Glutamic acid bacillus, treating agent and application of treating agent in degradation of polycyclic aromatic hydrocarbon
Through the combination of Glutamicibacter soil ENR6 and Mycolicobacterium pallens PBY9, the problem of poor degradation of PAHs by a single strain was solved, and efficient and economical PAHs degradation was achieved, which significantly improved the degradation rate and and reduce costs.
Patent Information
- Application Number
- CN202411385638.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2024-12-17
AI Technical Summary
The prior art has toxic problems caused by poor effect of a single strain, low environmental survival rate, difficulty in achieving complete mineralization and accumulation of intermediate metabolites when degrading polycyclic aromatic hydrocarbons. In addition, traditional physicochemical repair methods consume a lot of energy, are costly and easy to use. Secondary pollution occurs.
The combination of Glutamicibacter soil ENR6 and Mycolicobacterium pallens PBY9 was used to cultivate it through liquid culture medium to form a bacterial group that synergistically degrades polycyclic aromatic hydrocarbons. The polycyclic aromatic hydrocarbons are used as the only carbon source to reduce the degradation cost.
The degradation rate of PAH is significantly improved, the degradation time is shortened, the degradation effect is better than that of a single strain, and there is no need for additional carbon source, reducing costs, and its synergistic degradation ability is verified through 16S rDNA identification and improved Oxford Cup method.
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Figure CN119144500A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of microorganisms, and in particular relates to a glutamicum bacillus, a treatment agent and an application in degrading polycyclic aromatic hydrocarbons. Background Art
[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.
[0003] Polycyclic aromatic hydrocarbons (PAHs) refer to a class of typical persistent organic pollutants containing two or more benzene rings that are fused in a linear, angular or clustered form. They include naphthalene, anthracene, phenanthrene, pyrene and other compounds, which are widely distributed in the environment. PAHs are mainly derived from the production of fossil fuels, coal gas and coal tar, wood processing, oil leakage and waste incineration. They are carcinogenic, teratogenic and induce mutations in organisms, endangering the ecological environment and human health. How to effectively remove PAHs in the environment and reduce their environmental risks has become a research hotspot.
[0004] Traditional physical and chemical remediation methods for PAHs are energy-intensive, costly, and prone to secondary pollution. Microorganisms, as important drivers of biogeochemical cycles, can use their own metabolism to achieve the migration and transformation of PAHs. The corresponding bioremediation technology is economical, environmentally friendly, and effective. Many bacterial species can degrade PAHs, and most of them are isolated from PAH-contaminated soil or sediments. Glutamicibacter is a common microorganism in soil environments, but no reports have yet found that Glutamicibacter strains have the ability to degrade PAHs.
[0005] The effect of single strain in remediating PAH pollution in the environment is often poor. On the one hand, the competition with indigenous microorganisms leads to low survival rate in the environment. On the other hand, it is difficult for a single degradation strain to achieve complete mineralization of PAHs. In addition, the accumulation of intermediate metabolites is toxic to the degradation strains, and the feedback inhibition of degradation activity affects the growth of the degradation strains and the degradation of PAHs. Compared with a single strain, microbial flora has stronger environmental adaptability and enhanced effect on the degradation of organic pollutants. It can effectively solve the metabolic bottleneck of a single strain and the secondary pollution of metabolites, and has greater application potential. Summary of the invention
[0006] In order to overcome the deficiencies in the prior art, the object of the present invention is to provide a glutamicum bacillus, a treatment agent and an application thereof in the degradation of polycyclic aromatic hydrocarbons. The glutamicum bacillus soil ENR6 provided by the present invention can achieve the degradation of high molecular weight polycyclic aromatic hydrocarbons, and the combination of glutamicum Glutamicibacter soil ENR6 and mycobacterium pallens PBY9 can synergistically degrade polycyclic aromatic hydrocarbons, and has the ability to efficiently degrade polycyclic aromatic hydrocarbons.
[0007] In order to achieve the above-mentioned objectives, in the first aspect, the present invention provides a Glutamicibacter soil ENR6, whose preservation number is CCTCC M 2024982, and which was preserved in the China Center for Type Culture Collection in Wuhan on May 17, 2024.
