Marine pantoea sp. capable of degrading pae and application thereof
By screening Aeromonas intermedius HJ02 from marine sediments, the problem of degradation of high molecular weight PAEs in marine environments has been solved, achieving efficient and stable bioremediation effects, and is suitable for the remediation of PAE-contaminated soil and water.
Patent Information
- Application Number
- CN202610499386.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-15
- Publication Date
- 2026-07-03
Smart Images
Figure CN122326465A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, and specifically relates to a marine aeromonas media strain that degrades phthalic acid esters (PAEs) and its applications. Background Technology
[0002] Phthalate esters (PAEs) are widely used as plasticizers and additives in industries such as plastic products, packaging materials, cosmetics, coatings, and medical devices. During their production, use, and disposal, PAEs are easily released into the environment through volatilization, leaching, and abrasion, and are currently widely detected in soil, water bodies, crops, and even human biological samples. PAEs are typical endocrine disruptors with estrogenic effects; long-term low-dose exposure can lead to endocrine disorders, carcinogenicity, or mutagenic risks, and are included in the key control list of my country's "New Pollutant Control Action Plan." The European Union and the US Environmental Protection Agency have listed PAE compounds such as dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) as priority controlled pollutants.
[0003] Bioremediation technology has become an important direction for the treatment of PAE pollution due to its advantages such as low cost, high safety, environmental friendliness, and strong sustainability. Microbial degradation is the main pathway for the reduction of PAEs in the environment. Currently, more than 80 PAE-degrading bacteria, covering approximately 36 genera, have been screened and reported from environmental samples such as farmland soil, activated sludge, and plant rhizosphere. These mainly include *Sphingomonas*, *Arthrobacter*, *Pseudomonas*, *Bacillus*, and *Rhodococcus*. Most degrading bacteria exhibit high degradation efficiency for low molecular weight PAEs (such as DMP, DEP, and DBP), and some strains show good environmental adaptability. Some strains can also synergistically degrade multiple PAEs, such as *Bacillus subtilis* 3C3, *Gordonia* sp. Dop5, and *Pseudomonas fluorescens* FS1.
[0004] However, PAE pollution in the environment often presents in complex forms, and high molecular weight PAEs (such as DEHP) are more difficult to degrade due to their strong hydrophobicity and low bioavailability. Currently, highly efficient multi-substrate degrading strains are still few in number. Most strains struggle to maintain stable growth and metabolic activity under adverse conditions such as high concentrations of pollution, complex substrates, or high salinity, leading to unstable or unsustainable remediation effects. Furthermore, PAE pollution in marine environments is becoming increasingly prominent. Currently reported marine PAE-degrading bacteria mainly include a few strains such as *Gordonia sihwaniensis* RL-BY03 and *Mycolicibacterium umphocaicum* RL-HY01, and overall resources remain scarce.
[0005] The existing PAEs-degrading bacteria still have the following shortcomings: (1) The strains have limited environmental adaptability to PAEs: Most strains are derived from freshwater or soil, and their survival rate and metabolic activity are significantly reduced in seawater / sediment environments with high salinity, low nutrition or high concentration of compound pollution, making it difficult to directly apply them to marine ecological restoration. (2) The ability to degrade high molecular weight PAEs is insufficient: Due to the strong hydrophobicity and low bioavailability of high molecular weight PAEs represented by DEHP, the conversion efficiency of existing strains is generally low, which limits the synergistic restoration of compound pollution. (3) The sustainability and stability of the restoration process need to be improved: Some microorganisms can only survive for a short period of time or have their growth inhibited in the presence of PAEs, making it difficult to play a continuous role in the restoration cycle, thus affecting the overall restoration efficiency.
[0006] Aeromonas is a genus of Gram-negative bacilli widely distributed in freshwater, seawater, sediments, and aquatic organisms. Most are facultative anaerobes with strong adaptability to aquatic environments. This genus has a large genome (approximately 4-5 Mbp), diverse metabolic pathways, and can produce various extracellular enzymes and bioactive substances, demonstrating strong potential for degrading organic pollutants. Studies have found that Aeromonas exhibits good performance in degrading petroleum hydrocarbons, polycyclic aromatic hydrocarbons, dyes, and some pesticides. However, research on its degradation of PAEs, especially high molecular weight PAEs (such as DEHP), is still very limited, and there are no systematic reports of Aeromonas strains derived from marine environments specifically for the remediation of PAE pollution in seawater.
