A composite bacterial community for degrading polycyclic aromatic hydrocarbons and its application

Abstract

The present invention provides a composite bacterial community for degrading polycyclic aromatic hydrocarbons and its application, belonging to the field of microbial technology. The composite bacterial community of the present invention comprises Fusarium solani E1 and Rhodococcus erythroxysporum E2; the preservation number of Fusarium solani E1 is CGMCC No: 41063, and the preservation number of Rhodococcus erythroxysporum E2 is CGMCC No: 29266. The present invention combines fungi and bacteria to synergistically degrade PAHs in the repair of PAHs. The composite bacterial community has better PAHs degradation effect and composite bacterial growth status than single bacteria, and has good effects on the degradation of high- and low-ring PAHs.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a composite bacterial community for degrading polycyclic aromatic hydrocarbons and an application thereof. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAHs) refer to persistent toxic pollutants composed of two or more benzene rings arranged in a linear, angular or clustered shape, mostly in the form of colorless or pale yellow crystals. According to the number of rings, PAHs can be divided into low-ring PAHs with 2-3 rings and medium- and high-ring PAHs with 4-7 rings.

[0003] The main physical properties of PAHs are semi-volatility and long-distance diffusion; high lipophilicity and low water solubility, and hydrophobicity increases with the increase of molecular weight. The chemical structure of PAHs is relatively stable, inert, not easy to be degraded, and has environmental persistence. PAHs are bioaccumulative and can be enriched through the food chain. The main hazards of PAHs are carcinogenicity, teratogenicity and mutagenicity, and the toxicity of PAHs with 4-7 rings is higher than that of PAHs with 2-3 rings. The PAHs remediation technologies in soil include: bioremediation, physical remediation, and chemical remediation. However, physical remediation, in essence, only transfers pollutants but cannot fundamentally remove pollutants, while chemical remediation is prone to secondary pollution. Bioremediation uses plants, animals, and microorganisms to absorb, metabolize, and transform PAHs in the soil. It has the characteristics of low price, simple operation, wide application range, and no secondary pollution. However, the methods of bioremediation are diverse and the degradation rate is not very ideal. Therefore, the development of a bioremediation method with high degradation efficiency and environmental protection for PAHs is a technical problem that needs to be solved at this stage. Summary of the invention

[0004] The present invention provides a composite bacterial community for degrading polycyclic aromatic hydrocarbons and its application. The present invention combines fungi and bacteria to synergistically degrade PAHs in the repair of PAHs. The composite bacterial community has better PAHs degradation effect and composite bacterial growth condition than single bacteria, and has good effects on the degradation of high- and low-ring PAHs.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The invention provides a composite bacterial community for degrading polycyclic aromatic hydrocarbons, comprising Fusarium solani E1 and Rhodococcus erythropolis E2; the Fusarium solani E1 is classified and named as Fusarium solani, with a preservation number of CGMCC No: 41063, and the Rhodococcus erythropolis E2 is classified and named as Rhodococcus erythropolis, with a preservation number of CGMCC No: 29266.

[0007] The invention provides a composite liquid bacterial agent for degrading polycyclic aromatic hydrocarbons. The composite liquid bacterial agent comprises a bacterial suspension of Fusarium solani E1 and Rhodococcus erythroxysporum E2.

[0008] Preferably, the bacterial suspension OD of the Fusarium solani E1 or Rhodococcus erythroxysporum E2 is 600 =0.05~0.15.

[0009] Preferably, the volume ratio of the mixture of the Fusarium solani E1 bacterial suspension and the Rhodococcus erythroxysporum E2 bacterial suspension is 1:9 to 9:1.

[0010] The present invention provides a preparation method of the composite liquid bacterial agent, comprising the following steps: inoculating Fusarium solani E1 or Rhodococcus rubervillei E2 strain into LB culture medium for cultivation, centrifuging to remove supernatant, inoculating the bacterial bodies into sterile water to obtain Fusarium solani E1 bacterial suspension or Rhodococcus rubervillei E2 bacterial suspension, and mixing the above bacterial suspensions to obtain.

[0011] Preferably, the culture temperature is 26°C to 30°C, and the culture time is 2 to 4 days.

[0012] The present invention provides the composite bacterial community and the application of the composite liquid bacterial agent in degrading polycyclic aromatic hydrocarbons.

