Decoloring novel sphingomonas with the functions of degrading petroleum and fixing nitrogen and application thereof

Abstract

The present application relates to a strain of decoloring novosphingobium with the functions of degrading petroleum and nitrogen fixation, and application thereof, wherein the decoloring novosphingobium is Novosphingobium decolorationis DG17-1, which is preserved in the China General Microbiological Culture Collection Center on December 12, 2025, with a preservation number of CGMCC No. 37061. The strain DG17-1 can efficiently degrade linear alkanes and aromatic hydrocarbons of C 12 ‑C 37 under an oxygen concentration of 0-21%. The strain DG17-1 couples nitrogen fixation and petroleum hydrocarbon degradation together, improves the growth amount through biological nitrogen fixation, realizes the degradation of broad-spectrum petroleum hydrocarbon components (especially fluorene), and has great application potential in the field of bioremediation of petroleum hydrocarbon contaminated environment.

Description

Technical Field

[0001] This invention belongs to the field of microbial and environmental engineering technology, specifically relating to a decolorizing neosphingosine bacterium with petroleum degradation and nitrogen fixation functions and its applications. Background Technology

[0002] Petroleum, as one of the most important energy sources in modern society, supports the development of various sectors such as industry, transportation, and agriculture. At the same time, my country's oil and gas fields and refining enterprises produce more than 800 million tons of oily sludge annually. 6 With oil sludge exceeding 20% ​​oil content, resource recovery and harmless treatment have become urgent priorities. Physicochemical treatment is economically feasible when the oil content of the sludge exceeds 20%. Among these methods, thermochemical washing is widely used and has achieved large-scale treatment and resource utilization of oil sludge, but problems remain such as incomplete recovery of petroleum hydrocarbons and high environmental risks from residual solids. Research shows that chemical cleaning of oily sludge from Daqing Oilfield No. 1, at a cleaning temperature of 78.68℃ and a chemical agent concentration of 0.84 g / L, is effective. -1 When the liquid-to-solid ratio is 9.40:1, the residual oil content in the sediment is still 3.96%. To achieve harmless treatment, microbial remediation technology, due to its economic, efficient, and environmentally friendly advantages, has become an ideal combined remediation method.

[0003] The microbial community structure in oil sludge undergoes alteration after thermal washing, with only microorganisms capable of tolerating extreme environments able to adapt and survive. Simultaneously, the large-scale introduction of petroleum leads to a severe imbalance in the carbon-to-nitrogen ratio of the oil sludge, making nitrogen a key factor limiting microbial growth, metabolism, and degradation efficiency. Nitrogen-fixing bacteria can convert molecular nitrogen into ammonia, thus becoming important "growth factors" for regulating nitrogen balance, promoting the growth of other microorganisms, and maintaining community function and stability. Therefore, targeted acquisition of dominant nitrogen-fixing and petroleum hydrocarbon-degrading bacteria with in-situ adaptability to this environment will be a key strategy for rapidly adapting to extreme environments, simultaneously alleviating nitrogen limitation, and degrading petroleum hydrocarbons, thereby improving the adaptability, functional stability, and remediation efficiency of bioremediation agents.

[0004] Novosphingobium is a type of Gram-negative bacterium capable of degrading various aromatic compounds, making it an excellent bioremediation agent for aromatic hydrocarbon-contaminated environments. CN102277312A discloses a low-temperature polycyclic aromatic hydrocarbon (PAH) degrading strain and its application in the bioremediation of groundwater at petroleum hydrocarbon-contaminated sites. CN112501080B discloses the application of *Novosphingobium clavatum* in the preparation of microbial agents for removing COD from petroleum wastewater.

[0005] Seventy-five validly published species of *Novosphingobium* have been reported. Genome-wide analysis revealed that only nine species had the *nifH* gene annotated, and three of these species were reported to possess nitrogenase activity, indicating that the nitrogen-fixing characteristics of the *Novosphingobium* genus are species-specific. Currently, no *Novosphingobium* species with dual functions of nitrogen fixation and petroleum hydrocarbon degradation have been reported. Summary of the Invention

[0006] To better explore the efficacy of *Sphingosine monocytogenes*, address environmental pollution caused by petroleum, and improve the efficiency of biological nitrogen fixation, this invention provides the following technical solution.

