Strain c q33 of heat and salt tolerant surfactant-producing bacteria and application thereof

Abstract

The application discloses a heat-resistant and salt-resistant surfactant-producing bacterium CQ33 and application thereof, and belongs to the technical field of environmental microorganisms and oil pollution treatment. Tepidicella The application provides a new functional strain which has heat resistance, salt resistance, broad-spectrum oil hydrocarbon degradation and biological surfactant production capacity, and has oil hydrocarbon degradation activity under the conditions of 30-60 DEG C and 1-9% NaCl, can degrade alkanes and polycyclic aromatic hydrocarbons, and can produce biological surfactants to improve the biological availability of oil hydrocarbons. The strain can be used for oil-contaminated soil remediation, oil-containing wastewater treatment and microbial oil production.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology and petroleum pollution control technology, and more specifically to a heat- and salt-resistant surfactant-producing bacterium CQ33 and its applications. Background Technology

[0002] Petroleum hydrocarbon pollution is widespread during oilfield extraction, transportation, refining, and storage. Its main components include straight-chain alkanes, branched-chain alkanes, cycloalkanes, and polycyclic aromatic hydrocarbons (PAHs), characterized by strong hydrophobicity, low bioavailability, and high environmental persistence. Microbial remediation technology has attracted widespread attention due to its advantages of being green, economical, and producing no secondary pollution.

[0003] Currently, numerous studies have reported on the genus *Pseudomonas* (…). Pseudomonas ), Bacillus spp. Bacillus ) and Rhodococcus spp. ( Rhodococcus The application of strains such as [list of strains] in petroleum hydrocarbon degradation. However, most of the petroleum hydrocarbon-degrading bacteria reported so far are mesophilic bacteria, whose optimal growth temperature is usually in the range of 25-35°C. Their activity decreases significantly or even becomes inactive in high-temperature environments such as oil reservoirs (40-70°C) or in scenarios such as high-temperature surfaces in summer and hot washing wastewater. In addition, oilfield produced water, oily sludge, and soils in saline-alkali areas are often accompanied by high salt stress (NaCl concentration can reach 1-5%). Mesophilic microorganisms are difficult to survive under high ionic strength due to cell dehydration, protein denaturation, and membrane structure damage, resulting in a sharp drop in bioremediation efficiency.

[0004] Tepidicella The genus *Syntheticus* is a group of Gram-negative, rod-shaped, aerobic or facultative anaerobic bacteria. Existing literature mainly reports their use in the isolation and identification of environmental samples or in the metabolism of specific metals / sulfur. However, the functions of this genus in the degradation of petroleum hydrocarbons, tolerance to extreme environments, and the production of biosurfactants have not yet been reported.

[0005] Therefore, developing a functional strain that can stably survive under high temperature and high salinity conditions and efficiently degrade petroleum hydrocarbons (while also possessing the ability to degrade alkanes and PAHs) is of great significance for expanding the technological boundaries of petroleum pollution bioremediation and promoting the application of microbial enhanced oil recovery (MEOR). Summary of the Invention

[0006] In view of this, the present invention provides a heat- and salt-resistant surfactant-producing bacterium CQ33 and its applications. It possesses the ability to withstand heat and salt, degrade broad-spectrum petroleum hydrocarbons, and produce biosurfactants. TepidicellaThis is a novel functional strain that exhibits petroleum hydrocarbon degradation activity under conditions of 30-60°C and 1-9% NaCl. It can degrade alkanes and polycyclic aromatic hydrocarbons and produce biosurfactants, thereby improving the bioavailability of petroleum hydrocarbons. This strain can be used for petroleum-contaminated soil remediation, oily wastewater treatment, and microbial enhanced oil recovery.

[0007] Preservation information: ( Tepidicella xavieri CQ33 is deposited at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO.31749 and deposit date of August 26, 2024.