[0008] In a second aspect, the present invention provides a treatment agent comprising the above-mentioned Bacillus glutamicum ENR6.
[0009] In some specific embodiments of the present invention, the treatment agent also includes Mycolicibacterium pallens PBY9, the preservation number of Mycolicibacterium PBY9 is CCTCC M 2024983, and it was deposited in the China Center for Type Culture Collection in Wuhan on May 17, 2024.
[0010] In some specific embodiments of the present invention, the ratio of live bacteria of Bacillus glutamicum ENR6 to Mycobacterium PBY9 is: (10-100):1.
[0011] In some specific embodiments of the present invention, Bacillus glutamicum ENR6 and Mycobacterium PBY9 are both inoculated into liquid culture medium for culture.
[0012] In some specific embodiments of the present invention, the liquid culture medium is a mineral culture medium.
[0013] In a third aspect, the present invention provides the use of the above-mentioned glutamicum bacteria or the treatment agent in the degradation of polycyclic aromatic hydrocarbons.
[0014] In some specific embodiments of the present invention, the polycyclic aromatic hydrocarbons are phenanthrene and pyrene.
[0015] In some specific embodiments of the present invention, the polycyclic aromatic hydrocarbon is pyrene.
[0016] In some specific embodiments of the present invention, the application is that the above-mentioned glutamicum bacteria or treatment agent degrades water or soil containing polycyclic aromatic hydrocarbons.
[0017] In some specific embodiments of the present invention, the concentration of pyrene is 50-500 mg / L, preferably 500 mg / L.
[0018] The beneficial effects of the present invention are:
[0019] 1. The present invention provides a glutamicum Bacillus ENR6, which can effectively degrade high molecular weight polycyclic aromatic hydrocarbon pyrene and has a good effect, enriching the bacterial resources for degrading polycyclic aromatic hydrocarbons.
[0020] 2. The present invention constructs a synthetic bacterial community by using two strains of Bacillus glutamicum ENR6 and Mycobacterium PBY9. It is found that the two bacteria have the ability to synergistically degrade the polycyclic aromatic hydrocarbon pyrene. Compared with single bacteria, they can significantly increase the degradation rate of pyrene and shorten the degradation time, showing a degradation effect significantly better than that of single strains of Bacillus glutamicum ENR6 or Mycobacterium PBY9. Moreover, the two strains are combined, with polycyclic aromatic hydrocarbons as the only carbon source, and there is no need to add an additional carbon source, which greatly reduces the degradation cost. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 To construct phylogenetic trees and morphological characteristics for the strains based on 16S rDNA sequences;
[0022] Among them, A is the phylogenetic tree of Bacillus glutamicum ENR6 based on 16S rDNA sequence, and a is the phylogenetic tree of Mycobacterium PBY9 based on 16S rDNA sequence;
[0023] Figure 2 The bacterial cells and colony images of Bacillus glutamicum ENR6 and Mycobacterium PBY9;
[0024] Among them, B is a Gram staining photo of Bacillus glutamicum ENR6 under an optical microscope, and b is a Gram staining photo of Mycobacterium PBY9 under an optical microscope; C is the colony morphology of Bacillus glutamicum ENR6 after culturing on LB medium for 24 hours, and c is the colony morphology of Mycobacterium PBY9 after culturing on LB medium for 72 hours; D is a degradation circle diagram formed by Bacillus glutamicum ENR6 after culturing on MSM solid medium with pyrene (500 mg / L) as the sole carbon source for 7 days, and d is a degradation circle diagram formed by Mycobacterium PBY9 after culturing on MSM solid medium with pyrene (500 mg / L) as the sole carbon source for 4 days;
[0025] Figure 3 Schematic diagram of the improved Oxford cup method for screening strain combinations that synergistically degrade PAHs;
[0026] Figure 4 This is a graph showing the co-culture results of the PBY9×ENR6 combination at different periods;
[0027] Figure 5This is a diagram of the co-culture results of different strain combinations;
[0028] Among them, A is the co-culture result of PBY9×PBY9 and PBY9×ENR6 combination, and B is the co-culture result of ENR6×ENR6 and PBY9×ENR6 combination;
[0029] Figure 6 This is the result of the confrontation between the strain PBY9 and ENR6 on plates;
[0030] Figure 7 The degradation graph of 100 mg / L pyrene after 5 days of cultivation with different strain ratios;
[0031] Figure 8 The degradation diagram of PBY9×ENR6 combination on different concentrations of pyrene;
[0032] Deposit of biological material under patent procedure:
[0033] Glutamicibacter soil, with the deposit number of CCTCC M 2024982, was deposited in China Center for Type Culture Collection on May 17, 2024, Wuhan University, Wuhan, China;
[0034] Mycolicibacterium pallens, with the deposit number of CCTCC M2024983, was deposited in the China Center for Type Culture Collection on May 17, 2024, located at Wuhan University, Wuhan, China. DETAILED DESCRIPTION
[0035] The present invention provides a glutamicum bacillus, a treatment agent and an application in the degradation of polycyclic aromatic hydrocarbons. A person skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired effect. It should be particularly noted that all similar substitutions and modifications are obvious to a person skilled in the art and are considered to be included in the present invention.
