =1.
2. Preparation of terpineol mother liquor
Accurately measuring quantitative terpineol, adding dichloromethane for mixing, fixing volume to obtain 1× 4 mg/L terpineol mother liquor, filtering with 0.45 μm filter membrane, placing into brown volumetric flask, and storing in refrigerator at 4deg.C in dark place.
3. Influence of different factors on degradation of terpineol by Pseudomonas sp.4
(1) Influence of pH
The prepared 100mL MSM culture medium is respectively adjusted to pH values of 5, 6, 7, 8, 9 and 10 by using pH values, and the culture medium is autoclaved by using a sterilizing pot (121 ℃ for 20 min); 1mL terpineol mother liquor was added to sterilized MSM medium and inoculated with Pseudomonas sp 4 (OD 600nm = 0.6); after sealing the sealing film, the mixture was put on a shaker (30 ℃ C., 150 rpm) for 2d of cultivation.
After the completion of the culture, the culture broth was collected, and the degradation rate of terpineol by Pseudomonas (Pseudomonas sp.) S4 was measured by the method of "GC-MS measurement" in example 1. Each treatment was set with 3 replicates, using MSM medium at each pH without inoculation as a blank.
(2) Influence of temperature
The prepared 100mL MSM culture medium is autoclaved (121 ℃ C., 20 min) by a sterilizing pot; then 1mL terpineol mother liquor is added into sterilized MSM medium in an ultra clean bench, and then Pseudomonas sp 4 is taken for inoculation (OD 600nm =0.6); after sealing the sealing film, the culture medium was placed in shaking tables at 20 ℃,25 ℃, 30 ℃, 35 ℃ and 40 ℃ respectively, and cultured for 2d with shaking at 150 rpm.
After the completion of the culture, the culture broth was collected, and the degradation rate of terpineol by Pseudomonas (Pseudomonas sp.) S4 was measured by the method of "GC-MS measurement" in example 1. With non-inoculated MSM medium as a blank, 3 replicates were set for each treatment.
(3) Influence of inoculation quantity on degradation capability of pinitol oil degradation bacteria
The prepared 100mL MSM culture medium is autoclaved (121 ℃ C., 20 min) by a sterilizing pot; then, 1mL of terpineol mother liquor was added to sterilized MSM medium in an ultra clean bench, and Pseudomonas sp.s4 was inoculated at respective inoculation amounts of OD 600nm＝0.2、OD600nm＝0.4、OD600nm＝0.6、OD600nm =0.8 and OD 600nm =1, sealed with a sealing film, and then placed on a shaker (30 ℃,150 rpm) for 2d cultivation.
After the completion of the culture, the culture broth was collected, and the degradation rate of terpineol by Pseudomonas S4 was measured by the method of "GC-MS measurement" in example 1, and 3 replicates were set for each treatment using the MSM medium without inoculation as a blank.
(4) Response surface test
Based on the detection results of the above 3 single-factor tests, the Box-Behnken Design is utilized, the influence of pH (X 1), temperature (X 2) and inoculation amount (X 3) on the degradation rate of terpineol is considered respectively, the specific grouping conditions are shown in table 2, and the optimal combination condition of Pseudomonas sp 4 degradation of terpineol is determined by a response surface method.
TABLE 2 Box-Behnken Design experimental factor level table and coding
And (3) using Design-Expert 8.0 software to draw a response curve graph to study the influence rule of single factors and interaction of the factors on the degradation rate of terpineol, using OD 600nm = 0.8 inoculation quantity as a fixed treatment condition, exploring the influence of temperature and pH on the degradation rate of terpineol, and manufacturing a corresponding three-dimensional response curve and contour graph.
4. Kinetics study of Pseudomonas sp.S 4 degradation of terpineol
The prepared 100mL MSM culture medium is autoclaved (121 ℃ C., 20 min) by a sterilizing pot; then, in an ultra-clean workbench, different amounts of terpineol mother liquor are added into a sterilized inorganic salt culture medium, so that the final concentration of terpineol is 100mg/L, 300mg/L, 600mg/L and 1000mg/L respectively, then Pseudomonas sp 4 is taken, and the culture is carried out according to the optimal combination condition obtained by a response surface test, and the culture is continued for 3d.
To analyze the kinetics of degradation of terpineol by Pseudomonas sp. The culture media were collected at 0h, 6h, 12h, 48h and 72h of incubation, respectively, and the terpineol content of the media was measured as in example 1, using non-sterile MSM media as a blank control, and 3 replicates were set per treatment.