[0008] Functional characteristics of strains

[0009] Strain CQ33 possesses the following functional characteristics: (1) Moderate thermophilicity: It has petroleum hydrocarbon degradation activity in the range of 30-60°C, and the optimal degradation temperature is 45-50°C; (2) Salt tolerance: It has petroleum hydrocarbon degradation activity in the range of 1-9% NaCl, and the optimal salinity is 1-3% NaCl; (3) Broad-spectrum degradation capability: It can grow and degrade using n-tetracosane (C24), naphthalene, phenanthrene, fluoranthene and crude oil as the sole carbon source; (4) Biosurfactant production capacity: It can endogenously produce glycolipid biosurfactants, reduce oil-water interfacial tension, and improve the bioavailability of petroleum hydrocarbons.

[0010] genetic basis Whole-genome sequencing and functional annotation showed that strain CQ33 carries a complete cluster of petroleum hydrocarbon degradation genes and a cluster of stress-resistance genes: Degradation gene clusters: including the naphthalene 1,2-dioxygenase gene cluster ( ndoA - ndoC ), salicylate 5-hydroxylase gene cluster ( nagG - nagH ), protocatechuic acid 4,5-dioxygenase gene cluster ( ligA - ligB ), Aromatic ring hydroxylation dioxygenase gene ( ndmA ) and cytochrome P450 monooxygenase gene ( p450 These correspond to the initial hydroxylation, aromatic ring cleavage, and alkane terminal oxidation functions of PAHs, respectively. Salt-tolerant gene clusters: including the complete tetrahydropyrimidine (ectoine) synthesis operon ( ectA - ectB - ectC - ectD ) and genes related to glycine betaine synthesis ( betA), maintain intracellular osmotic pressure balance by accumulating compatible solutes; Heat-resistant gene clusters: including molecular chaperones DnaK - DnaJ - GrpE system( dnaK , dnaJ , grpE ) and chaperone protein GroEL ( groEL ), to maintain the correct folding of proteins and intracellular homeostasis under high temperature conditions.

[0011] To achieve the above objectives, the present invention adopts the following technical solution: A heat- and salt-resistant surfactant-producing bacterium ( Tepidicella xavieri The strain is CQ33, with the preservation number CGMCC NO.31749.

[0012] This invention also provides the application of the above-mentioned bacterium CQ33 in the degradation of petroleum hydrocarbons.

[0013] This invention also provides the application of the above-mentioned bacterium CQ33 in the production of biosurfactants.

[0014] This invention also provides the application of the above-mentioned bacterium CQ33 in the remediation of petroleum-contaminated soil, treatment of oily wastewater, or microbial oil recovery.

[0015] The present invention also provides a petroleum hydrocarbon degradation microbial agent comprising the above-mentioned strains.

[0016] Preferably, the bacterial agent is an immobilized bacterial agent.

[0017] The present invention also provides a method for degrading petroleum hydrocarbons, wherein the above-mentioned strain or the above-mentioned bacterial agent is inoculated into a petroleum hydrocarbon-contaminated system and cultured at 30-60°C and 1-9% NaCl for degradation treatment.

[0018] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a heat- and salt-resistant surfactant-producing bacterium CQ33 and its application, achieving the following technical effects: 1. No public reports have been seen yet. Tepidicella These bacteria possess a broad spectrum of petroleum hydrocarbon degradation capabilities and adaptability to extreme environments. 2. Effective degradation under high temperature and high salt conditions was achieved: Specifically, under conditions of 50°C and 1-3% NaCl, the degradation rate of n-tetracosane by this strain can reach 64.2% in 7 days, and the degradation rate of PAHs such as phenanthrene and fluoranthene can reach more than 58%. 3. This strain also has the ability to produce endogenous surfactants: by producing glycolipid biosurfactants, it can improve the bioavailability of hydrophobic substrates in situ without the need for the addition of exogenous chemical surfactants; 4. This strain exhibits excellent scalability for engineering applications: it can be used as a core functional strain to construct composite treatment systems with other functional strains (such as surfactant-producing strains), or prepared as an immobilized bacterial agent for oil-contaminated soil remediation, high-temperature oily wastewater treatment, and microbial enhanced oil recovery. Core displacement experiments showed that it can increase oil recovery by 9.21%. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 The attached figure shows the morphological characteristics and molecular identification of the strain provided by the present invention, wherein A: colony; B: ordinary light microscope (1000×); C: electron microscope; D: phylogenetic tree (Neighbor-Joining).

