Beijerinckia sp. with high efficiency of anaerobic petroleum degradation and application thereof

Abstract

This invention discloses a strain of *Bebrilia marinum* with highly efficient anaerobic petroleum degradation capabilities and its applications, belonging to the field of environmental microbiology technology. Strain AC-6 was obtained from oil-contaminated sediment through enrichment, domestication, isolation, purification, and screening. It grows and metabolizes under high-salt anaerobic conditions using petroleum as the sole carbon source, exhibiting both excellent salt tolerance and highly efficient anaerobic petroleum degradation capabilities. Under optimal conditions for anaerobic petroleum degradation, the strain achieves a petroleum degradation rate of up to 90.62%. The *Bebrilia marinum* provided by this invention can be applied to the anaerobic biological treatment of oily wastewater from ship bilges, providing an excellent microbial germplasm resource for the microbial treatment of high-salt, oily wastewater.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, specifically relating to a strain of Betella hygroscopica with highly efficient anaerobic petroleum degradation capabilities and its applications. Background Technology

[0002] During ship operation, oily bilge water inevitably accumulates in the engine room and bilge, a mixture of fuel oil, lubricating oil, hydraulic oil leaks, seawater, fresh water, and detergents. This type of wastewater is complex, containing not only high concentrations of dispersed oil, emulsified oil, and dissolved oil, but also large amounts of suspended solids, surfactants, and salts, making it a typical example of difficult-to-treat oily industrial wastewater. According to the International Convention for the Prevention of Pollution from Ships (MARPOL Convention) and relevant port state control regulations, oily bilge water must be treated to meet the discharge standard of an oil content below 15 mg / L before being discharged into the sea, which places extremely high demands on treatment technology.

[0003] Currently, the main method for treating oily wastewater in ship bilges is the onboard oil-water separator, whose core technologies focus on physicochemical methods such as gravity separation, coalescence, adsorption, and membrane filtration. However, in actual operation, it often faces prominent problems such as difficulty in demulsifying highly emulsified oils, easy clogging of filter elements and membrane components, frequent replacement of consumables, and low efficiency in removing dissolved oils. Furthermore, the separated waste oil and oily waste still need to be stored in special containers and sent ashore for treatment, increasing ship operating costs and space requirements. Biological treatment technology has received widespread attention due to its ability to ultimately mineralize petroleum hydrocarbons into carbon dioxide and water, and its green and gentle treatment process. However, existing ship biological treatment solutions are mostly based on aerobic activated sludge or biofilm methods, which have problems such as high aeration energy consumption, generation of large amounts of residual sludge, poor tolerance to high concentrations of petroleum hydrocarbons and salinity in bilge water, and the need for continuous operation, which is difficult to maintain during ship port stays.

[0004] Currently, utilizing microorganisms to degrade oily wastewater is considered the most promising green treatment technology. Reported research on petroleum-degrading bacteria has largely focused on aerobic microorganisms, such as Pseudomonas and Acinetobacter. However, in ship bilges, the bottom of oil-water separators, or sediments, oxygen diffusion is extremely limited, often resulting in strictly anaerobic or very low-oxygen conditions, where aerobic bacteria can hardly grow or metabolize very slowly. Very few specific strains have been reported that can efficiently perform anaerobic metabolism using petroleum as the sole carbon source, hindering the large-scale application of anaerobic bioremediation technology. Therefore, it is essential to screen for specialized microbial strains that possess both high salt tolerance and efficient anaerobic petroleum degradation capabilities to provide an efficient and feasible anaerobic biological treatment solution for oily wastewater from ship bilges. Summary of the Invention

[0005] This invention provides a strain of Betella hygroscopica with highly efficient anaerobic petroleum degradation capabilities and its applications to solve the above-mentioned problems.

[0006] The first aspect of this invention provides a strain of *Cobetia marina* with highly efficient anaerobic petroleum degradation capabilities. This strain is named *Cobetia marina* AC-6, and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.38131, deposited on March 23, 2026, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] Furthermore, the 16S rDNA sequence of strain AC-6 is shown in SEQ ID NO. 1.