Fitting the content (C) and the time (t) of terpineol in the Pseudomonas sp.) S4 in the culture mediums with different culture times by adopting a primary degradation dynamics model, and drawing degradation dynamics curves of different concentrations of the terpineol, wherein the calculation method of the primary degradation dynamics model is shown in a formula (2), and the calculation method of a degradation half-life model is shown in a formula (3):
formula (2): lnC = a+kt;
Equation (3): t 1/2 = 0.693/k;
Wherein C is the terpineol content in the culture medium at time t (h), k is the first order rate constant, a is the constant, t is the culture time, and t 1/2 is the biodegradation half-life of terpineol.
2. Experimental results
1. Degradation rate of terpineol by Pseudomonas sp.s4 under influence of different factors
(1)pH
As shown in a in fig. 4, in the range of pH 5 to 10, the ability of Pseudomonas (sp.) S4 to degrade terpineol is significantly affected by pH, and when pH is 5 to 8, the rate at which Pseudomonas (sp.) S4 degrades terpineol increases with increasing pH, and when pH is 8 to 10, the degradation rate of Pseudomonas (sp.) S4 on terpineol decreases with increasing pH; the degradation rate of pseudomonad (Pseudomonas sp.) S4 on terpineol is highest up to 92% when pH is 8, and is sequentially lower to 61% and 73% when pH is 5 and 10, and higher than 75% when pH is 7-9.
The results show that Pseudomonas sp.s4 inhibits the degradation efficiency of terpineol in both acidic and strongly alkaline environments, and has good degradation capacity for terpineol in weakly alkaline environments, and the optimal pH range for degrading terpineol is 7-9.
(2) Temperature (temperature)
As shown in B in fig. 4, the temperature is 20 to 40 ℃, the ability of Pseudomonas (sp.) S4 to degrade terpineol is significantly affected by temperature, when the temperature is 20 to 30 ℃, the rate of Pseudomonas (sp.) S4 degradation of terpineol increases with increasing temperature, and when the temperature is 30 to 40 ℃, the rate of Pseudomonas (sp.) S4 degradation of terpineol decreases with increasing temperature; the degradation rate of the pseudomonad (Pseudomonas sp.) S4 on terpineol is highest up to 93% when the temperature is 30 ℃, the degradation rate of the terpineol is as low as 63% when the temperature is 20 ℃, and the degradation rate of the terpineol is higher than 75% when the temperature is 25-40 ℃.
The above results show that the optimum temperature range for the degradation of terpineol by Pseudomonas sp.S 4 is 25℃to 40 ℃.
(3) Inoculation amount
As shown in C in fig. 4, the capacity of Pseudomonas sp 4 to degrade terpineol is significantly affected by the inoculation amount, the inoculation amount is OD 600nm =0.2-0.8, the degradation rate increases with the increase of the inoculation amount, and when the inoculation amount is OD 600nm =0.8, the degradation rate of Pseudomonas sp 4 to terpineol is highest, up to 93%; the inoculation amount is OD 600nm =0.8-1, and the degradation rate of the Pseudomonas sp 4 to terpineol is slightly reduced; when the inoculation amount is OD 600nm =0.4-1, the degradation rate of terpineol is higher than 75%.
The above results indicate that the optimum inoculation amount for the degradation of terpineol by Pseudomonas sp.s4 is OD 600nm = 0.4-1.
2. Response surface test results
TABLE 3 Box-Behnken test design and results of terpineol degradation
/>
Note that: the data in the table are mean ± standard deviation (n=3); the same column containing the same letter indicates that the difference is not significant (P > 0.05).
Table 4 Box-Behnken test design and results of terpineol degradation
Note that: r 2 = 0.9916 (correction R 2 =0.9807), coefficient of Variation (CV) =1.98%; p <0.05 indicates that the factors under investigation have a significant effect.
The response surface test results are shown in table 3, the response surface results are subjected to a quadratic polynomial model regression analysis by taking the degradation rate of terpineol as a response value, the results shown in table 4 are obtained, and a regression equation of the degradation rate of terpineol shown in formula (4) is fitted:
Formula (VI) (4)：Y＝94.00+3.20×X1+0.0150×X2-19.58×X1 2-7.45×X2 2-2.30×X3 2;
Wherein Y represents a response value (degradation rate of terpineol), and X 1、X2 and X 3 represent independent variables of 3 of pH, temperature and inoculum size, respectively.
