Method for constructing microbiome for repairing organic-heavy metal combined pollution and application of microbiome

Abstract

The invention relates to the technical field of environment-friendly microbial remediation, and particularly discloses a method for constructing a microbiome for organic-heavy metal combined pollution remediation and application of the microbiome, the microbiome comprises Pseudomonas putida, Bacillus cereus, Paracoccus denitrificans and Streptomyces rochei, the number ratio of Pseudomonas putida to Bacillus cereus to Paracoccus denitrificans to Streptomyces rochei is 2: 1: 1: 1, and the construction method comprises the following steps: screening and constructing a functional microbiome to obtain multiple strains; respectively carrying out growth curve determination, gas chromatography or high performance liquid chromatography detection and minimum inhibitory concentration experiment evaluation on the dominant strains, preferably selecting four dominant strains according to experiment results, and constructing a functional microbiome with a synergistic metabolic network; under laboratory conditions, a multi-component mixed culture mode is adopted, co-culture experiments are carried out according to different proportions, the organic matter removal rate, the heavy metal removal rate and flora growth are monitored, and the optimal combination proportion is determined by integrating the results, so that the combined pollution adaptive microbiome is formed; the composite pollution adaptive microbiome is verified through a soil small-scale experiment.

Description

Technical Field

[0001] The present invention relates to the field of environmentally friendly microbial remediation technology, and in particular to a composite pollution control technology that utilizes functional microbial assemblies to synergistically degrade organic pollutants and heavy metals in soil or water. Background Art

[0002] In typical contaminated sites (such as petrochemical plants, electroplating processing areas, and chemical enterprise sites), the long-term discharge of organic solvents and heavy metal waste liquids often forms a complex pollution of organic matter (such as benzene series, polycyclic aromatic hydrocarbons, etc.) and heavy metals (such as chromium, lead, and cadmium, etc.). This type of complex pollution is highly toxic, highly mobile, and difficult to degrade, posing a serious threat to groundwater safety and ecosystem stability. Traditional remediation methods such as high-temperature thermal desorption, chemical oxidation / reduction, and acid washing are effective in removing pollutants, but often face the following problems:

[0003] It is difficult to achieve the coordinated removal of organic matter and heavy metals, and there are problems such as "only organic matter is degraded while heavy metals remain" or "heavy metals are fixed while organic matter is not degraded".

[0004] For example, Chinese patent CN117655090B discloses a microbial composite agent for degrading organic pollutants. Although it has a good remediation effect on organic pollutants, it does not involve the coordinated treatment of heavy metals, that is, it focuses on the treatment of a single pollutant, while some heavy metal remediation methods rely on plant absorption or chemical fixation, and cannot degrade associated organic matter. For the "organic + heavy metal" complex pollution scenario that is prevalent in reality, there is a lack of a unified and effective remediation system, that is, there is a problem of a single remediation target and difficulty in achieving coordinated degradation and transformation. For another example, the combined remediation method proposed in Chinese patent CN104056852B, although it takes organic-heavy metal pollution into consideration, adopts a combination of chemistry and microorganisms. The process is complex and has poor stability. It is not suitable for application in production enterprises, and exogenous agents may cause secondary pollution, which is not conducive to the ecological safety of the site and production coordination. That is, there is a problem that the remediation method relies on exogenous intervention, the process is complex, and the ecological risk is high. Chinese patent CN109055263A proposes a plant + fungus coordinated remediation method, but it relies on the plant growth cycle, the remediation cycle is long, and it is difficult to cope with acute pollution scenarios.

[0005] At the same time, the lack of systematic screening and optimization of microbial functions also leads to poor performance in real-site applications.

[0006] Therefore, there is an urgent need to construct methods and applications of microbial communities for the remediation of organic-heavy metal complex pollution, and to achieve efficient, safe, and in-situ treatment of organic and heavy metal complex pollution by constructing new microbial remediation strategies. Summary of the Invention

[0007] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method and application of constructing a microbial group for remediation of organic-heavy metal composite pollution.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] Microbial groups for remediation of organic-heavy metal complex pollution include Pseudomonas putida, Bacilluscereus, Paracoccus denitrificans and Streptomyces rochei.

