CN115386520A - Rhodococcus pyridinivorans RL-GZ01 strain and application thereof - Google Patents
Rhodococcus pyridinivorans RL-GZ01 strain and application thereof Download PDFInfo
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- CN115386520A CN115386520A CN202211033339.2A CN202211033339A CN115386520A CN 115386520 A CN115386520 A CN 115386520A CN 202211033339 A CN202211033339 A CN 202211033339A CN 115386520 A CN115386520 A CN 115386520A
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- phthalate
- dehp
- rhodococcus
- rhodococcus pyridinivorans
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- 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
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- 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
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- 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
- C02F2101/10—Inorganic compounds
- C02F2101/105—Phosphorus compounds
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- 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
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
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- 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
- C02F2101/34—Organic compounds containing oxygen
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- 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
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- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention provides a Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) RL-GZ01 strain and application thereof. The Rhodococcus pyridinivorans RL-GZ01 strain is preserved in the Guangdong province microorganism culture collection center at 20 months 4 in 2022, and the preservation number is GDMCC No:62401. the strain can effectively degrade DBP, BBP, DEP, DMP, DEHP, DCHP and DNOP, and can treat NH in a nitrogen-containing medium containing DEHP 4 + ‑N、NO 2 ‑ The denitrogenation rate of-N is as high as 95.0% and 100%,and has the capability of synchronous nitrification and denitrification, and can effectively repair the environment polluted by PAEs and nitrogen and phosphorus at the same time.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a Rhodococcus pyridinivorans RL-GZ01 strain and application thereof.
Background
Plasticizers are commonly used as additives in the preparation of plastics, and are mainly classified into Phthalic Acid Esters (PAEs), etc., and PAEs contain various substances, such as di (2-ethylhexyl) phthalate, dicyclohexyl phthalate, dimethyl phthalate, di-n-butyl phthalate, diethyl phthalate, di-n-octyl phthalate, and butyl benzyl phthalate, which are bonded to polymers through weak secondary bonds, and thus are easily released from plastics and permeate into the environment. Plastic products are widely used in various fields of human production and life due to good physicochemical properties and low use cost, but as more and more plastics are buried or discarded in the environment, more plasticizers are released into the environment. According to the research, PAEs can interfere the endocrine system of organisms, and cause the occurrence of diseases such as the reduction of the number of sperms, the stoppage of the formation of the sperms, the reduction of reproductive capacity, the hyperplasia of uterine mucosa tissues and the like; for example, di (2-ethylhexyl) phthalate has strong developmental toxicity, neurotoxicity, toxicity causing multiple organ canceration and the like, and the residue of the visible plasticizer causes no small harm to human beings and other organisms in the nature.
PAEs are degraded mainly by biodegradation of aerobic and anaerobic bacteria due to low photolysis and hydrolysis efficiency in natural environment. At present, a large number of efficient PAEs degrading strains are separated from various environments including mangrove forest, soil, constructed wetland, river, activated sludge of wastewater treatment plants, compost remediation samples, heavy metal polluted soil and the like, and the strains mainly comprise sphingosine bacteria, gordonia bacteria, mycobacteria, rhodococcus and the like. For example, CN111575197A discloses a rhodococcus pyridinivorans XB with a removing capability for dibutyl phthalate and di-n-octyl phthalate, but the removing capability of the rhodococcus pyridinivorans XB for dibutyl phthalate and di-n-octyl phthalate is only 27.5% and 21.2%, respectively, and the removing effect is not ideal. Most of the PAEs degrading strains obtained by separation at present are from land, only a few strains belong to marine microorganisms, and the fate trend and ecological restoration research of PAEs in a marine ecosystem are relatively few. The marine microorganisms capable of efficiently degrading PAEs obtained by separation are of great significance for understanding the transformation mechanism of PAEs in a marine ecosystem and establishing a bioremediation technology of PAEs polluted sea areas.
In addition, nitrogen pollution in the environment is mainly caused by inorganic nitrogen and organic nitrogen, and the inorganic nitrogen mainly includes ammonia Nitrogen (NH) 4 + -N), nitrate Nitrogen (NO) 3 - N), nitrous Nitrogen (NO) 2 - -N), mainly from the wastewater discharged after the treatment of urban domestic sewage, the wastewater discharged from fertilizer plants, etc.; the organic nitrogen mainly comprises urea, protein, organic alkali and the like, and is mainly derived from pesticide loss, livestock excrement and the like in the tanning industry, the printing and dyeing industry and the agricultural production process. At present, denitrification is mainly carried out by a biological denitrification technology, and the technology is divided into a traditional biological denitrification technology and a novel biological denitrification technology according to the process technology and the difference of denitrifying bacteria. In the traditional biological denitrification process, the two processes of nitrification and denitrification are generally carried out separately, namely, the aquaculture wastewater enters an aerobic zone for nitrification after being ammoniated and then flows into an anoxic zone with denitrifying bacteria for denitrification. In recent years, more and more novel biological denitrification technologies, such as shortcut nitrification and denitrification, anaerobic ammonia oxidation, synchronous nitrification and denitrification and the like, have emerged, and compared with the traditional biological denitrification, the technology has many advantages, such as realization of nitrification, denitrification and the like in the same space. The prior art provides a Rhodococcus pyridinivorans CPZ-24 strain which has synchronous nitrification and denitrification and can be applied to a novel biological denitrification technology, but the nitrate nitrogen removal capacity of the strain is only 66.74%. Selecting novel denitrifying microorganismsHeterotrophic nitrification aerobic denitrifying bacteria are important for denitrification and environmental protection, and are beneficial to the sustainable development of the habitat.