[0021] Figure 2 The attached figure shows the 7-day degradation rate of different petroleum hydrocarbon substrates by strain CQ33 provided by this invention.

[0022] Figure 3 The attached figure shows the degradation rate under different conditions provided by the present invention, where A: C24 concentration; B: temperature; C: NaCl concentration; D: pH.

[0023] Figure 4 The attached figure shows the growth status of CQ33 provided by this invention when different petroleum hydrocarbons are used as the sole carbon source.

[0024] Figure 5 The attached figure shows the detection and analysis diagram provided by the present invention, wherein A: oil expulsion ring of strain CQ33; B: Fourier transform infrared spectroscopy of surfactant components; C: thin layer chromatography analysis.

[0025] Figure 6 The attached figure shows the effect of CQ33 strain immobilization on crude oil degradation.

[0026] Figure 7 The attached figure shows the displacement effect of the produced water core after treatment provided by the present invention.

[0027] Figure 8 The attached figure is a complete genome circle diagram of strain CQ33 provided by the present invention.

[0028] Figure 9 The attached figure is a schematic diagram of the key genes and metabolic pathways for CQ33 petroleum hydrocarbon degradation provided by the present invention. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] This invention discloses a heat- and salt-resistant surfactant-producing bacterium CQ33 and its applications.

[0031] Example 1 Screening and identification of strain CQ33 1.1 Strain source and enrichment Produced water samples were taken from the No. 7 Oil Production Plant of Changqing Oilfield in Shaanxi Province. After centrifugation at 3000 r / min for 10 min to remove large particulate impurities, 10 mL of the supernatant was inoculated into 100 mL of inorganic salt medium (MSM) containing 1% (v / v) crude oil. The MSM medium composition was: NaNO3 6 g / L, KCl 0.2 g / L, MgSO4 0.5 g / L, Na2HPO4 5.13 g / L, NaH2PO4 0.76 g / L, glycerol 2 mL / L, and trace elements 0.2 mL / L. The Erlenmeyer flasks were placed in a constant temperature shaker at 55°C and 160 r / min for 7 days for enrichment.

[0032] 1.2 Separation and Purification Take the enriched culture medium and serially dilute it to 10⁻⁶ with sterile physiological saline. - ³-10 -6 Spread 100 μL of each culture onto MSM agar plates (with 15-20 g / L agar added), with three replicates for each dilution. Incubate the plates upside down at 50°C for 3-5 days. Pick single colonies with different morphologies and streak them repeatedly on the MSM plates 2-3 times until pure single colonies are obtained, which are named CQ33.

[0033] 1.3 Morphological characteristics and molecular identification CQ33 was inoculated onto LB agar plates (10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, 15-20 g / L agar), and incubated at 45°C for 36 h. Colony morphology was observed: colonies were small, milky white, raised, translucent, smooth, and had regular edges. Figure 1 (A) Ordinary optical microscope ( Figure 1 (B) and scanning electron microscope (SEM) Figure 1 (C) Observation shows that the bacterial cells are rod-shaped.

[0034] PCR amplification of the 16S rRNA gene was performed using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The products were sequenced and compared with the NCBI database. Results showed that strain CQ33 was similar to... Tepidicellaxavieri It showed the highest homology, and phylogenetic tree analysis confirmed that it belonged to... Tepidicella genus ( Figure 1 (D).

[0035] Example 2 Petroleum hydrocarbon degradation performance of strain CQ33 Strain CQ33 was inoculated into MSM medium with each substance as the sole carbon source and cultured for 7 days at 50°C, 1% NaCl, pH 7.0, and 160 r / min. The concentration of residual petroleum hydrocarbons was determined by GC-MS, and the degradation rate was calculated.

