Facultative anaerobic petroleum degrading bacterium LH1 and application thereof
By developing the facultative anaerobic petroleum degradation bacteria Vagococcus sp.LH1, the existing facultative anaerobic petroleum degradation bacteria have been solved, and the effect of rapid degradation of petroleum pollutants under aerobic and anaerobic conditions is achieved at a lower temperature.
Patent Information
- Application Number
- CN202510198870.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-24
- Publication Date
- 2025-05-30
AI Technical Summary
Existing facultative anaerobic petroleum degradation bacteria take a long time or higher temperature to degrade petroleum pollutants, have low degradation efficiency and poor environmental adaptability.
A new facultative anaerobic petroleum degradation bacteria Vagococcus sp.LH1 was developed. This strain can rapidly degrade petroleum pollutants under both aerobic conditions and anaerobic conditions at 30°C.
Under the optimal degradation conditions (2% bacterial inoculation amount, 1% salinity, pH=7.0, temperature 30℃), the degradation rate of 2% crude oil under aerobic conditions reached 54.12%, and 64.32% after 28 days; under anaerobic conditions, the degradation rate reached 30.58%.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microbial remediation, and specifically relates to a facultative anaerobic petroleum-degrading bacterium Rhizococcus fluvii LH1 and an application thereof. Background Art
[0002] As one of the most important energy resources in the world, oil leaks and spills are inevitable during its mining, transportation, refining and use, and the resulting environmental pollution cannot be underestimated. Oil components are complex, including aliphatic hydrocarbons, aromatic hydrocarbons, colloids, asphaltene and other substances. Among them, high molecular weight oil compounds such as long-chain alkanes and aromatic hydrocarbons containing benzene rings, colloids and asphaltene have complex structures and are extremely difficult to degrade. Oil pollutants are environmentally persistent, difficult to degrade and have "three-hazard" effects, posing a serious threat to the ecological environment and human health.
[0003] Microbial remediation technology is an economical, efficient and eco-friendly remediation method that can completely mineralize pollutants and has great application potential in the degradation of petroleum pollutants. High-efficiency petroleum-degrading bacteria play an important role in the remediation of petroleum pollution. Some existing petroleum-degrading bacteria are suitable for anaerobic or anoxic conditions, and some are suitable for aerobic conditions. For example, Liang Laifeng et al. found that Pseudomonas GD-2 can degrade crude oil in a culture medium with crude oil as a carbon source under anoxic conditions at 50°C, producing surfactants, organic acids and gases; Li Xiang et al. found that the strain Klebsiella sp.LZ6 had a degradation rate of 33% for 50 mg / L pyrene after anaerobic culture for 30 days. Facultative anaerobic petroleum-degrading bacteria can survive and metabolize in an alternating aerobic and anaerobic environment, and can play a role in different polluted environments. For example, Shao Zongze's research showed that the degradation rate of pyrene by Halomonastitanicae PA16_9 under anaerobic conditions was 58% in 3 months, and the degradation rate under aerobic conditions was 74.5%. However, existing facultative anaerobic petroleum-degrading bacteria often require a long time (about 20 days to 3 months) or a high temperature (above 45°C), have low degradation efficiency and poor environmental adaptability. Therefore, it is urgent to develop efficient facultative anaerobic petroleum-degrading bacteria that can quickly degrade petroleum pollutants. Summary of the invention
[0004] The purpose of the present invention is to provide a novel facultative anaerobic petroleum efficient degradation bacterium Vagococcus sp. LH1 and its application, so as to achieve efficient degradation of petroleum pollutants under aerobic and anaerobic conditions.
[0005] In order to achieve the above-mentioned purpose of the present invention, the following technical scheme is adopted:
[0006] The present invention provides a facultative anaerobic petroleum-degrading bacterium. The strain is a Gram-positive bacterium named Vagococcus sp. LH1, which is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (CGMCC), with the preservation number CGMCC No. 32265, the preservation date being October 18, 2024, and the preservation address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.
[0007] Vagococcus sp. LH1 is a Gram-positive bacterium, white and translucent, round, with irregular edges, opaque in the center, moist and smooth on the surface, and slightly convex.