[0013] Preferably, the polycyclic aromatic hydrocarbons include polycyclic aromatic hydrocarbons including phenanthrene, benzo[a]anthracene and benzo[a]pyrene.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention screens out Fusarium solani E1 and Rhodococcus rubervillei E2 for the first time. Rhodococcus rubervillei E2 is an actinomycete with good biological toxicity resistance and PAHs degradation potential. The fungus Fusarium solani E1 can not only absorb and store polycyclic aromatic hydrocarbons in lipid vesicles, but also degrade a variety of PAHs including phenanthrene, benz[a]anthracene, and benzo[a]pyrene. The present invention combines the above-mentioned fungi with bacteria to synergistically degrade in PAHs repair. The composite bacterial community is better than a single bacterium in terms of PAHs degradation effect and composite bacterial growth status, and has a good effect on the degradation of high- and low-ring PAHs, and has a wide range of application prospects.

[0016] Biological deposit information

[0017] Fusarium solani E1, classified as Fusarium solani, with Latin name Fusarium solani, was deposited at the General Microbiology Center of China Culture Collection Administration on December 8, 2023. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No: 41063.

[0018] Rhodococcus erythropolis E2, classified as Rhodococcus erythropolis, with a Latin name of Rhodococcus erythropolis, was deposited at the General Microbiology Center of China Culture Collection on December 8, 2023. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No: 29266. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Antagonism test results of strains.

[0020] Figure 2 Phylogenetic tree of identified degrading bacteria A (Fusarium solani E1).

[0021] Figure 3 Phylogenetic tree of identified degrading bacteria B (Rhodococcus erythropolis E2).

[0022] Figure 4 Growth curves of degrading bacteria at different strain ratios.

[0023] Figure 5 Degradation rate results of fluoranthene under different strain ratios. DETAILED DESCRIPTION

[0024] The present invention provides a composite bacterial community for degrading polycyclic aromatic hydrocarbons, including Fusarium solani E1 and Rhodococcus erythropolis E2; the Fusarium solani E1 is classified and named as Fusarium solani, with a preservation number of CGMCC No: 41063; the Rhodococcus erythropolis E2 is classified and named as Rhodococcus erythropolis, with a preservation number of CGMCC No: 29266. The Rhodococcus erythropolis E2 of the present invention is an actinomycete, which has good biological toxicity resistance and PAHs degradation potential; the Fusarium solani E1 is a fungus, which can not only absorb and store polycyclic aromatic hydrocarbons in lipid vesicles, but also degrade a variety of PAHs including phenanthrene, benz[a]anthracene, and benzo[a]pyrene.

[0025] The present invention provides a composite liquid bacterial agent for degrading polycyclic aromatic hydrocarbons, wherein the composite liquid bacterial agent comprises a bacterial suspension of Fusarium solani E1 and Rhodococcus rubervillei E2. The OD value of the bacterial suspension of Fusarium solani E1 or Rhodococcus rubervillei E2 of the present invention is 600 =0.05~0.15, preferably OD 600 =0.08 to 0.13, more preferably OD600 =0.1. The volume ratio of the mixture of the Fusarium solani E1 suspension and the Rhodococcus erythroxysporum E2 suspension of the present invention is 1:9 to 9:1, preferably 2:8 to 8:2, and more preferably 7:3.

[0026] The present invention provides a method for preparing the composite liquid bacterial agent, comprising the following steps: inoculating Fusarium solani E1 or Rhodococcus rubervillei E2 strain into LB medium for cultivation, centrifuging to remove the supernatant, inoculating the bacterial body in sterile water to obtain Fusarium solani E1 bacterial suspension or Rhodococcus rubervillei E2 bacterial suspension, and mixing the above bacterial suspensions to obtain. The culture temperature of the present invention is 26°C to 30°C, and the culture time is 2 to 4 days.

[0027] The present invention also provides a composite solid bacterial agent for degrading polycyclic aromatic hydrocarbons. The composite solid bacterial agent can be obtained by mixing E1 bacterial powder of Fusarium solani and E2 bacterial powder of Rhodococcus erythroxysporum with certain activity.

[0028] The present invention also provides the use of the composite bacterial community, the composite liquid bacterial agent and the composite solid bacterial agent in degrading polycyclic aromatic hydrocarbons. The polycyclic aromatic hydrocarbons described in the present invention include polycyclic aromatic hydrocarbons including phenanthrene, benzo[a]anthracene and benzo[a]pyrene. The composite bacterial community described in the present invention can degrade polycyclic aromatic hydrocarbons in soil or sewage or other substances containing polycyclic aromatic hydrocarbons.

