Low-temperature-resistant alkyl polycyclic aromatic hydrocarbon degrading bacterium and application thereof in remediation of combined polluted groundwater

Abstract

The invention relates to a low-temperature-resistant alkyl polycyclic aromatic hydrocarbon degrading bacterium and application of the low-temperature-resistant alkyl polycyclic aromatic hydrocarbon degrading bacterium in remediation of combined polluted groundwater, the low-temperature-resistant alkyl polycyclic aromatic hydrocarbon degrading bacterium is named as Novosphingobium sp. SAF and is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC M 20253008, and the preservation time is December 25, 2025. Compared with the prior art, the strain Novosphingobium sp. SAF can degrade alkyl polycyclic aromatic hydrocarbons such as methylnaphthalene, 1-methylphenanthrene and the like, non-alkyl polycyclic aromatic hydrocarbons such as naphthalene, phenanthrene, pyrene and the like, and heterocyclic aromatic hydrocarbons such as dibenzofuran and the like under the condition of 10-28 DEG C, has the performance of efficiently degrading various aromatic pollutants at low temperature, and is beneficial to solving the problems of tailing, rebound and the like in underground water remediation of petrochemical polluted sites.

Description

Technical Field

[0001] This invention belongs to the field of environmental pollution microbial remediation technology, and relates to a low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacterium and its application in the remediation of groundwater with complex pollution. Background Technology

[0002] Groundwater systems, as a crucial resource for maintaining ecological balance, are frequently threatened by non-aqueous liquid organic pollutants (NAPLs). NAPLs exist in the underground environment in the form of droplets or films within soil pores, exhibiting strong concealment and persistence. While physical extraction techniques can effectively remove free-phase NAPLs in existing contaminated site remediation, they are less effective at removing residual NAPLs bound by capillary forces, often exhibiting a long-term "tailing" phenomenon. After extraction ceases, pollutant concentrations tend to rebound rapidly. Alkylated polycyclic aromatic hydrocarbons (A-PAHs), as highly toxic and carcinogenic components of NAPLs, have become a key bottleneck restricting the achievement of remediation standards due to their continuous dissolution in groundwater. These problems mainly stem from the difficulty of fundamentally reducing residual pollution sources using physical methods. In recent years, in-situ bioremediation to degrade key pollutant components in residual NAPLs has been considered a priority for achieving green and sustainable groundwater remediation. The formation of A-PAHs is multi-source, originating not only from the natural combination of the parent polycyclic aromatic hydrocarbon (PAHs) and alkyl side chains in crude oil, but also more widely from the process of underground anaerobic microorganisms metabolizing petroleum hydrocarbons, which promotes the binding of alkyl side chains to the parent PAH structure. This leads to the continuous accumulation of A-PAHs in the contaminated system and their significant characteristic features. In petroleum-contaminated sites, the detection rate of A-PAHs is high, and their content can even account for more than 98% of the total PAH content. Among them, bicyclic and tricyclic A-PAHs, represented by methylnaphthalene and 1-methylphenanthrene, are the most widely distributed, and their abundance in groundwater NAPLs is often an order of magnitude higher than that of the corresponding non-alkyl PAHs.

[0003] Compared to the 16 PAHs prioritized for monitoring by the U.S. Environmental Protection Agency (EPA) (naphthalene, indo[1,2,3-cd]pyrene, acenaphthene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, benzo[ghi]perylene), A-PAHs exhibit stronger bioaccumulation and environmental persistence. Due to the introduction of alkyl substituents, the water solubility of these chemicals decreases, and their hydrophobicity increases, making them more prone to accumulation in organisms and thus inducing higher biotoxic effects. Studies show that methylphenanthrene is 2-5 times more effective than its non-alkylphenanthrene counterparts in activating human aryl hydrocarbon receptors (AhR), leading to a significant increase in the risks of genotoxicity and carcinogenicity. Furthermore, due to the oxygen-deficient underground environment and the stable chemical structure of A-PAHs, the natural degradation pathways of these pollutants are blocked, posing a long-term and severe challenge to groundwater ecological security.