[0007] In a first aspect, the present invention provides a decolorizing neosphingobium strain with functions of petroleum degradation and nitrogen fixation. The decolorizing neosphingobium strain is (Novosphingobium decolorationis) DG17-1, which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No.37061 and deposit date of December 12, 2025.

[0008] The decolorizing neosphingomonas DG17-1, isolated from oily mud in Dagang, Tianjin, possesses dual functions of nitrogen fixation and petroleum hydrocarbon degradation, and can degrade C. 12 ~C 37 It contains straight-chain alkanes and 17 aromatic hydrocarbons. The decolorizing neosphingoblast DG17-1 exhibits high nitrogenase activity in environments with oxygen concentrations ranging from 0% to 15%.

[0009] In a second aspect, the present invention provides a microbial agent comprising the decolorizing neosphingosine bacterium described in the first aspect.

[0010] Preferably, the microbial agent is a nitrogen-fixing microbial fertilizer or a petroleum degradation agent.

[0011] Furthermore, the microbial agent also includes excipients.

[0012] Thirdly, the present invention provides a method for culturing the decolorized neosphingosine bacteria described in the first aspect, wherein the oxygen concentration in the culture environment of the decolorized neosphingosine bacteria is 0-15%, for example: 0%, 2%, 4%, 6%, 8%, 10%, 12%, 15%.

[0013] Preferably, the petroleum content in the culture medium of the decolorized neosphingolipid bacteria is ≤5%, for example: 1%, 2%, 3%, 4%, 5%.

[0014] Furthermore, the surfactant content in the culture medium is 0-2%, for example: 0%, 1%, 2%.

[0015] Furthermore, the surfactants include, but are not limited to, Tween-80 and rhamnolipid.

[0016] Furthermore, the culture medium includes, but is not limited to, LB medium, nitrogen-free basal medium, and nitrogen-containing basal medium.

[0017] Furthermore, the formulation of the nitrogen-free basal culture medium is as follows: sodium pyruvate 12.22 g, K2HPO4 1.0 g, MgSO4·7H2O 0.2 g, FeSO4·7H2O 0.05 g, NaMoO4·2H2O 0.001 g, H2O 1 L, pH 7.0.

[0018] Furthermore, the formulation of the nitrogen-based basal medium is as follows: sodium pyruvate 12.22 g, K2HPO4 1.0 g, MgSO4·7H2O 0.2 g, FeSO4·7H2O 0.05 g, NaMoO4·2H2O 0.001 g, NH4SO4 2 g, H2O 1 L, pH 7.0.

[0019] Furthermore, the culture temperature of the decolorized neosphingosine bacteria is 25~35℃, for example: 25℃, 28℃, 30℃, 33℃, 35℃.

[0020] Fourthly, the present invention provides the application of the decolorizing neosphingosine bacteria described in the first aspect or the bacterial agent described in the second aspect in the degradation of petroleum.

[0021] Preferably, the conditions for degrading petroleum are either nitrogen-free or nitrogen-containing.

[0022] Preferably, the petroleum contains C 12 -C 37 Straight-chain alkanes and aromatic hydrocarbons.

[0023] Furthermore, the aromatic hydrocarbon comprises one or more of the following: naphthalene, 2-methylnaphthalene, 1-methylnaphthalene, acenaphthene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthene, chrysoprase, benzo[b / k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthene, indo[1,2,3,cd]pyrene, or benzo[g,h,i]perylene.

[0024] Fifthly, the present invention provides the application of the decolorizing neosphingosine bacteria described in the first aspect or the bacterial agent described in the second aspect in the preparation of nitrogen-fixing microbial fertilizers.