[0036] The embodiments of the present invention will be described in detail below in conjunction with examples. The following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.
[0037] Example
[0038] 1. Experimental materials:
[0039] Oxford cup, 5000 mg / L pyrene acetone stock solution, 200 μL, 1 mL, 5 mL pipette tip, 50 mL conical flask, tweezers, test strain.
[0040] Preparation of 5000 mg / L pyrene acetone stock solution: Weigh 0.25 g of pyrene into a 50 mL volumetric flask, add acetone to fully dissolve it, then dilute to the mark, transfer to a brown bottle, and store at 4°C.
[0041] 1. Preparation of Experimental Materials
[0042] (1) Test strains
[0043] The PAHs-degrading strains were Glutamicibacteresoil ENR6 and Mycolicibacterium eriumpallens PBY9. Both strains were isolated and purified from petroleum-contaminated soil around Dongying oil wells. Both strains were deposited in China Center for Type Culture Collection.
[0044] Table 1. ENR6 of Bacillus glutamicum and PBY9 of Mycobacterium tumefaciens
[0045]
[0046] (2) Test culture medium
[0047] S liquid culture medium: 10 g / L glucose, 4 g / L yeast extract, 4 g / L peptone, 4 g / L K2HPO4, 2 g / LKH2PO4, 0.5 g / L MgSO4·7H2O, pH=7.0, 115°C, high pressure steam sterilization for 15 min.
[0048] Mineral medium (MSM): 0.5 g / LNaNO3, 1.0 g / LKH2PO4, 0.02 g / LCaCl2, 0.2 g / L MgSO4, 0.5 g / L(NH4)2SO4, 1.0 g / LNaH2PO4, pH=6.5, 121°C, high pressure steam sterilization for 20 min.
[0049] LB medium: 5 g yeast powder, 10 g tryptone, 10 g sodium chloride, add deionized water to 1 L, 121°C, high-pressure steam sterilization for 20 min.
[0050] 2. Experimental methods:
[0051] 1. Strain identification
[0052] The two strains obtained by screening were identified, and strain ENR6 was identified as Glutamicibacter soil by observation of colony morphology and bacterial morphology and 16SrDNA identification. After 24 hours of culture on LB medium, the colony of Glutamicibacter soil ENR6 was convex, with regular edges, light yellow, and opaque; the bacteria were rod-shaped, Gram-positive, and formed obvious degradation circles after 7 days of culture on MSM solid medium with pyrene (500 mg / L) as the only carbon source. Strain PBY9 was identified as Mycolicibacter eriumpallens by observation of colony morphology and bacterial morphology and 16SrDNA identification. After culturing for 72 hours on LB medium, the colonies of Mycobacterium (Mycolicibacteriumpallens) PBY9 were low-raised, with regular edges, orange, and opaque; the bacteria were long rod-shaped, Gram-positive, and formed obvious degradation zones after culturing for 4 days on MSM solid medium with pyrene (500 mg / L) as the sole carbon source.