The total phosphorus also causes serious pollution to the environment, and is divided into inorganic phosphorus and organic phosphorus, wherein the inorganic phosphorus comprises phosphate, polyphosphate and the like, and the organic phosphorus mainly refers to phosphorus elements contained in biological components such as protein and the like. The accumulation of nitrogen and phosphorus in the environment can cause eutrophication of water bodies, further seriously affect the ecological balance, even threaten the health of human beings and aquatic animals, and increase the cost of water treatment. The biological treatment method is widely applied to the removal of total phosphorus in the environment, and has the advantages of low cost, high efficiency, less secondary pollution and the like compared with the traditional physical method and chemical method. The biological treatment method is mainly characterized in that the phosphorus in the sewage is absorbed through the self-growth metabolism of microorganisms, the phosphorus which is more than the self-growth requirement is gathered in a short time and stored in cells, and the purpose of removing the phosphorus is achieved. The prior art provides a Rhodococcus ruber HDRR2Y strain with phosphate removing capability, the average removal rate of the strain on phosphate is 36.3-44.9%, and the degradation effect on phosphate is not ideal, so that the separation, development and practical application of phosphorus removing bacteria are crucial to the restoration of eutrophic water.
Therefore, the microbial strain capable of degrading PAEs and clearing nitrogen and phosphorus is found, and has considerable necessity for environment restoration.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a Rhodococcus pyridinivorans RL-GZ01 strain and application thereof, and simultaneously realizes efficient removal of phthalate substances, total nitrogen and total phosphorus in the environment and remediation of the polluted environment.
The invention mainly aims to provide a Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) RL-GZ01 strain.
The invention also aims to provide application of the Rhodococcus pyridinivorans RL-GZ01 strain in degrading phthalate substances and/or repairing phthalate substance polluted environments.
It is a further object of the present invention to provide the use of said Rhodococcus pyridinivorans RL-GZ01 strain for the elimination of total nitrogen and/or the remediation of nitrogen-contaminated environments.
It is still another object of the present invention to provide the use of said Rhodococcus pyridinivorans RL-GZ01 strain for the elimination of total phosphorus and/or the remediation of phosphorus-contaminated environments.
The invention also aims to provide application of the Rhodococcus pyridinivorus RL-GZ01 strain in preparation of products for degrading phthalate substances and/or eliminating total nitrogen and/or total phosphorus.
Still another object of the present invention is to provide a microbial inoculum.
It is yet another object of the present invention to provide a biological cleanser.
It is still another object of the present invention to provide a biodegradation agent.
The invention provides a Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) RL-GZ01 strain, which is preserved in Guangdong province microorganism strain preservation center in 2022 in 4 and 20 days, wherein the preservation number is GDMCC No:62401, the storage address is building No. 59, building No. 5 of the Mingzhou Jielizhou 100 yard.
The Rhodococcus pyridinivorans RL-GZ01 strain is a gram-positive strain; the thalli is rod-shaped, has no flagellum and no spore; the colony is orange and round, the edge is smooth, and the surface is provided with protrusions; the hydrogen sulfide reaction is positive; the denitrification experiment shows obvious blue and is positive; a large amount of bubbles are generated in the catalase test, and the catalase test is positive; urease test also showed positive; V-P, methyl red, indole, gelatin and glucose fermentation tests show that the test is negative; the tetracycline-resistant peptide has no resistance to Amp, kan and tetracycline.
The Rhodococcus pyridinivorans RL-GZ01 strain is separated from sludge collected from mangrove in the intertidal zone of the seaside park of Zhanjiang province, guangdong province, and can completely degrade 100mg/L of di-n-butyl phthalate, butyl benzyl phthalate, diethyl phthalate and dimethyl phthalate within 12 hours and 100mg/L of di (2-ethylhexyl) phthalate, dicyclohexyl phthalate and di-n-octyl phthalate within 24 hours. Based on the above, the application of the Rhodococcus pyridinivorans RL-GZ01 strain in degrading phthalate substances and/or repairing phthalate substance polluted environments is also within the protection scope of the invention.
Preferably, the phthalate ester substance is one or more of di (2-ethylhexyl) phthalate, dicyclohexyl phthalate, di-n-octyl phthalate, di-n-butyl phthalate, butyl benzyl phthalate, diethyl phthalate or dimethyl phthalate.
Rhodococcus pyridinivorans RL-GZ01 strain has aerobic heterotrophic nitrification ammonia Nitrogen (NH) 4 + -N), and aerobic denitrification of nitrate Nitrogen (NO) 3 - -N), nitrous Nitrogen (NO) 2 - -N) of the cells: culturing Rhodococcus pyridinivorans RL-GZ01 strain in nitrogen source culture medium only containing ammonia nitrogen, nitrite nitrogen or nitrate nitrogen for 72 hours, and then culturing NH 4 + -N、NO 2 - -N、NO 3 - The denitrogenation rates of-N can reach 93.6%, 77.3% and 77.0% respectively. In nitrogen-containing medium containing 100mg/L DEHP, rhodococcus pyridinivorans RL-GZ01 strain can completely remove DEHP and remove NH 4 + -N、NO 2 - The denitrogenation rate of-N is as high as 95.0% and 100%. Based on this, the application of the Rhodococcus pyridinivorans RL-GZ01 strain in the elimination of total nitrogen and/or the remediation of nitrogen-contaminated environments should also be within the scope of the present invention.
Preferably, the nitrogen is inorganic nitrogen.
Further preferably, the inorganic nitrogen is NH 4 + -N、NO 2 - -N or NO 3 - -one or more of N.
After the culture is carried out for 12 hours in urban wastewater containing DEHP and nitrogen and phosphorus, the Rhodococcus pyridinivorus RL-GZ01 strain can thoroughly remove the DEHP with the concentration of 5 mg/L; after culturing for 84h, the clearance rate of total nitrogen in the urban wastewater reaches 95.4%, and the total phosphorus is reduced from 10.89mg/L to about 2.37mg/L, which indicates that the Rhodococcus pyridinivorans RL-GZ01 strain can not only effectively degrade PAEs, but also effectively remove nitrogen and phosphorus in the environment; in the degradation of NH 4 + In the process of-N, doWith the occurrence of NO 2 - -N or NO 3 - Accumulation of-N shows that the Rhodococcus pyridinivorans RL-GZ01 strain has synchronous nitrification and denitrification capabilities and can effectively repair the environment polluted by PAEs and nitrogen and phosphorus simultaneously, so that the application of the Rhodococcus pyridinivorans RL-GZ01 strain in the environment of removing total phosphorus and/or repairing phosphorus pollution and the application of the Rhodococcus pyridinivorans RL-GZ01 strain in the preparation of products for degrading phthalate substances and/or removing total nitrogen and/or removing total phosphorus are both within the protection range of the invention.