[0025] The beneficial effects of this invention are: The decolorizing neosphingobium decolorationis DG17-1 of this invention can couple nitrogen fixation and petroleum hydrocarbon degradation together, increasing growth through biological nitrogen fixation and achieving the degradation of a broad spectrum of petroleum hydrocarbon components (especially fluorene), which has great application potential in the field of bioremediation of petroleum hydrocarbon-polluted environments. Attached Figure Description

[0026] Figure 1 The figure shows the changes in microbial community structure and abundance of the microbial community containing strain DG17-1 in samples at different time periods. Figure 2 The figure shows the nitrogenase activity of strain DG17-1 under different carbon source treatments; Figure 3 The figure shows the nitrogenase activity of strain DG17-1 after culturing for 48 h at different oxygen concentrations; Figure 4 The image shows the effect of strain DG17-1 on hydrocarbons (fluorene, C). 16 C 28 The degradation rate of naphthalene.

[0027] Culture preservation for patent procedures: The decolorizing neosphingobium is (Novosphingobium decolorationis) DG17-1, with accession number CGMCC NO.37061 and accession date of December 12, 2025.

[0028] Preservation institution: China General Microbiological Culture Collection Center (CGMCC).

[0029] Address of the depository: No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing, China, 100101, China. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to embodiments and accompanying drawings. The advantages and features of the present invention will become clearer as the description unfolds. However, it should be understood that the embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention.

[0031] It should be noted that, unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art. Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0032] The culture medium involved in this invention: R2A medium: yeast extract 0.5 g; glucose 0.5 g; acid-hydrolyzed casein 0.5 g; soluble starch 0.5 g; tryptone 0.5 g; sodium pyruvate 0.3 g; K2HPO4 0.3 g; MgSO4·7H2O 0.05 g; H2O 1 L; pH 7.0.

[0033] LB medium: NaCl 10 g; tryptone 10 g; yeast extract 5 g; H2O 1 L; pH 7.0.

[0034] Nitrogen-free basal medium: Sodium pyruvate 12.22 g; K2HPO4 1.0 g; MgSO4·7H2O 0.2 g; FeSO4·7H2O 0.05 g; NaMoO4·2H2O 0.001 g; H2O 1 L; pH 7.0.

[0035] Nitrogen-containing basal medium: Sodium pyruvate 12.22 g; K2HPO4 1.0 g; MgSO4·7H2O 0.2 g; FeSO4·7H2O 0.05 g; NaMoO4·2H2O 0.001 g; NH4SO4 2 g; H2O 1 L; pH 7.0.

[0036] Petroleum degradation medium: 2% petroleum is added to LB medium; for the group with added petroleum emulsifier Tween-80, an additional 2% Tween-80 is added.

[0037] Petroleum degradation medium (nitrogen-free): 2% petroleum is added to a nitrogen-free basal medium.

[0038] Petroleum degradation medium (nitrogen-containing): 2% petroleum is added to a nitrogen-containing basal medium.

[0039] Example 1: Strain Screening and Identification Oil-contaminated soil samples were collected from near the Dagang Oilfield in Tianjin. After thermal elution pretreatment, the oil content was 42,400 mg / kg. The samples were enriched and isolated using a nitrogen-free semi-solid medium to obtain strain DG17-1. 16S rRNA gene sequencing confirmed that it shared 100% homology with the standard strain of *Novosphingobium decolorationis*, KCTC82134=MCCC 1K04799 (Chen X, Dong B, Chen T, et al. *Novosphingobium decolorationis* sp. nov., an aniline blue-decolourizing bacterium isolated from East Pacific sediment. Int J Syst Evol Microbiol 2021,71:5001.). The strain DG17-1 was identified as *Novosphingobium decolorationis* and named *Novosphingobium decolorationis* DG17-1.