[0007] Therefore, screening marine aeromonas strains with good seawater adaptability and high efficiency in degrading high molecular weight PAEs (such as DEHP) is of great scientific significance and application value for promoting the bioremediation of PAE pollution in the marine environment. Summary of the Invention
[0008] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a marine intermediate aeromonas strain that degrades PAEs and its applications.
[0009] The objective of this invention is achieved through the following technical solution:
[0010] A marine aeromonas media strain that degrades PAEs, named Aeromonas media HJ02, was isolated and purified from coral reef sediments in the Beibu Gulf of Guangxi near Weizhou Island.
[0011] The preservation information of Aeromonas media HJ02 is as follows: depositary institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC), deposit date: August 8, 2025, deposit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, accession number: GDMCC NO: 66828.
[0012] The colony morphology of Aeromonas intermedius HJ02 is as follows: the cells are rod-shaped, smooth, and uniform in size, which conforms to the typical morphological characteristics of bacteria of the genus Aeromonas.
[0013] A biological agent comprising at least one of the above-mentioned Aeromonas intermedius HJ02, Aeromonas intermedius HJ02 bacterial solution, and Aeromonas intermedius HJ02 powder.
[0014] Furthermore, the Aeromonas intermediate HJ02 powder is obtained by centrifuging the Aeromonas intermediate HJ02 bacterial solution, collecting the bacterial cells, adding a protectant, mixing thoroughly, and then freeze-drying.
[0015] Preferably, the protective agent includes at least one of trehalose, sucrose, and glycerol.
[0016] Preferably, the mass ratio of the bacterial cells to the protectant is 1:1 to 4.
[0017] The application of the aforementioned Aeromonas intermediate HJ02 or biological agents in the degradation of PAEs and / or PAE intermediate metabolites.
[0018] Preferably, the PAEs include at least one of di(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), diethyl phthalate (DEP), and dimethyl phthalate (DMP);
[0019] Preferably, the intermediate metabolites of the PAEs include at least one of protocatechuic acid (PCA) and phthalic acid (PA).
[0020] The present invention also provides the application of the above-mentioned Aeromonas intermedius HJ02 or biological agent in the remediation of environmental media contaminated with PAEs and / or PAE intermediate metabolites.
[0021] Preferably, the environmental medium includes soil or water.
[0022] As a preferred embodiment, Aeromonas intermedius HJ02 or a biological agent is inoculated into soil contaminated with PAEs and / or PAE intermediate metabolites to degrade PAEs in the soil.
[0023] As a preferred embodiment, Aeromonas intermedius HJ02 or a biological agent is inoculated into water bodies contaminated with PAEs and / or PAE intermediate metabolites to degrade PAEs in the water.
[0024] Preferably, the degradation time is 12–120 h; more preferably 12–36 h.
[0025] Preferably, the degradation temperature is 28±2℃;
[0026] Preferably, the degradation rotation speed is 120-180 rpm; more preferably 150 rpm.
[0027] The present invention has the following advantages and effects compared with the prior art:
[0028] This invention isolates and purifies a strain of Aeromonas media, named Aeromonas media HJ02, from coral reef sediments in the Beibu Gulf of Guangxi, specifically targeting phthalic acid esters (PAEs). This strain exhibits significant advantages in environmental adaptability and PAE degradation activity, efficiently degrading various PAEs and their intermediate metabolites. It maintains favorable growth kinetics throughout a 0-120 h culture period. With an initial DEHP concentration of 300 mg / L, the strain significantly reduces DEHP residues in the culture system, achieving a 91.73% degradation rate after 24 h. This invention is simple, safe, and economical, providing a highly efficient and stable microbial resource for PAE remediation and possessing significant practical value. Therefore, this invention demonstrates superior stability and practical value in the bioremediation of PAE-contaminated environments, particularly those contaminated with DEHP. Attached Figure Description
[0029] Figure 1 This is a scanning electron microscope (SEM) image of strain HJ02.
[0030] Figure 2 This is a phylogenetic tree of strain HJ02 constructed based on the 16S rRNA gene sequence.
[0031] Figure 3 This is a graph showing the substrate profile analysis of bacteria HJ02 on different phthalic acid esters (PAEs) and their metabolic intermediates (300 mg / L, cultured at 28℃ and 150 rpm for 24 h).
[0032] Figure 4 This is a growth curve of Aeromonas intermedius HJ02 in 300 mg / L DEHP-MSM medium.