Preferably, the total phosphorus is inorganic phosphorus.
Preferably, the product is one or more of a microbial inoculum, a biological cleaning agent and a biological degradation agent.
Therefore, the invention also provides a microbial inoculum, a biological cleaning agent and a biological degradation agent, which contain the Rhodococcus pyridinivorus RL-GZ01 strain and/or bacterial liquid thereof.
The invention has the following beneficial effects:
the Rhodococcus pyridinivorans RL-GZ01 strain is obtained by screening and separating, and can completely degrade 100mg/L DBP, BBP, DEP and DMP within 12 hours, completely degrade 100mg/L DEHP, DCHP and DNOP within 24 hours, and has excellent degradation capability on phthalate substances; to NH 4 + -N、NO 2 - -N、NO 3 - The denitrification rates of-N can reach 93.6%, 77.3% and 77.0%, respectively, for NH in the nitrogen-containing medium containing DEHP 4 + -N、NO 2 - The denitrification rate of N is improved to 95.0%, 100% and the complete degradation capability of DEHP is maintained; the Rhodococcus pyridinivorans RL-GZ01 strain has synchronous nitrification and denitrification capability, and can effectively repair the environment polluted by PAEs and nitrogen and phosphorus at the same time.
Drawings
FIG. 1 is a structural diagram of Rhodococcus pyridinivorans RL-GZ01 strain under an electron microscope.
FIG. 2 shows the colony morphology of Rhodococcus pyridinivorans RL-GZ01 strain.
FIG. 3 is a phylogenetic tree of Rhodococcus pyridinivorans RL-GZ01 strain.
FIG. 4A shows the degradation of different PAEs by Rhodococcus pyridinivorans RL-GZ01 strain, and FIG. 4B shows the PAEs concentration of a control group without bacteria.
FIG. 5 shows the denitrification of various nitrogen sources by Rhodococcus pyridinivorans RL-GZ01.
FIG. 6 shows the denitrification of Rhodococcus pyridinivorans RL-GZ01 strain in a medium containing 100mg/L DEHP in different nitrogen sources.
FIG. 7 shows the degradation of DEHP by Rhodococcus pyridinivorans RL-GZ01 in a medium containing 100mg/L DEHP in different nitrogen sources.
FIG. 8 shows NH concentration of Rhodococcus pyridinivorans RL-GZ01 at 20mg/L, 60mg/L and 200mg/L 4 + Degradation curves at N concentration for different concentrations of DEHP.
FIG. 9 shows NH of Rhodococcus pyridinivorans RL-GZ01 strain at 500mg/L 4 + Degradation curves at N concentration for different concentrations of DEHP.
FIG. 10 shows Rhodococcus pyridinivorans RL-GZ01 strain at different concentrations of DEHP versus NH concentration of 20mg/L 4 + The denitrification of N.
FIG. 11 shows Rhodococcus pyridinivorans RL-GZ01 strain at different concentrations of DEHP versus NH concentration of 60mg/L 4 + Denitrification of N.
FIG. 12 shows Rhodococcus pyridinivorans RL-GZ01 strain at different concentrations of DEHP against NH at a concentration of 200mg/L 4 + In the graph, CK1 represents a control group to which no bacteria were added when the DEHP concentration was 5mg/L, CK2 represents a control group to which no bacteria were added when the DEHP concentration was 20mg/L, and CK3 represents a control group to which no bacteria were added when the DEHP concentration was 50 mg/L.
FIG. 13 shows Rhodococcus pyridinivorans RL-GZ01 strain at different concentrations of DEHP versus NH at a concentration of 500mg/L 4 + In the graph, CK1 represents a control group without adding bacteria at a DEHP concentration of 5mg/L, CK2 represents a control group without adding bacteria at a DEHP concentration of 20mg/L, and CK3 represents a control group without adding bacteria at a DEHP concentration of 50 mg/L.
FIG. 14 is the experimental result of the effect of Rhodococcus pyridinivorans RL-GZ01 strain on COD value in experimental urban wastewater.
FIG. 15 shows the degradation of TP in experimental municipal wastewater by Rhodococcus pyridinivorans RL-GZ01.
FIG. 16 shows the effect of Rhodococcus pyridinivorans RL-GZ01 strain on TN and NH in experimental urban wastewater 4 + -N、NO 3 - -N、NO 2 - -removal of N.
FIG. 17 shows the degradation of DEHP in experimental municipal wastewater by Rhodococcus pyridinivorans RL-GZ01.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Inorganic salt medium (MSM): 1.0g/L NH 4 NO 3 ，20g/L NaCl，0.5g/L(NH 4 ) 2 SO 4 ，0.05g/L CaCl 2 ，0.5g/L KH 2 PO 4 And 1.5g/L K 2 HPO 4 ，pH＝7.0±0.2。
LB culture medium: 10.0g/L peptone, 5.0g/L NaCl,10.0g/L yeast extract, pH =7.0 ± 0.2.
The solid culture medium is prepared by adding agar into corresponding culture medium according to the proportion of 15 g/L.
Heterotrophic Nitrification Medium (HNM) (per liter of distilled water): (NH) 4 ) 2 SO 4 0.66g, 4.72g of sodium succinate 2 PO 4 0.50g，Na 2 HPO 4 0.50g，MgSO 4 ·7H 2 0.20g of O, 30.00g of NaCl, 2.00mL of trace element solution and pH =7.5.
Nitrite Denitrification Medium (NDM) (per liter of distilled water): naNO 2 0.28g, sodium succinate 3.16g 4 ·7H 2 O 0.20g，CaCl 2 0.01g，EDTA 0.07g，KH 2 PO 4 0.50g，Na 2 HPO 4 0.50g，FeSO 4 0.01g, 2.00mL of trace element solution, and pH =7.5.
