Pseudomonas zhaodongensis, microbial agent thereof and application of pseudomonas zhaodongensis in degradation of deep-sea bottom oil

Abstract

The invention discloses pseudomonas zhaodongensis, a microbial agent of the pseudomonas zhaodongensis and application of the pseudomonas zhaodongensis to degradation of deep-sea bottom oil, and belongs to the technical field of marine environmental pollution treatment. The pseudomonas zhaodongensis provided by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the pseudomonas zhaodongensis is CGMCC No.29239. The bacterium serves as a main petroleum degrading bacterium and is compounded with dimethyl mercaptopropionic acid inner salt to form a microbial agent capable of efficiently degrading petroleum in a high-pressure and low-temperature environment, and the microbial agent is used for biodegradation of deep-sea sedimentary oil. The microbial agent has the characteristics of simple formula, low cost, simple use steps and the like, and has good application prospect and market value.

Description

Technical Field

[0001] The invention belongs to the technical field of marine environmental pollution control, and specifically relates to a strain of Pseudomonas zhaodongensis, a bacterial agent thereof and application thereof in degrading deep-sea bottom oil. Background Art

[0002] During the process of offshore oil extraction and transportation, offshore oil spills occur from time to time, which seriously threaten the marine ecological environment. After an oil spill, in addition to floating oil, a part of the oil will also form bottom oil under certain conditions. Although this part of the bottom oil is not visible on the sea surface, it causes serious harm to marine fish and benthic ecology of marine sediments. After the oil spill, common oil recovery is mainly based on physical methods, including diver collection, vacuum pump underwater suction recovery, and capture adsorption. In addition, chemical methods often use chemical treatment agents to treat oil spills, but chemical dispersants are prone to secondary pollution. However, for the deep-sea environment, due to its high pressure and water depth restrictions, the above two methods are not suitable for oil pollution remediation in this environment. Biological methods have the characteristics of low cost and no secondary pollution, and are an important means to completely remove marine oil spills in shallow sea oil spill remediation. However, the low temperature environment of the deep sea will affect the activity of microbial enzymes, community structure, growth and reproduction, etc., thereby affecting the degradation effect of oil. Pressure is also an important factor restricting deep-sea life. Under deep-sea pressure, the conformation and supramolecular structure of the molecular system in microorganisms will change, and metabolic activities will be affected. As for petroleum hydrocarbon metabolism, the growth and metabolic rate of petroleum hydrocarbon degrading strains under pressure conditions (>5MPa) are usually lower than those under normal pressure conditions. The types and abundance of petroleum degrading bacteria under different pressure conditions will show significant differences, and the ability of microbial communities to degrade petroleum hydrocarbons under high pressure is also weakened. Therefore, the use of biological methods to degrade bottom oil requires overcoming the adverse effects of deep-sea pressure and low temperature on microorganisms and providing a solution that is resistant to high pressure and low temperature. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention develops a Zhaodong Pseudomonas and uses it as the main petroleum-degrading bacteria. By compounding it with sulfide (dimethylthiopropionic acid inner salt), a microbial agent is formed that can efficiently degrade petroleum in a high-pressure and low-temperature environment.

[0004] The technical solution of the present invention is as follows:

[0005] The invention provides a Pseudomonas zhaodongensis GQ10, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.29239.

[0006] The above-mentioned Zhaodong Pseudomonas GQ10 can degrade petroleum and is resistant to low temperatures. Based on this, the present invention provides the following applications of the above-mentioned Zhaodong Pseudomonas GQ10:

[0007] The application of the above-mentioned Pseudomonas zhaodongensis GQ10 in the degradation of petroleum;

[0008] The application of the above-mentioned Pseudomonas zhaodongensis GQ10 in the degradation of petroleum in a low temperature environment;

[0009] The above-mentioned application of Pseudomonas zhaodongensis GQ10 in the degradation of deep-sea bottom oil.

