Gordonia and application thereof

Abstract

The invention provides Gordonia and application thereof, the Gordonia is preserved in Guangdong Microbial Culture Collection Center in July 2024, and the preservation number is GDMCC NO.64865. The Gordonia has been preserved in Guangdong Microbial Culture Collection Center. The Gordonia can be used for degrading 3OCB, 6OCB, 6CB, 5CHB, 3eCHB, 5cHdFCP, 5cHCaACP, 5BzodFCP and the like, and the Gordonia shows an efficient degradation capability on the 3OCB, the 6OCB, the 5CHB, the 3eCHB, the 5cHCaACP and the 5BzodFCP. The Gordonia disclosed by the invention has a good prospect in the biodegradation and repair process of cyano liquid crystal monomer pollutants.

Description

Technical Field

[0001] The invention belongs to the technical field of biological treatment of environmental organic pollutants, and relates to a Gordonia strain capable of degrading a variety of cyano liquid crystal monomer pollutants and application thereof. Background Art

[0002] Liquid crystal monomers (LCMs) are a class of synthetic organic chemicals. As a special functional material, they are widely used in liquid crystal displays of various electronic products. LCMs are filled between the polarizers of the product by physical addition rather than chemical bonding. They are easily released into the surrounding environment during the processing, use, recycling, treatment and disposal of the product. Evidence shows that thousands of LCMs have been detected in the environment. According to their differences in structure and functional groups, they include three categories: cyano LCMs, fluorinated LCMs and biphenyl analogs. Among them, because cyano LCMs contain benzene rings and cyano functional groups in their molecular structure, more and more studies have shown that cyano LCMs have environmental persistence, bioaccumulation and biological toxicity, and have become a new pollutant that has attracted much attention. Therefore, the ecological risks and health and safety effects of cyano LCMs in the environment have attracted widespread attention from environmental researchers.

[0003] Microbial degradation technology has the advantages of environmental friendliness and low cost, making it one of the safest, most economical and effective methods for remediation of organic pollutants. However, there are relatively few studies on the microbial degradation of cyano-LCMs, especially the screening and application of microbial strains that can efficiently degrade cyano-LCMs. Gordonia is a type of microorganism widely present in the environment, which has been proven to be able to degrade a variety of organic pollutants. For details, see "Biodegradation and metabolic pathway of phenanthrene by a newly isolated bacterium Gordonia sp." published by Zhimao Mai et al. and "Resting for viability: Gordonia polyisoprenivorans ZM27, a robust generalist for petroleum bioremediation under hypersaline stress" published by Renzhang Lin et al. However, no research has reported the degradation and transformation ability of Gordonia on cyano-LCMs.

[0004] The present invention aims to screen and identify a new strain of Gordonia with the ability to efficiently degrade cyano-LCMs, and explore its degradation mechanism, so as to provide a new solution for the biodegradation of cyano-LCMs. Through extensive screening of environmental samples and combined with modern molecular biology techniques, the present invention successfully isolated a new strain of Gordonia, which exhibited a high efficiency in degrading cyano-LCMs. This study not only enriched the degradation function of the genus Gordonia, but also provided new strain resources and theoretical basis for the microbial degradation of organic pollutants. Summary of the invention

[0005] The purpose of the present invention is to provide a Gordonia strain capable of degrading multiple cyano LCMs and application thereof.

[0006] The technical solution of the present invention:

[0007] A strain of Gordonia, classified and named Gordonia sp.QZP3, was screened and isolated from soil samples from an electronic waste dismantling site in Qingyuan City, Guangdong Province. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 12, 2024, with the deposit number GDMCC NO.64865.

[0008] This strain of Gordonia has the ability to degrade cyano LCMs, including 4-Propoxy-4'-cyanobiphenyl (3OCB), 4'-(Hexyloxy)-4-biphenylcarbonitrile (6OCB), 4-hexylbiphenylcarbonitrile (6CB), 4-(trans-4-pentylcyclohexyl)benzonitrile (4-(trans-4-pentylcyclohexyl)benzonitrile (4-(trans-4-pentylcyclohexyl)benzonitrile, ... The invention discloses a variety of cyano LCMs, including 4-[trans-4-[(E)-1-propenyl]cyclohexyl]benzonitrile (5CHB), 4-[trans-4-[(E)-1-Propenyl]cyclohexyl]benzonitrile (3eCHB), 2,6-difluoro-4-(trans-4-pentylcyclohexyl)benzonitrile (5cHdFCP), p-cyanophenyl trans-4-pentylcyclohexanecarboxylate (5cHCaACP) and 4-cyano-3,5-difluorophenyl4-pentylbenzoate (5BzodFCP).