The results in table 4 show that the coefficient R 2 = 0.9916, demonstrating that Pseudomonas sp 4 has a very high correlation with the observed and predicted degradation rates of terpineol; the correction decision coefficient is 0.9807, and the correction decision coefficient has better explanation effect on the change of the response value of the Pseudomonas sp.S4; the Coefficient of Variation (CV) is 1.98%, which shows that the obtained regression equation can better reflect the actual measurement degradation rate of terpineol, and the fitting effect of the model is very remarkable (P < 0.0001). In addition, it can be seen that pH (X 1) and temperature (X 2) have a significant linear effect on the terpineol degradation rate of Pseudomonas sp 4 (P < 0.05), while the quadratic term coefficients X 11 and X 22 indicate that the linear effect of Pseudomonas sp 4 on the terpineol degradation rate reaches a very significant level (P < 0.0001), whereas the linear effect of other factors on the degradation rate of Pseudomonas sp 4 is not significant (P > 0.05).
The three-dimensional response surface and the contour plot are shown as A and B in FIG. 5, respectively, and it can be seen that the degradation rate of pseudomonad (Pseudomonas sp.) S4 on terpineol increases as the temperature increases from 20 ℃ to 30 ℃; however, when the temperature exceeds 30 ℃, the degradation rate of terpineol by strain S4 starts to decrease gradually, which is consistent with the previous experimental results; when the pH was maintained between 7 and 8, the degradation efficiency of strain S4 on terpineol was highest, and was also consistent with the previous experimental results. The optimal combination conditions for the final prediction of the degradation of terpineol by Pseudomonas sp.) S4 are: the degradation rate of terpineol is predicted to be 95% at ph=7, temperature 30 ℃, inoculation amount OD 600nm =0.7. Under the optimal combination condition, 3 repeated experiments prove that the average value of the degradation rate of the terpineol is 95%, and the predicted result is true and reliable.
3. Kinetics of degradation of terpineol by Pseudomonas sp.4
As shown in fig. 6, overall, as the initial concentration of terpineol in the MSM medium increases, the degradation rate of pseudomonas (pseudomonasp.) S4 decreases, probably because high concentrations of terpineol can inhibit the growth of pseudomonas (pseudomonasp.) S4 to some extent, specifically: when the concentration of terpineol is less than or equal to 300mg/L, the terpineol can be completely degraded under the condition that Pseudomonas sp.S4 is cultured for 24 hours; when the initial concentration of terpineol is increased to 1000mg/L, the degradation rate of the terpineol by Pseudomonas sp.) S4 is reduced by 79 percent at 24 hours, but the terpineol can still be fully used as the only carbon source and energy source for growth, and the degradation process has no obvious delay effect. Indicating that Pseudomonas sp.S4 is somewhat tolerant to high concentrations of terpineol.
TABLE 5 degradation kinetics parameters of terpineol at different initial concentrations
Note that: c is the terpineol content in the culture medium at time t (h), t is the culture time, and R 2 represents the determination coefficient of the degree of correlation; the data are averaged over 3 replicates in the table.
In order to explore the degradation kinetics of Pseudomonas sp 4 on terpineol, the present example uses a first order degradation kinetics model to fit the terpineol content (C) and the incubation time (t) in the culture medium at different initial concentrations, the fit results of the degradation kinetics fit model and the half-life model are shown in table 5, and it is seen that when Pseudomonas sp 4 degrades terpineol, the half-life of Pseudomonas sp is proportional to the substrate concentration, wherein the half-lives of 100mg/L, 300mg/L, 600mg/L and 1000mg/L correspond to the substrate concentrations of 4.46h, 7.18h, 10.5h and 12.73h in order, and the degradation rate constants k are 0.1553d -1、0.0965d-1、0.066d-1 and 0.0544d -1 in order.
Example 3 identification of Pseudomonas sp. S4 Metabolic surfactant
1. Experimental method
1. Blue gel plate experiment
Cetyltrimethylammonium bromide (CTAB) 0.5% (w/v), methine blue 0.002% (w/v), beef extract 0.5% (w/v), peptone 1.0% (w/v), naCl 0.5% (w/v) and agar powder 2.0% (w/v) were fixed to a volume of 1L with sterilized water, pH was adjusted to 7, and after sterilization at 120℃for 25min, the plate was poured to obtain a blue gel plate. The blue gel plate contains CTAB and methylene blue and can react with glycolipid surfactant secreted by microorganism.