[0010] The present invention provides a microbial agent, which comprises the above-mentioned Pseudomonas zhaodongensis GQ10 and dimethylmercaptopropionic acid inner salt.

[0011] The preparation method of the microbial agent comprises the following steps:

[0012] The Zhaodong Pseudomonas GQ10 was inoculated into a liquid inorganic salt culture medium containing petroleum, and fermented to make the OD value of the bacterial liquid 0.5-0.7, and then dimethylmercaptopropionic acid inner salt was added to obtain a microbial agent.

[0013] In the above-mentioned method for preparing the microbial agent, the formula of the liquid inorganic salt culture medium is as follows: KH2PO4 1.0 g / L, K2HPO4 0.5 g / L, FeSO4·7H2O 0.01 g / L, CaCl2 0.1 g / L, (NH4)2SO4 1.5 g / L, MgSO4 0.2 g / L, 1 L of aged sea water, and the pH is adjusted to 7.6.

[0014] In the above-mentioned method for preparing the microbial agent, the final concentration of the added dimethylmercaptopropionic acid inner salt in the bacterial solution is selected from 0 to 500 nmol / L, preferably 100 nmol / L.

[0015] The present invention provides an application of the above-mentioned microbial agent in degrading petroleum in a high-pressure and low-temperature environment; the high-pressure and low-temperature environment is selected from deep-sea sediments; the deep-sea sediments can be further selected from sandy sediments or muddy sediments; specifically, the present invention provides an application of the above-mentioned microbial agent in degrading deep-sea bottom petroleum.

[0016] In the present invention, the high pressure is selected from ≥10MPa; the low temperature is selected from ≤10°C.

[0017] In the above application, the dosage of the microbial agent is selected from 10 to 100 mL / kg of sediment, preferably 80 mL / kg of sediment.

[0018] The beneficial effects of the present invention are:

[0019] The present invention provides a Zhaodong Pseudomonas GQ10, which can degrade petroleum and is resistant to low temperatures. After the bacteria is compounded with dimethylmercaptopropionic acid inner salt, the degradation of petroleum in deep-sea sediments under high pressure and low temperature conditions can be effectively achieved. Therefore, the two can be prepared into a microbial agent for biodegradation of deep-sea sediment oil. The microbial agent has the characteristics of simple formula, low cost and simple use steps, and can achieve the degradation of petroleum in sandy and muddy sediment substrates, and has good application prospects and market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the colony picture of strain GQ10 on 2216E medium;

[0021] Figure 2 is the degradation rate of crude oil by strain GQ10 under high pressure environment;

[0022] Figure 3 The effect of different concentrations of dimethylmercaptopropionic acid salt on the degradation rate of diesel;

[0023] Figure 4 This is the effect of GQ10 bacterial solution inoculation concentration on the oil degradation rate. DETAILED DESCRIPTION

[0024] In the present invention, the materials used are as follows:

[0025] 2216E culture medium: 5 g / L peptone, 1 g / L yeast powder, 0.01 g / L FeSO4·7H2O, 20 g / L agar powder added to solid culture medium, 1 L Chenhai water, adjust the pH to 7.5-7.6.

[0026] 1 / 50 2216E solid culture medium: peptone 0.1g / L, yeast powder 0.02g / L, FeSO4·7H2O 0.01g / L, solid culture medium plus agar powder 20g / L, 1L of aged sea water, adjust the pH to 7.5-7.6.

[0027] Liquid inorganic salt culture medium: KH2PO4 1.0g / L, K2HPO4 0.5g / L, FeSO4·7H2O 0.01g / L, CaCl20.1g / L, (NH4)2SO4 1.5g / L, MgSO4 0.2g / L, 1L of aged sea water, adjust the pH to 7.6.

