Mixed flora for degrading polycyclic aromatic hydrocarbon and application thereof

Abstract

The invention provides a mixed flora for degrading polycyclic aromatic hydrocarbon and application of the mixed flora. The mixed flora comprises bacillus aryabhattai with the preservation number of CCTCC No: M 2024702, brucella with the preservation number of CCTCC No: M2024701 and bacillus thuringiensis with the preservation number of CCTCC No: M 202470700. According to the mixed flora disclosed by the invention, degradation targets cover typical low-ring, medium-ring and high-ring polycyclic aromatic hydrocarbons, all-directional efficient degradation of the polycyclic aromatic hydrocarbons can be realized, and particularly, the degradation efficiency of benzo [a] pyrene (BaP) can reach 87%.

Description

Technical Field

[0001] The invention relates to the field of soil environmental microorganisms, and in particular to a mixed bacterial community for degrading polycyclic aromatic hydrocarbons and applications thereof. Background Art

[0002] Polycyclic Aromatic Hydrocarbons (PAHs) refer to aromatic hydrocarbons containing two or more benzene rings. PAHs can be divided into low-ring (2-3 rings), medium-ring (4 rings) and high-ring (5 rings and above) according to their molecular weight. PAHs are toxic, genotoxic, mutagenic and carcinogenic, and can cause multiple hazards to the human respiratory system, circulatory system and nervous system. Therefore, they are identified as the main organic pollutants that affect human health.

[0003] The remediation technologies for PAH-contaminated soil mainly include physical remediation, chemical remediation, phytoremediation and microbial remediation. At present, there are 16 PAHs that are subject to priority control at home and abroad. Among them, low-ring PAHs (phenanthrene, anthracene, etc.) are more active than medium- and high-ring PAHs, and are more volatile and degradable. Existing technologies are mainly based on the biodegradation of medium- and low-ring PAHs. There are few biodegradation technologies for high-ring benzopyrene, and the degradation time is long and the degradation rate is low.

[0004] CN110734877A discloses a kind of highly efficient degradation bacteria of polycyclic aromatic hydrocarbons and its application, and the degradation rate of pyrene and benzo[a]pyrene by the highly efficient degradation bacteria is more than 73% in 75 days. It is of great significance to restore the polluted environment ecosystem. However, there are many kinds of polycyclic aromatic hydrocarbons in the environment, and there are 16 kinds of polycyclic aromatic hydrocarbons that have received attention and priority control at home and abroad. The highly efficient degradation bacteria can only degrade high-ring polycyclic aromatic hydrocarbons, and do not involve medium- and low-ring polycyclic aromatic hydrocarbons. The application range is narrow and the degradation time is long. Therefore, the field urgently needs to find strains or mixed bacterial communities with better degradation effects as microbial agents to better treat polycyclic aromatic hydrocarbons. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a mixed bacterial community for degrading polycyclic aromatic hydrocarbons and its application, wherein the mixed bacterial community significantly improves the degradation rate of polycyclic aromatic hydrocarbons, especially benzopyrene.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention screened three PAH-degrading strains from farmland soil heavily contaminated by PAHs, which are:

[0008] The strain PAHA-e (Priestia aryabhattai) is deposited in the China Center for Type Culture Collection. The strain name is Bacillus aryabhattai. The deposit address is Wuhan University, Wuhan, China. The deposit date is April 18, 2024. The deposit number is CCTCC No: M 2024702.

[0009] The strain PAHB-d (Brucella sp.) is deposited in the China Center for Type Culture Collection. The strain name is Brucella. The deposit address is Wuhan University, Wuhan, China. The deposit date is April 18, 2024. The deposit number is CCTCC No: M 2024701.

[0010] The strain PAHc-a (Bacillus sp.) is deposited in the China Center for Type Culture Collection. The strain name is Bacillus PAHc-a. The deposit address is Wuhan University, Wuhan, China. The deposit date is April 18, 2024. The deposit number is CCTCCNo:M 2024700.

[0011] In a second aspect, the present invention provides a mixed bacterial community for degrading polycyclic aromatic hydrocarbons, wherein the mixed bacterial community comprises the strain PAHA-e, the strain PAHB-d and the strain PAHc-a.