[0040] The 16S rRNA sequence of *Novosphingobium decolorationis* DG17-1 is as follows:

[0041] Example 2: Nitrogenase Activity Analysis of Strain DG17-1 1. Nitrogenase assay conditions: (1) Select strain DG17-1 and inoculate it into R2A solid medium, activating it twice at 30℃. Transfer it to liquid medium and incubate at 30℃ and 200 rpm for 14 hours. Harvest the bacteria, wash the cells with sterile water, and centrifuge. Wash the cells with nitrogen-free medium to adjust the initial OD. 600 =0.2, inoculate 5 mL into a 15 mL anaerobic tube, and incubate at 30°C.

[0042] (2) After replacing the gas above the anaerobic tube with nitrogen, and adjusting the oxygen concentration, inject 10% acetylene (or directly inject 10% acetylene for nitrogen-free semi-solid culture). After culturing for a period of time, take 100 μL of gas sample and determine the ethylene content in the gas using gas chromatography (SQ-204 gas chromatograph, FID detector, 2-meter packed column, filled with GDX502).

[0043] SQ-204 Gas Chromatography Determination Conditions: Carrier: N2; Amplifier range: 10-11 A / mV; Column temperature: 70℃; Detector temperature: 150℃.

[0044] Ethylene content in the sample (nmol / hr.OD) 600 = (Sample ethylene peak area * Sample gas volume (mL)) / 1 nmol ethylene gas standard peak area * 0.1 * hr * OD 600 (3) Calibration of the standard peak area of ​​ethylene Calculating the volume of 1 nmol of gas under the experimental conditions using the formula PV=nRT, at a room temperature of 23℃ and an atmospheric pressure of 762 mmHg, the volume of 1 mol of gas is 22.21 mL, and the volume of 1 nmol of gas is 22.2 × 10⁻¹⁰. -6 mL. Two 100 mL reaction flasks filled with water are capped with rubber stoppers (the reaction is carried out underwater to ensure no air is inside). 100 mL of water is expelled by gas, resulting in a gas volume of 100 mL in each flask. Then, 2.22 mL of gas is taken from reaction flask 1 and 1 mL of gas is drawn from reaction flask 2. 2.22 mL of gas is taken from an ethylene standard gas bag and injected into reaction flask 1. After mixing, 1 mL of the mixed gas is injected into reaction flask 2. This 100 mL gas contains 0.0222 mL of ethylene. 100 μL of this mixture is then analyzed by gas chromatography; its peak area is the standard peak area of ​​1 nmol of ethylene gas.

[0045] 2. Characteristics of nitrogenase under different carbon source enrichment conditions Using oily sludge from Dagang, Tianjin as a sample, the sample stored for 15 days was diluted, enriched and cultured in a nitrogen-free semi-solid medium, and nitrogenase activity was determined using sodium pyruvate and hexadecane as the sole carbon sources, respectively.

[0046] The results showed that strain DG17-1 was enriched in both sodium pyruvate and hexadecane treatment groups. Only strain DG17-1 was enriched with hexadecane as the sole carbon source, indicating that DG17-1 has high potential in nitrogen fixation and promoting petroleum hydrocarbon degradation.

[0047] 3. Analysis of nitrogen fixation capacity of strain DG17-1 under different carbon sources and oxygen concentrations (1) Analysis of nitrogen fixation capacity of strain DG17- under different carbon sources Following the nitrogenase assay method, after activating the DG17-1 strain, it was inoculated into semi-solid nitrogen-free medium containing the same number of carbon moles in different carbon sources (sodium pyruvate, mannose, and glucose), and cultured statically at 30°C for 7–14 days. After the appearance of a clear growth zone, the ethylene content was measured.

[0048] like Figure 2 As shown, strain DG17-1 exhibits the highest nitrogenase activity when sodium pyruvate is used as the carbon source.

[0049] (2) Analysis of nitrogen fixation capacity of strain DG17-1 under different oxygen concentrations Following the method for nitrogenase activity assay, strain DG17-1 was activated and inoculated into a liquid nitrogen-free medium with sodium pyruvate as the carbon source. Different oxygen concentrations (0%, 5%, 10%, 15%, 21%) were set in the anaerobic tubes, and the culture conditions were set at 30℃ and 200 rpm for 48 h. Samples were then taken to determine nitrogenase activity.