[0008] The screening process of the petroleum-degrading bacterium is as follows: Collect petroleum-contaminated soil from the Dagang Oilfield in Tianjin. Weigh 10 g of the soil sample and place it in 90 mL of sterilized MSM culture medium, add 0.5% crude oil (v:v), and enrich and culture at 150 rpm and 30 °C for 7 days. Then, aspirate 10% of the enriched bacterial solution and transfer it to 90 mL of fresh sterilized MSM medium, add 1% crude oil (v:v), and culture at 150 rpm and 30 °C for 7 days. Repeat the above process. After multiple concentration gradient domestication, an enriched bacterial solution with a tolerance to a crude oil concentration of 5% is finally obtained. Then, dip the inoculation loop into the enriched bacterial solution and streak and purify it on the LB solid medium plate to obtain single colonies of the petroleum-degrading bacterium;
[0009] Among them, the components of the MSM medium are: K 2 HPO 4 1.0 g / L, KH 2 PO 4 1.0 g / L, NH 4 NO 3 1.0 g / L, MgSO 4 ·7H 2 O 0.2 g / L, CaCl 2 ·2H 2 O 0.02 g / L, FeSO 4 ·7H 2 O 0.02 g / L, and the pH of this medium is 7.0;
[0010] The components of the LB solid medium are 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar powder, and 1 L of ultrapure water, and the pH is adjusted to 7.0 - 7.4.
[0011] Regarding the application of the above-mentioned facultative anaerobic petroleum-degrading bacterium, Vagococcus sp. LH1 is used in petroleum degradation. The degradation conditions are: the bacterial inoculation amount is 1 - 5%, the salinity is 1 - 2%, pH = 6.8 - 8.0, the temperature is 30 °C, and the degradation rate of crude oil reaches more than 50% under aerobic conditions and more than 30% under anaerobic conditions.
[0012] The optimal degradation conditions of Vagococcus sp. LH1 for crude oil are a bacterial inoculation amount of 2%, a salinity of 1%, pH = 7.0, and a temperature of 30°C. Under the optimal degradation conditions, in MSM medium under aerobic conditions, for 2% crude oil, at 150 rpm for 10 days, the degradation rate is 54.12%, and the degradation rate reaches 64.32% after 28 days; under anaerobic conditions, for 2% crude oil, at 150 rpm for 10 days, the degradation rate is 30.58%.
[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0014] The present invention provides a novel facultative anaerobic petroleum-degrading bacterium, Vagococcus sp. LH1. This strain can rapidly degrade petroleum pollutants under both aerobic and anaerobic conditions at a relatively low temperature of 30°C. The optimal degradation conditions of strain LH1 for crude oil are a bacterial inoculation amount of 2%, a salinity of 1%, pH = 7.0, and a temperature of 30°C. Under the optimal degradation conditions, under aerobic conditions, for 2% crude oil, at 150 rpm for 10 days, the degradation rate of Vagococcus sp. LH1 is 54.12%, and the degradation rate reaches 64.32% after 28 days; under anaerobic conditions, for 2% crude oil, at 150 rpm for 10 days, the degradation rate of Vagococcus sp. LH1 reaches 30.58%.
[0015] The present invention provides a cost-effective and environmentally friendly microbial remediation technology for petroleum pollution. The remediation process and operation flow are simple, and it has good application prospects in the field of petroleum pollutant degradation. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 Colony morphology diagram of Vagococcus sp. LH1
[0017] Figure 2 Phylogenetic tree of Vagococcus sp. LH1
[0018] Figure 3 Comparison diagram of the effect of bacterial inoculation amount on the degradation of crude oil by Vagococcus sp. LH1
[0019] Figure 4 Comparison diagram of the effect of salinity on the degradation of crude oil by Vagococcus sp. LH1
[0020] Figure 5 Comparison diagram of the effect of pH on the degradation of crude oil by Vagococcus sp. LH1
[0021] Figure 6 Comparison diagram of the effect of temperature on the degradation of crude oil by Vagococcus sp. LH1.
[0022] Figure 7 Degradation effect of Vagococcus sp. LH1 on crude oil under optimal degradation conditions.
[0023] Figure 8 Degradation effect of Vagococcus sp. LH1 on crude oil under anaerobic conditions. Specific implementation mode
[0024] The present invention will be further described below in conjunction with embodiments and the accompanying drawings, but this is not intended to limit the protection scope of the present application.