Denitrification Medium (DM) (per liter of distilled water): KNO 3 1.00g, sodium succinate 4.68g 4 ·7H 2 O 0.20g，CaCl 2 0.01g，EDTA 0.07g，KH 2 PO 4 0.50g，Na 2 HPO 4 0.50g，FeSO 4 0.01g, naCl 30.00g, trace element solution 2.00mL, and pH =7.5.
Trace element solution (per liter of distilled water): EDTA.2Na 57.10g, znSO 4 ·7H 2 O 3.90g，CaCl 2 ·2H 2 O 7.00g，MnCl 2 ·4H 2 O 1.00g，FeSO 4 ·7H 2 O 5.00g，(NH4) 6 Mo 7 O 24 ·4H 2 O 1.10g，CuSO 4 ·5H 2 O 1.60g，CoCl 2 ·6H 2 O1.60g, pH6.0, and sterilizing at 121 deg.C for 30 min.
NH 4 + -N、NO 3 - -N、NO 2 - The determination and analysis methods of the N-nitrogen element are all referred to national standards: NH (NH) 4 + The determination and analysis of-N are carried out according to water quality-determination of ammonia nitrogen-Nessler reagent spectrophotometry (GB HJ 535-2009); NO 3 - Determination and analysis of N according to "determination of Water quality-nitrate Nitrogen-ultraviolet Spectrophotometry" (GB HJ/T346-2007); NO 2 - The determination and analysis of-N are based on the "determination of Water quality-nitrite Nitrogen-spectrophotometry" (GB 7493-87).
The detection method of the total phosphorus adopts an ammonium molybdate method; the detection method of Chemical Oxygen Demand (COD) adopts a chromium method; the detection method of Total Nitrogen (TN) adopts a nano reagent method.
The steps for detecting the concentration of the phthalate ester substances in the following examples are as follows:
(1) And (3) extraction: adding n-hexane with equal volume to the sample, extracting for 10min by ultrasonic oscillation (40 KHz), standing for 1 hr, collecting the upper layer organic solvent, and filtering with 0.22 μm organic filter membrane.
(2) Detecting the concentration of the phthalate ester substances by using a gas chromatograph:
a. detection conditions of a gas chromatograph:
the column was a WondaCap 5 column (GL Sciences Inc, japan.25mm. Times.30 m. Times.0.25 μm);
the detector is an Electron Capture Detector (ECD);
the sample introduction amount is 1 mu L, and the flow dividing ratio is 49:1, split-flow sample injection;
the temperature of the sample introduction chamber is 300 ℃;
the column temperature is constant at 280 ℃;
the temperature of the detector is 300 ℃;
the carrier gas is high-purity nitrogen (99.999%);
the flow rate of the carrier gas is 2mL/min;
data were obtained and analyzed by the software Labsolutions (Version 5.90, SHIMADZU, japan).
b. The standard curve establishing method comprises the following steps:
and establishing a standard curve of the relation between the concentration of the phthalate ester substance and the detection peak area by adopting an external standard method. Accurately preparing phthalate ester substance standard substances with the concentrations of 20mg/L, 40mg/L, 60mg/L, 80mg/L and 100mg/L, wherein each concentration is 3 parts, detecting the standard substance with each concentration according to the detection conditions of the gas chromatograph, thereby establishing a standard curve between the concentration of the standard substance and the peak area, and the fitting result is shown in table 1.
TABLE 1 Standard Curve fitting results
(3) The degradation rate of the phthalate ester is calculated by the following formula:
example 1 isolation, identification and preservation of strains
1. Isolation of the Strain
The method comprises the following steps: collecting sludge from the mangrove in intertidal zone of the seaside park of Zhanjiang city, guangdong province, packaging with a brown sample bottle, marking, and storing in a refrigerator at 4 deg.C for later use.
Step two: enriching bacteria by a unique carbon source method, specifically, taking di (2-ethyl) hexyl phthalate (DEHP) with the final concentration of 100mg/L as a unique carbon source, weighing 5g of a sample from the stored sludge, adding the sample into 50mL of MSM culture medium containing 100mg/L DEHP, and culturing for 5 days in a shaking table at 30 ℃ and 180rpm to obtain an enriched culture solution 1.
Step three: taking the enrichment culture solution 1 of the second step according to the proportion of 10% (v/v), inoculating into 10mL MSM culture medium containing 200mg/L DEHP, and placing in a shaking table to culture for 5 days at 30 ℃ and 180rpm to obtain an enrichment culture solution 2.
Step four: taking the enrichment culture solution 2 of the third step according to the proportion of 10% (v/v), inoculating into 10mL MSM culture medium containing 500mg/L DEHP, and placing in a shaking table to culture for 5 days at 30 ℃ and 180rpm to obtain an enrichment culture solution 3.
Step five: taking 3 1mL of the enrichment culture solution obtained in the step four as a reserved bacterial solution to store the strain; the remaining 9mL were treated with n-hexane at 1: the extraction ratio of 1 (v/v), the DEHP concentration is detected by using a gas chromatograph, the DEHP degradation rate is calculated, and the degradation rate is more than 95 percent.
Step six: placing the reserved bacterial liquid corresponding to the enrichment culture solution in the fifth step into a centrifuge, centrifuging for 5min at 5000rpm, washing the cell sediment by Phosphate Buffered Saline (PBS), and repeating the centrifugation and washing for three times. The cells were resuspended in 1mL of MSM liquid medium, and then, the cell suspension was spread on solid MSM medium containing 500mg/L DEHP and 0.01g/L Tween 80, cultured at 30 ℃, and the colony status was checked every day.
Step seven: selecting single colonies with hydrolytic halos on a solid MSM culture medium and good growth on the solid MSM culture medium, respectively culturing in 10mL LB enrichment culture medium for 24 hours, respectively taking 1mL culture solution, centrifuging for 5min at the rotating speed of 5000rpm, collecting thalli, washing by using PBS buffer solution, repeating the centrifugation and washing for three times, inoculating the thalli into 10mL MSM culture medium containing 100mg/L DEHP, and performing shake culture at 30 ℃ and 180rpm for 5 days;
step eight: respectively reserving 1mL of bacterial liquid, and remaining 9mL of bacterial liquid, using normal hexane to perform reaction according to the weight ratio of 1: extraction was carried out at a ratio of 1 (v/v), and the degradation rate of DEHP was again measured by gas chromatography.