[0004] However, current research on bioremediation of polycyclic aromatic hydrocarbons (PAHs) mainly focuses on non-alkyl PAHs (naphthalene, anthracene, fluoranthene, benzo[b]fluoranthene, and benzo[a]pyrene, for which limits have been clearly set in the Groundwater Quality Standard (GB / T 14848-2017), and naphthalene is also included in my country's Priority Controlled Chemicals List (First Batch). Research on efficient remediation of A-PAHs is relatively lacking, with only a few reports on A-PAH-degrading strains. However, these studies are often significantly affected by the growth environment, especially in practical scenarios such as low temperatures and complex pollution, where degradation is inhibited, resulting in a significant decrease in efficiency. For example, Sphingobium Although strain sp. MP9-4 has the ability to degrade 1-methylphenanthrene, its reaction conditions are strictly limited to a laboratory isothermal environment of 28°C, and it can only treat 9.5 mg / L of 1-methylphenanthrene within 4 days; while CN106190922A discloses... Novosphingobium Although *Sp. sp.* exhibits a degradation rate of 7.1 mg / L / d under ideal conditions at 28℃, according to the thermodynamic laws of microbial metabolism, its activity will decrease exponentially under actual low-temperature (20℃) and mixed pollution conditions in groundwater, and it is easily inhibited by substrate competition, leading to incomplete remediation. Currently, experiments generally focus on the optimal growth temperature of around 28℃, failing to reflect its degradation efficiency under common low-temperature environments in groundwater and soil. This disconnect between single pollutants in ambient temperature environments (28℃-30℃) and the actual scenarios of low temperatures (10℃-20℃) and mixed pollution in groundwater and deep soil often results in these strains losing their engineering application value in practical site remediation due to inhibited metabolic activity.

[0005] Based on this, CN102277312A disclosed a strain Novosphingobiumsp. can degrade various 2-3 ring aromatics in diesel fuel systems under low-temperature conditions. However, based on the characteristics of groundwater NAPLs pollution, residual pollutants not only include 2-3 ring aromatics but may also contain more complex components such as high-ring aromatics and heterocyclic aromatics. Compared to 2-3 ring aromatics, high-molecular-weight polycyclic aromatics with 4 rings or more have more stable molecular structures due to the increased number of aromatic rings, making them more difficult to undergo effective initial oxygenation and ring-opening reactions under low-temperature conditions. Heterocyclic aromatics, due to the introduction of heteroatoms such as oxygen, require not only the destruction of aromatic ring structures but also the breaking of key heteroatom phases during degradation, placing higher demands on the specificity and synergistic ability of the metabolic enzyme system, as disclosed in patent CN107881126A. Martelella sp. can efficiently degrade phenanthrene, but not pyrene. Currently, there is a lack of methods for degrading phenanthrene at low temperatures. Novosphingobium Reports indicate that *Sp. sp.* has degraded A-PAHs, non-alkyl PAHs, and heterocyclic PAHs. Therefore, developing strains capable of overcoming low-temperature environmental barriers and possessing broad-spectrum substrate degradation capabilities has become a critical technological bottleneck that urgently needs to be addressed in the remediation of NAPLs contaminated groundwater. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing A-PAHs degrading strains, such as narrow substrate spectrum and low temperature limitation, and to provide a low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacterium and its application in the remediation of groundwater with complex pollution.

[0007] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of this invention is to provide a low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacterium, named Novosphingobium sp. SAF, deposited at the China Center for Type Culture Collection, accession number CCTCC M20253008, on December 25, 2025.