[0028] 1. Isolation and identification of bacterial strains:

[0029] The strain was isolated from the surface sediments at 18.00°N, 117.00°E in the northeastern part of the South China Sea at a depth of 3951m. The isolation steps were as follows:

[0030] 1 g of sediment sample was washed with sterile saline for 10 -2 , 10 -3 , 10 -4 After dilution, 100 μL of the supernatant was spread on 1 / 50 2216E solid medium and placed in an inverted culture at 10°C. After 14 days, well-growing colonies were selected and inoculated and streaked in 4 areas on a sterilized 2216E solid plate and placed at 10°C for culture. This operation was repeated several times until a single colony was cultured and named GQ10.

[0031] Morphological characteristics:

[0032] The color of strain GQ10 on 2216E medium is orange-yellow, nearly round, with wavy edges, smooth surface, and opaque; Figure 1 shown.

[0033] Genetic characteristics:

[0034] (1) DNA extraction by boiling method

[0035] Pick a single strain and inoculate it on a 2216E slant solid medium. After growing for 48 hours, add 200 μL of filtered sterilized TE buffer solution and vortex to elute the bacteria. Collect the bacteria into a 1.5 mL centrifuge tube, boil it in 100°C water bath for 10 minutes, immediately freeze it at -20°C for 20 minutes, centrifuge it at 4°C and 12000 rpm for 10 minutes, and take the supernatant as a DNA template.

[0036] (2) 16S rDNA PCR amplification and sequence determination

[0037] The amplification system is: 20μL T3 Super PCR Mix, 1μL primer 27F, 1μL primer 1492R, 3μL DNA template. The primer sequences are as follows:

[0038] Primer 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1)

[0039] Primer 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO: 2)

[0040] PCR amplification program: 98°C pre-denaturation for 2 min, 30 cycles (98°C denaturation for 10 s, 55°C annealing for 10 s, 72°C extension for 25 s), 72°C final extension for 2 min. After amplification, the samples were sent to Qingke Sequencing Co., Ltd. for bidirectional sequencing.

[0041] The sequencing results are as follows:

[0042] 16S rDNA:

[0043]

[0044] (3) Sequence alignment

[0045] The bidirectional sequences obtained by sequencing were spliced ​​to obtain the complete sequence and submitted to the EzBioCloud website for comparison. The comparison results showed that the strain GQ10 had the greatest similarity with the Pseudomonas zhaodongensis standard strain NEAU-ST5-21 (T), reaching 99.75%. The bacteria were finally identified as Pseudomonas zhaodongensis. The strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101) on December 5, 2023, with the deposit number CGMCC No. 29239.

[0046] 2. Stress protection effect of dimethylmercaptopropionic acid inner salt

[0047] In order to verify the effect of pressure in the deep-sea environment on the degradation of crude oil by GQ10 and to determine the pressure-protective effect of dimethylmercaptopropionic acid salt on strain GQ10, 0.2 mL of Pseudomonas zhaodongensis GQ10 bacterial liquid (a single colony of GQ10 on 2216E solid culture medium was inoculated into a liquid inorganic salt culture medium and fermented in a constant temperature shaker at 28°C at 150 rpm / min until the OD value of the bacterial liquid was 0.6) was cultured in a liquid inorganic salt culture medium under the conditions of 1% (v / v) crude oil concentration, 10 MPa / 0.1 MPa, and 10°C temperature for 30 days, and the degradation rate was determined using an infrared oil meter.

[0048] The test results are as follows Figure 2 As shown:

[0049] In the absence of dimethylmercaptopropionic acid salt, pressure significantly inhibited the degradation of crude oil by the strain, and the degradation rate dropped from 25.45% to 10.51%. The degradation rate after adding dimethylmercaptopropionic acid salt at 0.1MPa was 22.44%, which did not produce a significant effect. However, under 10MPa conditions, the degradation rate increased significantly after adding dimethylmercaptopropionic acid salt, from 10.51% to 18.05%. This shows that in a high-pressure environment, dimethylmercaptopropionic acid salt relieved the inhibitory effect of pressure and promoted the degradation of petroleum hydrocarbons in crude oil by strain GQ10.