Step nine: and according to the detection result of the gas chromatography in the step eight, taking the reserved bacterial liquid of the sample with the DEHP degradation rate of more than 95%, placing the reserved bacterial liquid in a centrifuge, centrifuging for 5min at 5000rpm, washing the cell sediment by Phosphate Buffered Saline (PBS), and repeating the centrifugation and washing for three times. The cells were resuspended in 1mL of MSM liquid medium, and then, the cell suspension was spread on solid MSM medium containing 500mg/L DEHP and 0.01g/L Tween 80, cultured at 30 ℃ and checked for colony status every day.
Step ten: and (3) firstly repeating the steps seven, eight and nine to purify the strains twice, and then repeating the steps seven and eight to purify the strains once, wherein from the results obtained in the step eight, the DEHP degradation rates of one strain in the three purification plates are all 100%, the degradation capability is stable, the bacterial colony is single, and the strain is named as the strain RL-GZ01.
2. Morphological characteristics of Strain RL-GZ01
The morphological characteristics of the strain RL-GZ01 were observed under an electron microscope, and the results are shown in FIG. 1, and it can be seen that the strain has a rod-shaped body, no flagella and no spores.
The colony morphology of the strain RL-GZ01 is shown in FIG. 2, and the colony is orange and round, has smooth edges and has a protrusion on the surface.
3. Physiological and biochemical characteristics of strain RL-GZ01
RL-GZ01 is a gram-positive strain; the hydrogen sulfide reaction is black, and the reaction is positive; obvious blue appears in the denitrification color development plate, and the plate is positive; a great amount of bubbles are generated in the catalase test, and the catalase test also shows positive results; urease test also showed positive; the fermentation test of V-P, methyl red, indole, gelatin and glucose has no corresponding positive reaction phenomenon, and the result is negative. In an antibiotic resistance test, sterile circular filter paper sheets respectively dripped with Amp, kan and tetracycline are placed in the middle of an LB culture medium which is fully coated with a strain RL-GZ01 by a coating plate method, the flat plate is placed in a constant temperature incubator at 30 ℃ for culturing for several days, the flat plate is observed, and a circle of non-growing bacteria surround the circular filter paper sheets, which indicates that the strains have no resistance to Amp, kan and tetracycline. The physiological and biochemical identification of the strain RL-GZ01 is shown in Table 2.
TABLE 2 physiological and biochemical identification results of strain RL-GZ01
4. 16S rRNA gene identification of strain RL-GZ01
Inoculating the strain RL-GZ01 into an LB culture medium, culturing for 12h at 30 ℃ and 180rpm, taking 1mL of Bacterial liquid, centrifugally collecting thalli, extracting Bacterial genome DNA by using a Bacterial genome extraction Kit (TaKaRaMiniBEST Bacterial Genomic DNA extraction Kit Ver.3.0), detecting the obtained gene DNA by using 0.8% agarose gel electrophoresis, determining the concentration of the Genomic DNA by using a Micro spectrophotometer (Micro Drop, BIO-DL, china), and storing the obtained Genomic DNA at-20 ℃ for later use.
A pair of universal primers was designed for amplification of 16S rDNA sequences: (27F.
PCR amplification was carried out using Premix TaqTM DNA polymerase (Takara, japan) using the extracted genomic DNA of strain RL-GZ01 as a template, the PCR product was detected by 1% Agarose Gel electrophoresis, purified with a DNA Purification recovery Kit (TaKaRaMiniBEST Agarose Gel DNA extraction Kit Ver.4.0), ligated to a pMD19-T vector, transformed into E.coli DH 5. Alpha. Competent cells, spread on an LB solid medium plate containing 0.1% (v/v) ampicillin, cultured at 37 ℃ for 12 hours, white colonies were picked up into a liquid LB medium, cultured with shaking at 37 ℃ and 180rpm for 12 hours, extracted with a Plasmid extraction Kit (TaKaRaMiniBEST Plasmid Purification Kit Ver.4.0), and sent to HakkenProducer Biolabs for sequencing. The sequencing results were analyzed by Blast alignment on NCBI website (http:// www. NCBI. Nlm. Nih. Gov /), and phylogenetic trees were constructed using MEGA software (version: 7.0), and the results are shown in FIG. 3.
The strain RL-GZ01 is identified as Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) by integrating the thallus morphology, physiological and biochemical characteristics and 16S rRNA gene sequence, and is named as Rhodococcus pyridinivorans RL-GZ01 strain.
5. Strain preservation
Rhodococcus pyridinivorans RL-GZ01 strain is preserved in Guangdong province culture collection center in 2022 at 20.4 months, with the preservation number being GDMCC No:62401, the storage address is: building No. 59, building No. 5 of the prefecture midroad No. 100 yard in Guangzhou city.
EXAMPLE 2 degradation of phthalate esters by Strain RL-GZ01
1. Experimental methods
(1) Preparing a seed solution: selecting Rhodococcus pyridinivorans RL-GZ01 single colony from LB plate, and performing shaking culture in LB liquid culture medium at 30 ℃ until OD is reached 600 =1.5, obtain seed liquid.
(2) Degradation experiment of phthalate ester substances:
taking OD 600 200 mu L of Rhodococcus pyridinovorus RL-GZ01 strain seed liquid is centrifuged for 4min at 5000rpm, bacterial precipitates are collected, the bacterial precipitates are fully suspended by 200 mu L of PBS, the operations of centrifugation and suspension are repeated for 3 times, and the bacterial precipitates are sequentially added into 10mL of MSM culture medium which takes one of di (2-ethylhexyl) phthalate (DEHP), dicyclohexyl phthalate (DCHP), di-n-octyl phthalate (DNOP), butyl Benzyl Phthalate (BBP), di-n-butyl phthalate (DBP), diethyl phthalate (DEP) and dimethyl phthalate (DMP) as a unique carbon source, the final concentration of the phthalate substances is 100mg/L, and then the culture is carried out for 60h under the conditions of constant temperature shaking at 30 ℃.