[0008] Furthermore, the colonies of strain AC-6 are round, with smooth surfaces, intact edges, viscous texture, and opaque yellow color.

[0009] A second aspect of the present invention provides a method for degrading petroleum using *Bebrillium hygroscopicum*, which has a highly efficient anaerobic petroleum degradation capability, comprising the following steps: S1. Strain activation: The AC-6 strain, frozen in glycerol at -20℃, was inoculated into sterilized liquid activation medium and cultured at 20℃ and 120 rpm for 24 h. After centrifugation to collect the bacterial cells, the culture was diluted with sterile water to OD200. 600 A bacterial suspension was prepared with a value of 0.08-0.12. S2. Anaerobic degradation culture: The bacterial suspension was inoculated into liquid MMC medium at an inoculation rate of 5% (v / v) and cultured under anaerobic conditions at 10-40℃ and 0-150 rpm for 48 h to complete petroleum degradation; the preferred conditions for anaerobic degradation culture were 20℃ and 120 rpm.

[0010] Furthermore, it also includes steps for detecting petroleum concentration and calculating degradation rate: Take 10 mL of the degraded culture medium, add 10 mL of petroleum ether, shake to mix, and let stand to separate the layers. Collect the upper petroleum ether phase. Add 10 mL of petroleum ether to the remaining aqueous phase, repeat the shaking extraction and standing to separate the layers, and extract twice in total. Combine the petroleum ether extracts obtained from the two extractions to determine the residual petroleum concentration in the culture medium. Combine this with the petroleum concentration of the blank control group after simultaneous culture to calculate the degradation rate of petroleum by the strain.

[0011] The third aspect of this invention provides the application of the aforementioned Cobetia marina AC-6, which has highly efficient anaerobic petroleum degradation capabilities, in the treatment of oily wastewater from ship bilges.

[0012] Furthermore, the initial concentration of petroleum in the ship's bilge oily wastewater is 1-20 mL / L, preferably 10 mL / L, and the wastewater salinity is 20-25 g / L.

[0013] Furthermore, the degradation treatment conditions are as follows: temperature is 10-40℃, preferably 20℃, and shaking speed is 0-150 rpm, preferably 120 rpm.

[0014] Furthermore, the inoculum amount of strain AC-6 is 1-15%, preferably 10%.

[0015] The beneficial effects of this invention are: (1) The *Hydrocotyle spp. AC-6* provided by this invention was isolated from activated sludge in the return pipe of residual sludge from a municipal wastewater treatment plant and obtained through domestication and screening. Under anaerobic conditions, its degradation efficiency for petroleum can reach up to 90.62%, which has a high efficiency in anaerobic degradation of petroleum hydrocarbons and fills the application gap of marine-derived anaerobic petroleum degradation strains.

[0016] (2) The *Hydrocotyle spp.* AC-6 provided by this invention can directly use petroleum as the sole carbon source for growth and metabolism, without the need for additional carbon sources such as glucose. This results in low culture costs and avoids the metabolic interference and secondary pollution risks caused by additional carbon sources.

[0017] (3) The *Betterella hygroscopica* AC-6 provided by this invention is tolerant to high-salt environments and can grow stably in oxygen-deficient environments such as ship bilge water and sediments as well as the bottom of oil-water separators, and degrade petroleum pollutants, making it suitable for the actual treatment of ship bilge oily wastewater. Attached Figure Description

[0018] 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 some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Colony morphology of Cobetia marina AC-6 on culture medium; Figure 2 Phylogenetic tree of Cobetia marina AC-6 based on 16S rDNA; Figure 3 This is the standard curve for petroleum in Example 2; Figure 4The degradation efficiency of petroleum by Cobetia marina AC-6 under different temperature conditions in Example 2; Figure 5 The degradation efficiency of petroleum by Cobetia marina AC-6 under different rotation speeds in Example 3; Figure 6 The degradation efficiency of petroleum by Cobetia marina AC-6 under different initial petroleum concentrations in Example 4 is shown. Figure 7 The degradation efficiency of petroleum by Cobetia marina AC-6 under different inoculum amounts in Example 5; Figure 8 The degradation efficiency of petroleum by Cobetia marina AC-6 in Example 6 within 48 hours; Figure 9 Example 7 shows the degradation efficiency of Cobetia marina AC-6 on petroleum in simulated ship bilge oily wastewater. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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, 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.