[0033] Figure 5 This is a graph showing the degradation effect of strain HJ02 on DEHP (300 mg / L). Detailed Implementation
[0034] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the materials and reagents used are commercially available.
[0035] Example 1: Obtaining and Identifying Strain HJ02
[0036] 1. Obtaining strain HJ02
[0037] Coral reef sediment samples were collected from the Weizhou Island area of the Beibu Gulf in Guangxi. Surface sediment samples were collected from the nearshore coral reef area at a depth of 0–10 cm below the sediment surface. Sterile sampling tools were used to collect the samples, which were then stored in sterile sampling bags or sterile centrifuge tubes and transported to the laboratory at 4°C. Strains were isolated within 24 hours.
[0038] Weigh 1.0 g of sediment sample and add it to 9 mL of sterile physiological saline (0.85% NaCl), shake thoroughly to mix, and prepare 10 -1 Diluent. Further perform 10-fold serial dilutions (10... -2 ~10 -6 ), 100 μL of samples at different dilutions were evenly spread on MSM solid medium plates and incubated at 28℃ for 24–72 h to obtain colonies of different morphologies.
[0039] Single colonies with different morphological characteristics were picked from the plates and subjected to multiple streak purification cultures until a pure strain with consistent morphology was obtained. The obtained pure strains were then inoculated into liquid culture medium for culture and preliminary screening was performed.
[0040] During the strain screening process, the obtained pure strains were inoculated into a screening medium using phthalate esters (PAEs) as the carbon source. This screening medium was an inorganic salt medium supplemented with 200 mg / L DEHP as the sole carbon source. The growth and pollutant removal capabilities of the strains in this culture system were observed, and strains with good growth capacity and degradation potential were further screened.
[0041] After multiple rounds of screening, a strain capable of stable growth and degradation in the presence of PAEs and their intermediate metabolites was obtained, designated as HJ02.
[0042] 2. Identification of strain HJ02
[0043] Morphological identification of strain HJ02: such as Figure 1 As shown, the bacteria observed by scanning electron microscopy (SEM) are rod-shaped, with smooth surfaces and uniform size, consistent with the typical morphological characteristics of bacteria in the genus *Bacillus*.
[0044] Physiological and biochemical characteristics of strain HJ02 were analyzed: the results are shown in Table 1.
[0045] Table 1. Physiological and biochemical characteristics of strain HJ02
[0046]
[0047] Molecular biological identification of strain HJ02: According to Bergey's Manual of Systematic Bacteria (9th Edition), and based on the 16S rDNA sequence of strain HJ02 (as shown in SEQ ID NO: 1), BLAST alignment was performed. The alignment results showed that strain HJ02 had the highest sequence similarity to strain Aeromonas media, with a sequence coverage of 100% and a sequence similarity of 99.86%. A phylogenetic tree was constructed using the 16S rDNA sequence of strain HJ02, and the results are as follows: Figure 2 As shown, strain HJ02 clustered with the type strain Aeromonas media into the same clade, exhibiting high phylogenetic similarity, indicating a clear taxonomic position.
[0048] In summary, based on the comprehensive morphological characteristics, physiological and biochemical characteristics, and 16S rDNA sequence analysis results, strain HJ02 is classified as *Aeromonas media*, and named *Aeromonas media HJ02*. Preservation information: Guangdong Provincial Microbial Culture Collection Center (GDMCC); deposit date: August 8, 2025; deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, Institute of Microbiology, Guangdong Academy of Sciences; accession number: GDMCC NO: 66828.
[0049] The 16S rDNA sequence of Aeromonas media HJ02 is shown in SEQ ID NO: 1:
[0050]
[0051] Example 2: Substrate profile analysis of Aeromonas intermedius HJ02
[0052] To evaluate the growth characteristics of strain HJ02 in the presence of PAEs and their intermediate metabolites, strain HJ02 was inoculated into culture systems containing different carbon sources. The carbon sources included: DMP, DBP, PA, PCA, DEHP, and DEP.
[0053] Aeromonas intermedius HJ02 was activated by streaking from preservation slant or glycerol preservation tubes and cultured at 28℃ for 24 h. Single colonies were picked and inoculated into liquid LB medium and cultured at 28℃ and 150 rpm with shaking for 12 h to obtain seed culture. The liquid LB medium consisted of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and pH 7.0–7.2.