[0053] The 16S rDNA sequence of Glutamicibatersoil ENR6 is shown in SEQ ID NO: 1:
[0054] GCGGGGGGGGGGTGCTTACACATGCAGTCGAACGATGAAGCCCAGCTTGCTGGGT
[0055] GGATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCCTGACTCTGGGA
[0056] TAAGCCCGGGAAACTGGGTCTAATACCGGATATGACTTTTGACCGCATGGTTGAGG
[0057] GTGGAAAGATTTATCGGTTGGGGATGGACTCGCGGCCTATCAGCTTGTTGGTGAGG
[0058] TAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCAC
[0059] ACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTG
[0060] CACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGG
[0061] GTTGTAAACCTCTTTCAGTAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGA
[0062] AGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTA
[0063] TCCGGATTTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTATCGTGAAA
[0064] GTCCGAGGCTCAACCTCGGATCTGCGGTGGGTACGGGCAGACTAGAGTGATGTAG
[0065] GGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACAC
[0066] CGATGGCGAAGGCAGGTCTCTGGGCATTTACTGACGCTGAGGAGCGAAAGCATGG
[0067] GGAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGTTGGGCACTAG
[0068] GTGTGGGGGACATTCCACGTTTTCCGCGCCGTAGCTAACGCATTAAGTGCCCCGCC
[0069] TGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACA
[0070] AGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTT
[0071] GACATGTTCCAGATCGCCTCAGAGATGGGGTTTCCCTTCGGGGCTGGTTCACAGGT
[0072] GGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA
[0073] GCGCAACCCTCGTTCCATGTTGCCAGCGGGTTATGCCGGGGACTCATGGGAGACT
[0074] GCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTAT
[0075] GTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAATGGGTTGCGATACTGTGAG
[0076] GTGGAGCTAATCCCTAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGAC
[0077] CCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACG
[0078] TTCCCGGGCCTTGTACACACCGCCCGTCAAGTCACGAAAGTTGGTAACACCCGAAGCCGATGGCCTAACCACCTTGTGTGGGGGGAGTCGGTCCGA。
[0079] The 16S rDNA sequence of Mycolicibacterium pallens PBY9 is shown in SEQ ID NO:2:
[0080] TGGGGGCGGCTAACCATGCAAGTCGAACGGAAAGTGCTCTTCGGAGTGACTCGAG
[0081] TGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTTGGGATAAGCCT
[0082] GGGAAACTGGGTCTAATACCGGATAGGACCACGGCCTTCATGGGTTGTGGTGGAA
[0083] AGCTTTTGCGGTGTGGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGG
[0084] CCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGTCCGGCCACACTGGG
[0085] ACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAAT
[0086] GGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTA
[0087] AACCTCTTTCAGCAGGGACGAAGCGCAAGTGACGGTACCTGTAGAAGAAGCACC
[0088] GGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGA
[0089] ATTACTGGGCGTAAAGAGCTCGTAGGTGGTTTGTCGCGTTGTTCGTGAAAACTCAC
[0090] AGCTTAACTGTGGGCGTGCGGGCGATACGGGCAGACTGGAGTACTGCAGGGGAG
[0091] ACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTG
[0092] GCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAG
[0093] CGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTG
[0094] GGTTTCCTTCCTTGGGATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGG
[0095] AGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGG
[0096] CGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATG
[0097] CACAGGACGCCGGCAGAGATGTCGGTTCCCTTGTGGCCTGTGTGCAGGTGGTGCA
[0098] TGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCA
[0099] ACCCTTGTCTCATGTTGCCAGCACGTTATGGTGGGGACTCGTGAGAGACTGCCGG
[0100] GGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCA
[0101] GGGCTTCACACATGCTACAATGGCCGGTACAAAGGGCTGCGATGCCGTGAGGTGG
[0102] AGCGAATCCTTTCAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCC
[0103] GTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTC
[0104] CCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCCTTGTGGGAGGGAGCCCGTCGAAAGAGA.
[0105] 2. Improved Oxford Cup Experiment Method:
[0106] (1) Preparation of bacterial suspension
[0107] 1% of the -80°C glycerol-stored Bacillus glutamicum ENR6 and Mycobacterium PBY9 were taken out and rejuvenated in 30 mL of LB medium and S medium, respectively. Then 1% of the rejuvenated bacterial suspension was transferred to new LB medium and S medium for activation culture, respectively, to serve as the experimental bacterial suspension.