[0025] Example 1: Screening and identification of petroleum hydrocarbon-degrading bacteria
[0026] (1) Enrichment and isolation of petroleum hydrocarbon-degrading bacteria
[0027] Collect petroleum-contaminated soil from Tianjin Dagang Oilfield. Weigh 10 g of soil sample and place it in 90 mL of sterilized MSM culture medium. Add 0.5% crude oil (v:v). After culturing at 150 rpm and 30 °C for 7 days, the first batch of enriched bacterial liquid is obtained; Pipette 10% of the first batch of enriched bacterial liquid and transfer it to fresh 90 mL of sterilized MSM medium. Add 1% crude oil (v:v). After culturing at 150 rpm and 30 °C for 7 days, the second batch of enriched bacterial liquid is obtained. After multiple concentration gradient acclimations, an enriched bacterial liquid with a crude oil tolerance concentration of 5% is finally obtained. Then, dip the enriched bacterial liquid with an inoculation loop and streak it on a solid LB medium plate for purification to obtain single colonies of petroleum-degrading bacteria.
[0028] Among them, the components of the MSM medium are: K 2 HPO 4 1.0 g / L, KH 2 PO 4 1.0 g / L, NH 4 NO 3 1.0 g / L, MgSO 4 ·7H 2 O 0.2 g / L, CaCl 2 ·2H 2 O 0.02 g / L, FeSO 4 ·7H 2 O 0.02 g / L, and the pH of this medium is 7.0;
[0029] The components of the LB solid medium are 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride, 15 g of agar powder, 1 L of ultrapure water, and the pH is adjusted to 7.0 - 7.4.
[0030] The morphology of the strain on the solid LB plate is as Figure 1 shown. The strain LH1 is white and translucent, round, with an irregular edge, an opaque center, a moist and smooth surface, and is slightly convex.
[0031] (2) Strain identification and preservation
[0032] The strain was subjected to 16S rRNA sequencing, and the obtained sequence was analyzed by BLAST with the sequences already existing in the NCBI database. Strains with similar homology were selected, and a phylogenetic tree was constructed using MEGA 11.0 software. As Figure 2 shown, the 16S rRNA sequencing results of the strain indicated that the strain was highly homologous (100%) to Vagococcus fluvialis strain GX21. The strain was named Vagococcus sp. LH1. The strain was sent to the General Microbiology Center of the China Committee for Culture Collection of Microorganisms for preservation, with the preservation number CGMCC No. 32265 and the preservation date of October 18, 2024.
[0033] Example 2: Exploration of the optimal degradation conditions of strain LH1 for crude oil
[0034] Under aerobic conditions, the single-factor batch experiment method was used to explore the optimal degradation conditions of strain LH1 for crude oil. Four factors were selected for the experiment, including the bacterial inoculation amount, salinity, pH value, and temperature. The strain LH1 was activated using liquid LB medium and cultured at 150 rpm for 10 days under different conditions, and the quality of the crude oil was measured. 1 ml of crude oil was added to each 50 ml of the medium, and the initial crude oil quality was 0.892 g.
[0035] Bacterial inoculation amount: Under the conditions of a temperature of 30 °C, a crude oil concentration of 2%, a salinity of 2%, and a pH of 7.0, five gradients of bacterial inoculation amounts of 0.5%, 1%, 2%, 5%, and 10% were set. The experimental results are as Figure 3 shown. As the inoculation amount increased, the degradation effect of the strain on petroleum decreased. When the inoculation amount was 2%, the degradation effect was the best, at 52.64%.
[0036] Salinity: Under the conditions of a bacterial inoculation amount of 2%, a temperature of 30 °C, a crude oil concentration of 2%, and a pH of 7.0, five gradients of salinities of 0.5%, 1%, 2%, 5%, and 10% were set. The experimental results are as Figure 4 shown. The strain had a degradation effect on crude oil under the conditions of a salinity of 0.5% - 10%. At a salinity of 1%, the degradation effect on crude oil was the best, at 54.12%.
[0037] pH value: Under the conditions of 2% bacterial inoculum, 30 °C temperature, 2% crude oil concentration, and 2% salinity, five gradients of pH were set at 5.0, 6.0, 7.0, 8.0, and 9.0. The experimental results are as Figure 5 shown. Under the conditions of pH 5.0 - 9.0, the strain can degrade petroleum, and when pH = 7.0, the degradation effect is the best, which is 52.64%.
[0038] Temperature: Under the conditions of 2% bacterial inoculum, pH 7.0, 2% crude oil concentration, and 2% salinity, five gradients of temperature were set at 20 °C, 25 °C, 30 °C, 35 °C, and 40 °C. The experimental results are as shown in 6. Temperature has a greater impact on the degradation rate of crude oil by the strain. When the temperature is 30 °C, the degradation rate of crude oil by the strain is the highest, which is 52.64%.