[0021] The culture medium used in this embodiment is as follows: Liquid MMC medium (inorganic salt medium): NaCl 24 g / L, MgSO4·7H2O 7.0 g / L, NH4NO3 1.0 g / L, KH2PO4 2.0 g / L, KCl 0.7 g / L, Na2HPO4 3.0 g / L, trace element solution, distilled water 1000 mL, pH 7.4-7.8. The trace element solution consists of: CaCl2 0.02 mg / L, CuSO4 0.005 mg / L, FeCl3·6H2O 0.5 mg / L, ZnSO4·7H2O 0.1 mg / L, MnCl2·4H2O 0.005 mg / L. Liquid activation medium (2216E liquid medium): peptone 5g / L, ferric phosphate 0.1g / L, yeast extract 1g / L, distilled water 1000mL, pH 7.4-7.8; Solid screening medium (2216E agar medium): peptone 5g / L, ferric phosphate 0.1g / L, yeast extract 1g / L, agar 15g / L, distilled water 1000mL, pH 7.4-7.8.

[0022] Example 1: Isolation and Identification of Strain AC-6 Activated sludge was collected from the residual sludge return pipe of a wastewater treatment plant in Dalian. The activated sludge was placed in a CSTR reactor and acclimated for 30 days. 10g of the acclimated reactor sludge was then taken, and 10mL of physiological saline was added. The mixture was vortexed for 5 minutes to ensure thorough mixing, and then allowed to stand. 10mL of the supernatant was added to 90mL of sterile liquid MMC medium, with 5mL of petroleum added as the sole carbon source. The mixture was then anaerobically cultured at 120 rpm and 20℃ for 7 days. After the culture was completed, 1mL of the enriched bacterial solution was collected and diluted with 9mL of sterile water to a concentration of 10. -1 Dilute stepwise to 10 using the same method. -2 10 -3 10 -4 10 -5 10 -6 10 -7 and 10 -8 A series of diluted bacterial solutions were obtained. Then, using the plate spreading method, 0.1 mL of the diluted bacterial solution was spread on 2216E solid medium. After 7 days of anaerobic culture at 20℃, colonies with clear plaques and large diameters were selected for streak isolation. After 4-6 generations of isolation and purification, relatively pure single colonies were obtained. Petroleum degradation experiments were conducted to screen out the strain with the highest petroleum degradation efficiency, and finally, Betella hygrophytes AC-6 was obtained.

[0023] Morphological identification of strain AC-6: based on colony morphology Figure 1 It can be seen that the colonies of strain AC-6 on solid selection medium are round, smooth, with intact edges, viscous texture, and opaque yellow color.

[0024] Physiological and biochemical identification of strain AC-6 was performed, and the results are shown in Table 1 below.

[0025] Table 1. Physiological and biochemical identification results of strain AC-6

[0026] In the table, "+" indicates a positive result for the physiological and biochemical reaction; "-" indicates a negative result for the physiological and biochemical reaction.