[0108] (2) Experimental steps: Pour 15 mL of 1.5% agar-containing MSM medium into a plate and cool it down to solidify to prepare the lower layer medium. Use tweezers to pick up an Oxford cup and place it on the lower plate. Generally, 4 to 6 cups are suitable.
[0109] Take a 50mL sterile conical flask, add 4.5mL of MSM medium with 0.8% agar concentration, 500μL of pyreneacetone stock solution and 100μL of Mycobacterium PBY9 bacterial suspension, mix well and pour onto a plate to form the upper culture medium.
[0110] After solidification, use tweezers to take out the Oxford cup again, and add 30 μL of glutamicum ENR6 bacterial suspension into the hole formed in the Oxford cup. Figure 3 As shown, degrading bacteria 1 is Mycobacterium PBY9, and degrading bacteria 2 is Bacillus glutamicum ENR6.
[0111] After the bacterial solution has been absorbed, place it upside down in an incubator and culture it at 30°C to observe the degradation of pyrene.
[0112] 3. Flat plate confrontation experiment
[0113] The activated glutamicum ENR6 and Mycobacterium PBY9 were crossed on a double-layer plate with pyrene (500 mg / L) as the sole carbon source to observe whether there was antagonism between the two bacteria at the overlapping point.
[0114] 4. Quantitative determination of pyrene degradation rate:
[0115] (1) Preparation of bacterial suspension
[0116] Transfer the activated glutamicum ENR6 and Mycobacterium PBY9 bacterial suspensions to a 50 mL centrifuge tube, centrifuge at 8000 rpm and 25°C for 5 min, and discard the supernatant. Wash the cells with sterilized MSM, centrifuge at 8000 rpm and 25°C for 5 min, discard the supernatant, and repeat the washing. Finally, resuspend the cells with a certain amount of MSM, and adjust the density of the bacterial suspension under a UV-visible spectrophotometer so that the number of cells in the PBY9 and ENR6 bacterial suspensions is 10, ... 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL, 10 10 CFU / mL. (2) Quantitative degradation experiment with different strain ratios
[0117] In a 50 mL conical flask containing sterilized MSM medium, 100 mg / L pyrene was used as the sole carbon source, and the above-prepared PBY9 and ENR6 bacterial suspensions were added to prepare 10 mL of degradation system. The initial number of PBY9 in the PBY9 single bacterial degradation system was 10 7CFU / mL; the initial number of ENR6 in the ENR6 single bacteria degradation system was 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL; PBY9:ENR6 in the co-culture degradation system were 1:1, 10:1, 100:1, 1:10, and 1:100, respectively. The abiotic control treatment was inoculated with an equal amount of sterilized MSM instead of bacterial suspension, and 3 replicates were set for each treatment. The above treatments were cultured at 30°C, 160rpm, and in the dark for 5 days.
[0118] (3) Quantitative degradation experiment of different concentrations of pyrene by strain combination
[0119] In a 50 mL conical flask containing sterilized MSM medium, 50 mg / L, 100 mg / L, 250 mg / L, and 500 mg / L pyrene were used as the sole carbon source, and the above-prepared PBY9 and ENR6 bacterial suspensions were added to prepare 10 mL degradation systems. Among them, the initial number of PBY9 in the PBY9 single bacterial degradation system was 10 7 CFU mL -1 ; The initial number of ENR6 in the ENR6 single bacteria degradation system is 10 9 CFU mL -1 ; In the co-culture degradation system of strains, PBY9:ENR6=1:100. The same amount of sterilized MSM was inoculated in the non-biological control treatment to replace the bacterial suspension. Three replicates were set for each treatment. The above treatments were cultured at 30°C and 160rpm in the dark. The 50mg / L pyrene concentration treatment was cultured for 2 days, the 100mg / L pyrene concentration treatment was cultured for 5 days, the 250mg / L pyrene concentration treatment was cultured for 7 days, and the 500mg / L pyrene concentration treatment was cultured for 10 days before sampling.