[0012] The present invention comprehensively considers three typical polycyclic aromatic hydrocarbons of low, medium and high concentrations, takes phenanthrene (Phe), pyrene (Pyr) and benzo[a]pyrene (BaP) as the sole carbon sources, screens efficient degradation bacteria, and forms a mixed bacterial community, thereby achieving the degradation of polycyclic aromatic hydrocarbons of different molecular weights, improving the degradation rates of the three polycyclic aromatic hydrocarbons of phenanthrene (Phe), pyrene (Pyr) and benzo[a]pyrene (BaP), especially significantly improving the degradation rate of benzopyrene, reducing the degradation time, and greatly improving the efficiency.

[0013] The three strains were selected from farmland soil heavily polluted by polycyclic aromatic hydrocarbons, among which strain PAHA-e, strain PAHB-d and strain PAHc-a have high decomposition efficiency for phenanthrene, pyrene and benzo[a]pyrene respectively; when these three monomer high-efficiency degradation bacteria form a composite bacterial community, the degradation efficiency can reach 87%.

[0014] As a preferred technical solution of the present invention, the mixing ratio of the strain Ae, strain Bd and strain Ca is 1:(0.8-1.2):(0.8-1.2), for example, it can be 1:1:1, 1:1:0.8, 1:1:1.2, 1:0.8:1, 1:1.2:1, 1:0.8:0.8, 1:1.2:1.2, 1:0.9:1.1, 1:0.95:1.15, 1:1.1:0.9, etc.

[0015] In a second aspect, the present invention further provides an application of the mixed bacterial system as described in the first aspect in degrading polycyclic aromatic hydrocarbons.

[0016] Preferably, the mixed bacterial strain can be used in soil or water sources contaminated by polycyclic aromatic hydrocarbons.

[0017] Preferably, the polycyclic aromatic hydrocarbons include any one or a combination of at least two of naphthalene, phenanthrene, anthracene, fluorene, fluoranthene, pyrene, benzo[a]anthracene, benzo[b]fluoranthene, and benzo[a]pyrene.

[0018] In a third aspect, the present invention also provides a degradation composition for treating polycyclic aromatic hydrocarbon pollution, wherein the degradation composition comprises a mixed bacterial community consisting of strain PAHA-e, strain PAHB-d and strain PAHc-a or contains metabolites produced by the mixed bacterial community.

[0019] Preferably, the degradation composition has a high degradation efficiency for high-, medium-, or low-ring polycyclic aromatic hydrocarbons in the soil. For example, the 9-day natural degradation rate for phenanthrene is at least 73%.

[0020] The 9-day natural degradation rate of the degradation composition on pyrene in soil is at least 71%.

[0021] The 9-day natural degradation rate of the degradation composition for benzo[a]pyrene in soil is at least 87%.

[0022] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The mixed bacterial community of the present invention comprises Bacillus arguta PAHA-e with a preservation number of CCTCC No: M 2024702, Brucella PAHB-d with a preservation number of CCTCC No: M 2024701 and Bacillus thuringiensis PAHc-a with a preservation number of CCTCC No: M 2024700. The degradation targets of the three strains cover low, medium and high polycyclic aromatic hydrocarbons, and the degradation range is more comprehensive; the 9-day natural degradation rate of phenanthrene by strain Ae is 67.46%, the 9-day natural degradation rate of pyrene by strain Bd is 49.54%, and the 9-day natural degradation rate of benzopyrene by strain Ca is 83.92%; meanwhile, the three monomer high-efficiency degradation bacteria form a composite bacterial community with high degradation efficiency, and the 9-day natural degradation rates of phenanthrene, pyrene and benzopyrene can reach 73.14%, 71.34% and 87.10% respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Figure 1 is the growth curve of phenanthrene-degrading bacteria (Aa, Ac and Ae); Figure I is the growth curve of Aa, Figure II is the growth curve of Ac, and Figure III is the growth curve of Ae.

[0026] Figure 2 Figure 1 is the growth curve of pyrene-degrading bacteria (Ba, Bb and Bd); Figure I is the growth curve of Ba, Figure II is the growth curve of Bb, and Figure III is the growth curve of Bd.

[0027] Figure 3 This is the growth curve of benzopyrene-degrading bacteria (Ca).

[0028] Figure 4 This is a physical picture of the colony morphology of the mixed strain.

[0029] Figure 5 It is a bar graph of the degradation rates of Phe, Pyr and BaP in the culture medium; Figure I is the degradation rate of Phe, Figure II is the degradation rate of Pyr, and Figure III is the degradation rate of BaP.

[0030] Figure 6 This is a bar graph showing the degradation rate of the composite bacterial community ABC-eba on a mixture of Phe, Pyr and BaP. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0032] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and technical means used were conventional methods and means in the field.

