Complex microbial inoculant for degrading polycyclic aromatic hydrocarbon and application of complex microbial inoculant

Abstract

The invention belongs to the technical field of polycyclic aromatic hydrocarbon degradation, and particularly relates to a complex microbial inoculant for degrading polycyclic aromatic hydrocarbon and application of the complex microbial inoculant. According to the present invention, the active component of the composite bacterial agent is Marinobacter sp.nov.LZ-6, and the preservation number of the composite bacterial agent is Marinobacter sp.nov.LZ-8, and the preservation number of the composite bacterial agent is Maricatenella alexandrii gen.nov.sp.nov.LZ-14T, and the preservation number of the composite bacterial agent is Marinobacter sp.nov.LZ-14T, and the preservation number of the composite bacterial agent is Marinobacter sp.nov.LZ-14T; and both the LZ-6 and the LZ-8 have the capability of degrading the polycyclic aromatic hydrocarbon pollutants. According to the present invention, the three bacterial strains such as LZ-6, LZ-8 and LZ-14T are ingeniously subjected to biological compounding, such that the biodegradation efficiency of the polycyclic aromatic hydrocarbon pollutants is significantly improved through the synergistic effect, and the innovative solution is provided for the efficient treatment of the refractory pollutants.

Description

Technical Field

[0001] This invention belongs to the field of polycyclic aromatic hydrocarbon (PAH) degradation technology, specifically relating to a composite microbial agent for degrading PAHs and its application. Background Technology

[0002] The severe environmental pollution caused by the petrochemical industry has become a key bottleneck restricting its sustainable development. In particular, petrochemical wastewater is rich in high concentrations of persistent polycyclic aromatic hydrocarbons (PAHs), organic pollutants with carcinogenic, teratogenic, and mutagenic effects, which not only severely pollute the environment but also pose a significant threat to human health. Therefore, developing efficient new technologies for petrochemical wastewater treatment is not only an urgent need to ensure the safety of the marine ecological environment upon which humanity depends, but also an essential path to achieving the green and sustainable development of the petrochemical industry.

[0003] Currently, among various petrochemical wastewater treatment technologies, biological methods, as a clean new technology developed based on biodegradation, show great promise due to their advantages such as low secondary pollution, high efficiency, and low energy consumption. However, to fully leverage the advantages of biological methods, obtaining highly active and superior bacterial strains and elucidating their functional mechanisms are undoubtedly necessary prerequisites and key factors for developing new biological treatment technologies. Unfortunately, research on multi-bacterial composite enhanced biological treatment processes targeting PAHs in petrochemical wastewater is still relatively limited, and increased research investment is urgently needed to achieve more breakthroughs and promote the innovation and development of petrochemical wastewater treatment technologies. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a composite microbial agent for degrading polycyclic aromatic hydrocarbons (PAHs), its preparation method, and its application.

[0005] In a first aspect, the present invention provides a compound microbial agent for degrading PAHs, wherein the active ingredient is: Marinobacter sp.nov.LZ-6、 Marinobacter sp.nov.LZ-8 and Maricatenella alexandrii gen.nov.,sp.nov.LZ-14 T .

[0006] This invention focuses on key technologies for the biological treatment of green petrochemical wastewater. Building upon previously successfully acquired novel species of marine algal functional microorganisms, it delves into and screens strains capable of efficiently degrading polycyclic aromatic hydrocarbons (PAHs), and significantly enhances their degradation efficiency through scientific biological compounding methods. This innovative approach not only opens up new research avenues for exploring the potential value of this unique resource of algal functional bacteria, but also injects new vitality into the development of green petrochemical wastewater treatment technologies. This invention utilizes LZ-6, LZ-8, and LZ-14... T By combining and optimizing these three strains, this invention further explores their practical application potential in the efficient biodegradation treatment of PAHs, opening up a new path for the reserve and research and development of new green petrochemical wastewater treatment technologies.

[0007] Furthermore, in the compound microbial agent, LZ-6, LZ-8, and LZ-14 T The ratio of live bacteria was 1.0:0.7~0.8:0.2~0.4.

[0008] Secondly, the present invention provides the application of the aforementioned compound microbial agent in the degradation of polycyclic aromatic hydrocarbons.

[0009] Furthermore, the PAHs include benzo[a]pyrene, benzo[b]fluoranthene, pyrene, and phenanthrene.

[0010] Fourthly, this aspect provides a method for degrading PAHs, comprising the following steps: The compound microbial agent was inoculated into the liquid to be treated, which contained polycyclic aromatic hydrocarbons.