[0029] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0030] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] 1. Screening of fungi and bacteria: Soil PAHs-degrading bacteria were enriched and cultured by gradually increasing the substrate concentration.

[0033] The culture medium used was as follows:

[0034] Liquid inorganic salt culture solution: K2HPO4·H2021.75g, Na2HPO4·12H2O 33.48g, KH2PO48.7g, NH4Cl 5.0g, MgSO422.5g, CaCl236.4g, FeCl30.25g, MnSO439.9mg, ZnSO4·H2O 42.8mg, (NH4)2MoO4·4H2O 34.7mg, distilled water 1000mL, adjusted to a pH close to that of the soil sample.

[0035] Inorganic salt culture medium for phenanthrene, acenaphthene and benzo[a]pyrene: Take the mother solutions of phenanthrene, acenaphthene and benzo[a]pyrene respectively, filter them through a 0.22μm filter membrane for sterilization, add the inorganic salt basal culture medium into a sterilized conical flask, and prepare screening culture medium of different concentrations. The preparation method of the mother solutions of phenanthrene, acenaphthene and benzo[a]pyrene is as follows: weigh phenanthrene, acenaphthene and benzo[a]pyrene respectively and dissolve them in a certain amount of acetone to make the final concentration of 1g / L.

[0036] Solid inorganic salt culture medium: Add 15 g agar powder to 1000 ml of phenanthrene, acenaphthene, and benzo[a]pyrene inorganic salt culture medium and sterilize under high pressure before use.

[0037] LB medium: 10 g of peptone, 5 g of yeast extract powder, 5 g of sodium chloride, 1 g of glucose, adjust the pH value to be close to the pH of the soil sample, 1000 mL of distilled water, and sterilize with high-pressure steam at 121°C for 30 min.

[0038] (1) 10 mL of soil extract from farmland soil that had been contaminated by PAHs for a long time near a coal-fired power plant in Weifang City (0-20 cm deep) was placed in 40 mL of liquid inorganic salt culture medium with phenanthrene, acenaphthene or benzo[a]pyrene as the sole carbon source (the initial concentration of the substrate was 2 mg / L), and glass beads were added. The soil was then placed in a constant temperature shaker at 180 r / min and incubated in the dark at 28°C for 7 days to enrich the microorganisms in the soil.

[0039] (2) Then, 25 mL of the first enrichment culture medium was transferred to a new inorganic salt liquid culture medium containing a higher concentration of phenanthrene, acenaphthene or benzo[a]pyrene for a second enrichment culture. The enrichment culture was repeated 5 times in total, and the concentration of phenanthrene, acenaphthene or benzo[a]pyrene transferred each time was 2, 4, 8, 16, and 32 mg / L. Finally, a bacterial culture with stable degradation ability of the three PAHs was obtained.

[0040] (3) The strains in the above three bacterial liquids were then screened in a solid inorganic salt culture medium by the dilution coating plate method, and the strains with good colony growth were selected and inoculated into a new solid inorganic salt culture medium for isolation and culture, and finally PAHs degrading bacteria A, degradation bacteria B and degradation bacteria C were obtained.

[0041] (4) The three types of degrading bacterial colonies screened out above were cultured in LB medium at 26°C to 30°C for 3 days, and then the cultured bacterial solution was mixed with 80% glycerol (water + glycerol) in a ratio of 1:3 and stored in a preservation tube at -80°C to obtain a seed bacterial solution.

[0042] 2. Antagonism Experiment

[0043] Activation of strains: The seed culture solution in step 1 of this example was cultured in LB medium at 26° C. to 30° C. for 3 days to obtain activated bacterial solutions of strains A, B, and C, respectively.

[0044] Antagonism experiment was conducted by plate streaking method: when the prepared solid fluoranthene inorganic salt solid culture medium was cooled to room temperature, it was poured into the culture dish (about 15-20mL per dish) according to the aseptic operation method, and used after cooling and solidification. Sterilize and cool the inoculation loop 2-3 times, pick a small amount of activated bacterial liquid with the inoculation loop and streak on the solid fluoranthene inorganic salt culture medium (single bacteria use the three-point method to streak, and multiple bacteria use the cross-line method to streak), and immediately burn the residual bacteria on the inoculation loop after streaking to prevent contamination by miscellaneous bacteria. The experimental strains were designed to be streaked in four ways: A, B, C, A+B, A+C, B+C, and A+B+C, with 3 parallels for each treatment. After streaking, it was placed in a 30℃ constant temperature incubator for 72 hours, and the bacterial growth at the intersection on the streaking plate was observed to preliminarily clarify the interaction relationship between the strains.