[0036] Sample pretreatment and determination methods: For alkane and crude oil samples: Add 10 mL of n-hexane extractant to 100 mL of the test solution, transfer to a separatory funnel, shake thoroughly, let stand for 5 min, collect the supernatant, and repeat the extraction at least three times. Combine the extracts and filter under reduced pressure, add 10 mL of n-hexane to make up to volume, and filter through a 0.22 μm oil-based microporous membrane.

[0037] For PAHs samples: use ethyl acetate as the extractant and acetone as the volume-fixing solvent, and the remaining steps are the same as above.

[0038] GC-MS detection conditions: The determination was performed using a GC-MS-TQ8040 system (Shimadzu co., Japan). The capillary column was SH-Rxi-5Sil-MS, the carrier gas was helium, the flow rate was 1 mL / min, the injection volume was 1 μL, the syringe temperature was 250°C, and the detector temperature was 310°C. The temperature program was: 80°C for 5 min, then increased to 290°C at a rate of 15°C / min. The chromatographic components were analyzed using GC-MSsolution 4.45 software and the NIST 2014 database. The degradation rate was calculated by comparing the peak area with that of the reference standard.

[0039] Preparation of standard curve: Prepare a 10 mg / L PAHs stock solution using acetone as solvent, and dilute it to 1, 2, 3, 5 and 10 mg / L standard solutions. After filtering through a 0.22 μm microporous membrane, detect the solutions under the above conditions. Prepare a standard curve with peak area as the x-axis and concentration as the y-axis.

[0040] The results showed that strain CQ33 exhibited significant degradation capabilities for straight-chain alkanes (C24) and various PAHs (phenanthrene, naphthalene, and fluoranthene), achieving a degradation rate of 64.2% for C24, 50.2%-58.51% for phenanthrene, naphthalene, and fluoranthene, and a degradation rate of 45.5% for crude oil, confirming its broad-spectrum degradation characteristics. Figure 2 ).

[0041] Example 3 Environmental adaptability analysis 3.1 Concentration adaptability In MSM medium with C24 as the sole carbon source, the effect of different concentrations of C24 on the degradation rate was investigated under conditions of 1% NaCl and pH 7.0. Strain CQ33 exhibited high degradation rates in C24 at concentrations ranging from 10 mg to 900 mg / L, with the highest degradation rate of 64.43% at 10 mg / L, and still reaching 42.4% at 900 mg / L. Figure 3 (A)

[0042] 3.2 Temperature adaptability In MSM medium with C24 (500 mg / L) as the sole carbon source, the effect of different temperatures on the degradation rate was investigated under conditions of 1% NaCl and pH 7.0. Strain CQ33 maintained high degradation activity in the temperature range of 40-60°C, with an optimal degradation temperature of 50°C. Even at 60°C, it maintained a degradation rate of 48.36%, demonstrating good moderate thermophilic characteristics. Figure 3 (B)

[0043] 3.3 Salinity adaptability Using C24 (500 mg / L) as the sole carbon source, the effect of different NaCl concentrations on the degradation rate was investigated at 50°C and pH 7.0. Strain CQ33 exhibited degradation activity in the range of 0-90 g / L (0-9%) NaCl, with an optimal salinity of 10-30 g / L (1-3%). Even at 70 g / L (7%) NaCl, it maintained a degradation rate of 44.1%. Figure 3 (C)

[0044] 3.4 pH adaptability Using C24 (500 mg / L) as the sole carbon source, the effect of different pH values ​​on the degradation rate was investigated at 50°C and 1% NaCl: Strain CQ33 maintains high activity in the pH range of 6.0-8.0, with an optimal pH of 7.0. Figure 3 (D).

[0045] 3.5 Growth status of strain CQ33 in different pollutants MSM medium with tetracosane (0.5 g / L), fluoranthene, naphthalene, and phenanthrene (10 mg / L) as the sole carbon sources, respectively, and a NaCl concentration of 1% and pH 7, was inoculated with 1% logarithmic-phase CQ33 bacterial suspension (OD). 600 =0.8), shaker speed 160 r / min, cultured at 50℃ with shaking, OD measured every 24 h. 600 value.