[0010] Preferably, the quantitative ratio of Pseudomonas putida, Bacillus cereus, Paracoccus denitrificans and Streptomyces rochei is 2:1:1:1.

[0011] A method for constructing a microbial group for remediation of organic-heavy metal composite pollution comprises the following steps:

[0012] Step 1: Screening and construction of functional microbial communities: Soil samples were collected from typical complex contaminated sites and cultured using a synthetic medium with organic matter as the sole carbon source and heavy metal ions added. A variety of strains were obtained through plate separation and purification culture.

[0013] Step 2: Perform growth curve analysis, gas chromatography or high-performance liquid chromatography (HPLC) testing, and minimum inhibitory concentration (MIC) experiments on the dominant strains. Based on the experimental results, four dominant strains are selected to construct a functional microbiome with a synergistic metabolic network.

[0014] Step 3: Under laboratory conditions, a multi-component mixed culture method was used to conduct co-culture experiments with different ratios. The organic matter removal rate, heavy metal removal rate, and bacterial growth were monitored. Based on the above results, the optimal combination ratio was determined to form a microbiome adapted to complex pollution.

[0015] Step 4: Verify the adaptive microbial community to complex pollution through soil pilot experiments.

[0016] Preferably, the organic matter is one or more of benzene series or polycyclic aromatic hydrocarbons.

[0017] Preferably, the heavy metal ions are Cr(VI), Pb 2+ and Cd 2+ One or more of .

[0018] Preferably, the preferred strains are Pseudomonas putida, Bacillus cereus, Paracoccus denitrificans and Streptomyces rochei, respectively.

[0019] Preferably, the Pseudomonas putida, Bacillus cereus, Paracoccus denitrificans and Streptomyces rochei are mixed in a quantitative ratio of 2:1:1:1.

[0020] The microbial agent is obtained using the above-mentioned construction method.

[0021] The application of the above-mentioned microbial agents in the remediation of soil contaminated by organic matter and heavy metals.

[0022] A soil remediation agent comprises the above-mentioned microbial agent.

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

[0024] 1: The present invention constructs a functional microbial group with both synergistic degradation and resistance. By screening indigenous bacteria in contaminated sites, multiple strains are selected to construct a synergistic metabolic network and electron transport chain to form a highly stable composite microbial group, which enhances the survival and repair ability of the bacterial community in a complex pollution environment, realizes the efficient degradation of organic matter and the simultaneous reduction, adsorption and fixation of heavy metals, and the overall removal rate is significantly higher than that of a single bacteria remediation system.

[0025] 2: The present invention optimizes the microbial combination ratio and culture conditions, adopts mixed culture, small-scale simulation and other means to systematically evaluate the synergistic effect of strains, optimizes the microbial combination ratio and inoculation method, and determines the optimal repair bacterial group configuration, thereby improving the stability of the bacterial community and the synergistic efficiency.

[0026] 3: The present invention is green, environmentally friendly, safe and controllable, with no toxic by-products and no secondary pollution in the whole process, which is in line with the green restoration concept and policy orientation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 In shake flasks, the degradation rate of ethylbenzene by different bacterial strain ratios;

[0028] Figure 2 Degradation rate of hexavalent chromium by different bacterial strain ratios in shake flasks;

[0029] Figure 3 In the soil test, the degradation rate of ethylbenzene by the microbiome;

[0030] Figure 4Degradation rate of hexavalent chromium by microbial communities in a soil pilot test. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0032] Example 1: Screening and performance evaluation of functional strains

[0033] Surface soil samples (0-20cm) were collected in a typical complex pollution chemical park. The bacterial flora in the soil was first enriched using LB culture medium. Then, an inorganic salt culture medium with ethylbenzene (1000mg / L) as the sole carbon source and Cr(VI) 50mg / L was added. After shaking at 28°C and 180rpm for 3 days, 20 functional bacteria were isolated using the dilution plate method. After 16SrRNA sequencing and functional evaluation, 4 dominant strains were screened out: strain A (Pseudomonas), strain B (Bacillus), strain C (Denitrifying Bacillus), and strain D (Actinomycetes). The OD of each strain was determined experimentally. 600 , benzene degradation rate, Cr(VI) minimum inhibitory concentration (MIC) and Cr(VI) removal rate are as follows:

[0034] Table 1. Degradation performance of 4 dominant bacteria strains

[0035]

[0036] The results showed that Pseudomonas strains had excellent ethylbenzene degradation ability and good Cr(VI) tolerance, but weak Cr(VI) reduction ability; although Bacillus strains did not have significant organic matter degradation ability, they had a very high Cr(VI) removal rate and were the main Cr(VI) reducing bacteria; Denitrifying Bacillus and Actinobacterium strains played a role in synergistic metabolism and population stability, respectively.

[0037] The four functional bacteria strains described in this invention were all derived from soil samples from typical complex contaminated sites. Full-length 16S rRNA gene amplification was performed on each of the pure culture strains using the universal primer pair:

[0038] 27F: AGAGTTTGATCMTGGCTCAG (forward);

[0039] 1492R:TACGGYTACCTTGTTACGACTT (reverse);

[0040] The amplified product was approximately 1450 bp. The complete 16S rRNA gene sequence was obtained by Sanger sequencing and BLAST comparison was performed using the NCBI database. The results are as follows:

[0041] Strain A: Its 16S sequence was 100% identical to Pseudomonas putida (RefSeq accession number NR_117678.1, DSM type strain 291), and was identified as Pseudomonas putida;

[0042] Strain B: Its 16S sequence was 100% identical to that of Bacillus cereus (RefSeq accession number NR_074540.1, ATCC type strain 14579), and was identified as Bacillus cereus;

[0043] Strain C: Its 16S sequence was 100% identical to that of Paracoccus denitrificans (RefSeq accession number NR_119264.1, DSM413 type strain), and was identified as Paracoccus denitrificans;

[0044] Strain D: Its 16S sequence was 100% identical to that of Streptomyces rochei (RefSeq accession number NR_041198.1, type strain NBRC12908), and was identified as Streptomyces rochei.

[0045] Example 2: Microbiome Ratio Optimization and Synergistic Effect Verification

[0046] The above four strains were cultured to the logarithmic phase (OD 600 =1.0), and mixed in different ratios to set three combinations: A:B:C:D = 1:1:1:1, A:B:C:D = 2:1:1:1, and A:B:C:D = 3:1:1:1. The mixed bacterial solution was centrifuged and added to an inorganic salt culture medium. 1000 mg / L ethylbenzene and 50 mg / L Cr(VI) were then added. Each treatment was repeated three times, and the pollutant removal effect was tested after 7 days of culture. Figure 1 、 Figure 2 shown.

[0047] The blank control (sterile treatment) failed to effectively remove ethylbenzene and Cr(VI), demonstrating the importance of microbial activity in the remediation process and excluding the influence of other non-microbial factors (such as chemical adsorption or physical removal) on pollutant removal. In terms of microbial group ratios, the ratio of 2:1:1:1 (Pseudomonas: Bacillus: Denitrifying Bacillus: Actinomycetes) showed the best remediation effect. This ratio achieved an ethylbenzene degradation rate of 85.6% to 88.1%, which was much higher than other ratios. For Cr(VI) removal, this ratio also showed the best effect, with removal rates fluctuating between 83.2% and 86.7%, which was much higher than the ratios of 1:1:1:1 and 3:1:1:1. The removal efficiency of the 1:1:1:1 ratio (equal proportions of each strain) and the 3:1:1:1 ratio (higher proportion of Pseudomonas) was relatively low: the 1:1:1:1 ratio had low ethylbenzene degradation and Cr(VI) removal rates, ranging from 61.3% to 65.2% for ethylbenzene degradation and 60.5% to 63.1% for Cr(VI), indicating insufficient synergy between the strains in this ratio. The 3:1:1:1 ratio was also unsatisfactory. Although ethylbenzene degradation increased (77.5% to 79.1%), Cr(VI) removal was low, at only 70.3% to 71.5%. This suggests that the excessively high proportion of Pseudomonas inhibited the reduction of Cr(VI) by Bacillus, resulting in insufficient removal capacity. Overall, the microbial composition with a 2:1:1:1 ratio performed best in the remediation of compound-contaminated soils, demonstrating the best ethylbenzene degradation and Cr(VI) reduction capabilities, with remediation results far superior to those achieved with other composition ratios. This suggests that Pseudomonas and Bacillus have a synergistic effect in compound-contaminated soil remediation, with Pseudomonas providing ethylbenzene degradation and Bacillus providing Cr(VI) reduction.