[0050] 3. Effect of dimethylmercaptopropionic acid salt on the degradation rate of petroleum hydrocarbons

[0051] In the initial stage of the experiment, diesel was used as a carbon source for petroleum hydrocarbons to study the effect of dimethylmercaptopropionic acid salt on the degradation rate of petroleum hydrocarbons. The general concentration of dimethylmercaptopropionic acid salt in the ocean is 20-200 nmol / L, and even in some sea areas it can reach the millimolar level. In order to study the effect of adding dimethylmercaptopropionic acid salt on the degradation rate of petroleum hydrocarbons within a general range, the following experiment was set up: liquid inorganic salt culture medium (total system 20mL), 0.2mL Zhaodong Pseudomonas GQ10 bacterial liquid, 1% diesel (v / v) and dimethylmercaptopropionic acid salt (final concentrations of 0nmol / L, 10nmol / L, 25nmol / L, 50nmol / L, 100nmol / L, 250nmol / L, 500nmol / L) were added to a sterile syringe, three parallel samples were set up for each group, and the diesel degradation rate was measured after culturing in a low-temperature and high-pressure (pressure of 10MPa, temperature of 10°C) hydrostatic autoclave for 7 days.

[0052] The test results are as follows Figure 3 As shown:

[0053] Under the condition of 0-25 nmol / L of dimethylmercaptopropionic acid inner salt, the diesel degradation rate did not change significantly; in the range of 25-100 nmol / L, the diesel degradation rate gradually increased and reached the maximum value at 100 nmol / L; at 100-250 nmol / L, the diesel degradation rate gradually decreased. Therefore, 100 nmol / L was selected as the optimal addition concentration of dimethylmercaptopropionic acid inner salt in subsequent experiments.

[0054] 4. Effect of bacterial inoculation concentration on oil degradation rate

[0055] At the above 100nmol / L as the optimal addition concentration of dimethylmercaptopropionic acid inner salt, the effect of bacterial solution inoculation concentration on the oil degradation rate was studied. The following experiment was set up: high-pressure sterilized marine sediments (total system 20mL) containing 1% (v / v) crude oil and GQ10 inoculation solution with a dimethylmercaptopropionic acid inner salt concentration of 100nmol / L (inoculation solution concentrations were 10mL / kg, 20mL / kg, 40mL / kg, 80mL / kg, and 100mL / kg, respectively) were added into a sterile syringe, and three parallel samples were set up in each group, and cultured in a low-temperature and high-pressure (pressure of 10MPa, temperature of 10°C) hydrostatic autoclave for 7 days. The crude oil degradation rate was measured after 7 days.

[0056] The test results are as follows Figure 4 As shown:

[0057] Under the condition of 100 nmol / L dimethylmercaptopropionic acid inner salt, the best effect was achieved when the concentration of the bacterial agent was 80 mL / kg sediment. Therefore, this concentration was selected as the optimal addition concentration in subsequent experiments.

[0058] Other materials used in the present invention, if not otherwise stated, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way.

[0059] Example 1

[0060] Preparation of microbial inoculants:

[0061] The Zhaodong Pseudomonas GQ10 was taken out of the ultra-low temperature refrigerator, cultured at 28°C for 4 days using 2216E medium, and after a single colony grew out, the colony was picked up with a sterile inoculation loop and put into a liquid inorganic salt medium containing 1% (v / v) petroleum, cultured at 28°C, 120rpm / min for about 24h, and the bacterial solution concentration was enriched to an OD value of 0.6. Subsequently, dimethylmercaptopropionic acid inner salt was added to make the final concentration reach 100nmol / L to obtain a microbial agent.

[0062] 1. High-pressure and low-temperature petroleum degradation test

[0063] Test 1:

[0064] The microbial agent prepared in Example 1 was used to degrade petroleum in sandy sediments. The sandy sediments used were collected from a 650-meter-deep area in the South China Sea.