[0008] The strain Novosphingobium sp. SAF was Gram-negative, lacked a capsule, and exhibited regular, round, yellow, opaque, and smooth colonies on Luria-Bertani medium. 16S rDNA sequencing was performed, and the sequence is shown in SEQ ID NO.1. BLAST alignment of the 16S rDNA sequence showed that the strain... Novosphingobium The nucleotide sequence of the 16S rDNA of *Sphingosine monophosphate* sp. SAF has greater than 99% homology with the nucleotide sequences of different strains of *Sphingosine monophosphate*. Therefore, the... Novosphingobium sp. SAF is a neosphingomonas.

[0009] NeosphingolipidsNovosphingobium The nucleotide sequence of the 16S rDNA of sp. SAF is shown in SEQ ID NO.1:

[0010] The second technical solution of the present invention is to provide an application of low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacteria as described in one of the above technical solutions in the remediation of groundwater with complex pollution.

[0011] In some specific embodiments, the low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacteria are used to degrade pollutants in situ in groundwater with complex pollution, including alkyl polycyclic aromatic hydrocarbons, non-alkyl polycyclic aromatic hydrocarbons, and heterocyclic aromatic hydrocarbons.

[0012] In some specific embodiments, the alkyl polycyclic aromatic hydrocarbon compound is selected from any one or more of methylnaphthalene, 1-methylphenanthrene, and 4-ethylbiphenyl.

[0013] In some specific embodiments, the non-alkyl polycyclic aromatic hydrocarbon compound is selected from any one or more of naphthalene, phenanthrene, and pyrene.

[0014] In some specific embodiments, the heterocyclic aromatic compound is dibenzofuran.

[0015] In some specific embodiments, the operating temperature of the low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacteria when degrading pollutants in in-situ in complex polluted groundwater is 10~28℃.

[0016] In some specific embodiments, the working pH of the low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacteria when degrading pollutants in in situ in groundwater with complex pollution is 6.0-9.0.

[0017] In some specific embodiments, the initial concentration of pollutants in the low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacteria when degrading pollutants in in-situ in groundwater with complex pollution is no greater than 80 mg / L.

[0018] The third technical solution of the present invention is to provide a product for degrading pollutants in groundwater with complex pollution, containing low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacteria as described in one of the above technical solutions, wherein the pollutants include alkyl polycyclic aromatic hydrocarbons, non-alkyl polycyclic aromatic hydrocarbons, and heterocyclic aromatic hydrocarbons.

[0019] In some specific embodiments, the alkyl polycyclic aromatic hydrocarbon compound is selected from any one or more of methylnaphthalene, 1-methylphenanthrene, and 4-ethylbiphenyl; The non-alkyl polycyclic aromatic hydrocarbons are selected from any one or more of naphthalene, phenanthrene, and pyrene; The heterocyclic aromatic compound is dibenzofuran.

[0020] In some specific embodiments, the product includes a microbial agent.

[0021] In some specific embodiments, the bacterial agent consists of low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacteria and a solvent, wherein the solvent may be physiological saline.

[0022] The fourth technical solution of the present invention provides a method for degrading pollutants in groundwater with complex pollution, comprising the following steps: The low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacteria described in one of the above technical solutions are inoculated into the complex polluted groundwater for degradation.

[0023] In some specific embodiments, the OD of the low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacteria in the composite polluted groundwater... 600nm The value is not less than 0.2.

[0024] In some specific implementations, the degradation temperature is 10~28℃, the pH is 6.0-9.0, and the initial concentration of pollutants is no more than 80mg / L.

[0025] Compared with the prior art, the present invention has the following advantages: (1) This invention involves extracting contaminated groundwater from a contaminated site in a petrochemical industrial park and screening out low-temperature alkyl polycyclic aromatic hydrocarbon degrading bacteria from it. Novosphingobium sp. SAF is capable of degrading alkyl polycyclic aromatic hydrocarbons such as methylnaphthalene and 1-methylphenanthrene, as well as non-alkyl polycyclic aromatic hydrocarbons such as naphthalene, phenanthrene, and pyrene, and heterocyclic aromatic hydrocarbons such as dibenzofuran, under conditions of 10-28℃ and pH 6-9. It has a broad substrate spectrum and strong degradation performance under low temperature conditions.