[0027] Molecular biological identification of strain AC-6: DNA was extracted from the selected strain using a bacterial genomic DNA extraction kit. PCR amplification of strain AC-6 was performed using universal 16S rDNA primers 27F and 1492R. The PCR reaction mixture consisted of: 2.0 μL genomic DNA (20 ng / μL), 15.0 μL Taq polymerase, 1.0 μL 27F primer (SEQ ID NO.2: 5'-AGAGTTTGATCCTGGCTCAG-3') (10 μM), 1.0 μL 1492R primer (SEQ ID NO.3: 5'-CTACGGCTACCTTGTTACGA-3') (10 μM), 12.0 μL ddH2O, and a total volume of 30.0 μL. The PCR amplification conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 58℃ annealing for 30 s; and 72℃ extension for 1 min. 30s; final extension at 72℃ for 7min; 35 cycles; after the reaction was completed, 3 μL of PCR product was taken for 1% agarose gel electrophoresis to confirm the PCR amplification fragment. The PCR product sequencing was performed by Qingdao NIO Biotechnology Co., Ltd.

[0028] The 16S rDNA sequence of this strain is 1450 bp in length, and its nucleotide sequence is SEQ ID NO.1.

[0029] The sequence was submitted to the NCBI database for BLAST alignment, and a phylogenetic tree was constructed using MEGA software. The phylogenetic analysis results of the strain are as follows: Figure 2 As shown. The results showed that this strain had the highest similarity to Cobetia marina KMM 3879 (GenBank accession number NR 113402.1), reaching 99%; based on its physiological and biochemical characteristics and phylogenetic position, this strain was identified as Cobetia marina.

[0030] Example 2: Effect of strain AC-6 on petroleum degradation rate at different temperatures This example investigates the anaerobic degradation effect of strain AC-6 on petroleum at different culture temperatures. The specific operation steps are as follows: S1. Strain activation: The AC-6 strain, which was frozen in glycerol at -20℃, was inoculated into sterilized liquid activation medium and cultured in a shaker at 20℃ and 120 rpm for 24 hours to allow the cells to grow to a certain abundance. After centrifugation, the cells were collected and diluted with sterile water to an OD600 value of approximately 0.1 to obtain a bacterial suspension for subsequent inoculation.

[0031] S2. Group Degradation Culture: Eight 250 mL Erlenmeyer flasks were used, divided into four groups of two flasks each. 100 mL of MMC culture medium and 1 mL of petroleum were added to each flask. At an inoculation rate of 5% (v / v), 5 mL of bacterial suspension was added to each flask containing petroleum-containing MMC culture medium. The four groups of flasks were then anaerobically cultured at 10℃, 20℃, 30℃, and 40℃, respectively, at 120 rpm for 48 hours. A blank control sample without bacterial suspension was also included. After the culture was completed, the culture medium in each flask was used as the test sample.

[0032] S3. Extraction: Every 24 h during the culture period, take 10 mL of culture medium into a separatory funnel, add 10 mL of petroleum ether, shake to mix thoroughly, let stand to separate the layers, take the supernatant, and collect the upper petroleum ether phase; then add 10 mL of petroleum ether to the remaining aqueous phase, shake to mix again, let stand to separate the layers, and collect the upper petroleum ether phase. That is, a total of two extractions are performed. Combine the two petroleum ether extraction phases as the extract to be tested, and the extraction is completed.

[0033] S4. Plotting the Petroleum Standard Curve: To quantitatively determine the petroleum degradation rate, a petroleum concentration-absorbance standard curve was established using ultraviolet spectrophotometry. The specific steps are as follows: Weigh 0.1 g (approximately 0.12 mL) of petroleum sample and dissolve it in petroleum ether. Transfer the solution to a 100 mL amber volumetric flask and dilute to volume to obtain a petroleum standard stock solution with a concentration of 1000 mg / L. Transfer 5 mL of the petroleum standard stock solution to a 50 mL amber volumetric flask and dilute to volume with petroleum ether to obtain an oil standard working solution with a concentration of 100 mg / L. Transfer 0 mL, 2.0 mL, 4.0 mL, 6.0 mL, 8.0 mL, 10.0 mL, and 12.0 mL of the oil standard working solution to 50 mL colorimetric tubes, dilute to volume with petroleum ether, and measure the absorbance at 225 nm in quartz cuvettes, corresponding to concentrations of 0 mg / L, 4 mg / L, 8 mg / L, 12 mg / L, 16 mg / L, 20 mg / L, and 24 mg / L, respectively. Plot a petroleum standard curve with oil concentration (mg / L) on the x-axis and absorbance on the y-axis. The petroleum standard curve is shown below. Figure 3 As shown.