[0011] Furthermore, the inoculation amount of the compound microbial agent is 10% to 20% of the volume of the liquid to be treated.

[0012] Furthermore, the method also includes the step of adding indole-3-acetic acid to the solution to be treated.

[0013] Furthermore, the concentration of indole-3-acetic acid in the solution to be treated is 10.0-30.0 mg / L.

[0014] Compared with the prior art, the present invention has the following beneficial effects: Polycyclic aromatic hydrocarbons (PAHs) are persistent pollutants that are highly toxic, structurally complex, and stable in the natural environment. Microbial degradation is considered one of the most promising approaches for remediating PAH pollution, primarily due to its low cost and lack of secondary pollution. Currently, there is a lack of efficient bioremediation systems for degrading high concentrations of PAHs, and their degradation mechanisms and pathways are also unclear. This invention screens and optimizes the bioprocess conditions of a three-strain combination, analyzing the impact of different combination methods on the degradation efficiency of PAHs and the degradation mechanisms of representative PAHs.

[0015] This bioprocess not only improves the degradation efficiency of polycyclic aromatic hydrocarbons (PAHs) but also provides important theoretical basis for subsequent bioremediation technologies. In practical applications, this bio-combination degradation technology holds promise as an efficient and environmentally friendly method for treating PAH pollution. With continued research, this technology is expected to be widely applied in more fields, contributing new strength to solving environmental pollution problems. Attached Figure Description

[0016] Figure 1 The degradation rate of benzo[a]pyrene in different groups varies with culture time. Detailed Implementation

[0017] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0018] Specific information about strains LZ-6 and LZ-8 involved in this invention is disclosed in the paper: Qi Min, Optimization of Fermentation Conditions for Extracellular Polysaccharide Production by Seven New Marine Algal Bacteria Species [D]. Zhejiang Ocean University, 2022. DOI:10.27747 / d.cnki.gzjhy.2022.000484. The classification and naming of strain LZ-6 is as follows: Marinobacter sp.nov.; strain LZ-8 is classified as Marinobacter sp.nov.; strain LZ-14 T Specific information is available in the paper: WenZhuo Z, HuiMin G, YaMing G, et al. Alexandriicola marinus gen. nov., sp. nov., a new member of the family Rhodobacteraceae isolated from marine phycosphere.[J]. Antonie van Leeuwenhoek, 2022, 115(4): 1-14. Strain LZ-14 T Category naming Maricatenella alexandrii gen.nov.,sp.nov.

[0019] The applicant promises to release the biological material to the public within twenty years from the date of application and provides a method for obtaining the biological material.

[0020] Example 1: Screening of candidate active strains for biological compounding.

[0021] Analysis of marine environmental microbial diversity and microbial species co-occurrence relationships based on marine surveys revealed the main factors influencing the core functional microbial groups. Through environmental microbiological diversity analysis of samples from the East China Sea, the following factors were identified: Marinobacter genus of bacteria and Mameliella genus, Qipengyuania The genus *Rhodobulariae*, a core functional microbial group, showed a significant positive correlation, suggesting a strong correlation between functional bacterial groups and their ecological functions.

[0022] Example 2: Selective isolation of IAA-producing strains 2216 medium is a commonly used conventional medium for isolating marine bacteria, but preliminary experiments showed that it was difficult to isolate rare new species of Rhodobulbs typical of marine algae. Therefore, this project developed a selective isolation medium for marine bacteria, RM, and analyzed its effectiveness in improving the low cultureability of Rhodobulbs. Using 2216 medium as a control, the isolation effects on algal bacteria of the marine dinoflagellate LZT09 were compared. The experimental results are shown in Table 1. It is evident that RM medium is significantly superior to the traditional 2216 medium in both the number of isolated Rhodobulbs and the number of newly discovered species. Therefore, RM medium is significantly effective in improving the isolation of dominant Rhodobulbs in the LZT09 algal community. An invention patent was applied for and obtained based on this, see CN202111376170.6.

[0023] Table 1. Comparison of separation effects between Marine 2216 medium and RM selective separation medium Note: Bacterial species are based on 16S rRNA gene sequencing and homology comparison, and the data are based on the results of 6 parallel experiments; 1 / 2 RM: the concentration of all culture medium components is 1 / 2 of the RM; 1.5RM: the concentration of all culture medium components is 1.5 times the RM.

[0024] Forty-two culturable bacterial strains were obtained using RM isolation medium, from which ten potential new species of Rhodobulbaceae were discovered. This represents 23.8% of the total isolated bacteria, significantly higher than the 11.1% probability of obtaining new species using conventional 2216 medium.