A control group (MSM medium with PAEs as the sole carbon source) was set without addition of bacteria, and 3 replicates were set for all experiments.
And detecting the concentration of the PAEs every 12h, and drawing a PAEs concentration histogram.
2. Results of the experiment
The degradation of different PAEs by the Rhodococcus pyridinivorans RL-GZ01 strain is shown in FIG. 4A, the concentration of the PAEs in a control group is shown in FIG. 4B, and the Rhodococcus pyridinivorans RL-GZ01 strain can completely degrade 100mg/L of di-n-butyl phthalate (DBP), butyl Benzyl Phthalate (BBP), diethyl phthalate (DEP) and dimethyl phthalate (DMP) when cultured for 12 h; when cultured for 24 hours, the Rhodococcus pyridinivorans RL-GZ01 strain can thoroughly degrade 100mg/L of di (2-ethylhexyl) phthalate (DEHP), dicyclohexyl phthalate (DCHP) and di-n-octyl phthalate (DNOP).
The results show that Rhodococcus pyridinivorans RL-GZ01 strain has the ability to completely degrade di (2-ethylhexyl) phthalate, dicyclohexyl phthalate, di-n-octyl phthalate, butyl benzyl phthalate, di-n-butyl phthalate, diethyl phthalate and dimethyl phthalate.
EXAMPLE 3 Denitrification Performance of Strain RL-GZ01
1. Experimental method
(1) Preparing a seed solution: picking single colony from LB plate to LB liquid culture medium, shaking culture at 30 deg.C until OD 600 =1.5, obtain seed liquid.
(2) And (3) denitrification experiment:
taking OD 600 200 mu L of Rhodococcus pyridinivorans RL-GZ01 strain seed liquid of =1.5, centrifuging for 4min under the condition of 5000rpm, collecting thalli, fully suspending with 1mL of HNM culture medium, NDM culture medium and DM culture medium respectively, repeating the operations of centrifuging and suspending for 3 times, supplementing 200mL of corresponding culture medium respectively, and culturing for 72h in a constant temperature shaking table at 30 ℃ and in the dark.
Three control groups (HNM medium, NDM medium, DM medium, respectively) were set without addition of bacteria, and 3 replicates were set for all experiments.
NH in each culture medium was detected every 12h 4 + -N、NO 2 - -N、NO 3 - -concentration of N and plotting a nitrogen content histogram.
2. Results of the experiment
The clearance of Rhodococcus pyridinivorans RL-GZ01 on different nitrogen sources is shown in FIG. 5, and it can be seen that the Rhodococcus pyridinivorans RL-GZ01 strain on NH after being cultured for 72 hours 4 + -N、NO 2 - -N、NO 3 - The denitrification rates of-N reach 93.6%, 77.0% and 77.3% respectively, and the Rhodococcus pyridinivorans RL-GZ01 strain is treated with NH respectively 4 + -N、NO 2 - -N、NO 3 - the-N shows good denitrification capability in the culture medium with the only nitrogen source, which indicates that the Rhodococcus pyridinivorus RL-GZ01 strain has good nitrification and denitrification capability, can effectively remove nitrogen in different forms, and can be used for repairing the environment polluted by nitrogen.
EXAMPLE 4 simultaneous denitrification and DEHP degradation Performance of Strain RL-GZ01
1. Experimental methods
(1) Preparing a seed solution: picking single colony from LB plate to LB liquid culture medium, shaking culture at 30 deg.C until OD 600 =1.5, obtain seed liquid.
(2) DEHP degradation experiment and denitrification experiment:
taking OD 600 200 mu L of Rhodococcus pyridinivorans RL-GZ01 strain seed liquid with the concentration of 1.5 is centrifuged for 4min at 5000rpm to collect thalli, then 200 mu L of HNM culture medium, NDM culture medium and DM culture medium are respectively used for full suspension, the operations of centrifugation and suspension are repeated for 3 times, 10mL of corresponding culture medium containing 100mg/L DEHP is respectively supplemented, and the shake cultivation is carried out for 60 hours at 30 ℃, 180rpm and under the dark condition.
Three control groups (10 mL of HNM medium, NDM medium, DM medium containing 100mg/L DEHP, respectively) were set without addition of bacteria, and 3 replicates were set for all experiments.
The degradation of DEHP and NH in each medium were measured every 12 hours 4 + -N、NO 2 - -N、NO 3 - N concentration and plotting a nitrogen content histogram and a DEHP degradation graph.
2. Results of the experiment
Denitrification of different nitrogen sources by Rhodococcus pyridinivorans RL-GZ01 strain in medium with different nitrogen sources containing 100mg/L DEHP is shown in FIG. 6, and degradation of DEHP by Rhodococcus pyridinivorans RL-GZ01 strain in medium with different nitrogen sources containing 100mg/L DEHP is shown in FIG. 7.
As can be seen, rhodococcus pyridinivorans RL-GZ01 strain 72h was cultured in a medium supplemented with DEHP 3 - The denitrogenation rate of-N is more than 60%, NH 4 + -N、NO 2 - The denitrification rates of-N and 100mg/L DEHP respectively reach 95.1% and 100%, and the degradation rate of 100mg/L DEHP reaches 100%, so that the Rhodococcus pyridinivorans RL-GZ01 strain has the capacity of degrading DEHP and denitrifying simultaneously.
In addition, compared to the HNM medium or NDM medium of example 3 containing no DEHP, the medium was adjusted to NH 4 + -N、NO 2 - The denitrification rate of-N is improved because the addition of DEHP increases the carbon source in the culture medium, changes the carbon-nitrogen ratio in the original culture medium, and influences the denitrification condition of the strain.
Example 5 Strain RL-GZ01 against different concentrations of NH 4 + Scavenging Properties of-N and DEHP
1. Experimental methods
(1) Preparing a seed solution: picking single colony from LB plate to LB liquid culture medium, shaking culture at 30 deg.C until OD 600 And =1.5, obtaining a seed liquid.