[0039] In summary, the optimal degradation conditions of the strain LH1 for crude oil are 2% bacterial inoculum, 1% salinity, pH = 7.0, and temperature 30 °C.
[0040] Example 3: Degradation performance of strain LH1 on crude oil
[0041] The strain LH1 was inoculated into a liquid LB medium and incubated in a constant-temperature shaker at 30 °C and 150 rpm until the logarithmic phase. The activated bacterial solution was centrifuged at 8000 r / min for 10 min, washed three times with 0.9% sterilized normal saline, washed and resuspended with 0.9% sterilized normal saline to prepare a bacterial suspension. According to the inoculation amount of 2% (v:v), the above bacterial suspension was inoculated into an MSM medium containing 2% crude oil concentration, and the treatment without bacteria was used as a blank control. The mass of crude oil was measured at 0, 7, 10, 14, 21, and 28 days.
[0042] The results are as Figure 7 shown. Under aerobic conditions, the degradation rate of crude oil by the strain LH1 was 54.12% in 10 days, and after 28 days, the degradation rate of crude oil by the strain LH1 reached 64.32%, indicating that the strain LH1 has a good degradation effect on crude oil.
[0043] Example 4: Degradation performance of strain LH1 on crude oil under anaerobic conditions
[0044] The method for preparing the bacterial suspension was the same as that in Example 3. According to the inoculation amount of 2% (v:v), the bacterial suspension was inoculated into an MSM medium containing 2% crude oil concentration, and the treatment without bacteria was used as a blank control. After evacuating the air in an anaerobic glove box and sealing it, it was transferred to a shaker at 30 °C and 150 rpm for culturing for 10 days, and then the mass of crude oil was measured.
[0045] The results are as Figure 8 shown. Under anaerobic conditions, the degradation rate of crude oil by the strain was 30.58%.
[0046] Those parts not described in the present invention are applicable to the prior art.
Claims
1. A facultative anaerobic petroleum-degrading bacterium, characterized in that: The name is Vagococcus sp.LH1, which is deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC), with the deposit number CGMCC No.32265, the deposit date is October 18, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.
2. A facultative anaerobic petroleum-degrading bacterium according to claim 1, characterized in that: Vagococcus sp.LH1 is white and translucent, round in shape, with irregular edges, opaque center, moist and smooth surface, and slightly convex.
3. The method for screening facultative anaerobic petroleum-degrading bacteria according to claim 1, characterized in that: Contaminated soil from Tianjin Dagang Oilfield was collected, 10 g of soil sample was weighed and placed in 90 mL of sterilized MSM culture medium, 0.5% crude oil (v:v) was added, and the first batch of enriched bacterial solution was obtained after culturing at 150 rpm and 30 ° C for 7 days; 10% of the first batch of enriched bacterial solution was taken and transferred to fresh 90 mL of sterilized MSM culture medium, 1% crude oil (v:v) was added, and the second batch of enriched bacterial solution was obtained after culturing at 150 rpm and 30 ° C for 7 days. After multiple concentration gradient acclimation, an enriched bacterial solution with tolerance to crude oil concentration of 5% was finally obtained, and then the enriched bacterial solution was dipped with an inoculation loop and streaked on an LB solid culture medium plate for separation and purification to obtain a single colony of petroleum-degrading bacteria; The components of the MSM culture medium are: K2HPO4 1.0 g / L, KH2PO4 1.0 g / L, NH4NO3 1.0 g / L, MgSO4·7H2O 0.2 g / L, CaCl2·2H2O 0.02 g / L, FeSO4·7H2O 0.02 g / L, and the pH of the culture medium is 7.0; The components of the LB solid culture medium are peptone 10g, yeast powder 5g, sodium chloride 10g, agar powder 15g, and ultrapure water 1L, and the pH is adjusted to 7.0-7.
4.
4. A use of a facultative anaerobic petroleum-degrading bacterium according to claim 1, characterized in that: Vagococcus sp. LH1 is used in petroleum degradation.
5. The use according to claim 4, characterized in that: The optimal conditions for the strain to degrade crude oil are 2% bacterial inoculum, 1% salinity, pH=7.0, and temperature 30°C; Under the optimal degradation conditions, aerobic conditions, the degradation rate of Vagococcus sp.LH1 in MSM medium for 2% crude oil was 54.12% at 150rpm in 10 days and 64.32% in 28 days; Under anaerobic conditions, the degradation rate of Vagococcus sp. LH1 in MSM medium for 2% crude oil reached 30.58% at 150rpm for 10 days.