A small amount of Pseudomonas sp.S4 was picked from the tube of the deposited strain, diluted and spread on a blue gel plate, and incubated in a constant temperature incubator at 37℃for 2d, and the color change of colonies on the plate was observed.
2. Preparation of fermentation broths
Fermentation broths were prepared according to the optimal combination of conditions (ph=7, temperature 30 ℃, inoculum size OD 600nm =0.7) for the degradation of terpineol by Pseudomonas sp.) S4 determined in example 2, as follows:
pseudomonas sp.s4 was inoculated in an amount of inoculation OD 600nm =0.7 into an MSM medium (pH=7) containing 100mg/L terpineol, and the culture was continuously carried out in a shaker at 30℃and 150rpm for 3d, and after the completion of the culture, the medium was collected to obtain a fermentation broth.
3. Determination of the emulsifying Properties
Adding liquid paraffin and fermentation liquor into a 10mL centrifuge tube according to the volume ratio of 1:1, culturing for 24h at 30 ℃, then oscillating for 1min by using a vortex oscillator, standing for 24h at room temperature, measuring the height of an emulsion layer, and according to the formula: emulsification ratio (%) =emulsion layer height/total liquid height×100%, and the emulsification ratio of Pseudomonas sp 4 is calculated. Liquid paraffin without fermentation broth was used as a control.
3. Extraction of surfactants
Taking 100mL of fermentation liquor, centrifuging at 6000rpm for 5min, collecting supernatant, placing into a triangular flask, adding hydrochloric acid to adjust pH to 2, sealing, and placing into a refrigerator at 4 ℃ for overnight precipitation. After precipitation, the supernatant was centrifuged at 8500rpm for 10min, the supernatant was discarded, the precipitate was collected, resuspended in methylene chloride and the surfactant produced by Pseudomonas sp.S 4 was obtained by rotary evaporation.
4. Fourier transform Infrared Spectroscopy (FTIR) analysis
Samples mixed with 1mg of surfactant and 100mg of KBr were analyzed using FTIR in the spectral range 4000-400 cm -1 to detect characteristic functional groups in the surfactant.
5. HPLC-TOF-MS/MS analysis
The extracted surfactant is identified by HPLC-TOF-MS/MS, the sample is dissolved in acetonitrile, the concentration of the sample is configured to be 15mg/mL, the sample is filtered by a microporous filter membrane with the thickness of 0.22 mu m, and 10 mu L of sample is taken for sampling. The column was DP-5 (0.25 μm. Times.0.25 mm. Times.30 m). Mobile phase a was methanol: water = 1:100, mobile phase B is formic acid: acetonitrile=1:100. The flow rate was 0.5mL/min.
6. Quantitative analysis of rhamnolipids
Analysis of rhamnolipids in extracted surfactants using anthrone-sulfuric acid chromogenic method, the basic principle is: under the action of concentrated sulfuric acid, rhamnolipid loses moisture to generate furfural, and then the fistulone and the furfural further react to generate blue-green furfural derivatives.
Adding 0.2g of allinone into 100mL of concentrated sulfuric acid as a color developing agent; preparing a rat Li Tangmu solution with the concentration of 100 ppm; injecting 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL and 0.9mL of mouse Li Tangmu liquid into a test tube respectively, adding enough water to enable the liquid volume to reach 1mL, adding 4mL of color developing agent, and uniformly mixing on ice; boiling the test tube in boiling water for 8min, and mixing on ice; the OD value of each sample at 620nm wavelength was measured with an ultraviolet spectrophotometer, and a standard curve of rhamnose concentration and OD 620nm was made.
Pseudomonas sp.4 is inoculated into MSM culture medium with terpineol as the only carbon source, placed in a shaking table at 30 ℃ and 150rpm for continuous culture for 24 hours, the culture medium is collected every 2 hours, surfactant is obtained by extraction according to the method of the embodiment, then OD value at 620nm wavelength is measured by using an anthrone-sulfuric acid chromogenic method, and the corresponding rhamnose concentration is obtained by substituting into a standard curve according to the formula: rhamnolipid content = rhamnose concentration x 3.4 (conversion coefficient of rhamnolipid to rhamnose), yield of rhamnolipid produced by Pseudomonas sp 4 at each time point is obtained by conversion.