[0050] like Figure 3 As shown, strain DG17-1 exhibits nitrogenase activity at oxygen concentrations ranging from 0% to 21%, with the nitrogenase activity decreasing in the following order: 5%, 10%, 0%, 15%, and 21%. This indicates that strain DG17-1 has strong oxygen adaptability and can retain nitrogen-fixing ability over a wide range of oxygen concentrations, demonstrating its potential value in biological nitrogen fixation.

[0051] Example 3: Determination of petroleum degradation ability of strain DG17-1 1. The petroleum degradation ability of strain DG17-1 was analyzed by gas chromatography (GC).

[0052] The activated strain DG17-1 was inoculated into LB medium supplemented with 2% petroleum (naphthalene concentration of 125 mg / mL and phenanthrene concentration of 56 mg / mL) and cultured at 30°C and 200 rpm for 15 days until the petroleum emulsified. 5 mL of n-hexane was added to the culture flask, and after thorough shaking, the entire mixture was transferred to a 250 mL separatory funnel. The mixture was shaken thoroughly, and after standing to separate the layers, the lower layer was discarded. The upper hexane extract was transferred to a clean glass petri dish, and the culture flask was rinsed with a small amount of n-hexane. The liquid was then poured into the separatory funnel. This process was repeated until all the petroleum in the flask and separatory funnel was extracted.

[0053] Transfer the petroleum extract from the petri dish to a 50 mL centrifuge tube, taking care to avoid aspirating impurities, and bring the volume to 10 mL with n-hexane. Transfer 1 mL to an Agilent sample vial and test the alkane content. Simultaneously, transfer another 1 mL to an Agilent sample vial, allow it to evaporate in a fume hood until the volume remains constant, dissolve it in 1 mL of a 1:1 benzene:dichloromethane solvent, and test the aromatic hydrocarbon content.

[0054] The conditions and procedures for gas chromatography (GC) determination of alkanes and aromatic hydrocarbons are as follows: GC conditions: HP-5MS column (30 m × 0.25 mm × 0.25 μm), N2 as carrier gas, hydrogen 30 mL / min, air 250 mL / min, nitrogen make-up flow rate 30 mL / min.

[0055] Alkane program: Inlet temperature 300℃, FID detector temperature 250℃, splitless. Heating conditions: Initial temperature 50℃, hold for 5 min, increase to 230℃ at 40℃ / min, increase to 320℃ at 20℃ / min, hold for 20 min (auto-inject 1 μL).

[0056] Aromatic hydrocarbon program: Inlet temperature 250℃, FID detector temperature 300℃, splitless. Heating conditions: Initial temperature 40℃, hold for 2 min, increase to 100℃ at 10℃ / min, increase to 250℃ at 15℃ / min, increase to 345℃ at 20℃ / min, hold for 3.25 min (auto-inject 1 μL).

[0057] Table 1. Alkane degradation rate of strain DG17-1

[0058] Table 2. Aromatic hydrocarbon degradation rate of strain DG17-1

[0059] As can be seen from Tables 1 and 2, strain DG17-1 is effective against C 12 ~C37 The degradation rates of straight-chain alkanes and aromatic hydrocarbons were 20.2–43.6% and 34.7–87.5%, respectively, with the latter exhibiting a higher degradation capacity for aromatic hydrocarbons. In the treatment group with the addition of emulsifier Tween-80, the petroleum degradation rate of strain DG17-1 was significantly increased, as was its degradation of C... 12 ~C 37 The degradation rates of straight-chain alkanes and aromatic hydrocarbons were 48.2–100% and 46.7–96.3%, respectively, indicating that the addition of emulsifiers increased the contact area between strain DG17-1 and petroleum droplets, thereby improving petroleum utilization efficiency.