[0034] S5. Absorbance Measurement: Measurements were performed using a UV spectrophotometer. At a wavelength of 225 nm, using the absorbance of petroleum ether as a reference, the absorbance of the extract was measured using a 10 mm quartz cuvette. If the absorbance value exceeded the range of the standard curve, the extract was serially diluted at a ratio of 1 / 10. Based on the standard curve and the corresponding dilution factor, the petroleum concentration in the extract was calculated. If the extract was diluted before measurement, it needed to be multiplied by the corresponding dilution factor to obtain the actual petroleum concentration in the extract. Combining the extraction dilution factor and the sample volume, the residual petroleum concentration in the corresponding culture medium was calculated. The degradation rate of petroleum by strain AC-6 was then calculated using the following formula:

[0035] In the formula, C0 is the concentration of petroleum in the culture medium of the blank control group without bacterial suspension; C1 is the concentration of petroleum in the culture medium of the experimental group inoculated with strain AC-6 48 hours after degradation (residual petroleum concentration).

[0036] Experimental results are as follows Figure 4 As shown, strain AC-6 exhibits the best petroleum degradation effect at a culture temperature of 20℃, with a petroleum degradation rate of 90.12% after 48 h of culture. This indicates that strain AC-6 of the present invention has the best petroleum degradation ability at 20℃.

[0037] Example 3: Effect of strain AC-6 on petroleum degradation rate under different rotation speeds This embodiment investigates the anaerobic degradation effect of strain AC-6 on petroleum at different shaking speeds. The specific operation steps are as follows: S1. Strain activation: The method is the same as in Example 2; S2. Anaerobic Degradation Culture in Groups: Twelve 250 mL Erlenmeyer flasks were used, divided into six groups of two flasks each. 100 mL of liquid MMC medium and 1 mL of petroleum were added to each flask. At a 5% (v / v) inoculation rate, 5 mL of bacterial suspension was added to each flask containing petroleum-containing liquid MMC medium. The six groups of flasks were then incubated at 20°C at rotation speeds of 0 rpm, 30 rpm, 60 rpm, 90 rpm, 120 rpm, and 150 rpm for 48 hours. A blank control sample without bacterial suspension was also included. After incubation, the culture medium in each flask was used as the test sample.

[0038] S3. Petroleum concentration determination and degradation rate calculation: The operation method is the same as in Example 2.

[0039] Experimental results are as follows Figure 5 As shown, strain AC-6 exhibited the best petroleum degradation effect when the shaker speed was 120 rpm, with a petroleum degradation rate of 90.23% after 48 h of cultivation.

[0040] Example 4: Effect of strain AC-6 on petroleum degradation rate under different initial petroleum concentrations This embodiment investigates the anaerobic degradation effect of strain AC-6 on petroleum under different initial petroleum concentrations. The specific operation steps are as follows: S1. Strain activation: The operation method is the same as in Example 2.

[0041] S2. Anaerobic Degradation Culture in Groups: Ten 250 mL Erlenmeyer flasks were used, divided into five groups, with two parallel flasks in each group. 100 mL of liquid MMC medium was added to each flask. 0.1 mL, 0.2 mL, 0.5 mL, 1 mL, and 2 mL of petroleum were added to the corresponding groups, respectively, to achieve petroleum concentrations of 1 mL / L, 2 mL / L, 5 mL / L, 10 mL / L, and 20 mL / L in the medium. 5 mL of bacterial suspension was inoculated into each flask containing petroleum-containing MMC medium at a 5% (v / v) inoculation rate. The five groups of flasks were anaerobically cultured at 20℃ and 120 rpm for 48 h. A blank control sample without bacterial suspension was also provided. After the culture was completed, the culture medium in each flask was used as the test sample.

[0042] S3. Petroleum concentration determination and degradation rate calculation: The operation method is the same as in Example 2.