2. Experimental results
1. Blue gel plate test results
After 2 days of incubation on blue gel plates, blue spots appear around the colonies, indicating that Pseudomonas sp.S 4 is able to produce a biosurfactant for glycolipids, as shown in FIG. 7A.
2. Results of measurement of emulsifying Properties
As shown in FIG. 7B, the fermentation broth of Pseudomonas sp.S 4 had a significant emulsifying effect on terpineol compared to the control group, and a stable emulsion layer was formed after 24 hours of culture. According to the measurement result, the height of the emulsion layer was 3.9cm, the total liquid phase height was 6cm, and the emulsion rate of Pseudomonas sp was calculated to be 65% based on this, indicating that the emulsion effect of the secreted surfactant was high.
3. FTIR analysis results
The infrared absorption spectrum of the surfactant produced by the growth of Pseudomonas sp.s4 is shown in FIG. 8, wherein 3422.51cm -1 is the stretching vibration peak of the N-H bond; 2929.04cm -1 is an aliphatic-CH 2 -symmetrical stretching vibration; 1638.9cm -1 is CO-N bond stretching vibration of amide, belonging to one of lipopeptides; 1543.24cm -1 there is a-CH 3 group; the presence of a C-O-C sugar acetal structure at 1229cm -1 confirms the presence of glycolipids; 1066cm -1 indicates the presence of a C-O bond. The above results indicate that the surface-active substances produced by pseudomonas (pseudomonasp.) S4 during metabolism belong to glycolipids.
4. HPLC-TOF-MS/MS analysis results
HPLC-TOF-MS/MS detects the surface active substances generated by Pseudomonas sp in the metabolic process of the Pseudomonas S4, and according to the generated negative ion mass spectrum signal diagram, as shown by A-C in FIG. 9, the main mass spectrum fragments are m/z 306, 302 and 452, and C 14H26O7 and C 14H22O7 belong to single rhamnolipid; c 20H36O11 is a bisrhamnolipid. Surfactants that demonstrate production of Pseudomonas S4 include monose rhamnolipids and bisrhamnolipids.
5. Quantitative analysis results of rhamnolipid
The standard curve of rhamnose concentration and OD 620nm is shown as a in fig. 10, the rhamnose concentration and OD 620nm are in a significant linear relationship, the regression equation is y=0.0072 x+0.3556, and the correlation coefficient R 2 is as high as 0.9902.
At the beginning of culture, terpineol appears transparent oily distributed on the surface of liquid, but gradually breaks down and emulsifies into small oil drops with the passage of time; meanwhile, the color of the liquid is changed, and after the liquid is cultured for 24 hours in a shaking flask, the fermentation liquid of terpineol becomes milky white, and no grease floats on the liquid surface.
The production of rhamnolipid by Pseudomonas sp.s4 during 24h of culture is shown as B in figure 10, and during degradation of terpineol, rhamnolipid accumulates in large amounts during the stationary phase of cell growth, up to 2.57g/L by 18 h; after 18h, even though the growth of Pseudomonas sp.s4 has entered the decay phase, a certain amount of rhamnolipid can still accumulate.
In summary, pseudomonas sp.s4 uses terpineol as a carbon source, breaks the interface between terpineol and water by secreting rhamnolipid in the growth process, increases the contact degree between the Pseudomonas sp and the terpineol, and promotes the uptake, metabolism and self proliferation of the terpineol.
EXAMPLE 4 Pseudomonas sp. S4 remediation of contaminated soil
1. Experimental method
1. Test soil
The test soil used in this example includes: 1. paddy soil from Guangzhou experimental farm was used to study the degradation rate of Pseudomonas sp 4 under experimental conditions; 2. mine soil taken from the mining area of the Guangxi nan Dan county is used for simulating the actual soil restoration environment. The partial physicochemical properties of the two soils are shown in Table 6.
TABLE 6 physicochemical Properties of Rice soil and mine soil
2. Preparation of terpineol contaminated soil
The terpineol is quantitatively measured and dissolved in methylene dichloride, 250g of paddy soil or 250g of mine soil is filled in a 500mL triangular flask, the mixture is uniformly mixed, the mixture is placed in a dark place, the methylene dichloride is naturally volatilized for 3 days (the solvent is volatilized continuously during the period of time), and the contaminated paddy soil with the terpineol concentration of 100 mg.kg -1 or the contaminated mine soil with the terpineol concentration of 100 mg.kg -1 is obtained.
