Mixed bacteria for degrading polycyclic aromatic hydrocarbons and application thereof

Abstract

The application provides a mixed bacterial population for degrading polycyclic aromatic hydrocarbons and application thereof, and the mixed bacterial population comprises Bacillus aryabhattai with a preservation number of CCTCC No:M 2024702, Brucella with a preservation number of CCTCC No:M 2024701 and Bacillus thuringiensis with a preservation number of CCTCC No:M 2024700. The mixed bacterial population of the application can degrade typical low, medium and high ring polycyclic aromatic hydrocarbons, and can realize all-round and efficient degradation of polycyclic aromatic hydrocarbons, and the degradation efficiency of benzo[a]pyrene (BaP) can reach 87%.

Description

Technical Field

[0001] This invention relates to the field of soil environmental microbiology, and in particular to a mixed bacterial community that degrades polycyclic aromatic hydrocarbons and its applications. Background Technology

[0002] Polycyclic aromatic hydrocarbons (PAHs) are aromatic hydrocarbons containing two or more benzene rings. Based on their molecular weight, PAHs can be classified into low-ring (2-3 rings), medium-ring (4 rings), and high-ring (5 rings and above). Due to their toxicity, genotoxicity, mutagenicity, and carcinogenicity, PAHs can cause various harms to the human respiratory, circulatory, and nervous systems, and are therefore considered major organic pollutants affecting human health.

[0003] Remediation technologies for polycyclic aromatic hydrocarbon (PAH) contaminated soil mainly include physical remediation, chemical remediation, phytoremediation, and microbial remediation. Currently, there are 16 types of PAHs that are prioritized for control both domestically and internationally. Among them, low-ring PAHs (such as phenanthrene and anthracene) are more reactive than medium- and high-ring PAHs, making them more volatile and degradable. Existing technologies mainly focus on the biodegradation of medium- and low-ring PAHs, while biodegradation technologies for high-ring PAHs (benzo[a]pyrene) are scarce, and those that exist have long degradation times and low degradation rates.

[0004] CN110734877A discloses a highly efficient polycyclic aromatic hydrocarbon (PAH) degrading bacterium and its application. This highly efficient bacterium achieves a 75-day degradation rate of over 73% for pyrene and benzo[a]pyrene. This is of great significance for restoring the ecosystem of polluted environments. However, there are many types of PAHs in the environment, and 16 types of PAHs are of concern and prioritized for control both domestically and internationally. This highly efficient degrading bacterium can only degrade high-ring PAHs and does not address medium- and low-ring PAHs, resulting in a narrow application range and a long degradation time. Therefore, there is an urgent need in this field to find strains or mixed bacterial communities with better degradation effects as microbial agents to better treat PAHs. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a mixed microbial community for degrading polycyclic aromatic hydrocarbons and its application, wherein the mixed microbial community significantly improves the degradation rate of polycyclic aromatic hydrocarbons, especially benzo[a]pyrene.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In the first aspect, this invention screened three polycyclic aromatic hydrocarbon (PAH) degrading strains from farmland soil heavily polluted with PAHs, namely:

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

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

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

[0011] Secondly, the present invention provides a mixed bacterial community for degrading polycyclic aromatic hydrocarbons, the mixed bacterial community including the strains PAHA-e, PAHB-d and PAHc-a.

[0012] This invention comprehensively considers three typical polycyclic aromatic hydrocarbons (PAHs): low, medium, and high molecular weight. Using phenanthrene (Phe), pyrene (Pyr), and benzo[a]pyrene (BaP) as the sole carbon source, it screens highly efficient degrading bacteria and forms a mixed bacterial community. This enables the degradation of PAHs of different molecular weights, improves the degradation rate of Phe, Pyrene, and benzo[a]pyrene (BaP), especially significantly improves the degradation rate of benzo[a]pyrene, reduces degradation time, and greatly enhances efficiency.