[0026] (2) The invention provides Novosphingobium sp. SAF can degrade 1-methylphenanthrene and methylnaphthalene, achieving the goal of removing complex pollutants under low-temperature groundwater conditions. It can open the ring of aromatics, avoid the metabolic accumulation of toxic intermediates, and can be applied to groundwater remediation. It can solve the tailing and rebound problems that exist when using physical extraction technology to treat groundwater with related pollutants. It has little negative impact on the environment and has good development and utilization prospects.

[0027] (3) The screening results obtained by this invention Novosphingobium sp. SAF maintained a degradation rate (6.67 mg / L / d) comparable to that of existing technologies under high-temperature conditions, even under harsh conditions of low temperature (20°C) and multiple substrates coexisting, while achieving the accumulation of non-toxic intermediate products. Attached Figure Description

[0028] Figure 1 Provided by the present invention Novosphingobium Colony morphology of sp. SAF (A) and scanning electron microscope (B); Figure 2 Provided by the present invention NovosphingobiumThe phylogenetic tree of sp. SAF; Figure 3 Provided by the present invention Novosphingobium Growth and degradation curves of sp. SAF at 20℃ for the degradation of 1-methylphenanthrene and methylnaphthalene; Figure 4 Provided by the present invention Novosphingobium Schematic diagram of the degradation rate of 1-methylphenanthrene and methylnaphthalene by sp. SAF at different temperatures; Figure 5 Provided by the present invention Novosphingobium Schematic diagram of the degradation rate of 1-methylphenanthrene and methylnaphthalene by sp. SAF at different pH values; Figure 6 Provided by the present invention Novosphingobium Schematic diagram of the degradation rates of 1-methylphenanthrene and methylnaphthalene by sp. SAF at different initial pollutant concentrations; Figure 7 Provided by the present invention Novosphingobium A schematic diagram illustrating the degradation effect of sp. SAF on naphthalene, phenanthrene, pyrene, dibenzofuran, and 4-ethylbiphenyl at 20℃; Figure 8 Provided by the present invention Novosphingobium Sp. SAF deduces the degradation pathways of two alkyl polycyclic aromatic hydrocarbons. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0031] Unless otherwise specified, the materials and processes described in the following embodiments or examples are conventional materials and processes used in the art to achieve the corresponding functions.

[0032] Example 1: This embodiment provides a highly efficient and stable methylnaphthalene and 1-methylphenanthrene degrading bacterium, which is *Sphingosine monocytogenes* (Neosphingosine monocytogenes). Novosphingobium sp.), strain named Novosphingobiumsp. SAF, this strain was Gram-negative, with a regular, round, yellow, opaque, and smooth morphology. 16S rDNA sequencing analysis was performed, and the obtained 16S rDNA sequence was BLAST-aligned. The alignment results showed that the strain... Novosphingobium The nucleotide sequence of the 16S rDNA of sp. SAF (GenBank accession number PX700584) is similar to that of *Sphingomonas* spp. ( Novosphingobium The nucleotide sequences of different strains (sp.) have greater than 99% homology.

[0033] Neosphingolipids Novosphingobium The nucleotide sequence of the 16S rDNA of sp. SAF is shown in SEQ ID NO.1:

[0034] The strain provided in this embodiment Novosphingobium sp. SAF is deposited at the China Center for Type Culture Collection, accession number CCTCC M 20253008, on December 25, 2025.

[0035] This embodiment also provides Novosphingobium One screening method for sp. SAF includes the following steps: (1) Add 0.5‰ (v / v) micronutrient mixture to basic inorganic salt culture medium to obtain SSDM liquid culture medium; The inorganic salt components in the basic inorganic salt culture medium include: 0.2 g / L NH4Cl, 7.95 g / L NaCl, 0.77 g / L MgCl2·6H2O, 1.05 g / L MgSO4·7H2O, 0.076 g / L CaCl2, 0.22 g / L KCl, 0.01 g / L NaHCO3, 0.026 g / L NaBr, and 0.25 g / L K2HPO4.