3. Preparation of bacterial suspension of Pseudomonas sp.S 4
Pseudomonas S4 is picked from the LB solid plate by an inoculating loop and inoculated into a liquid LB culture medium, and shake-cultured for 24h (30 ℃ C., 150 rpm); the bacterial liquid was collected, centrifuged (4 ℃,3500 rpm), and the precipitate was collected to obtain bacterial cells, which were resuspended in 0.9% sterilized physiological saline to prepare a bacterial suspension having a concentration of OD 600nm =1.
4. Preparation of biochar immobilized microbial inoculum
Single colonies of Pseudomonas sp 4 were picked up, inoculated into 100mL of LB medium and cultured for 24 hours, then bacterial liquid was collected, centrifuged (4 ℃,3500 rpm) to remove supernatant, the precipitate was collected to obtain bacterial cells, the bacterial cells were washed with 0.9% sterilized physiological saline, centrifuged again, and then the bacterial cells were resuspended with 5mL of physiological saline to prepare bacterial suspension having a concentration of OD 600nm =1.
Weighing 25g of biochar, adding into a sterilized tissue culture bottle, pouring 5mL of bacterial suspension, uniformly mixing, centrifuging at 1000r/min for 10min after the biochar fully adsorbs Pseudomonas sp S4, collecting precipitate, and drying in a baking oven at 30 ℃ to obtain the biochar immobilized microbial agent for later use.
The morphology of Pseudomonas sp.s4 immobilized before and after charcoal was examined by scanning electron microscopy. As shown in a in fig. 11, the surface of the biochar before bacteria fixation is not smooth and has irregular folds and gaps; as shown in B in fig. 11, after the bacteria are immobilized, the surface morphology of the biochar is not changed significantly, and the degrading bacteria are captured in the folds and gaps on the surface of the biochar; under the high power electron microscope, the form of the Pseudomonas (Pseudomonas sp.) S4 on the surface of the biochar is clearly visible and the biochar is rich, and the biochar itself is not obviously changed, which indicates that the Pseudomonas (Pseudomonas sp.) S4 is successfully fixed to the biochar.
5. Terpineol contaminated soil remediation test
TABLE 7 grouping set up for terpineol contaminated soil remediation experiments
Note that: the concentration of the bacterial suspension of Pseudomonas sp.s4 was OD 600nm =1; the initial concentration of rhamnolipid and CTAB is 200mg/L, and the solvent is water.
Seven treatment groups were set up in this experiment, each treatment was formulated using rhamnolipid, CTAB, and bacterial suspension of contaminated soil (contaminated rice soil or contaminated mine soil) prepared in this example, pseudomonas sp. S4, and biochar immobilized bacterial agent according to the information shown in table 7, with 6 replicates per treatment group.
Each treatment group was incubated at 30℃and sterile water was added to each treatment group to maintain the water content of the soil at 20% (v/w), and incubated for 11d. During the incubation, 1 time is sampled every 2d, 1g is sampled, the terpineol content in the contaminated soil of each treatment group is detected by referring to the method of "degradation of terpineol" in example 1, and the degradation rate of terpineol by Pseudomonas sp.s4 is counted.
6. Microbial diversity analysis
After the culture is finished, collecting soil samples of the CK group, the A group, the B group, the C group, the E group and the F group, and constructing a small fragment library by adopting a double-End sequencing (Paired-End) method on the basis of Illumina NovaSeq sequencing platforms to carry out high-throughput sequencing on 16S rDNA of the microbial community. Clustering or denoising and species annotation and abundance analysis are performed through Reads splicing and screening, alpha diversity analysis (ALPHA DIVERSITY) is performed according to sequencing amounts and sequencing depths of different samples by utilizing Mothur software and an R language tool, and the index value of Simpson, the index value of Shannon and the index value of Chao1 are calculated to evaluate the richness of the soil microbial community and the integrity of the community diversity.