[0013] The three strains were screened from farmland soil heavily contaminated with polycyclic aromatic hydrocarbons. Among them, strains PAHA-e, PAHB-d, and PAHc-a showed high decomposition efficiency for phenanthrene, pyrene, and benzo[a]pyrene, respectively. When these three highly efficient monomeric degrading bacteria were combined into a complex microbial community, the degradation efficiency could reach 87%.

[0014] As a preferred technical solution of the present invention, the mixing ratio of 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] Secondly, the present invention also provides an application of the mixed bacterial strain as described in the first aspect in the degradation of polycyclic aromatic hydrocarbons.

[0016] Preferably, the polycyclic aromatic hydrocarbons include any one or a combination of at least two of the following: naphthalene, phenanthrene, anthracene, fluorene, fluoranthene, pyrene, benzo[a]anthracene, benzo[b]fluoranthene, and benzo[a]pyrene.

[0017] Thirdly, the present invention also provides a degradation composition for treating polycyclic aromatic hydrocarbon pollution, the degradation composition comprising a mixed microbial community consisting of strains PAHA-e, PAHB-d and PAHc-a or containing metabolites produced by the mixed microbial community.

[0018] Preferably, the degradation composition exhibits high degradation efficiency for high, medium, or low-ring polycyclic aromatic hydrocarbons (PAHs) in soil. For example, the 9-day natural degradation rate of phenanthrene is at least 73%.

[0019] The degradation composition exhibits a 9-day natural degradation rate of at least 71% for pyrene in soil;

[0020] The degradation composition exhibits a 9-day natural degradation rate of at least 87% for benzo[a]pyrene in soil.

[0021] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] The mixed microbial community described in this invention includes *Bacillus aureus* PAHA-e (CCTCC No.: M 2024702), *Brucella* PAHB-d (CCTCC No.: M 2024701), and *Bacillus thuringiensis* PAHc-a (CCTCC No.: M 2024700). These three strains degrade targets covering low, medium, and high polycyclic aromatic hydrocarbons (PAHs), providing a more comprehensive degradation range. Specifically, strain Ae exhibits a 9-day natural degradation rate of 67.46% for phenanthrene, strain Bd a 9-day natural degradation rate of 49.54% for pyrene, and strain Ca a 9-day natural degradation rate of 83.92% for benzo[a]pyrene. Furthermore, these three highly efficient individual degrading bacteria form a complex microbial community with high degradation efficiency, achieving 9-day natural degradation rates of 73.14%, 71.34%, and 87.10% for phenanthrene, pyrene, and benzo[a]pyrene, respectively. Attached Figure Description

[0024] Figure 1The growth curves are for phenanthrene-degrading bacteria (Aa, Ac, and Ae); Figure I shows the growth curve for Aa, Figure II shows the growth curve for Ac, and Figure III shows the growth curve for Ae.

[0025] Figure 2 The growth curves are for pyrene-degrading bacteria (Ba, Bb, and Bd); Figure I shows the growth curve for Ba, Figure II shows the growth curve for Bb, and Figure III shows the growth curve for Bd.

[0026] Figure 3 The growth curve of benzo[a]pyrene degrading bacteria (Ca) is shown.

[0027] Figure 4 This is a photograph of the colony morphology of the mixed bacterial strains.

[0028] Figure 5 The bar chart shows the degradation rates of Phe, Pyr, and BaP in the culture medium; Figure I shows the degradation rate of Phe, Figure II shows the degradation rate of Pyr, and Figure III shows the degradation rate of BaP.

[0029] Figure 6 A bar chart showing the degradation rate of a mixture of Phe, Pyr, and BaP by the complex microbial community ABC-eba. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely 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 determined by the claims.

[0031] In the following embodiments, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the art; unless otherwise specified, all experimental methods and techniques used were conventional methods and techniques in the art.

[0032] Example 1 Screening of highly efficient degrading bacteria

[0033] A suitable amount of soil was collected from farmland with heavy polycyclic aromatic hydrocarbon pollution around chemical plants and brought back to the laboratory. After removing stones and plant debris, the soil was passed through a 10-mesh sieve, freeze-dried, and stored in brown glass bottles at 4°C for later use.