[0065] Specific experimental process:

[0066] After sediment collection, 15g of sediment and about 1% (g / g) crude oil were added to each of 6 20mL sterile syringes. 1.2mL of bacterial agent was added to three of the syringes, and the same volume of inorganic salt culture medium was added to the other three as a control. The syringes were placed in a 10MPa, 10℃ autoclave for 35 days. The crude oil content was measured using an infrared oil meter, and the crude oil degradation rates of the experimental and control groups were calculated.

[0067] The test results are shown in Table 1:

[0068] Table 1 Oil degradation rate in sandy sediments (%)

[0069] Group Crude oil concentration before degradation (mg / g) Crude oil concentration after degradation (mg / g) Degradation rate (%) Control group 1 11.85 8.43 28.87 Control group 2 12.08 9.04 25.16 Control group 3 11.65 8.69 25.41 Experimental Group 1 12.46 5.86 52.99 Experimental Group 2 12.85 6.24 51.44 Experimental Group 3 11.98 6.56 45.25

[0070] As shown in Table 1, the degradation rate of the experimental group is much higher than that of the control group, which indicates that the microbial agent of the present invention has an excellent petroleum degradation rate.

[0071] Test 2:

[0072] The microbial agent prepared in Example 1 was used to degrade petroleum in sandy sediments. The sandy sediments used were collected from an area at a depth of 757 meters in the South China Sea.

[0073] The specific experimental process is as described in Experiment 1 above.

[0074] The test results are shown in Table 2:

[0075] Table 2 Oil degradation rate in sandy sediments

[0076] Group Crude oil concentration before degradation (mg / g) Crude oil concentration after degradation (mg / g) Degradation rate (%) Control group 1 13.09 8.83 32.51 Control group 2 12.59 9.30 26.08 Control group 3 12.10 7.24 40.19 Experimental Group 1 12.35 5.24 57.55 Experimental Group 2 11.56 3.80 67.12 Experimental Group 3 13.10 3.87 70.45

[0077] As shown in Table 2, the degradation rate of the experimental group is much higher than that of the control group, which indicates that the microbial agent of the present invention has an excellent petroleum degradation rate.

[0078] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A Pseudomonas zhaodongensis GQ10, characterized in that: The strain is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number being CGMCC No.29239.

2. Use of the Pseudomonas zhaodongensis GQ10 described in claim 1 in degrading petroleum in a low-temperature environment.

3. A microbial agent, characterized in that: The microbial agent comprises the Pseudomonas zhaodongensis GQ10 described in claim 1 and dimethylmercaptopropionic acid inner salt.

4. The method for preparing the microbial agent according to claim 3, characterized in that: Here are the steps: The Zhaodong Pseudomonas GQ10 was inoculated into a liquid inorganic salt culture medium containing petroleum, and fermented to make the OD value of the bacterial liquid 0.5-0.7, and then dimethylmercaptopropionic acid inner salt was added to obtain a microbial agent.

5. The preparation method according to claim 4, characterized in that: The formula of the liquid inorganic salt culture medium is as follows: KH2PO4 1.0g / L, K2HPO4 0.5g / L, FeSO4·7H2O 0.01g / L, CaCl2 0.1g / L, (NH4)2SO4 1.5g / L, MgSO4 0.2g / L, 1L of aged sea water, adjust the pH to 7.

6.

6. The preparation method according to claim 4, characterized in that: The final concentration of the added dimethylmercaptopropionic acid inner salt in the bacterial solution is selected from 0 to 500 nmol / L.

7. The preparation method according to claim 4, characterized in that: The final concentration of the added dimethylmercaptopropionic acid salt in the bacterial solution was 100 nmol / L.

8. Use of the microbial agent according to claim 3 in degrading deep-sea petroleum.

9. The use according to claim 8, characterized in that: The high pressure is selected from ≥10 MPa; the low temperature is selected from ≤10°C.

10. The use according to claim 8, characterized in that: The dosage of the microbial agent is selected from 10 to 100 mL / kg of sediment.

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