2. Experimental results
1. Results of terpineol contaminated soil remediation test
As shown in A in FIG. 12, after 11d of co-incubation culture, the terpineol content in the contaminated rice soil (CK group) without any treatment was reduced from 100mg/kg to 78mg/kg, and the total degradation rate of terpineol was 22%; in contaminated rice soil (group A) repaired by adding only Pseudomonas (Pseudomonas sp.) S4, the terpineol content is reduced from 100mg/kg to 39mg/kg, and the total degradation rate of terpineol is 61%; in the polluted rice soil (group B) which is repaired by adding only rhamnolipid, the terpineol content is reduced from 100mg/kg to 72mg/kg, and the total degradation rate of the terpineol is 28%; adding Pseudomonas sp 4 and rhamnolipid into polluted rice soil (group C) to be repaired together, wherein the terpineol content is reduced from 100mg/kg to 13mg/kg, and the total degradation rate of the terpineol is 87%; in contaminated rice soil (group D) co-repaired by addition of Pseudomonas sp 4 and cationic surfactant CTAB, growth was inhibited due to the influence of CTAB-sol toxicity on Pseudomonas sp 4 growth, and the total degradation rate of terpineol was only 33%. The result shows that the Pseudomonas sp 4 can obviously reduce the content of terpineol in the polluted rice soil, and can generate a synergistic effect with rhamnolipid, the effect of repairing the polluted rice soil together is further obviously improved, and the degradation of the terpineol in the polluted rice soil by the Pseudomonas sp is inhibited by CTAB (CTAB).
As shown in B in fig. 12, after 11d of co-incubation culture, the total degradation rate of terpineol in contaminated rice soil (CK group) without any treatment was only 17.5%; compared with the CK group, the content of terpineol in polluted mine soil (E group) repaired by adding only Pseudomonas sp is obviously reduced, the degradation rate of terpineol is in an ascending trend along with the time, and the total degradation rate of terpineol reaches 48.5% after 11d of co-incubation culture; compared with the E group, the content of terpineol in polluted mine soil (F group) repaired by adding the biochar immobilized microbial inoculum is further remarkably reduced, the degradation rate is lower in the initial days, the physical adsorptivity and the mass transfer performance of the biochar are gradually developed along with the increase of time, the time of adaptation of Pseudomonas sp 4 to the environment is reduced, the high-efficiency degradation capability is promoted, the degradation capability is kept stable, the degradation rate of the terpineol is gradually increased and reversely exceeded, and the total degradation rate of the terpineol reaches 67.8 percent. The Pseudomonas sp is shown to be capable of remarkably reducing the terpineol content in polluted mine soil, and the effect of restoring polluted rice soil after being adsorbed and fixed by biological carbon is further remarkably improved.
The results show that the Pseudomonas sp.4 can repair terpineol-polluted soil, can exert synergistic effect when being combined with rhamnolipid or biochar, and has more remarkable repair effect.
2. Microbial diversity analysis results
TABLE 8 results of Alpha diversity index analysis of bacteria in each treatment group
As shown in Table 8, the index value of Shannon, chao1 and Simpson of the soil microorganisms in the remaining treatment groups were significantly increased compared to the CK group, wherein the index value of Shannon and Chao1 of Pseudomonas (sp.) S4-modified contaminated mine soil was the highest and the index value of Simpson of the charcoal immobilized microbial inoculum was the highest. The Pseudomonas sp is shown to promote the abundance and uniformity of bacteria in polluted soil and improve the diversity of microorganisms when the Pseudomonas sp is used for degrading terpineol.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

Claims (10)

1. Pseudomonas sp.) S4, characterized in that said Pseudomonas sp 4 has the deposit number GDMCC No:63948, which was deposited at the cantonese collection of microorganism strains on the 10 th month 31 of 2023.
2. Use of Pseudomonas sp for degrading terpineol.
3. Use of Pseudomonas sp for repairing terpineol contaminated soil.
4. Use of Pseudomonas sp in combination with a biosurfactant for repairing terpineol contaminated soil.
5. Use of Pseudomonas sp in combination with biochar for repairing terpineol contaminated soil.
6. The use according to any one of claims 2 to 5, characterized in that the Pseudomonas sp is Pseudomonas sp. S4 according to claim 1.
7. A formulation for degrading terpineol, characterized in that the formulation comprises Pseudomonas sp according to claim 1, S4.
8. The formulation of claim 7, further comprising a biosurfactant or biochar.
9. The formulation of claim 8, wherein the biosurfactant comprises rhamnolipids.
10. A method of remediating terpineol contaminated soil, characterized in that the terpineol contaminated soil is treated with a formulation according to any of claims 7-9.