[0034] Take 10.0 g of the soil sample and place it in a 250 mL Erlenmeyer flask, maintain 65% field capacity, and incubate at 25℃ for 7 days to restore soil microbial activity.

[0035] Weigh 1.0 g of the above soil sample or 5 mL of soil suspension and add it to an Erlenmeyer flask containing 50 mL of inorganic salt liquid culture medium. Then add phenanthrene (Phe), pyrene (Pyr), and benzo[a]pyrene (BaP) to make the concentrations of Phe, Pyr, and BaP in the culture medium 2.0 mg·L⁻¹. -1 At 30℃ and 180 r·min -1 Cultured in a shaker in the dark for 7 days.

[0036] Take 5 mL of the above culture medium and inoculate it again into a new inorganic salt culture medium (Phe, Pyr, and BaP concentrations of 5.0 mg·L⁻¹). -1 With the culture conditions and time remaining unchanged, repeat the above operations.

[0037] The concentrations of Phe, Pyr, and BaP were 2.0, 5.0, 10.0, and 20.0 mg·L, respectively. -1 This is done to acclimate the microbial community in the soil sample.

[0038] Take the last bacterial culture and dilute it serially to 10⁻⁶ using phosphate-buffered saline (PBS) as the diluent. -1 10 -2 10 -3 10 -4 10 -5 10 -6 and 10 -7 times.

[0039] Take 0.1 mL of the diluted bacterial culture and spread it evenly on a tryptone yeast powder solid medium plate. Incubate at 30℃ for 24-72 h and observe the colony growth.

[0040] Selected colonies were isolated and purified multiple times on tryptone yeast powder solid culture medium plates, and the resulting bacterial population was finally preserved as slant culture.

[0041] Through enrichment and domestication, as well as separation and purification processes at different concentration gradients, three phenanthrene-degrading bacteria, three pyrene-degrading bacteria, and one benzo[a]pyrene-degrading bacteria were finally screened out and numbered Aa, Ac, Ae, Ba, Bb, Bd, and Ca, respectively.

[0042] The isolated strains were subjected to physiological and biochemical tests, and the results are shown in Table 1.

[0043] Table 1

[0044]

[0045] Note: "+" indicates a positive result; "-" indicates a negative result.

[0046] Example 2 Identification of Highly Efficient Degrading Bacteria

[0047] The concentration of the strains was determined by turbidimetric assay and growth curves were plotted. The screened degrading bacteria were inoculated into tryptone yeast powder liquid medium and incubated at 30℃ and 180 r·min. -1 The culture was carried out in a shaker in the dark, and the results were measured every 2 hours at a wavelength of 600 nm using a UV spectrophotometer.

[0048] The growth curves of the 7 degrading bacteria are shown below. Figure 1 , Figure 2 and Figure 3 As shown, the OD600 of the seven degrading bacteria changed in an "S" shape over time, which is consistent with the general growth pattern of the strains.

[0049] Figure 1 The figures show 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. The figures show that the lag phases of strains Aa, Ac, and Ae in liquid culture medium are 6 h, 10 h, and 4 h, respectively. During this period, the strains are in their adaptation phase, the cells have not yet divided, and the bacterial solution is relatively clear. The logarithmic growth phases of strains Aa, Ac, and Ae are 6–20 h, 10–22 h, and 4–12 h, respectively. During this phase, the cells adapt to the new growth environment and begin to replicate, leading to a rapid exponential increase in cell number and a gradual turbidity in the bacterial solution.

[0050] As time goes on, cells begin to die due to lack of nutrients and increase of harmful substances in the culture medium. This stage is called the death phase, which is reflected in the growth curve as a decrease in biomass. The death phase of strains Aa, Ac and Ae occurs at 20h, 22h and 12h respectively.

[0051] According to the growth curve, strain Ae has the shortest lag phase, indicating that this strain is more adaptable than the other two strains and can be considered as one of the candidate strains for the complex microbial community.