[0009] Beneficial effects of the present invention: The present invention provides a strain of Gordonia sp. QZP3, with a deposit number of GDMCC NO: 64865. The Gordonia sp. QZP3 provided by the present invention is domesticated and screened from soil samples of electronic waste dismantling sites in Qingyuan City, Guangdong Province. It has been verified that the strain can degrade a variety of cyano LCMs including 3OCB, 6OCB, 6CB, 5CHB, 3eCHB, 5cHdFCP, 5cHCaACP and 5BzodFCP. Among them, 3OCB, 6OCB, 5CHB, 3eCHB, 5cHCaACP and 5BzodFCP show efficient degradation ability (the degradation efficiency of 1.0 mg / L after 7 days of cultivation is as high as 90%). It can be seen that the Gordonia sp. QZP3 of the present invention has good application prospects in the bioremediation of cyano LCMs pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is a colony morphology diagram of Gordonia QZP3 in Example 1;

[0011] Figure 2 This is the phylogenetic tree diagram of Gordonia QZP3 in Example 1;

[0012] Figure 3 This is a graph showing the degradation characteristics of Gordonia QZP3 to different concentrations of 6OCB in Example 2;

[0013] Figure 4 This is a diagram showing the effect of system pH on the degradation of 6OCB by Gordonia QZP3 in Example 3;

[0014] Figure 5 This is a diagram showing the effect of the bacterial dosage on the degradation of 6OCB by Gordonia QZP3 in Example 4;

[0015] Figure 6 This is the degradation kinetics of different cyano LCMs by Gordonia QZP3 in Example 5. DETAILED DESCRIPTION

[0016] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and technical solutions.

[0017] Example 1

[0018] Screening and identification of strains

[0019] (1) Screening of strains

[0020] Weigh 5 g of soil from an e-waste site and add it to 200 mL of an inorganic salt culture medium containing 1.0 mg / L 6OCB, and culture it in a constant temperature shaker at 30°C and 160 rpm for 14 days; then, aspirate 20 mL of the culture solution and transfer it to 200 mL of a new inorganic salt culture medium containing 2.0 mg / L 6OCB, and culture it under the same conditions for 14 days; repeat the above transfer culture 6 times, and the concentrations of 6OCB in the acclimation process are 1.0, 2.0, 4.0, 6.0, 8.0 and 10.0 mg / L, respectively.

[0021] Take 1.0 mL of the 6th acclimatization culture medium and dilute it with sterile water for 10 4 ~10 6 times, and evenly spread them on beef extract peptone solid culture medium plates, then place the plates in a 30°C constant temperature incubator for inverted culture until clear colonies grow on the plates. Use an inoculation loop to pick up an appropriate amount of colonies with different morphologies and streak them on the same solid plate, and purify each colony with different morphologies until a single colony is obtained. Use an inoculation loop to pick up an appropriate amount of single colonies and inoculate them into 100mL beef extract peptone liquid culture medium, place them in a constant temperature shaker at 30°C and 160rpm for enrichment culture, mix the enriched culture solution with 50% glycerol in a 15mL centrifuge tube, obtain the strain preservation solution, and store it in a -20°C refrigerator for use.

[0022] 0.1 mL of the above strain preservation solution was taken and inoculated into 100 mL of beef extract peptone liquid medium, placed in a constant temperature shaker at 30°C and 160 rpm for enrichment culture, and the bacterial cells were collected by centrifugation at 4°C and 6500 rpm, washed with inorganic salt medium 3 times, and resuspended in inorganic salt medium to obtain the bacterial suspension of each strain. An appropriate amount of bacterial suspension was added to 10 mL of inorganic salt medium containing 1.0 mg / L 6OCB, and placed in a constant temperature shaker at 30°C and 160 rpm for 7 days. The above degradation solution was extracted 3 times with a mixed solvent of analytical pure hexane and dichloromethane (volume ratio of 1:1), and the organic phases of the 3 extractions were collected and mixed into a chicken heart bottle, evaporated to near dryness at 40°C, and fixed to volume with 10 mL of chromatographic pure acetonitrile. 1.0 mL was taken into an injection bottle, and the concentration of residual 6OCB was quantitatively analyzed by high performance liquid chromatography, and the degradation efficiency of 6OCB was calculated.

[0023] The inorganic salt culture medium used comprises: 1.5 g / L KH2PO4, 3.0 g / L K2HPO4, 0.5 g / L NaCl, 1.0 g / L (NH4)2SO4, 2.0 mL / L trace element stock solution, pH 7.0-7.2. The trace element stock solution comprises: 4 g / L MgSO4, 4 g / L ZnSO4, 1 g / L CuSO4, 1 g / L MnSO4, 1 g / L FeSO4·7H2O and 1 g / L CaCl2.

[0024] The beef extract peptone solid culture medium used above comprises: 3.0 g / L beef extract, 10.0 g / L peptone, 5.0 g / L NaCl, 20.0 g / L agar, and the pH value is 7.0-7.2.