CN202410122942.0A 2024-01-29 2024-01-29 Pseudomonas and application thereof in degradation of terpineol and restoration of polluted soil Pending CN118126871A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202410122942.0A CN118126871A (en) 2024-01-29 2024-01-29 Pseudomonas and application thereof in degradation of terpineol and restoration of polluted soil

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202410122942.0A CN118126871A (en) 2024-01-29 2024-01-29 Pseudomonas and application thereof in degradation of terpineol and restoration of polluted soil

Publications (1)

Publication Number Publication Date
CN118126871A true CN118126871A (en) 2024-06-04

Family

ID=91236903

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202410122942.0A Pending CN118126871A (en) 2024-01-29 2024-01-29 Pseudomonas and application thereof in degradation of terpineol and restoration of polluted soil

Country Status (1)

Country Link
CN (1) CN118126871A (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4490471A (en) * 1981-12-11 1984-12-25 Ciba-Geigy Corporation Microorganisms of the genus Pseudomonas and process for degrading compounds which contain methyl groups in aqueous solutions
KR20070063200A (en) * 2005-12-14 2007-06-19 안동대학교 산학협력단 Novel strain pseudomonas sp. ks-2p useful for degradation of endosulfan and toxic endosulfan sulfate
CN115247139A (en) * 2021-09-13 2022-10-28 浙江海洋大学 Pseudomonas capable of utilizing methyl mercaptan as unique carbon source and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4490471A (en) * 1981-12-11 1984-12-25 Ciba-Geigy Corporation Microorganisms of the genus Pseudomonas and process for degrading compounds which contain methyl groups in aqueous solutions
KR20070063200A (en) * 2005-12-14 2007-06-19 안동대학교 산학협력단 Novel strain pseudomonas sp. ks-2p useful for degradation of endosulfan and toxic endosulfan sulfate
CN115247139A (en) * 2021-09-13 2022-10-28 浙江海洋大学 Pseudomonas capable of utilizing methyl mercaptan as unique carbon source and application thereof

Similar Documents

Publication Publication Date Title
CN108486006B (en) A kind of preparation method and application of greasy filth oil degradation complex enzyme
CN112625981B (en) Serratia marcescens and application thereof
CN110938569B (en) Microbial agent for hexavalent chromium pollution treatment and hexavalent chromium pollution treatment method
KR102564256B1 (en) Microorganism producing sulfur and method for preparing sulfur using the same
CN104830708A (en) Crude oil degrading bacteria strain and application thereof
CN110904004A (en) Bacterium for producing trehalose hydrolase and breeding method and application thereof
CN109868231B (en) Cupriavidus (Cupriavidus) and application thereof
CN110551662B (en) Arthrobacter strain ZJ-H and application thereof
CN107523513B (en) Compound bacterium capable of rapidly degrading 17 beta-estradiol and preparation method and application thereof
CN109929785B (en) Bacterium capable of degrading 2, 6-dimethylphenol and microbial inoculum produced by same
CN106635872B (en) One plant of Mo Haiwei bacillus and its application
Badis et al. Characterization and biodegradation of soil humic acids and preliminary identification of decolorizing actinomycetes at Mitidja plain soils (Algeria)
CN118126871A (en) Pseudomonas and application thereof in degradation of terpineol and restoration of polluted soil
CN106834189B (en) Biosurfactant producing bacterium and application thereof
CN114703088B (en) Bacillus licheniformis and application thereof
CN113832081B (en) Bacterial strain with hexavalent chromium reduction capacity and application thereof
CN112239731A (en) Surface active bacterial strain for degrading petroleum hydrocarbon and application thereof
CN113801821B (en) Novel mycobacterium alfa WCJ and application thereof in degrading organic pollutants
CN116925947A (en) Bacillus subtilis capable of generating surfactant and degrading crude oil and application thereof
Tian et al. Characterization of a Biosurfactant-producing Strain Rhodococcus sp. HL-6
CN108841742B (en) Saline-alkali-tolerant bacillus strain ZH-1 and preparation method and application thereof
KR20020044733A (en) Microorganism consortia for the degradation of diesel and process for preparation thereof
CN113337425B (en) Gordoniella for producing long-carbon-chain mycolic acid and application thereof
Pan et al. Characterization of aerobic bacteria involved in degrading polyethylene glycol (PEG)-3400 obtained by plating and enrichment culture techniques
CN111440742B (en) Pseudomonas aeruginosa mutant strain for petroleum hydrocarbon degradation

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