[0033] Example 1 Screening of efficient degradation bacteria

[0034] An appropriate amount of soil was collected from farmland soil heavily contaminated by PAHs around chemical companies and brought back to the laboratory. Stones and plant debris were removed and the soil was passed through a 10-mesh sieve. After freeze-drying, the soil was stored in a brown glass bottle and refrigerated in a 4°C refrigerator for later use.

[0035] Take 10.0 g of the test soil sample in a 250 mL conical flask, maintain 65% of the field water capacity, and culture it at 25°C for 7 days to restore the activity of soil microorganisms.

[0036] Weigh 1.0 g of the above soil sample or 5 mL of soil suspension and add it into a conical flask containing 50 mL of inorganic salt liquid culture medium. Then add phenanthrene (Phe), pyrene (Pyr) and benzo[a]pyrene (BaP) drugs respectively to make the concentration of Phe, Pyr and BaP in the culture medium reach 2.0 mg·L -1 , at 30℃, 180r·min -1 The cells were cultured in a shaker in the dark for 7 days.

[0037] 5 mL of the above culture medium was taken and inoculated into new inorganic salt culture medium (Phe, Pyr and BaP concentrations were 5.0 mg·L -1 ), the culture conditions and time remain unchanged and the above operation is repeated.

[0038] The concentrations of Phe, Pyr and BaP were 2.0, 5.0, 10.0 and 20.0 mg·L -1 , in order to complete the domestication of the bacterial flora in the soil sample.

[0039] Take the last bacterial culture solution, use phosphate buffered saline (PBS) as diluent, and dilute it to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 times.

[0040] Take 0.1 mL of the diluted bacterial culture solution and spread it evenly on a tryptone yeast powder solid culture medium plate, culture it at 30°C for 24 to 72 hours, and observe the growth of the colonies.

[0041] The well-grown colonies were selected and separated and purified multiple times on tryptone yeast powder solid culture medium plates, and finally the obtained bacterial colonies were preserved on slants.

[0042] Through enrichment and acclimatization as well as separation and purification at different gradient concentrations, three phenanthrene-degrading bacteria, three pyrene-degrading bacteria and one benzopyrene-degrading bacteria were finally screened out and numbered Aa, Ac, Ae, Ba, Bb, Bd and Ca respectively.

[0043] The isolated strains were tested for physiological and biochemical indicators, and the results are shown in Table 1.

[0044] Table 1

[0045] Measurement indicators Aa Ac Ae Ba Bb B Ca Gram stain + + + + + + + Catalase + + + + + + + Methyl red + - + - + - + VP determination + - - - - - + Starch hydrolysis - + - - - - - Denitrification - - - - - - + Urease + + + - + + + Indole + + + + + + +

[0046] Note: “+” means positive; “-” means negative.

[0047] Example 2 Identification of highly efficient degradation bacteria

[0048] The turbidity method was used to determine the concentration of the strain and draw a growth curve. The selected degrading bacteria were inoculated into tryptone yeast powder liquid culture medium and grown at 30℃ and 180r·min. -1 The culture was cultured in a shaker in the dark and measured every 2 hours using an ultraviolet spectrophotometer at a wavelength of 600 nm.

[0049] Growth curves of 7 strains of degrading bacteria. The results are as follows Figure 1 , Figure 2 and Figure 3 The OD600 of the 7 degrading bacteria showed an "S"-shaped change over time, which was consistent with the general growth law of the strains.

[0050] Figure 1 These are the growth curves of three phenanthrene-degrading bacteria (Aa, Ac, and Ae), where Figure I represents strain Aa, Figure II represents strain Ac, and Figure III represents strain Ae. It can be seen from the figure that the lag phases of strains Aa, Ac, and Ae in liquid culture medium are 6h, 10h, and 4h, respectively. During this period, it is the adaptation stage of the strains, the cells do not divide, and the bacterial solution is relatively clear; the logarithmic growth phases of strains Aa, Ac, and Ae are 6-20h, 10-22h, and 4-12h, respectively. During this stage, the cells adapt to the new growth environment and begin to replicate, and then the number of cells grows rapidly exponentially, and the bacterial solution gradually becomes turbid;

[0051] As time goes by, cells begin to die due to lack of nutrients and increase of harmful substances in the culture medium. This stage is called the decay phase, which is manifested as a decrease in biomass on the growth curve. The decay phase of strains Aa, Ac and Ae occurs at 20h, 22h and 12h.