[0036] The solute components in the trace element mixture include: 0.15 g / L ZnSO4·7H2O, 0.26 g / L MnSO4·H2O, 0.03 g / L CoCl2·6H2O, 4.5 g / L FeSO4·7H2O, 0.02 g / L NiCl2·6H2O, 0.01 g / L CuCl2, 0.1 g / L Na2MoO4·2H2O, and 0.06 g / L H3BO3.

[0037] (2) A first mixed solution containing bacteria was prepared by adding 100 mL of SSDM liquid culture medium to groundwater contaminated by a chemical plant in Northeast China. The volume ratio of the contaminated groundwater to the SSDM liquid culture medium was 1:5. Then, 20 μL of methylnaphthalene (solvent N,N-dimethylformamide) at a concentration of 10 g / L and 200 μL of 1-methylphenanthrene (solvent N,N-dimethylformamide) at a concentration of 1 g / L were injected into the solution using a syringe. After 3-5 days in a constant temperature shaking incubator at 20℃, 10% of the first culture solution was transferred to another fresh SSDM liquid culture medium to obtain the second mixed solution. The solution was cultured and inoculated repeatedly under the same conditions until the nth mixed solution was obtained, where n=6. (3) Dilute and spread the obtained sixth mixture on LB solid culture plates, incubate in an incubator at 20°C for 5-7 days, and then streak on LB medium plates to separate the culture, thus obtaining alkyl and non-alkyl polycyclic aromatic hydrocarbon degrading bacteria.

[0038] The streak plating isolation process is as follows: Take 0.1 mL of the sixth mixture and add it to 0.9 mL of sterile SSDM medium, mix well, and the mixture is 10 mL. -1 Gradually diluted to 10 -2 10 -3 10 -4 10 -5 The culture was spread on LB agar plates, with three replicates for each gradient. Single colonies were then picked up with an inoculation loop and isolated from the LB agar plates. This process was repeated 2-3 times to obtain single colonies. The obtained single colonies were then inoculated into the degradation system to verify their ability to degrade 1-methylphenanthrene and methylnaphthalene. If the bacteria were observed to reduce the concentration of 1-methylphenanthrene and methylnaphthalene, they were identified as 1-methylphenanthrene and methylnaphthalene degrading bacteria.

[0039] The morphology of this single colony is as follows: Figure 1 As shown, the colonies are regular in shape, round, yellow, opaque and smooth.

[0040] The 16S rDNA sequence obtained was subjected to BLAST alignment. The alignment results showed that the nucleotide sequence of the strain's 16S rDNA was similar to that of *Neosphingosporidium* spp. Novosphingobium The nucleotide sequences of different strains (sp.) have greater than 99% homology.

[0041] Figure 2 The strain was displayed Novosphingobium The phylogenetic relationship between sp. SAF and other bacteria in the genus *Neosphomonas*, and ( Novosphingobium sp. B2580 (ON077593.1) is on the same branch of the phylogenetic tree, indicating that they are closely related.

[0042] Example 2 This embodiment provides Novosphingobium Study on the degradation ability of sp. SAF for 1-methylphenanthrene and methylnaphthalene 20 mL of SSDM liquid medium containing methylnaphthalene and 1-methylphenanthrene (concentration ratio 1:1) at a total concentration of 20 mg / L was placed in a 150 mL serum bottle, and the bacterial strain was inoculated. Novosphingobium After sp. SAF, the strains in this degradation system Novosphingobium sp. SAF initial content OD 600nm The value was 0.2. To prevent methylnaphthalene from evaporating, the container was sealed and incubated at 20°C, 150 rpm, and in the dark with shaking. Samples were taken periodically to determine the residual concentration of the contaminant.