[0043] Experimental results are as follows Figure 6 As shown, strain AC-6 exhibited the best petroleum degradation effect at a petroleum concentration of 10 mL / L, achieving a petroleum degradation rate of 89.63% after 48 h of cultivation.

[0044] Example 5: Effect of strain AC-6 on petroleum degradation rate under different inoculum amounts This embodiment investigates the anaerobic degradation effect of strain AC-6 on petroleum under different inoculum amounts. The specific operation steps are as follows: S1. Strain activation: The operation method is the same as in Example 2.

[0045] S2. Group Degradation Culture: Eight 250 mL Erlenmeyer flasks were used, divided into four groups, with two parallel flasks in each group. 100 mL of liquid MMC medium and 1 mL of petroleum were added to each flask. Inoculation amounts of bacterial suspension were 1 mL, 5 mL, 10 mL, and 15 mL for each flask in the corresponding group, respectively, at inoculation rates of 1% (v / v), 5% (v / v), 10% (v / v), and 15% (v / v). The four groups of flasks were anaerobically cultured at 20℃ and 120 rpm for 48 h. A blank control sample without bacterial suspension was also provided. After the culture was completed, the culture medium in each flask was used as the test sample.

[0046] S3. Petroleum concentration determination and degradation rate calculation: The operation method is the same as in Example 2.

[0047] Experimental results are as follows Figure 7 As shown, when the inoculum amount is 10% (v / v), strain AC-6 has the best petroleum degradation effect, and the petroleum degradation rate can reach 90.53% after 48 hours of cultivation.

[0048] Example 6: Determination of petroleum degradation rate by strain AC-6 under optimal conditions In this embodiment, under the optimal culture conditions obtained above, the anaerobic degradation efficiency of strain AC-6 on petroleum was determined. The specific operation steps are as follows: S1. Strain activation: The operation method is the same as in Example 2.

[0049] S2. Anaerobic Degradation Culture: Add 100 mL of MMC medium to a 250 mL Erlenmeyer flask, sterilize at 121 °C for 15 min, and cool to room temperature. Add 1 mL of petroleum (10 mL / L concentration). Inoculate the flask with 10 mL of bacterial suspension at a 10% (v / v) inoculation rate. Incubate anaerobically at 20 °C and 120 rpm for 48 h. A blank control sample without bacterial suspension is also provided. After incubation, the culture medium in the Erlenmeyer flask is the test sample.

[0050] S3. Petroleum concentration determination and degradation rate calculation: The operation method is the same as in Example 2.

[0051] After the culture was completed, the trends of degradation efficiency and cell growth over time were as follows: Figure 8 As shown, strain AC-6 has a petroleum degradation rate of 90.62%, indicating that strain AC-6 provided by the present invention has a highly efficient anaerobic degradation ability for petroleum.

[0052] Example 7: Determination of petroleum degradation rate in simulated ship bilge oily wastewater by strain AC-6 under optimal conditions The simulated ship bilge oily wastewater consisted of 100 mL of natural seawater, 1 mL of petroleum, and 0.1 g of ammonium nitrate, with petroleum serving as the carbon source and ammonium nitrate as the nitrogen source. The concentration of the nitrogen source was 100 mg / L (as N), and the salinity was 24 g / L.

[0053] This embodiment measures the anaerobic degradation effect of strain AC-6 on simulated ship bilge oily wastewater under optimal conditions. The specific operating steps are as follows: S1. Strain activation: The operation method is the same as in Example 2.

[0054] S2. Anaerobic Degradation Culture: Add 100 mL of natural seawater to a 250 mL Erlenmeyer flask, sterilize at 121℃ for 15 min, and cool to room temperature. Then add 1 mL of petroleum and 0.1 g of ammonium nitrate to prepare simulated ship bilge oily wastewater. Inoculate the simulated ship bilge oily wastewater with 10 mL of bacterial suspension at an inoculation rate of 10% (v / v) and anaerobic culture at 20℃ and 120 rpm for 48 h. A blank control sample without bacterial suspension is also provided. After the culture is complete, the culture medium in the Erlenmeyer flask is the sample to be tested.