[0025] The beef extract peptone liquid culture components used include: 3.0g / L beef extract, 10.0g / L peptone, 5.0g / L NaCl, and pH is 7.0-7.2.

[0026] (2) Identification of strains

[0027] After the above screening and testing, a strain capable of degrading 6OCB was obtained. The colonies of the strain grown on beef extract peptone solid medium were orange, with neat edges, smooth surface, and round shape ( Figure 1 ). Further testing the strain's ability to degrade different cyano LCMs under the same conditions revealed that the strain had the ability to degrade a variety of cyano LCMs, including 3OCB, 6OCB, 6CB, 5CHB, 3eCHB, 5cHdFCP, 5cHCaACP, and 5BzodFCP.

[0028] The 16S rRNA gene sequence was compared with the NCBI database and found to have the highest similarity with Gordonia spp. Its phylogenetic tree is shown in Figure 2 . Therefore, the strain was identified as Gordonia and named Gordonia sp. QZP3. It was deposited in Guangdong Microbiological Culture Collection Center on July 12, 2024, with the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC NO: 64865.

[0029] The 16S rRNA gene sequence of Gordonia QZP3 is shown below:

[0030]

[0031]

[0032] Example 2

[0033] Degradation characteristics of Gordonia QZP3 on different concentrations of 6OCB

[0034] 0.1 mL of the preservation solution of Gordonia QZP3 was taken and inoculated into 100 mL of beef extract peptone liquid medium, and then placed in a constant temperature shaker at 30°C and 160 rpm for enrichment culture. The bacterial cells were collected by centrifugation at 4°C and 6500 rpm, and the bacteria were washed three times with inorganic salt medium and resuspended in inorganic salt medium to obtain a certain concentration of bacterial suspension. An appropriate amount of bacterial suspension was inoculated into 10 mL of inorganic salt medium containing 0.25, 0.5, 1.0, 2.0, 3.0, 5.0 and 10.0 mg / L 6OCB, respectively. The initial bacterial dose (OD 600 ) is 0.2. Three parallel samples were set up for each treatment. After sealing with a breathable sealing film, it was placed in a constant temperature shaker at 30°C and 160rpm for enrichment culture for 7 days for degradation performance test. The above degradation liquid was extracted 3 times with a mixed solvent of analytical grade n-hexane and dichloromethane (volume ratio of 1:1), and the 3 extracted organic phases were collected and mixed in a chicken heart bottle, evaporated to near dryness at 40°C, and the volume was made up with 10mL of chromatographic grade acetonitrile. 1.0mL was taken into the injection bottle, and the concentration of residual 6OCB was quantitatively analyzed by high performance liquid chromatography, and the degradation efficiency of 6OCB at different initial concentrations was calculated. The results are shown in Figure 3 As shown:

[0035] according to Figure 3It can be seen that the initial concentration of 6OCB significantly affects its degradation efficiency. When the initial 6OCB is 1.0 mg / L, the maximum degradation efficiency (95.15%) is obtained; when the initial concentration of 6OCB is 1.0 mg / L, its degradation efficiency decreases slightly; and when the initial concentration increases to 2.0, 3.0, 5.0 and 10.0 mg / L, its degradation efficiency decreases significantly to 73.64%, 47.50%, 27.98% and 20.72%, respectively. Therefore, the optimal concentration range of 6OCB degradation by Gordonia QZP3 is 0.25-1.0 mg / L.

[0036] Example 3

[0037] Effect of system pH on degradation of 6OCB by Gordonia QZP3

[0038] The strains were enriched and cultured according to the method steps of Example 2, and a certain concentration of bacterial suspension was prepared by centrifugation and washing. Different amounts of bacterial suspension were inoculated in inorganic salt medium containing 1.0 mg / L 6OCB at pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10, respectively. The initial bacterial dose (OD 600 ) were 0.2 respectively. Three parallel samples were set up for each treatment. After sealing with a breathable sealing film, it was placed in a constant temperature shaker at 30°C and 160rpm for enrichment culture for 7 days for degradation performance test. The above degradation liquid was extracted 3 times with a mixed solvent of analytical grade n-hexane and dichloromethane (volume ratio of 1:1), and the 3 extracted organic phases were collected and mixed in a chicken heart bottle, rotary evaporated at 40°C to near dryness, and fixed to volume with 10mL of chromatographic grade acetonitrile. The concentration of residual 6OCB was quantitatively analyzed by high performance liquid chromatography, and the degradation efficiency of 6OCB under different pH conditions was calculated. The results are shown in Figure 4 As shown:

[0039] according to Figure 4 It can be seen that the pH value of the system has a great influence on the degradation of 6OCB by Gordonia QZP3. Too high or too low system pH significantly affects the degradation efficiency of 6OCB by the strain. For example, when the system pH decreases from 6.0 to 4.0, its degradation efficiency decreases significantly from 92.50% to 47.50%; and when the pH increases from 8.0 to 10, it decreases significantly from 92.41% to 62.37%. Therefore, the optimal pH adaptation range of Gordonia QZP3 is 6.0-8.0.