[0043] Figure 3 The display shows that at a temperature of 20℃, Novosphingobiumsp. SAF can completely degrade 1-methylphenanthrene and methylnaphthalene with an initial mixed concentration of 20 mg / L within 3 to 4 days, indicating that it can adapt to actual groundwater temperature and maintain its degradation activity against alkyl polycyclic aromatic hydrocarbons.

[0044] Example 3 This embodiment provides Novosphingobium Degradation conditions of sp. SAF Will Novosphingobium After sp. SAF was expanded in LB medium, it was centrifuged at 10000 r / min for 5 min, resuspended in 0.9% physiological saline, and washed to remove yeast extract and other carbon sources. This process was repeated twice without changing the total volume. The absorbance was measured at 600 nm. The sample was then added to a 150 mL serum bottle containing 20 mL of SSDM medium containing methylnaphthalene and 1-methylphenanthrene. The degradation system of the strain... Novosphingobium sp. SAF initial content OD 600nm The value was 0.2. To prevent methylnaphthalene from volatilizing, the container was sealed and incubated at 150 rpm in the dark. Temperature, pH, and initial contaminant concentration were varied. Samples were taken after 96 h, and the residual concentration was determined by liquid chromatography.

[0045] 1. Different temperatures Degradation conditions: pH 7.0, total concentration of 20 mg / L of methylnaphthalene and 1-methylphenanthrene (concentration ratio 1:1); temperature: 10℃, 15℃, 20℃, 28℃; The results show that: Figure 4 The different temperatures of aromatic hydrocarbon degrading strains provided in this invention Novosphingobium A schematic diagram illustrating the degradation rate of alkyl polycyclic aromatic hydrocarbons by sp. SAF. Within the experimentally set temperature range, the strain... Novosphingobium The degradation efficiency of *Sp. SAF* exhibits a clear temperature response characteristic: when the temperature is in the range of 15℃ to 28℃, it shows extremely strong stability in the degradation of methylnaphthalene, with degradation rates exceeding 99.0%; simultaneously, its degradation ability for 1-methylphenanthrene is also at a high level, with degradation rates exceeding 77%. This temperature range shows high compatibility with the temperature ranges of most contaminated sites in the natural environment. At a low temperature of 10℃, the strain... Novosphingobium sp. SAF still exhibits certain degradation activity: the degradation rate of methylnaphthalene can reach 55.2%~59.6%, and the degradation rate of 1-methylphenanthrene is about 20%, which breaks through the limitation of insufficient low-temperature activity of most degrading bacteria and demonstrates excellent adaptability to low-temperature environment.

[0046] 2. Different pH values: Degradation conditions: Total concentration of 20 mg / L methylnaphthalene and 1-methylphenanthrene (concentration ratio 1:1); Temperature: 20℃; pH: 6.0, 7.0, 8.0, 9.0; The results show that: Figure 5 The different pH values ​​of the aromatic hydrocarbon degrading strains provided in this invention Novosphingobium Schematic diagram of the degradation rate of alkyl polycyclic aromatic hydrocarbons by sp. SAF. (Strain) Novosphingobium sp. SAF exhibits broad pH tolerance: it maintains high efficiency in degrading methylnaphthalene and 1-methylphenanthrene in weakly acidic, neutral, and weakly alkaline environments ranging from pH 6.0 to 9.0. Specifically, in a weakly acidic environment of pH 6.0 or a neutral environment of pH 7.0, the strain... Novosphingobium sp.SAF has a 100% degradation rate for methylnaphthalene and a degradation rate of over 98.5% for 1-methylphenanthrene. In a weakly alkaline environment with pH 8.0 or 9.0, its degradation rate for both pollutants remains at 100%. This characteristic makes it suitable for most polluted water pH ranges, making its application more flexible.