[0046] Tetracosane was dissolved in n-hexane to prepare a stock solution, and fluoranthene, naphthalene, and phenanthrene were dissolved in acetone to prepare stock solutions. The solutions were prepared and used immediately and stored at 4°C in the dark.

[0047] Strain CQ33 exhibited typical growth characteristics of "adaptation-growth-decline" in phenanthrene, naphthalene, fluoranthene, and tetracosane. When phenanthrene or naphthalene was used as the sole carbon source, the adaptation period (OD) was 0-24 h. 600 (almost unchanged), then entered the growth period and reached 192 h (OD) 600 0.340) and 168 h (OD) 600 The biomass reached its peak at 0.351; when using fluoranthene as a carbon source, the adaptation period was relatively long, with slow growth from 0 to 96 hours, followed by rapid growth after 96 hours, reaching its highest biomass (OD) at 192 hours. 600 0.393); When using tetracosane as a carbon source, hydrophobicity caused dissolution interference in the 0-24 h measurement values, and adaptive growth inhibition (OD) appeared from 24-48 h. 600 It decreased to 0.151, gradually recovered after 48 hours, and peaked at 216 hours (OD). 600 0.428). All treatment groups entered a decline phase after reaching their peak values, mainly due to carbon source depletion and nutrient deficiency. The tetracosane treatment may also have been inhibited by the accumulation of metabolic waste. Figure 4 ).

[0048] Example 4 Identification of biosurfactants 4.1 Determination of the activity of the oil drain ring Strain CQ33 was inoculated into LB liquid medium and cultured at 50°C and 160 r / min for 24 h. Then, it was transferred to MSM medium containing 3 mL of vegetable oil at a 4% inoculation rate and cultured at 50°C and 160 r / min for 168 h. The fermentation supernatant was centrifuged at 8000 r / min for 1 min, and the supernatant was used for oil expulsion experiments.

[0049] Add 20 mL of water to the petri dish. Take 1 mL of the mixture of Sudan III and liquid paraffin (100 mL of liquid paraffin + 0.1 g of Sudan III) and spread it evenly on the water surface. After the liquid paraffin has spread evenly and no longer diffuses, take 10 μL of the supernatant of the centrifuged bacterial solution and drop it into the center of the paraffin. Observe the formation of the oil drainage ring and use ImageJ software to measure the diameter of the oil drainage ring.

[0050] The results showed that the supernatant formed a clear oil-draining ring with a diameter of 7.062 cm on the liquid paraffin-Sudan III oil film, indicating that the strain had strong surface activity. Figure 5 (A)

[0051] 4.2 Fourier Transform Infrared Spectroscopy (FTIR) Analysis After inoculating strain CQ33 into an inorganic salt medium and culturing for 72 h, the pH of the fermentation broth was adjusted to 2 with concentrated hydrochloric acid, and the mixture was centrifuged at 8000 r / min for 30 min. The supernatant was collected. Then, an equal volume of the supernatant was extracted with an extractant of chloroform:methanol = 2:1. After shaking and mixing, the mixture was placed in a separatory funnel and allowed to stand for separation. The lower layer was collected, and the upper layer was extracted and recovered again. The lower organic phases were combined and evaporated in a rotary evaporator. The volume was then adjusted to 10 mL with methanol to obtain the crude extract of the surfactant.

[0052] The major chemical functional groups in the crude surfactant extract were analyzed using a Fourier transform infrared spectroscopy (FIR) instrument (Bruker, INVENIO, Germany). Samples were dropped onto the instrument's cell window in thicknesses of 0.01–1 mm, and 64 scans were performed on each sample, recording data from 4000–400 cm⁻¹. - ¹ Spectrum within the range, with a resolution of 4 cm⁻¹ - ¹. Baseline correction and integration of the spectra were performed using OMNIC 8.2 software.