[0052] Figure 2 The figures show the growth curves 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. The figures show that the lag phases of strains Ba, Bb, and Bd are 12h, 10h, and 8h, respectively; the logarithmic growth phases are 12–24h, 10–24h, and 8–20h, respectively; and the death phases occur at 24h, 24h, and 10h. Based on the growth curves, strain Bd has the shortest lag phase, indicating that it has stronger adaptability than the other two strains and could be considered as one of the candidate strains for the complex bacterial community.

[0053] Figure 3The growth curve of benzo[a]pyrene degrading bacterium Ca is shown. The lag phase of this strain is 6 hours, the logarithmic growth phase is 6–12 hours, and the death phase occurs at 12 hours. The growth curve indicates that strain Ca has the shortest lag phase, suggesting strong adaptability and making it a potential candidate strain for complex bacterial cultures.

[0054] The selected strains were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The resulting 16S rDNA sequence was then uploaded to the National Center for Biotechnology Information (NCBI) database and subjected to BLAST comparison to obtain the strain sequence with the highest similarity to the strain.

[0055] The 16S rDNA sequence of strain Ae showed the highest similarity to Bacilus aryabhattai, with 100% homology; the 16S rDNA sequence of strain Bd showed the highest similarity to Brucella sp., with 100% homology; and the 16S rDNA sequence of strain Ca showed the highest similarity to Bacillus thuringiensis, with 100% homology.

[0056] Strains Ae, Bd, and Ca were preserved and named Bacillus PAHA-e, Brucella PAHB-d, and Bacillus PAHc-a, respectively.

[0057] Example 3: Antagonistic Characteristics of Complex Microbial Communities

[0058] Antagonism between strains mainly refers to the phenomenon where two or more different strains are inoculated onto the same culture medium in a petri dish. During the growth of the strains, the different strains resist each other's invasion, forming a dark antagonistic line visible to the naked eye at the interface between the strains. If no antagonistic line appears in the antagonism experiment, it indicates that there is no competitive relationship between the strains.

[0059] Strains Ae, Bd, and Ca were randomly combined and streaked onto solid culture medium for 3 days. Growth was observed as follows: Figure 4 As shown, the three strains are growing well and have similar growth rates, indicating that there is no antagonistic relationship between them. That is, the three strains will not inhibit each other's growth under the same environmental conditions.

[0060] Example 4 Degradation characteristics of single strains of Phe, Pyr and BaP

[0061] (1) Preparation of bacterial suspension

[0062] The selected optimal degrading bacteria, Phe, Pyr, and BaP, were inoculated into sterilized tryptone yeast extract liquid medium and placed in a shaking incubator at 30°C and 180 r·min.-1 After culturing for 24 hours under the specified conditions, the culture medium was aliquoted into 100 mL sterile centrifuge tubes and centrifuged at 4000 r·min at room temperature. -1 Centrifuge for 10 min, collect the wet bacterial cells at the bottom of the tube, then wash repeatedly with sterile physiological saline (at least 3 times), and then prepare a bacterial suspension with physiological saline to achieve its OD value. 600 It is 1.0.

[0063] (2) Vaccination

[0064] The bacterial suspension was inoculated into 100 mL of Phe, Pyr, and BaP solutions at a concentration of 50 mg·L⁻¹, respectively, at a 5% inoculation rate. -1 In sterile tryptone yeast powder liquid culture medium, at 30℃ and 180 r·min -1 Under the conditions of shaking culture for 3 days, 6 days, and 9 days;

[0065] Meanwhile, a liquid culture medium of uninoculated tryptone yeast powder was set up as a control and three experimental replicates were performed. Samples were taken at 3, 6 and 9 days of culture for analysis to determine the degradation rate of Phe, Pyr and BaP by individual strains.

[0066] (3) Degradation rate calculation

[0067] like Figure 5 As shown, the degradation rates of Phe, Pyr, and BaP in the culture medium increased significantly with the extension of culture time. Figure I shows that the natural degradation rates of Phe on days 3, 6, and 9 were 2.34%, 6.76%, and 10.58%, respectively, while the degradation rates increased to 9.02%, 47.42%, and 67.46% on days 3, 6, and 9 after adding strain Ae. Figure II shows that the natural degradation rates of Pyr on days 3, 6, and 9 were 1.76%, 4.58%, and 7.05%, while the degradation rates increased to 23.28%, 36.58%, and 49.54% on days 3, 6, and 9 after adding strain B-. Figure III shows that the natural degradation rates of BaP on days 3, 6, and 9 were 0.63%, 2.15%, and 4.31%, while the degradation rates increased to 17.80%, 63.94%, and 83.92% on days 3, 6, and 9 after adding strain Ca.