[0045] The results showed that the antagonistic test results of different strains were shown in Figure 1 ,Depend on Figure 1 (a~c) It can be seen that the surface morphology of degrading bacteria A is relatively rough, with white hairy edges; the surface morphology of degrading bacteria B is relatively smooth, yellow, and has neat and regular edges; the surface morphology of degrading bacteria C is wrinkled, with milky white raised edges. Figure 1 (d~g) From the strain antagonism experiment, it can be seen that the strains grew well at the intersection of the line culture between the degrading bacteria A (white hairy state) and the degrading bacteria B (smooth state). The strains at the intersection of the line culture between the degrading bacteria B and C, the degrading bacteria A and C, and the degrading bacteria A, B, and C became one degrading bacterium, indicating that the degrading bacteria A and B did not produce an antagonistic reaction. Therefore, the degrading bacteria A and B were selected to construct the composite bacterial community.

[0046] 3. Identification of strains

[0047] The selected degradation strains A and B were tested at Sangon Biotech (Shanghai) Co., Ltd. After extraction of genomic DNA of the two degradation bacteria, PCR amplification, gel electrophoresis, purification and recovery, and sequencing analysis, the two strains were molecularly identified using primers 27F (SEQ ID NO.1: AGTTTGATCMTGGCTCA G), 1492R (SEQ ID NO.2: GGTTACCTTGTTACGACTT) and ITS1 (SEQ ID NO.3: TCCGTAGGTGAACCTGCGG), ITS4 (SEQ ID NO.4: TCC TCCGCTTATTGATATGC). Sequence alignment was performed using the NCBI database, and the phylogenetic tree of each strain was constructed using MEGA-X software and the neighbor-joining method ( Figure 2-Figure 3 ), the confidence of each branch of the developmental tree was obtained by 1000 repetitions of the resampling method. It was concluded that the degrading bacteria A was Fusarium solani E1, and the degrading bacteria B was Rhodococcus erythropolis E2.

[0048] Example 2 Construction and activity verification of composite bacterial flora

[0049] (1) Take the acclimated degrading bacteria A and B bacterial liquid (the activated bacterial liquid prepared in step 2 of Example 1) and centrifuge them at 8000 r / min for 5 min. Discard the supernatant and wash the bacterial cells twice with sterile water. The obtained bacterial cells are inoculated with sterile water and the OD value is adjusted so that the OD value of the bacterial suspension is 600 is 0.1;

[0050] (2) The optimal ratio experiment of composite bacterial community was carried out according to the volume ratio of degradation bacteria A: B of 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0. Composite degradation bacterial community was constructed by composite bacterial liquid with different ratios, and 1% of the volume of the culture medium was inoculated into an inorganic salt culture medium with fluoranthene as the sole carbon source (the concentration of fluoranthene was set to 3 mg / L);

[0051] The preparation of an inorganic salt culture medium in which fluoranthene is the sole carbon source comprises the following steps: (1) an inorganic salt culture medium: 1.5 g KH2PO4, 4 g Na2HPO4, 1 g NH4Cl, 0.02 g CaCl2, 1 g NaNO3, 0.2 g MgSO4·7H2O, 0.03 g FeSO4·7H2O, 1 mL trace elements (each 100 mL solution comprises: 0.25 mg CoCl2·6H2O, 0.37 mg (NH4)6-Mo7O 24·4H2O, 4.0mg CuSO4·5H2O, 7mg H3BO3, 4.3mg MnSO4·5H2O, 4.3mg ZnSO4·7H2O), dilute to 1000mL with deionized water, sterilize at 121℃ with high pressure steam for 20min. (2) Prepare fluoranthene mother solution: weigh 15mg fluoranthene and dissolve it in 50mL acetone to make the final concentration 300mg / L. (3) Fluoranthene inorganic salt culture medium: measure the above fluoranthene mother solution and sterilize it through 0.22μm filter membrane, then add inorganic salt basal culture medium to make the final mass concentration of fluoranthene 3mg / L, sterilize at 121℃ with high pressure steam for 30min.