[0025] Based on the systematic optimization of selective enrichment and isolation media for algal bacteria, IAA-producing functional bacteria were screened from culturable algal bacteria, and a new bacterial species, LZ-14, was obtained. T As preliminary candidate active strains, LZ-14 was analyzed using polyphasic taxonomy techniques. T The taxonomic classification of strain LZ-14 was analyzed. T Identified asMaricatenella alexandrii gen.nov., sp. nov.

[0026] Example 3: Study on the biocombination of three strains and their performance in degrading PAHs (1) Optimization of conditions for biological compounding An optimization experiment was conducted using a 5L fermentation culture system for petrochemical wastewater to combine three strains (LZ-6, LZ-8, and LZ-14) for degradation. Factors such as strain inoculation amount, culture medium nutrients (carbon source, nitrogen source, and inorganic salts, etc.), IAA concentration and addition time were selected. The experimental parameters of the three-strain biological compound degradation system for PAHs were optimized by quantitative analysis and comparison of PAHs degradation capacity, and the optimal compound reaction conditions were obtained. The results are shown in Table 2.

[0027] Table 2. Optimal process parameters for the three-strain combination obtained through optimization. (2) Comparative analysis of degradation efficiency under biological compounding conditions The degradation rate of 200 mg / L benzo[a]pyrene (added at the beginning) was analyzed by comparing the biocombination of different degrading bacteria. Results are as follows: Figure 1 As shown, the ability of the three-strain combination to degrade benzo[a]pyrene was significantly improved. With prolonged treatment time, the residual concentration of benzo[a]pyrene decreased significantly. Table 3 shows the degradation rate and half-life of benzo[a]pyrene after 10 days of cultivation. It can be seen that the biodegradation rates of benzo[a]pyrene under LZ-6 alone, LZ-8 alone, two-strain combination, and three-strain combination conditions were 0.027 d. -1 0.025 d -1 0.054 d -1 and 0.077 d -1 The degradation rate of benzo[a]pyrene in the tri-strain combination was approximately 2.92 times that under the action of a single strain, indicating that the tri-strain combination significantly enhanced the degradation efficiency of benzo[a]pyrene.

[0028] Table 3 Comparison of degradation rates and half-lives of benzo[a]pyrene under different conditions The degradation efficiency of PAHs under different strain combinations was compared. As shown in Table 4, the results indicate that the PAH degradation efficiency was highest under the three-strain combination condition. The degradation efficiencies of the four PAHs, including benzo[a]pyrene, benzo[b]fluoranthene, pyrene, and phenanthrene, were 1.71–1.84 mg / L·d⁻¹. -1 0.21-0.22 mg / L·d -1 3.88-4.09 mg / L·d-1 and 4.02-4.16 mg / L·d -1 .

[0029] Table 4 Comparison of degradation efficiency of four PAHs under different conditions The constructed three-strain compound system showed stable operation after 6 months of continuous operation.

[0030] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0031] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. A compound microbial agent for degrading polycyclic aromatic hydrocarbons, characterized in that, Its active ingredient is Marinobacter sp.nov.LZ-6、 Marinobacter sp.nov.LZ-8 and Maricatenella alexandrii gen.nov.,sp.nov.LZ-14 T .

2. The compound microbial agent according to claim 1, characterized in that, The compound microbial agent contains LZ-6, LZ-8, and LZ-14. T The ratio of live bacteria was 1.0:0.7~0.8:0.2~0.

4.

3. The application of the composite microbial agent according to claim 1 in the degradation of polycyclic aromatic hydrocarbons.

4. The application of the compound microbial agent according to claim 3 in the degradation of polycyclic aromatic hydrocarbons, characterized in that, The polycyclic aromatic hydrocarbons include benzo[a]pyrene, benzo[b]fluoranthene, pyrene, and phenanthrene.

5. A method for degrading polycyclic aromatic hydrocarbons, characterized in that, Includes the following steps: The compound microbial agent of claim 1 is inoculated into the liquid to be treated containing polycyclic aromatic hydrocarbons.

6. The method for degrading polycyclic aromatic hydrocarbons according to claim 5, characterized in that, The inoculation amount of the compound microbial agent is 10% to 20% of the volume of the liquid to be treated.

7. The method for degrading polycyclic aromatic hydrocarbons according to claim 6, characterized in that, It also includes the step of adding indole-3-acetic acid to the solution to be treated.

8. The method for degrading polycyclic aromatic hydrocarbons according to claim 7, characterized in that, The concentration of indole-3-acetic acid in the solution to be treated is 10.0-30.0 mg / L.

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