[0068] This indicates that strains Ae, Bd, and Ca can significantly degrade Phe, Pyr, and BaP in the culture medium.

[0069] Example 5: Degradation of mixed pollutants, including phenanthrene, pyrene, and benzo[a]pyrene, by the complex microbial community ABC-eba

[0070] The selected optimal degrading bacteria, Phe, Pyr, and BaP, were used to prepare OD. 600 The bacterial suspension was prepared at a concentration of 1.0, and then mixed evenly at a volume ratio of 1:1:1 to construct the composite bacterial community ABC-eba solution.

[0071] Add the ABC-eba complex solution at a 5% inoculation rate to 100 mL of a mixture of Phe, Pyr, and BaP at a concentration of 50 mg·L⁻¹. -1 In sterile tryptone yeast powder liquid culture medium, at 30℃ and 180 r·min -1 The culture was shaken and cultured for 3, 6, and 9 days under the specified conditions. A liquid culture medium containing uninoculated tryptone yeast powder was set up as a control and three experimental replicates were performed. Samples were taken at 3, 6, and 9 days of culture for analysis to detect the degradation rate of mixed pollutants Phe, Pyr, and BaP by the complex microbial community ABC-eba.

[0072] Figure 6 The figure shows the degradation of a mixture of Phe, Pyr, and BaP by the complex microbial community ABC-eba. As can be seen from the figure, the degradation capacity of the complex microbial community ABC-eba for the mixed pollutants increases to varying degrees over time.

[0073] The degradation rates of Phe by the compound microbial community ABC-eba were 37.84%, 66.04%, and 73.14% during the experimental period; 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%.

[0074] The degradation rates of each strain and the degradation rates of the mixed bacterial community are shown in Table 2 below.

[0075] Table 2

[0076]

[0077] Compared with single strains, the degradation of Phe, Pyr and BaP was increased by 5.68%, 21.8% and 3.18%, respectively, with the composite microbial community ABC-eba showing the most significant improvement in the degradation rate of Pyr.

[0078] The applicant declares that the above description is only a specific embodiment 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 conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A mixed bacterial community for degrading polycyclic aromatic hydrocarbons, characterized in that, The mixed microbial community includes Priestia aryabhattai strain PAHA-e, Brucella sp. strain PAHB-d, and Bacillus thuringiensis strain PAHc-a; The preservation number of the strain PAHA-e is CCTCC No: M 2024702; The preservation number of the strain PAHB-d is CCTCC No: M 2024701; The preservation number of the strain PAHc-a is CCTCC No:M 2024700.

2. The mixed microbial community according to claim 1, characterized in that, The mixing ratio of strain PAHA-e, strain PAHB-d and strain PAHc-a is 1:(0.8-1.2):(0.8-1.2).

3. The application of the mixed microbial community as described in claim 1 or 2 in the degradation of polycyclic aromatic hydrocarbons, wherein the polycyclic aromatic hydrocarbons are phenanthrene, pyrene, or benzo[a]pyrene.

4. A degradation composition for treating polycyclic aromatic hydrocarbon pollution, characterized in that, The polycyclic aromatic hydrocarbon is phenanthrene, pyrene, or benzo[a]pyrene; The degradation composition comprises the mixed microbial community as described in claim 1 or 2.

5. The degradation composition according to claim 4, characterized in that, The degradation composition exhibits a 9-day natural degradation rate of at least 73% for phenanthrene in soil; The degradation composition exhibits a 9-day natural degradation rate of at least 71% for pyrene in soil; The degradation composition exhibits a 9-day natural degradation rate of at least 87% for benzo[a]pyrene in soil.

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