[0053] The analysis results show that 3440 cm - A broad peak of hydroxyl (-OH) appears near ¹, at 1731 cm⁻¹. - A strong absorption peak appears near the carbonyl group (C=O) of the ester, at 1077 cm⁻¹. - ¹ An absorption peak for glycosidic bonds (COC) appears near 2924 cm⁻¹. - ¹ and 2854 cm - A characteristic peak of long-chain alkyl (-CH2 / -CH3) appears at position ¹. These characteristics collectively confirm that the biosurfactant produced by strain CQ33 belongs to the glycolipid class (…). Figure 5 (B)

[0054] 4.3 Thin-layer chromatography (TLC) validation 10 μL of purified biosurfactant was pipetted onto the starting line of a chromatography plate. The developing solvent was chloroform / methanol / water: 65 / 15 / 2 (v / v / v). The plate was placed in the chromatography rod and developed. Development was stopped and the plate was removed when the solvent reached 1.0 cm above the top of the plate. After the plate dried, a colorimetric reagent was evenly sprayed onto the plate, and the plate was heated in a 100℃ oven for 5-10 min. Then, a colorimetric reagent, such as phenol-sulfuric acid reagent, was evenly sprayed onto the silica gel plate. The composition of the biosurfactant was preliminarily determined based on the colorimetric results. The results showed that brown spots appeared after phenol-sulfuric acid spraying, further confirming that it was a glycolipid biosurfactant. Figure 5 (C)

[0055] Example 5 Validation of the application potential of strain CQ33 in mixed culture immobilization system To further verify the application potential of this strain, immobilization methods can be used, such as immobilizing the bacterial cells using sodium alginate embedding.

[0056] Specifically, the bacterial suspension was mixed with a sodium alginate solution of a certain concentration and then added dropwise to a calcium chloride solution to form immobilized particles, which were used in petroleum hydrocarbon degradation experiments.

[0057] Experimental results show that, compared with the free bacterial system, immobilization treatment can improve the stability and degradation efficiency of the bacterial cells to a certain extent, indicating that this strain has good potential for engineering applications.

[0058] CQ33 and the known petroleum hydrocarbon degrading bacterium CQ42 (CGMCC NO.31748) were cultured to the logarithmic growth phase (OD2). 600 (The concentration is approximately 0.8), and then mix them at a 1:1 volume ratio to obtain a mixed culture medium.

[0059] Preparation of immobilized particles: Sterile acrylic fiber (ACF) immobilization material was first immersed in a 2% (w / v) sodium alginate solution, and then immersed in a mixed bacterial culture medium. At room temperature, 150 mL of a 2% (w / v) CaCl2 solution was slowly added dropwise to the immobilized material, and allowed to stand for 4 h for cross-linking and curing. After repeating the above process once, the immobilized material was placed in the mixed bacterial culture medium and aerated overnight at 30°C using an aeration pump.

[0060] Degradation experiment: Synthetic produced water containing 0.5 g / L crude oil (total salinity 30 g / L) was added to the crude oil produced water treatment unit, and purification was carried out at 50°C with an aeration pump maintaining a rate of approximately 0.04 m³ / h. Simultaneously, single-strain CQ33, single-strain CQ42, free mixed bacterial suspension, and a blank control without inoculation were set up. After 7 days, the crude oil content in each system was measured, and the degradation rate was calculated.

[0061] The results showed that the degradation rate of the blank control (CK) was only 4.5%, the degradation rate of single-strain CQ33 was 45.5%, the degradation rate of single-strain CQ42 was 38.2%, and the degradation rate of the mixed-strain free suspension was 62.2%. After the mixed bacteria were prepared into immobilized particles using the sodium alginate-CaCl2 encapsulation method, the degradation rate was increased to 85.25%. Figure 6 ).

[0062] This embodiment is only used to illustrate that strain CQ33 can synergistically construct a composite treatment system with other functional strains to further improve the treatment efficiency of complex crude oil, and does not constitute a limitation on the scope of protection of this invention.

[0063] Example 6 Core displacement experiment (evaluation of microbial enhanced oil recovery potential) To evaluate the application potential of the microbial system containing strain CQ33 in microbial enhanced oil recovery (MEOR), core displacement simulation experiments were conducted.