[0040] Example 4

[0041] Effect of bacterial dosage on the degradation of 6OCB by Gordonia QZP3

[0042] The strains were enriched and cultured according to the method steps of Example 2, and a certain concentration of bacterial suspension was prepared by centrifugation and washing. Different amounts of bacterial suspension were inoculated into 10 mL of inorganic salt medium containing 1.0 mg / L 6OCB, so that the initial bacterial dose (OD 600 ) were 0.05, 0.1, 0.15, 0.2, 0.3 and 0.4 respectively. Three parallel samples were set up for each treatment. After sealing with a breathable sealing film, it was placed in a constant temperature shaker at 30°C and 160rpm for enrichment culture for 7 days for degradation performance test. The above degradation liquid was extracted three times with a mixed solvent of analytical grade n-hexane and dichloromethane (volume ratio of 1:1), and the three extracted organic phases were collected and mixed in a chicken heart bottle, evaporated to near dryness at 40°C, and fixed to volume with 10mL of chromatographic grade acetonitrile. The concentration of residual 6OCB was quantitatively analyzed by high performance liquid chromatography, and the degradation efficiency of 6OCB under different bacterial dosage conditions was calculated. The results are shown in Figure 5 As shown:

[0043] according to Figure 5 It can be seen that when the amount of Gordonia QZP3 (OD 600 ) is less than 0.15, the degradation efficiency of 6OCB decreases significantly; and when the bacterial dosage is greater than 0.4, the degradation efficiency of 6OCB also decreases. Therefore, the optimal bacterial dosage for Gordonia QZP3 to degrade 6OCB is 0.15-0.3.

[0044] Example 5

[0045] Degradation kinetics of different cyano-LCMs by Gordonia QZP3

[0046] The strains were enriched and cultured according to the method and steps of Example 2, and a certain concentration of bacterial suspension was prepared by centrifugation and washing. An appropriate amount of bacterial suspension was inoculated into 10 mL of inorganic salt medium containing 1.0 mg / L 3OCB, 6OCB, 6CB, 5CHB, 3eCHB, 5cHdFCP, 5cHCaACP and 5BzodFCP, respectively, so that the initial bacterial dose (OD 600 ) is 0.2. The control group was not added with bacterial suspension, and the different pollutant treatment groups were sampled on the 1st, 2nd, 3rd, 5th and 7th days, respectively. Three parallel samples were set up at each sampling point. After sealing with a breathable sealing film, it was placed in a constant temperature shaker at 30°C and 160rpm for incubation. The above degradation liquid was extracted three times with a mixed solvent of analytical grade n-hexane and dichloromethane (volume ratio of 1:1), and the three extracted organic phases were collected and mixed in a chicken heart bottle, rotary evaporated at 40°C to near dryness, and fixed to volume with 10mL of chromatographic grade acetonitrile. The concentration of residual different cyano LCMs was quantitatively analyzed by high performance liquid chromatography, and the degradation efficiency of cyano LCMs at different time points was calculated, and the degradation kinetic curve was drawn. The results are shown in the figure Figure 6 As shown:

[0047] according to Figure 6 It can be seen that the degradation efficiency of Gordonia QZP3 for 3OCB, 6OCB, 5CHB, 3eCHB, 5cHCaACP and 5BzodFCP reached 96.22%, 94.57%, 100%, 98.45%, 100% and 100%, respectively; at the same time, the degradation efficiency of 6CB and 5cHdFCP also reached 55.37% and 75.70%, respectively. It can be seen that Gordonia QZP3 can degrade a variety of cyano LCMs.

[0048] Based on the above content, it can be seen that the Gordonia QZP3 provided by the present invention can degrade a variety of cyano LCMs, and has a high efficiency in degrading a variety of LCMs including 3OCB, 6OCB, 5CHB, 3eCHB, 5cHCaACP and 5BzodFCP.

[0049] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Gordonia sp. QZP3 capable of degrading a variety of cyano liquid crystal monomer pollutants, characterized in that: This Gordonia was deposited in the Guangdong Provincial Microbiological Culture Collection Center in July 2024, with the collection number GDMCCNO.64865.

2. Use of the Gordonia described in claim 1 in degrading cyano liquid crystal monomer pollutants in water environments.

3. The use according to claim 3, characterized in that: The cyano liquid crystal monomer contaminants include 3OCB, 6OCB, 6CB, 5CHB, 3eCHB, 5cHdFCP, 5cHCaACP and 5BzodFCP.

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