[0052] According to the growth curve, strain Ae has the shortest lag phase, indicating that this strain has stronger adaptability than the other two strains and can be used as one of the candidate strains for the composite bacterial community.

[0053] Figure 2 Figure 1 is the growth curve of three pyrene-degrading bacteria (Ba, Bb and Bd), where Figure I represents strain Ba, Figure II represents strain Bb, and Figure III represents strain Bd. It can be seen from the figure that the lag phase of strains Ba, Bb and Bd are 12h, 10h and 8h respectively, the logarithmic growth phase is 12-24h, 10-24h and 8-20h respectively, and the decay phase appears at 24h, 24h and 10h. According to the growth curve, strain Bd has the shortest lag phase, indicating that the strain has stronger adaptability than the other two strains and can be used as one of the candidate strains for the composite flora.

[0054] Figure 3This is the growth curve of benzopyrene-degrading bacteria Ca. The lag phase of the strain is 6 hours, the logarithmic growth phase is 6 to 12 hours, and the decay phase appears at 12 hours. According to the growth curve, the lag phase of strain Ca is the shortest, indicating that the strain has strong adaptability and can be used as one of the candidate strains for the composite bacterial community.

[0055] The screened strains were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the obtained 16S rDNA sequences were uploaded to the National Center for Biotechnology Information (NCBI) database of the United States, and then BLAST comparison was performed to obtain the strain sequence with the highest similarity to the strain.

[0056] The 16S rDNA sequence of strain Ae is most similar to Bacilus aryabhattai, with a homology of 100%; the 16S rDNA sequence of strain Bd is most similar to Brucella sp, with a homology of 100%; the 16S rDNA sequence of strain Ca is most similar to Bacillus thuringiensis, with a homology of 100%.

[0057] The strains Ae, Bd and Ca were deposited separately and their deposit names were Bacillus PAHA-e, Brucella PAHB-d and Bacillus PAHc-a.

[0058] Example 3 Antagonistic properties of composite flora

[0059] The antagonism between strains mainly refers to the process of inoculating two or more different strains into the culture medium of the same culture dish. During the growth of the strains, different strains resist each other's invasion, and a dark antagonism line visible to the naked eye will be formed at the junction of the strains. If no antagonism line appears in the antagonism experiment, it indicates that there is no competition between the strains.

[0060] Strains Ae, Bd and Ca were randomly combined and streaked on solid medium for 3 days. Figure 4 As shown, the growth state is good and the growth momentum is similar, indicating that there is no antagonistic relationship between the strains, that is, the three strains will not inhibit each other's growth under the same environmental conditions.

[0061] Example 4 Degradation characteristics of Phe, Pyr and BaP by a single strain

[0062] (1) Preparation of bacterial suspension

[0063] The selected optimal Phe, Pyr and BaP degradation bacteria were inoculated into the sterilized tryptone yeast powder liquid culture medium and placed in a shaking incubator at 30°C and 180 r·min -1After culturing for 24 h, the culture solution was divided into 100 mL sterile centrifuge tubes and centrifuged at 4000 r / min at room temperature. -1 Centrifuge for 10 minutes, collect the wet bacteria at the bottom of the tube, then wash repeatedly with sterilized saline (at least 3 times), and then prepare it into a bacterial suspension with saline to make its OD 600 is 1.0.

[0064] (2) Vaccination

[0065] According to the inoculum volume of 5%, the bacterial suspension was inoculated to 100 mL of Phe, Pyr and BaP concentrations of 50 mg·L -1 Sterile tryptone yeast powder liquid medium was used at 30℃ and 180r·min -1 The shaking culture was carried out for 3, 6 and 9 days under the conditions;

[0066] At the same time, a tryptone yeast powder liquid culture medium without bacteria was set as a reference and three experimental replicates. Samples were taken for analysis at 3d, 6d and 9d of culture to determine the degradation rates of Phe, Pyr and BaP by individual strains.

[0067] (3) Calculation of degradation rate

[0068] like Figure 5 As shown, with the extension of culture time, the degradation rates of Phe, Pyr and BaP in the culture medium increased significantly.