(2) DEHP degradation experiment and denitrogenation experiment:
preparation of different NH 4 + HNM medium at N and DEHP concentrations: first of all, NH is prepared 4 + HNM medium with final N concentrations of 20mg/L, 60mg/L, 200mg/L and 500mg/L, 3 parallel samples are arranged at each concentration, DEHP is added into each parallel sample respectively, so that the final DEHP concentrations are 5mg/L, 20mg/L and 50mg/L, and 12 groups of medium are finally obtained.
Taking OD 600 200 μ L of Rhodococcus pyridinivorans RL-GZ01 strain seed liquid, centrifuging at 5000rpm for 4min to collect bacterial precipitate, suspending with 200 μ L of the above 12 groups of HNM medium, repeating centrifuging and suspending for 3 times, and repeatingThe cells were each supplemented with 10mL of the corresponding medium and shake-cultured at 30 ℃ and 180rpm in the dark for 60 hours. The specific test design is shown in table 3:
TABLE 3
The control group without added bacteria of the above 12 tests was set up, and 3 replicates were set up for all tests.
The DEHP degradation and NH were measured every 12 hours 4 + N concentration, plotting DEHP degradation curves and histograms of nitrogen content.
2. Results of the experiment
Rhodococcus pyridinovorus RL-GZ01 strain NH at 20mg/L, 60mg/L and 200mg/L 4 + The degradation curves of DEHP at different concentrations (5 mg/L, 20mg/L, 50 mg/L) at N concentration are shown in FIG. 8, in which only 3 curves of the experimental group, i.e. different NH values, are visible 4 + At the N concentration, the DEHP degradation curves at the same concentration are overlapped, which shows that when NH is generated 4 + When the concentration of N is 20mg/L, 60mg/L and 200mg/L, the degradation capability of the strain RL-GZ01 on DEHP is not influenced by NH 4 + Influence of the N concentration, the concentration of DEHP all dropping to 0 after 24h of incubation; in addition, only three curves of the control group are shown in the figure, because the detection finds that the concentrations of DEHP in the experiment process are quite close to each other for the control group with the same initial concentration of DEHP, so that when data are processed, the DEHP degradation results of the control group with the same initial concentration of DEHP are plotted into one curve by means of averaging, and the data are conveniently read from the figure.
500mg/L NH of Rhodococcus pyridinivorans RL-GZ01 strain 4 + The degradation curves for DEHP at different concentrations (5 mg/L, 20mg/L, 50 mg/L) at N concentration are shown in FIG. 9, and it can be seen that the concentration of DEHP at 5mg/L, 20mg/L drops to 0 after 24h of incubation; after 36h of incubation, the DEHP concentration, at a concentration of 50mg/L, was reduced to 0. Indicating that the Rhodococcus pyridinivorans RL-GZ01 strain has NH of 500mg/L 4 + The complete degradability towards DEHP is maintained at N concentration.
The results in FIGS. 8 and 9 show that the NH concentration is different 4 + The Rhodococcus pyridinivorus RL-GZ01 strain has complete degradation capability on DEHP with the concentration of 5mg/L, 20mg/L and 50mg/L under the condition of N concentration, but NH 4 + The concentration of-N influences the rate of DEHP degradation, particularly at NH 4 + The concentration of-N reaches 500mg/L and above, with NH 4 + The degradation rate of DEHP also slows down with increasing N concentration.
Rhodococcus pyridinovorus RL-GZ01 strain under the condition of different concentrations (5 mg/L, 20mg/L and 50 mg/L) of DEHP to NH with the concentration of 20mg/L 4 + The denitrification of-N is shown in FIG. 10, which shows that NH concentration of 20mg/L is present in the presence of DEHP 4 + The concentration of N reaches a stable value after 24 hours, and approaches 0; indicating NH at 20mg/L 4 + N Environment, addition of DEHP at different concentrations degrades NH against Rhodococcus pyridinivorans RL-GZ01 strain 4 + The performance of-N has no significant effect.
Rhodococcus pyridinovorus RL-GZ01 strain under the condition of different concentrations (5 mg/L, 20mg/L and 50 mg/L) of DEHP to NH with the concentration of 60mg/L 4 + The denitrification of-N is shown in FIG. 11, which shows that the concentration of NH is 60mg/L under the condition that the DEHP concentration is 5mg/L and 20mg/L 4 + -the concentration after N24 hours is substantially close to 0; at a DEHP concentration of 50mg/L, a concentration of 60mg/L NH 4 + The concentration after 24 hours of-N is about 15mg/L, and after 36 hours, the concentration is close to 0.
Rhodococcus pyridinivorans RL-GZ01 strain under the condition of different concentrations (5 mg/L, 20mg/L and 50 mg/L) of DEHP to NH with the concentration of 200mg/L 4 + The denitrification of-N is shown in FIG. 12, which shows that after 72 hours of incubation under conditions of different concentrations of DEHP, the concentration of NH was 200mg/L 4 + The concentration of-N is reduced to about 20 mg/L.
Rhodococcus pyridinivorans RL-GZ01 strain under the condition of different concentrations (5 mg/L, 20mg/L and 50 mg/L) of DEHP to NH with the concentration of 500mg/L 4 + The denitrification of-N is shown in FIG. 13, which shows that the DEHP was present at different concentrationsThen, after 72 hours of culture, NH was added at a concentration of 500mg/L 4 + The concentration of N is reduced to about 65 mg/L.
The results in FIGS. 10, 11, 12, and 13 show that Rhodococcus pyridinivorans RL-GZ01 strain at concentrations of 20mg/L, 60mg/L, 200mg/L, and 500mg/L NH under different concentrations of (5 mg/L, 20mg/L, and 50 mg/L) DEHP 4 + N has excellent scavenging ability, but the concentration of DEHP affects the denitrification efficiency, particularly in NH 4 + At lower N concentrations (20 mg/L), the addition of DEHP at different concentrations had little effect on denitrification; when NH is present 4 + When the concentration of N is higher (60 mg/L), the denitrification rate is reduced along with the DEHP concentration reaching 50 mg/L; but when NH 4 + When the concentration of-N is very high (200 mg/L and 500 mg/L), the denitrification condition can not obviously change due to the change of DEHP concentration. This is because the change in the carbon-nitrogen ratio affects the denitrification efficiency.