[0064] Strains CQ33 and CQ42 (CGMCC NO.31748) were mixed at a 1:1 ratio. The logarithmic growth phase bacterial solution was inoculated into simulated crude oil produced water (crude oil 0.5 g / L, total mineralization 30 g / L) at an inoculation rate of 1% (v / v). The solution was purified for 7 days at 50°C using an aeration pump at a rate of approximately 0.04 m³ / h to obtain a biological treatment solution for oil displacement.

[0065] A porosity of 12.98% and a permeability of 3.802 × 10⁻⁶ were selected. -3 μm 2 Natural core samples were dried to constant weight at 60°C, then vacuum-saturated with formation water. After oil injection and aging, pure water flooding was first performed until the outlet water cut exceeded 98%, establishing residual oil saturation. The oil displacement test was conducted under annular pressure of 5 MPa and temperature of 35°C. The above-mentioned biological treatment fluid was injected at a rate of 0.3 mL / min, and the cumulative oil production was recorded when the outlet water cut reached above 98%.

[0066] The results showed that, compared with the control group that only underwent pure water flooding, the produced water displacement after the above-mentioned mixed bacterial treatment could increase the oil recovery rate by 9.21%. Figure 7 The results indicate that the microbial system containing strain CQ33 can effectively exert functions such as surfactant production, viscosity reduction, and improvement of oil-water interface properties under in-situ reservoir conditions, and has application potential in the field of microbial enhanced oil recovery.

[0067] Example 7 Whole genome sequencing and analysis of key functional genes 7.1 Genome Sequencing and Assembly Genomic DNA was extracted from strain CQ33 and whole-genome sequencing was performed using the Illumina sequencing platform. After quality control, the raw data were assembled using SOAPdenovo v2.04, protein-coding gene (CDS) prediction was performed using Glimmer 3.0 and GeneMarkS 4.3, and non-coding RNA prediction was performed using tRNA-scan-SE 2.0 and barrnap 0.9. The predicted CDS sequences were BLAST-aligned with the NR, COG, GO, and KEGG databases to obtain gene functional annotation information.

[0068] Whole-genome sequencing results showed that the total genome length of CQ33 was 2,768,389 bp, with a GC content of 65.78%, encoding 2,642 genes. Figure 8 ).

[0069] The results of genome functional annotation showed that the strain contains a variety of functional genes related to the degradation of petroleum hydrocarbons, including but not limited to alkane monooxygenase, aromatic hydrocarbon dioxygenase and cytochrome P450 monooxygenase, thereby endowing it with the ability to degrade alkanes and polycyclic aromatic hydrocarbons.

[0070] Meanwhile, this strain also contains a variety of environmental adaptation-related genes, including osmolarity regulation-related genes (such as compatible solute synthesis and transport-related genes) and heat shock protein encoding genes, which enable it to maintain stable physiological activity under high salt and high temperature conditions.

[0071] In addition, this strain contains functional genes related to the synthesis of biosurfactants, which can generate biosurfactants in situ during degradation, thereby reducing the oil-water interfacial tension and improving the bioavailability of petroleum hydrocarbons.

[0072] 7.2 Petroleum hydrocarbon degradation-related gene clusters Genome annotation results showed that strain CQ33 carries multiple complementary key genes for aromatic hydrocarbon and alkane degradation pathways (Table 1). The presence of these gene clusters explains, at the molecular level, the broad-spectrum degradation ability of strain CQ33 for PAHs such as naphthalene, phenanthrene, and fluoranthene, as well as long-chain alkanes (C24). ndoB / C and nahC This constitutes the core upstream pathway for naphthalene degradation. ligA / B Provides meta-cleavage ability for protocatechuic acid. ndmA p450 expands the substrate spectral coverage.