[0069] Figure I shows that the natural degradation rates of Phe on the 3rd, 6th and 9th days were 2.34%, 6.76% and 10.58%, while the degradation rates on the 3rd, 6th and 9th days were increased to 9.02%, 47.42% and 67.46% after adding strain Ae;

[0070] Figure II shows that the natural degradation rates of Pyr substances were 1.76%, 4.58% and 7.05% on the 3rd, 6th and 9th days, and the degradation rates increased to 23.28%, 36.58% and 49.54% on the 3rd, 6th and 9th days after adding strain Bd;

[0071] Figure III shows that the natural degradation rates of BaP substance were 0.63%, 2.15% and 4.31% on the 3rd, 6th and 9th days. After adding strain Ca, the degradation rates on the 3rd, 6th and 9th days increased to 17.80%, 63.94% and 83.92%.

[0072] This indicates that strains Ae, Bd and Ca can significantly degrade Phe, Pyr and BaP in the culture medium.

[0073] Example 4 Degradation of mixed pollutants of phenanthrene, pyrene and benzopyrene by composite bacterial consortium ABC-eba

[0074] The selected best Phe, Pyr and BaP degradation bacteria were made into OD 600 The mixture was mixed evenly in a volume ratio of 1:1:1 to construct a composite bacterial community ABC-EBA solution.

[0075] The composite bacterial community ABC-eba solution was added to 100 mL of sterile tryptone yeast powder liquid culture medium with a mixed concentration of Phe, Pyr and BaP of 50 mg·L-1 at a 5% inoculum dose, and cultured with shaking at 30°C and 180 r·min-1 for 3, 6 and 9 days. At the same time, uninoculated tryptone yeast powder liquid culture medium was set as a reference and 3 experimental replicates. Samples were taken for analysis at 3, 6 and 9 days of culture to detect the degradation rate of the composite bacterial community ABC-eba on the mixed pollutants of Phe, Pyr and BaP.

[0076] Figure 6 This is the degradation of a mixture of Phe, Pyr and BaP by the composite bacterial community ABC-eba. As can be seen from the figure, with the extension of time, the degradation ability of the composite bacterial community ABC-eba on mixed pollutants has improved to varying degrees.

[0077] During the test period, the degradation rates of Phe by the composite bacteria ABC-eba were 37.84%, 66.04% and 73.14%; the degradation rates of Pyr were 36.96%, 52.50% and 71.34%; and the degradation rates of BaP were 23.16%, 86.42% and 87.10%.

[0078] The degradation rates of each strain and mixed flora are shown in Table 2.

[0079] Table 2

[0080]

[0081] Compared with a single strain, the degradation of Phe, Pyr and BaP increased by 5.68%, 21.8% and 3.18%, respectively. Among them, the composite bacterial community ABC-eba had the most significant improvement in the degradation rate of Pyr.

[0082] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A polycyclic aromatic hydrocarbons-degrading bacterium, characterized in that: The strain is strain PAHA-e (Priestia aryabhattai), and the deposit number is CCTCC No: M 2024702.

2. A polycyclic aromatic hydrocarbons-degrading bacterium, characterized in that: The strain is strain PAHB-d (Brucellas p.), and its deposit number is CCTCC No: M 2024701.

3. A polycyclic aromatic hydrocarbons-degrading bacterium, characterized in that: The strain is strain PAHc-a (Bacillus sp.), and its preservation number is CCTCC No: M 2024700.

4. A mixed bacterial community for degrading polycyclic aromatic hydrocarbons, characterized in that: The mixed bacterial system comprises the strain PAHA-e, strain PAHB-d and strain PAHc-a described in claims 1-3.

5. The mixed bacterial flora according to claim 4, characterized in that The mixing ratio of the strain PAHA-e, strain PAHB-d and strain PAHc-a is 1:(0.8-1.2):(0.8-1.2).

6. Use of the mixed bacterial community as claimed in claim 4 or 5 in degrading polycyclic aromatic hydrocarbons.

7. The use according to claim 6, characterized in that: The polycyclic aromatic hydrocarbons include any one of naphthalene, phenanthrene, anthracene, fluorene, fluoranthene, pyrene, benzo[a]anthracene, benzo[b]fluoranthene, and benzo[a]pyrene, or a combination of at least two of them.

8. A degradation composition for treating polycyclic aromatic hydrocarbon pollution, characterized in that: The degradation composition comprises the mixed bacterial community as claimed in claim 4 or contains metabolites produced by the mixed bacterial community.

9. The degradation composition according to claim 8, characterized in that The 9-day natural degradation rate of the degradation composition on phenanthrene in soil is at least 73%; The 9-day natural degradation rate of the degradation composition for pyrene in soil is at least 71%; The 9-day natural degradation rate of the degradation composition for benzo[a]pyrene in soil is at least 87%.

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