[0054] Seed culture was inoculated into 100 mL Erlenmeyer flasks containing 20 mL of inorganic salt medium at a 1% (v / v) inoculation rate. The target compound was added to the medium as the sole carbon source, with a final concentration of 300 mg / L. The culture was incubated at 28 °C with shaking at 150 rpm. Odionation precipitates were measured every 6 hours over 120 h. 600 .
[0055] Using a blank control group (CK) as a reference, the growth of bacterial cells was measured after a certain period of culture, and the result was expressed as optical density OD. 600 The value represents the growth level of the strain, and the growth status of strain HJ02 was analyzed. The results are as follows: Figure 3 As shown, strain HJ02 was able to maintain growth after 24 h of culture under various PAEs and their intermediate metabolites. Among them, the strain showed a relatively high growth level under the conditions of PCA, PA, DBP and DEHP as carbon sources, indicating that the strain has a strong tolerance and potential utilization ability to PAEs and their key metabolic intermediates.
[0056] Furthermore, the growth curve of strain HJ02 in the presence of PAEs was analyzed. The results are as follows: Figure 4 As shown, during the 0-120 h culture period, the growth curve of strain HJ02 exhibited obvious adaptation, growth and stationary phases.
[0057] Example 3: Application of Aeromonas intermedius HJ02 in the degradation of DEHP
[0058] DEHP, which is widely used and poses a high risk of pollution in the environment, was selected as a representative PAE to verify the degradation ability of strain HJ02. The blank control group (CK) was used as a reference.
[0059] A single colony of HJ02 was inoculated into 20 mL of LB liquid medium and cultured at 28°C and 150 rpm for 12 h with shaking to obtain a seed culture. The seed culture was then inoculated at a 10% (v / v) inoculation rate into a 100 mL Erlenmeyer flask containing 20 mL of inorganic salt medium. DEHP was added to the medium to a final concentration of 300 mg / L. The flask was then cultured at 28°C and 150 rpm with shaking for 24 h. After culture, the supernatant was collected by centrifugation, extracted with an organic solvent, and the residual DEHP content was determined by GC-MS. The degradation rate was then calculated.
[0060] Strain HJ02 was inoculated into a culture system with an initial DEHP concentration of 300 mg / L. After culturing for a certain period under suitable temperature and oxygen conditions, the removal ratio was calculated by detecting the residual DEHP in the system. Figure 5 As shown, compared with the initial concentration, the DEHP content in the system decreased significantly after the culture was completed, and the DEHP degradation rate was 91.73%, indicating that the *Pseudomonas lipolyticis* HJ02 can effectively transform and remove DEHP.
[0061] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A marine aeromonas intermediate strain that degrades PAEs, characterized in that, The specimen, named Aeromonasmedia HJ02, was deposited on August 8, 2025, at the Guangdong Provincial Microbial Culture Collection Center, Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC NO: 66828.
2. A biological agent, characterized in that, It contains at least one of Aeromonas intermedius HJ02, Aeromonas intermedius HJ02 bacterial solution, and Aeromonas intermedius HJ02 powder as described in claim 1.
3. The use of Aeromonas intermedius as described in claim 1 or the biological agent as described in claim 2 in the degradation of PAEs and / or PAE intermediate metabolites.
4. The application of Aeromonas intermedius as described in claim 1 or the biological agent as described in claim 2 in the remediation of environmental media contaminated with PAEs and / or PAE intermediate metabolites.
5. The application according to claim 4, characterized in that: The environmental media mentioned include soil or water.
6. The application according to claim 5, characterized in that: Aeromonas intermediate or biological agents are inoculated into soil contaminated with PAEs and / or PAE intermediate metabolites to degrade PAEs in the soil.
7. The application according to claim 5, characterized in that: Aeromonas intermediate or biological agents are inoculated into water bodies contaminated with PAEs and / or PAE intermediate metabolites to degrade PAEs in the water.
8. The application according to claim 7, characterized in that: The degradation time is 12–120 h; And / or, the degradation temperature is 28±2℃; And / or, the degradation rotation speed is 120-180 rpm.
9. The application according to any one of claims 3 to 8, characterized in that: The PAEs include at least one of di(2-ethylhexyl) phthalate, dibutyl phthalate, diethyl phthalate, and dimethyl phthalate.
10. The application according to any one of claims 3 to 8, characterized in that: The intermediate metabolites of PAEs include at least one of protocatechuic acid and phthalic acid.