[0047] 3. Different initial pollutant concentrations Degradation conditions: pH 7.0; temperature: 20℃; 20 / 40 / 60 / 80 mg / L of methylnaphthalene and 1-methylphenanthrene (concentration ratio 1:1); The results show that: Figure 6 The different initial contaminant concentrations provided in this invention affect the strains Novosphingobium A schematic diagram illustrating the degradation rate of alkyl polycyclic aromatic hydrocarbons by sp. SAF. Under a total concentration of 20 mg / L, the strain... Novosphingobium sp. SAF exhibited a near 100% degradation rate for both pollutants, demonstrating extremely strong pollutant removal capabilities; even at a concentration increased to 40 mg / L, the strain... Novosphingobium sp. SAF maintained a near 100% degradation rate of methylnaphthalene and approximately 70% degradation of 1-methylphenanthrene. With further increases in concentration (60–80 mg / L), although the inhibitory effect of the substrate on degradation gradually became apparent, the strain… Novosphingobium sp. SAF maintained a degradation rate of over 28% for methylnaphthalene, which was superior to the degradation performance of 1-methylphenanthrene during the same period. Overall, this strain... Novosphingobium sp. SAF not only has a high efficiency in degradation at low concentrations, but also exhibits stronger tolerance and degradation advantages for methylnaphthalene, making it suitable for the remediation of residual NAPLs in groundwater.

[0048] Example 4 This embodiment also provides strains Novosphingobium The degradation effect of sp. SAF on other polycyclic aromatic hydrocarbons. To investigate the effect of the strain... NovosphingobiumThe ability of sp. SAF to degrade a broad substrate spectrum will allow the strain to... Novosphingobium The bacterial suspensions prepared from *Sp. SAF* were added to a basic inorganic salt medium containing 10 mg / L naphthalene, phenanthrene, pyrene, dibenzofuran, and 4-ethylbiphenyl. Novosphingobium sp. SAF initial OD 600nm All values ​​were 0.2, and the initial conditions were set as follows: pH 7.0, temperature 20°C, and shaker speed 150 rpm. On day 4, residual pollutants were extracted from the degradation system with ethyl acetate, and the residual concentration was determined by liquid chromatography.

[0049] The concentration of the mother liquor for naphthalene, phenanthrene, methylnaphthalene, pyrene, 4-ethylbiphenyl, and dibenzofuran was 10 g / L, and the solvent was N,N-dimethylformamide. 20 μL of the contaminant mother liquor was added to every 20 mL SSDM system. The results showed that... Figure 7 The aromatic hydrocarbon degrading strain provided in this invention Novosphingobium A schematic diagram illustrating the degradation rate of polycyclic aromatic hydrocarbons in sp. SAF. Novosphingobium sp. SAF showed degradation effects on five polycyclic aromatic hydrocarbons within 4 days at 20℃. Among them, Novosphingobium sp. SAF can completely degrade four substrates—naphthalene, phenanthrene, dibenzofuran, and 4-ethylbiphenyl—at a concentration of 10 mg / L, achieving a degradation rate of 100%. For pyrene, a high-cyclic aromatic hydrocarbon, the degradation rate also reaches approximately 38% after 4 days, indicating that the strain... Novosphingobium sp. SAF has a broad substrate degradation spectrum, capable of degrading not only alkyl polycyclic aromatic hydrocarbons (PAHs), but also 2-4 ring PAHs without substituents, and oxygen-containing heterocyclic aromatic hydrocarbons. CN106190922A discloses a 1-methylphenanthrene-degrading bacterium. Novosphingobium sp. 1MP25 has not been reported to degrade multiple pollutants at a low temperature of 20°C.