[0073]

[0074] 7.3 Genes related to salt tolerance and osmotic adaptation Strain CQ33 carries a complete tetrahydropyrimidine synthesis operon ( ectA - ectB - ectC - ectD This gene cluster represents a classic molecular mechanism by which Gram-negative bacteria resist hyperosmolar stress. Its presence explains the physiological basis for the strain in Example 3 maintaining optimal degradation activity at 0.5-1% NaCl (5-10 g / L)—by accumulating compatible solutes to balance intracellular and extracellular osmotic pressures, while simultaneously protecting the conformational stability of the degradation enzyme system under high ionic strength (Table 2).

[0075]

[0076] 7.4 Heat-resistant related genes The existence of the aforementioned heat shock protein network explains, at the molecular level, the heat resistance mechanism of strain CQ33, which maintains 48.36% degradation activity at its optimum temperature of 50°C and at 60°C. dnaK-dnaJ-grpE It constitutes the core protein quality control system of prokaryotes and can identify and repair denatured petroleum hydrocarbon degrading enzymes (such as dioxygenases) under high temperature conditions. groEL The central fold cavity provides a refolding microenvironment for denatured peptides (Table 3).

[0077]

[0078] 7.5 Genes related to biosurfactants (Table 4) In the genome idnO / rhlG The presence of this gene provides genetic evidence for the endogenous production of glycolipid biosurfactants by strain CQ33 (as confirmed by FTIR and TLC in Example 4). This gene is involved in the reductive modification of fatty acid tail chains, which is one of the key steps in the synthesis of glycolipid surfactants such as rhamnolipids.

[0079]

[0080] 7.6 Summary of Gene-Phenomenon Associations Genome-wide functional annotation results showed a high degree of consistency between the genetic background and phenotypic characteristics of strain CQ33. Regarding petroleum hydrocarbon degradation, the annotated naphthalene dioxygenase gene cluster (…) ndoA-ndoC ), salicylate hydroxylase gene cluster ( nagG-nagH ), protocatechuic acid cyclic cleavage enzyme gene cluster ( ligA-ligB ) and broad-spectrum aromatic hydrocarbon hydroxylase gene ndmA The gene p450, along with the cytochrome P450 monooxygenase gene, corresponds to its broad-spectrum degradation ability of alkanes and polycyclic aromatic hydrocarbons, respectively, explaining the degradation phenotype of this strain at the molecular level. Figure 9 ).

[0081] Meanwhile, the strain carries a tetrahydropyrimidine synthesis operon ( ectA-ectB-ectC-ectDGenes related to glycine betaine synthesis betA This provides the genetic basis for maintaining osmotic balance and enzyme system stability under salt stress; while the molecular chaperone system ( dnaK-dnaJ-grpE-groEL The presence of [a specific substance] explains its heat-resistant mechanism for maintaining degradation activity under high-temperature conditions. Furthermore, idnO / rhlG The detection of genes related to the synthesis of glycolipid biosurfactants further confirms the genetic potential of this strain for endogenous biosurfactant production. The precise correspondence between these genes and phenotypes supports the feasibility and reliability of strain CQ33 in extreme reservoir environments from a microscopic regulatory mechanism perspective.

[0082] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0083] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A heat- and salt-resistant surfactant-producing bacterium ( Tepidicella xavieri CQ33, characterized in that, The strain has the preservation number CGMCC NO.31749.

2. The application of the bacterium CQ33 described in claim 1 in the degradation of petroleum hydrocarbons.

3. The application of the strain CQ33 of claim 1 in the production of biosurfactants.

4. The application of the bacteria CQ33 of claim 1 in the remediation of petroleum-contaminated soil, treatment of oily wastewater, or microbial oil recovery.

5. A petroleum hydrocarbon degradation microbial agent, characterized in that, It contains the strain described in claim 1.

6. The microbial agent as described in claim 5, characterized in that, The bacterial agent is an immobilized bacterial agent.

7. A method for degrading petroleum hydrocarbons, characterized in that: The strain described in claim 1 or the bacterial agent described in claim 5 or 6 is inoculated into a petroleum hydrocarbon-contaminated system and cultured at 30-60°C and 1-9% NaCl for degradation treatment.

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