[0060] The above results indicate that the production of surfactants can be increased by altering the metabolic pathway of strain DG17-1, or by combining strain DG17-1 with other strains to construct synthetic communities and compound microbial agents, thereby improving the efficiency of petroleum degradation.

[0061] 2. Analysis of the petroleum degradation ability of strain DG17-1 under nitrogen-free and nitrogen-containing culture conditions. Following the method for determining petroleum hydrocarbon content, strain DG17-1 was activated and inoculated into a 2% petroleum basal medium. The cultures were incubated at 30℃ and 200 rpm for 25 days, and the petroleum hydrocarbon components were extracted from the culture broth. The results showed that under nitrogen-free and nitrogen-containing conditions, the degradation rates of straight-chain alkanes by strain DG17-1 were 19.4–57.2% and 36.0–65.3%, respectively (Table 3), and the degradation rates of aromatic hydrocarbons were 1.8–82.9% and 1.8–100%, respectively (Table 4). Strain DG17-1 exhibited higher utilization of aromatic hydrocarbons. These results indicate that DG17-1 can couple nitrogen fixation and petroleum hydrocarbon degradation, increasing growth through biological nitrogen fixation, thereby achieving petroleum hydrocarbon degradation.

[0062] Table 3 Alkane degradation rate of strain DG17-1 in basal medium

[0063] Table 4. Aromatic hydrocarbon degradation rate of strain DG17-1 in basal medium.

[0064] 3. Analysis of the hydrocarbon degradation ability of strain DG17-1 (single component) After activating strain DG17-1, it was inoculated into four hydrocarbon components (fluorene, C4, C5, C6, C4, C6 ... 16 C 28 Naphthalene was cultured in a nitrogenous basal medium at 30°C and 200 rpm for 30 days, then extracted and analyzed. Figure 4The results showed that strain DG17-1 could completely degrade fluorene. Furthermore, this strain also exhibited high degradation activity for straight-chain alkane compounds (hexadecane), with a degradation rate reaching 85.8%. These results indicate that strain DG17-1 possesses a broad spectrum of petroleum hydrocarbon component utilization capabilities, especially demonstrating excellent efficiency in the degradation of polycyclic aromatic hydrocarbons (fluorene), and thus has high application potential in the field of bioremediation of petroleum hydrocarbon-contaminated environments.

[0065] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A decolorizing neosphingosine bacterium with petroleum degradation and nitrogen fixation functions, characterized in that, The decolorizing neosphingobium is (Novosphingobium decolorationis) DG17-1, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37061 and deposit date of December 12, 2025.

2. A microbial agent, characterized in that, The bacterial agent comprises the decolorizing neosphingosine bacterium as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent is a nitrogen-fixing microbial fertilizer or a petroleum degradation agent.

4. The method for culturing decolorizing neosphingolipids according to claim 1, characterized in that, The oxygen concentration in the culture environment of the decolorized neosphingosine bacteria is 0-15%.

5. The cultivation method according to claim 4, characterized in that, The petroleum content in the culture medium of the decolorized neosphingolipid bacteria is ≤5%.

6. The cultivation method according to claim 4, characterized in that, The surfactant content in the culture medium is 0-2%.

7. The application of the decolorizing neosphingosine bacterium of claim 1 or any of the bacterial agents of claims 2 to 3 in the degradation of petroleum, preferably, the conditions for the degradation of petroleum are nitrogen-free or nitrogen-containing.

8. The application according to claim 7, characterized in that, The petroleum contains C 12 -C 37 Straight-chain alkanes and aromatic hydrocarbons.

9. The application according to claim 8, characterized in that, The aromatic hydrocarbon comprises one or more of the following: naphthalene, 2-methylnaphthalene, 1-methylnaphthalene, acenaphthene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthene, chrysoprase, benzo[b / k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthene, indo[1,2,3,cd]pyrene, or benzo[g,h,i]perylene.

10. The use of the decolorizing neosphingosine bacterium of claim 1 or the bacterial agent of any one of claims 2 to 3 in the preparation of nitrogen-fixing microbial fertilizer.

Images