[0050] Example 5 This embodiment provides strains Novosphingobium Studies on the metabolic pathways of sp. SAF for 1-methylphenanthrene and methylnaphthalene Extraction was performed using ethyl acetate as the extractant under neutral and acidic conditions (pH=4~5). Novosphingobium Sp. SAF-degraded SSDM medium was extracted 2-3 times. The extracts were combined, dried with anhydrous sodium sulfate, and rotary evaporated to near dryness. The extracts were redissolved in acetonitrile, and derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide at a volume ratio of 5:3. After filtration through a 0.22 μm organic filter, the extracts were derivatized at 60 °C for 1 h. Qualitative analysis was performed using GC-MS / MS (TQ8050, Shimadzu, Japan).

[0051] The mass spectrometry data of the intermediate product were compared with the standard NIST 2020 library data for single-strain analysis. Novosphingobium The degradation pathway of 1-methylphenanthrene and methylnaphthalene by sp. SAF is a process of side chain / cyclic hydroxylation-ring-carboxylation. For example... Figure 8 As shown, when 1-methylphenanthrene is used as a substrate, the degradation pathway may involve oxidation of the methyl group followed by oxidative decarboxylation, leading to the detection of monocyclic hydroxycarboxylic acids. Alternatively, a dihydroxy group may be introduced into the phenanthrene ring via dioxygenase, followed by ring-opening enzymes to dismantle the polycyclic structure, resulting in the detection of 5-methyl-1,2-dinaphthol and 3-methylsalicylic acid. When methylnaphthalene is used as a substrate, methyl-substituted salicylic acid and salicylic acid without methyl substituents are detected, suggesting that the degradation process involves the introduction of a dihydroxy group into the naphthalene ring via dioxygenase or the introduction of a hydroxyl group into the naphthalene ring via monooxygenase, followed by ring-opening and oxidation reactions to convert the product into a small molecule carboxylic acid.

[0052] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacterium, characterized in that, Named Novosphingobium sp. SAF, deposited at the China Center for Type Culture Collection, accession number CCTCC M 20253008, on December 25, 2025.

2. The application of the low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacteria as described in claim 1 in the remediation of groundwater with complex pollution.

3. The application according to claim 2, characterized in that, The low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacteria are used to degrade pollutants in situ in groundwater with complex pollution, including alkyl polycyclic aromatic hydrocarbons, non-alkyl polycyclic aromatic hydrocarbons, and heterocyclic aromatic hydrocarbons.

4. The application according to claim 3, characterized in that, The alkyl polycyclic aromatic hydrocarbon compound is selected from any one or more of methylnaphthalene, 1-methylphenanthrene, and 4-ethylbiphenyl; The non-alkyl polycyclic aromatic hydrocarbons are selected from any one or more of naphthalene, phenanthrene, and pyrene; The heterocyclic aromatic compound is dibenzofuran.

5. The application according to claim 3, characterized in that, The operating temperature of the low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacteria when degrading pollutants in in-situ in complex-polluted groundwater is 10~28℃.

6. The application according to claim 3, characterized in that, The working pH of the low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacteria when degrading pollutants in in-situ in complex-contaminated groundwater is 6.0-9.

0.

7. The application according to claim 3, characterized in that, The initial concentration of pollutants in the in-situ degrading groundwater containing complex pollutants by the low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacteria is no greater than 80 mg / L.

8. A product for degrading pollutants in complexly polluted groundwater, characterized in that, It contains the low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacteria as described in claim 1, wherein the pollutants include alkyl polycyclic aromatic hydrocarbons, non-alkyl polycyclic aromatic hydrocarbons, and heterocyclic aromatic hydrocarbons.

9. A method for degrading pollutants in groundwater with complex contamination, characterized in that, Includes the following steps: The low-temperature resistant alkyl polycyclic aromatic hydrocarbon degrading bacteria as described in claim 1 were inoculated into the complex polluted groundwater for degradation.

10. The method according to claim 9, characterized in that, The OD of low-temperature alkyl polycyclic aromatic hydrocarbon degrading bacteria in the compound-polluted groundwater 600nm The value should not be lower than 0.2, the degradation temperature should be 10~28℃, the pH should be 6.0-9.0, and the initial concentration of pollutants should not be greater than 80 mg / L.

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