[0120] (4) Extraction and determination of pyrene
[0121] An equal volume of methanol was added to the culture medium, and the mixture was ultrasonically extracted for 30 min and filtered through a 0.22 μm PTFE membrane. The residual pyrene in the culture system was determined by UHPLC. A Waters (ACQUITY UPLC H-Class) detection system and a BEH C18 column (1.7 μm × 2.1 × 100 mm) were used. The mobile phase was water and methanol, with a volume ratio of water: methanol = 5:95, an injection volume of 10 μL, a column temperature of 40 °C, and a mobile phase flow rate of 0.5 mL min -1 , UV detection wavelength is 254nm. The recovery rate of pyrene by this extraction and detection method is 100.7±1.33%.
[0122] 3. Experimental Results and Analysis
[0123] 1. Improved Oxford Cup Experiment
[0124] Figure 3 Schematic diagram of the independently created improved Oxford Cup method for screening strain combinations for synergistic degradation of PAHs. Figure 3 A shows that on the double-layer plate with PAHs as the only carbon source, the lower layer is MSM medium with 1.5% agar concentration, and PAHs and glutamicum ENR6 are evenly distributed in the upper MSM medium. One side of the Oxford cup well is not added with bacteria or with degradation strain 1 as a control, and the other side of the Oxford cup well is added with Mycobacterium PBY9 as an experimental group. Figure 3 B shows that the degradation circle first appeared around the holes of the Oxford cup in the experimental group, indicating that the two strains of degrading bacteria have the effect of synergistically degrading PAHs.
[0125] 2. Improved Oxford Cup Experiment on Synergistic Degradation of Pyrene by PAHs-Degrading Bacteria
[0126] (1) Experiment on synergistic degradation of pyrene by PBY9 and ENR6
[0127] In this experiment, PBY9 bacterial solution and pyrene mixed MSM culture medium were used to prepare the upper culture medium with a pyrene concentration of 500 mg / L. No bacteria were added to one side of the Oxford cup well as a control, and ENR6 bacterial solution was added to the other side of the well to form a PBY9×ENR6 combination, and cultured at 30°C.
[0128] Figure 4 The results of PBY9×ENR6 co-culture for 4, 7, and 10 days. Figure 4 It can be seen that on the double-layer plate with pyrene as the only carbon source, the degradation zone first appeared around the Oxford cup well where the ENR6 bacterial solution was added, and the degradation zone gradually expanded with the increase of time, indicating that PBY9 and ENR6 can synergistically degrade pyrene.
[0129] (2) Optimization experiment of PBY9 and ENR6 synergistically degrading pyrene
[0130] In this experiment, the upper culture medium with a pyrene concentration of 500 mg / L was prepared using PBY9 bacterial solution and pyrene mixed MSM culture medium. PBY9 bacterial solution was added to one side of the Oxford cup well to form a PBY9×PBY9 combination as a control, and ENR6 bacterial solution was added to the other side well to form a PBY9×ENR6 combination. The upper culture medium with a pyrene concentration of 500 mg / L was prepared using ENR6 bacterial solution and pyrene MSM culture medium. ENR6 bacterial solution was added to the Oxford cup well to form an ENR6×ENR6 combination as a control, and the PBY9×ENR6 combination was also constructed on the other side.
[0131] Figure 5 The results of co-culture of different strains are shown in Table 1. Figure 5A shows that both PBY9×PBY9 and PBY9×ENR6 combinations can degrade pyrene, but the pyrene degradation zone around PBY9×ENR6 combination appears earlier. After the same culture time (10 days), the pyrene degradation zone around PBY9×ENR6 combination is larger, indicating that the PBY9×ENR6 combination degrades pyrene faster than the PBY9×PBY9 combination. Figure 5 B. The degradation rate of pyrene by the PBY9×ENR6 combination was significantly higher than that by the ENR6×ENR6 combination. The above results further indicate that PBY9 and ENR6 have a synergistic effect in degrading pyrene.
[0132] 3. Flat plate confrontation experiment
[0133] Figure 6 This is the result of the PBY9 and ENR6 plate confrontation. Figure 6 It can be seen that there is no antagonism between PBY9 and ENR6 at the cross point on the double-layer plate with pyrene as the only carbon source.