[0048] Example 3: Soil remediation small-scale test verification

[0049] The composite microbial composition was prepared in a ratio of 2:1:1:1 to prepare a bacterial agent. The bacterial agent was diluted to 1×10 8 The CFU / g dose was evenly applied to the contaminated soil sample (ethylbenzene 1000mg / kg, Cr(VI) 50mg / kg), maintaining 30% water holding capacity, setting 100g of contaminated soil for each treatment, three replicates for each treatment, and incubating at room temperature for 28 days. The monitoring results are as follows Figure 3 、 Figure 4 As shown. The blank control (sterile treatment) failed to remove ethylbenzene and Cr(VI), demonstrating that microbial activity is key to contaminant removal. The 2:1:1:1 ratio performed best in ethylbenzene degradation and Cr(VI) removal, consistently achieving high removal rates in each replicate, demonstrating the high efficiency of this microbial composition. After 28 days, 86.5% of the ethylbenzene was removed; hexavalent chromium was completely removed by day 14.

[0050] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art, according to the technical solution and inventive concept of the present application, makes equivalent replacement or change within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A microbial group for remediation of organic-heavy metal composite pollution, characterized in that: Including Pseudomonas putida, Bacillus cereus, Paracoccus denitrificans and Streptomyces rochei.

2. The microbial group for remediation of organic-heavy metal composite pollution according to claim 1, characterized in that: The number ratio of Pseudomonas putida, Bacillus cereus, Paracoccus denitrificans and Streptomyces rochei is 2:1:1:

1.

3. A method for constructing a microbial group for remediation of organic-heavy metal composite pollution, characterized in that: The steps include: Step 1: Screening and construction of functional microbial communities: Soil samples were collected from typical complex contaminated sites and cultured using a synthetic medium with organic matter as the sole carbon source and heavy metal ions added. A variety of strains were obtained through plate separation and purification culture. Step 2: Perform growth curve analysis, gas chromatography or high-performance liquid chromatography (HPLC) testing, and minimum inhibitory concentration (MIC) experiments on the dominant strains. Based on the experimental results, four dominant strains are selected to construct a functional microbiome with a synergistic metabolic network. Step 3: Under laboratory conditions, a multi-component mixed culture method was used to conduct co-culture experiments with different ratios. The organic matter removal rate, heavy metal removal rate, and bacterial growth were monitored. Based on the above results, the optimal combination ratio was determined to form a microbiome adapted to complex pollution. Step 4: Verify the adaptive microbial community to complex pollution through soil pilot experiments.

4. The method for constructing a microbial group for remediation of organic-heavy metal composite pollution according to claim 3, characterized in that: The organic matter is one or more of benzene series or polycyclic aromatic hydrocarbons.

5. The method for constructing a microbial group for remediation of organic-heavy metal composite pollution according to claim 3, characterized in that: The heavy metal ions are Cr(VI), Pb 2+ and Cd 2+ One or more of .

6. The method for constructing a microbial group for remediation of organic-heavy metal complex pollution according to claim 3, characterized in that: The preferred strains are Pseudomonas putida, Bacillus cereus, Paracoccus denitrificans and Streptomyces rochei.

7. The method for constructing a microbial group for remediation of organic-heavy metal composite pollution according to claim 3, characterized in that: The Pseudomonas putida, Bacillus cereus, Paracoccus denitrificans and Streptomyces rochei were mixed in a quantitative ratio of 2:1:1:

1.

8. A microbial agent, characterized in that: Obtained using the construction method described in any one of claims 3-7.

9. Use of the microbial agent according to claims 1 and 2 in the remediation of soil contaminated by organic matter and heavy metals.

10. A soil remediation reagent, characterized in that: The method comprises the microbial agent according to claim 8.

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