Example 6 Performance of the strain RL-GZ01 in urban wastewater for degrading PAEs, removing nitrogen and removing phosphorus simultaneously
1. Experimental method
(1) Preparing a seed solution: picking single colony from LB plate to LB liquid culture medium, shaking culture at 30 deg.C until OD 600 =1.5, obtain seed liquid.
(2) Co-degradation experiments:
the urban wastewater is sampled from a Zhanjiang Xixia mountain wastewater factory, the initial pH is 7.13, the total phosphorus concentration is 10.89mg/L, the total nitrogen concentration is 74.51mg/L, the ammonia nitrogen concentration is 70.91mg/L, the DEHP concentration is 0.13mg/L, and the COD is 622.4mg/L. Before the experiment, the concentration of DEHP in the urban wastewater is adjusted to 5mg/L in a mode of manually adding DEHP, and the rest is unchanged to obtain experimental urban wastewater, and the experimental urban wastewater is placed in a refrigerator at 4 ℃ for later use.
Taking OD 600 200 mu L of Rhodococcus pyridinivorans RL-GZ01 strain seed liquid of 1.5, centrifuging for 4min at 5000rpm to collect thalli, fully suspending 200 mu L of the experimental urban wastewater, repeating the operations of centrifuging and suspending for 3 times, adding 10mL of experimental urban wastewater, and performing shake culture at 30 ℃, 180rpm and in the dark for 84 hours. And setting a control group (experimental urban wastewater) without bacteria.
The DEHP concentration is detected every 6h, and the COD, TP and NH are detected every 12h 4 + -N、NO 3 - -N、NO 2 - N, TN, DEHP concentrations, each detection was performed in 3 replicates.
2. Results of the experiment
The change of COD value of the urban wastewater is shown in FIG. 14, and it can be seen that the COD value of the urban wastewater is reduced from 622.4mg/L to 101.0mg/L by Rhodococcus pyridinivorans RL-GZ01 strain after being cultured for 84 hours, and the COD value of the control group has no obvious change within 84 hours.
The degradation condition of the Rhodococcus pyridinivorans RL-GZ01 strain on TP of the experimental urban wastewater is shown in FIG. 15, and it can be seen that the Rhodococcus pyridinivorans RL-GZ01 strain can reduce the total phosphorus concentration of the experimental urban wastewater from 10.89mg/L to 2.37mg/L after being cultured for 84 hours, and the phosphorus removal rate reaches 78.23%.
Rhodococcus pyridinivorans RL-GZ01 strain for testing TN and NH of urban wastewater 4 + -N、NO 3 - -N、NO 2 - The degradation condition of the-N is shown in figure 16, and therefore, after the Rhodococcus pyridinivorans RL-GZ01 strain is cultured for 84 hours, the ammonia nitrogen concentration of the experimental urban wastewater can be reduced from 70.91mg/L to 2.2mg/L, and the removal rate reaches 96.9%; after being cultured for 84 hours, the Rhodococcus pyridinivorans RL-GZ01 strain can reduce the total nitrogen concentration of the experimental urban wastewater from 74.51mg/L to 3.4mg/L, and the removal rate reaches 95.4%. Meanwhile, the change condition of nitrate nitrogen and nitrite nitrogen in the experimental urban wastewater is the same as that of a control group, and no obvious accumulation occurs. The Rhodococcus pyridinivorans RL-GZ01 strain is shown to have the capability of synchronous nitrification and denitrification.
The degradation condition of the Rhodococcus pyridinivorans RL-GZ01 strain on DEHP in the experimental urban wastewater is shown in FIG. 17, and therefore the Rhodococcus pyridinivorans RL-GZ01 strain can completely degrade 5mg/L DEHP in the experimental urban wastewater.
The results show that the Rhodococcus pyridinivorans RL-GZ01 strain has good adaptability in urban wastewater, has the capabilities of degrading PAEs, removing nitrogen and removing phosphorus simultaneously, and can effectively repair the complex environment polluted by phthalate substances and/or nitrogen and phosphorus.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) RL-GZ01 strain is preserved in Guangdong province microorganism culture collection center at 20 days 4 months 2022, and the preservation number is GDMCC No:62401, the storage address is building No. 59, building No. 5 of the Mingzhou Jielizhou 100 yard.
2. The use of the Rhodococcus pyridinivorans RL-GZ01 strain of claim 1 for degrading phthalate and/or remediating phthalate-contaminated environments.
3. The use according to claim 2, wherein the phthalate is one or more of di (2-ethylhexyl) phthalate, dicyclohexyl phthalate, di-n-octyl phthalate, di-n-butyl phthalate, butyl benzyl phthalate, diethyl phthalate or dimethyl phthalate.
4. Use of the Rhodococcus pyridinivorans RL-GZ01 strain according to claim 1 for the elimination of total nitrogen and/or the remediation of nitrogen-contaminated environments.
5. Use of the Rhodococcus pyridinivorans RL-GZ01 strain of claim 1 for the elimination of total phosphorus and/or the remediation of phosphorus-contaminated environments.
6. Use of the Rhodococcus pyridinivorans RL-GZ01 strain according to claim 1 for the preparation of a product for degrading phthalate and/or scavenging total nitrogen and/or total phosphorus.
7. The use of claim 6, wherein the product is one or more of a microbial inoculum, a biological cleaner, and a biological degradation agent.
8. A microbial preparation comprising the Rhodococcus pyridinovorans RL-GZ01 strain according to claim 1 and/or a bacterial solution thereof.
9. A biological detergent characterized by containing the Rhodococcus pyridinivorans RL-GZ01 strain according to claim 1 and/or a bacterial solution thereof.
10. A biodegradation agent comprising the Rhodococcus pyridinovorans RL-GZ01 strain according to claim 1 and/or a bacterial solution thereof.
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