[0134] 4. Quantitative determination experiment of pyrene degradation rate
[0135] (1) Quantitative degradation experiment with different strain ratios
[0136] Figure 7 The initial concentration of pyrene was 100 mg / L, and the initial number of ENR6 in the single bacterial degradation system was 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL and the initial number of PBY9 was 10 7 CFU / mL, and the residual amount of pyrene after 5 days of culture in the co-culture degradation system of strains when the ratio of ENR6:PBY9 was 100:1, 10:1, 1:1, 1:10, and 1:100, respectively. Figure 7 It can be seen that the degradation rate of pyrene in the single bacterial treatment was 42.0-7.7%. When the ratio of ENR6:PBY9 was 10:1 and 1:100, the degradation rate of pyrene in the strain combination treatment was 55.6% and 73.9%, respectively, which was significantly higher than the degradation rate of pyrene in all treatments. This shows that the ENR6:PBY9 ratio of 10:1 and 1:100 can show significantly better pyrene degradation ability than a single bacterial strain. (2) Quantitative degradation experiment of different concentrations of pyrene by strain combination
[0137] Figure 8 The initial numbers of PBY9 and ENR6 in the single bacterial degradation system were 10 7 CFU mL -1 and 10 9 CFU mL-1 , the degradation of 50mg / L, 100mg / L, 250mg / L, and 500mg / L pyrene when ENR6:PBY9=100:1 in the co-culture degradation system of the strains. Figure 8 It can be seen that under different pyrene concentration conditions, the PBY9×ENR6 combination showed efficient pyrene degradation ability. When the pyrene concentration was 50 mg / L (2 days of culture), 100 mg / L (5 days of culture), 250 mg / L (7 days of culture) and 500 mg / L (10 days of culture), the degradation rates of pyrene by the PBY9×ENR6 combination were 80.4%, 72.5%, 89.2% and 92.0%, respectively. Moreover, with the increase of pyrene concentration, the synergistic effect of the strain combination on pyrene degradation was more significant. When the pyrene concentration was 100-500 mg / L, the PBY9×ENR6 combination showed a significantly better pyrene degradation ability than the single strain.
[0138] In summary, the improved Oxford cup method for screening bacteria co-culture for synergistic degradation of PAHs is feasible. In this study, plate confrontation and ultra-high performance liquid chromatography verified that the PBY9 and ENR6 strain combination had a significantly higher degradation rate of pyrene than either PBY9 or ENR6 alone.
[0139] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A Glutamicibacter soil ENR6, whose preservation number is CCTCC M 2024982, and was deposited in the China Center for Type Culture Collection in Wuhan on May 17, 2024.
2. A treatment agent, characterized in that The method comprises the Bacillus glutamicum ENR6 as claimed in claim 1.
3. The treatment agent according to claim 2, characterized in that The treatment agent also includes Mycolicibacteriumpallens PBY9, the preservation number of Mycobacterium PBY9 is CCTCC M 2024983, and it was preserved in the China Center for Type Culture Collection in Wuhan on May 17, 2024.
4. The treatment agent according to claim 3, characterized in that The ratio of live bacteria of Bacillus glutamicum ENR6 to Mycobacterium PBY9 is (10-100:)1.
5. The treatment agent according to claim 3, characterized in that Both Bacillus glutamicum ENR6 and Mycobacterium PBY9 were inoculated into liquid culture medium for culture.
6. The treatment agent according to claim 5, characterized in that The liquid culture medium is a mineral culture medium.
7. Use of the glutamicum bacteria according to claim 1 or the treatment agent according to any one of claims 2 to 6 in degrading polycyclic aromatic hydrocarbons.
8. The use according to claim 7, characterized in that The polycyclic aromatic hydrocarbons are phenanthrene and pyrene, preferably pyrene.
9. The use according to claim 8, characterized in that The concentration of pyrene is 50-500 mg / L.
10. The use according to claim 7, characterized in that The application is that the glutamicum bacteria or the treating agent degrades water or soil containing polycyclic aromatic hydrocarbons.