[0052] (3) The culture medium containing the composite degrading bacteria was placed in a 28°C constant temperature shaking incubator for culture, and the OD of the culture medium was measured every 12 hours. 600 The degradation rate was calculated based on the residual concentration of fluoranthene. The growth of composite degrading bacterial communities and the degradation rate of fluoranthene were observed under the same culture conditions and different treatments (three replicates were set for each treatment). The optimal composite bacterial community was obtained by comparing the degradation effects of composite bacterial communities with different ratios on fluoranthene.

[0053] (4) Take a 5 ml sample under sterile conditions on a clean bench and place it in a sterilized centrifuge tube. After the bacterial solution is filtered through a 0.22 μm organic filter membrane, its absorbance is measured at a wavelength of 230 nm using an ultraviolet spectrometer. The concentration of fluoranthene in the sample at the time is calculated using the constructed fluoranthene concentration curve.

[0054] Take 8 ml of sample under sterile conditions on a clean bench, place it in a sterilized centrifuge tube, shake it thoroughly, rinse the glass cuvette twice with the bacterial solution, and measure its OD value at a wavelength of 600 nm using an ultraviolet spectrometer.

[0055] The growth curves of the degrading bacteria at different ratios of the strains are shown in Figure 4 The OD values ​​of the degrading bacteria in each ratio showed an increasing trend over time. When the ratio of degrading bacteria A to B was 7:3, 6:4, and 5:5, they grew better in fluoranthene medium, and the OD values ​​were 0.538, 0.467, and 0.451 after 84 hours of culture, respectively.

[0056] The degradation rate of fluoranthene at 84h under different strain ratios is shown in Figure 5 , the degradation rates of fluoranthene by different strain ratios were different. When the ratio of degradation bacteria A to B was 0:10 and 10:0, the degradation rates of fluoranthene were 24.4% and 31.8%, respectively. Degradation bacteria A and B had a degradation effect on fluoranthene. When the ratio of degradation bacteria A to B was 7:3, the degradation rate of fluoranthene was the highest, at 44.5%, followed by the ratios of 6:4 and 5:5. Figure 4 and Figure 5It can be seen that when the ratio of degrading bacteria A and B is 7:3, they grow better in fluoranthene culture medium. After 84 hours of culture, the OD value increases from 0.01 to 0.538. In the composite bacterial community construction experiment, the degradation rate is positively correlated with the OD value. When the degrading bacteria are in a ratio of 7:3, the strain growth is the best and the degradation effect of fluoranthene is also the best.

[0057] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composite bacterial community for degrading polycyclic aromatic hydrocarbons, characterized in that: The invention comprises Fusarium solani E1 and Rhodococcus erythropolis E2; the Fusarium solani E1 is classified and named as Fusarium solani, with a preservation number of CGMCC No: 41063; the Rhodococcus erythropolis E2 is classified and named as Rhodococcus erythropolis, with a preservation number of CGMCC No: 29266.

2. A composite liquid bacterial agent for degrading polycyclic aromatic hydrocarbons, characterized in that: The composite liquid bacterial agent comprises the bacterial suspension of Fusarium solani E1 and Rhodococcus erythroxysporum E2 as claimed in claim 1.

3. The composite liquid bacterial agent as claimed in claim 2, wherein: The bacterial suspension of Fusarium solani E1 or Rhodococcus erythroxysporum E2 has an OD600 value of 0.05 to 0.

15.

4. The composite liquid bacterial agent according to claim 3, characterized in that: The volume ratio of the mixture of the Fusarium solani E1 bacterial suspension and the Rhodococcus erythroxysporum E2 bacterial suspension is 1:9 to 9:

1.

5. The method for preparing the composite liquid bacterial agent according to any one of claims 2 to 4, characterized in that: The method comprises the following steps: inoculating the Fusarium solani E1 or Rhodococcus rubervillei E2 strain into LB culture medium for cultivation, removing the supernatant by centrifugation, inoculating the bacterial bodies into sterile water to obtain a Fusarium solani E1 bacterial suspension or a Rhodococcus rubervillei E2 bacterial suspension, and mixing the above bacterial suspensions to obtain the method.

6. The preparation method according to claim 5, characterized in that: The culture temperature is 26° C. to 30° C., and the culture time is 2 to 4 days.

7. Use of the composite bacterial consortium according to claim 1 and the composite liquid bacterial agent according to any one of claims 2 to 4 in degrading fluoranthene.

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