[0055] S3. Petroleum concentration determination and degradation rate calculation: The operation method is the same as in Example 2.

[0056] Degradation effect such as Figure 9 As shown, after 48 h of cultivation, strain AC-6 achieved a degradation rate of 90.62% for petroleum in simulated ship bilge oily wastewater, indicating that this strain is suitable for oily wastewater treatment in high-salinity seawater systems and has good engineering application potential.

[0057] In summary, the *Berates hygroscopicus* AC-6 isolated in this invention exhibits highly efficient anaerobic degradation capabilities for petroleum. By sequentially optimizing the culture temperature, shaking speed, initial petroleum concentration, and inoculum size, the optimal conditions for the anaerobic degradation of petroleum by strain AC-6 were determined to be a culture temperature of 20℃, a shaking speed of 120 rpm, an initial petroleum concentration of 10 mL / L, and an inoculum size of 10%. Under these optimal conditions, strain AC-6 achieved a petroleum degradation rate of 90.62% after 48 hours of anaerobic culture, making it suitable for the anaerobic biological treatment of high-salt ship bilge oily wastewater.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of *Berateella hygroscopica* with highly efficient anaerobic petroleum degradation capabilities, characterized in that... The *Cobetia marina* strain was named *Cobetia marina* AC-6, with accession number CGMCC No. 38131.

2. The *Bebrilia hygroscopica* strain with highly efficient anaerobic petroleum degradation capability according to claim 1, characterized in that, The 16S rDNA sequence of strain AC-6 is shown in SEQ ID NO.

1.

3. The *Hydrocotyle spp.* with highly efficient anaerobic petroleum degradation capability according to claim 1, characterized in that, The colonies of strain AC-6 are round, with a smooth surface, intact edges, a viscous texture, and an opaque yellow color.

4. A method for degrading petroleum using *Cobetiamarina*, a bacterium with highly efficient anaerobic petroleum degradation capabilities as described in claim 1, characterized in that... Includes the following steps: S1, strain activation: the strain AC-6 stored in glycerol at -20℃ was inoculated into sterilized liquid activation medium, and cultured at 20℃ in a 120 rpm shaker for 24h. After centrifugal collection of the bacterial cells, the bacterial cells were diluted with sterile water to OD 600 The value was 0.08-0.12, and a bacterial suspension was prepared; S2. Anaerobic degradation culture: The bacterial suspension was inoculated into liquid MMC medium at an inoculum of 5% and cultured under anaerobic conditions at 10-40℃ and 0-150 rpm for 48 h to complete the degradation of petroleum.

5. The method for degrading petroleum according to claim 4, characterized in that, It also includes steps for petroleum concentration detection and degradation rate calculation: Take 10 mL of the degraded culture medium, add 10 mL of petroleum ether, shake to mix, and let stand to separate the layers. Collect the upper petroleum ether phase. Add 10 mL of petroleum ether to the remaining aqueous phase, repeat the shaking extraction and standing to separate the layers, and extract twice in total. Combine the petroleum ether extracts obtained from the two extractions to determine the residual petroleum concentration in the culture medium. Combine this with the petroleum concentration of the blank control group after simultaneous culture to calculate the degradation rate of petroleum by the strain.

6. The application of Cobetiamarina AC-6, a bacterium with highly efficient anaerobic petroleum degradation capabilities as described in claim 1, in the treatment of oily wastewater from ship bilges.

7. The application according to claim 6, characterized in that, The initial concentration of petroleum in the ship's bilge oily wastewater is 1-20 mL / L, and the salinity of the wastewater is 20-25 g / L.

8. The application according to claim 6, characterized in that, The degradation treatment conditions are: temperature 10-40℃, shaking speed 0-150 rpm.

9. The application according to claim 6, characterized in that, The inoculum size of strain AC-6 is 1-15%.

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