Crude oil degradation microbial composition, microbial agent and preparation method and application thereof

Abstract

The invention belongs to the technical field of microbial water treatment, and provides a produced water crude oil degradation microbial composition, a microbial agent and a preparation method and application thereof. The microbial composition for degrading the crude oil in the produced water is prepared from Acinetobacter sp.DQ1 (Acinetobacter sp.DQ1), bacillus (Bacillus sp.X2), bacillus paramycoides (Bacillus paramycoides X1) and enterobacter (Enterobacter sp.BR1), and the microbial composition for degrading the crude oil in the produced water is prepared from the following raw materials: the bacillus (Bacillus paramycoides X1), the enterobacter (Enterobacter sp.BR1) and the enterobacter (Enterobacter sp.DQ1), the bacillus (Bacillus sp.X2) and the enterobacter (Enterobacter sp.BR1). The microbial agent comprises a microbial composition and a carrier, after the microbial composition and the microbial agent are subjected to shake cultivation or biological contact oxidation reaction HRT treatment in produced water with the oil content of 10-500 mg / L, the sulfur content of 20-200 mg / L and the salt content of 1.0-3.0%, the crude oil degradation rate can reach 90% or above.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial water treatment and provides a crude oil degradation microbial composition, a bacterial agent and a preparation method and application thereof. Background Art

[0002] As oilfield exploitation continues, the amount of produced water continues to increase, causing serious harm to the environment; due to different strata and oil production processes, the composition of produced water is very complex. After the produced crude oil is dehydrated, the water generally contains a certain amount of oil, sulfide, organic phenol, cyanide, solid particles, and added demulsifiers, flocculants and other chemical agents, which brings great difficulties to the treatment of oil production wastewater. Realizing economical and efficient treatment of produced water is one of the key points to ensure cost reduction and efficiency improvement in oilfield production.

[0003] Conventional produced water treatment uses the old physical and chemical methods of oil separation, flocculation, and filtration, which have problems such as high reagent costs, large residual sludge production, and high disposal costs, resulting in high costs for produced water treatment. In contrast, biological treatment technology with crude oil degradation bacteria as the core has the advantages of good effect, simple operation, low sludge production, and can degrade oil into harmless CO2 and H2O through a series of self-metabolism reactions. It has a wide range of application value in the field of produced water treatment.

[0004] Chinese invention patent application CN109321504A has developed a composite functional petroleum hydrocarbon degradation microbial agent, which is prepared by fluorescent phosphate-solubilizing, round brown nitrogen-fixing bacteria and petroleum hydrocarbon degradation bacteria. The degradation rate of petroleum-contaminated soil or sludge with an oil content of 3-10% in a high saline-alkali environment can reach 21-50% within 30 days.

[0005] Chinese invention patent application CN107674869A uses attapulgite as an immobilized carrier to prepare an attapulgite-immobilized low-temperature crude oil degradation bacterial agent, which can efficiently degrade crude oil pollutants in the soil under low temperature conditions. In the oily soil samples inoculated with the attapulgite-immobilized crude oil degradation bacterial agent, the degradation rate can reach 82.54% after 30 days of degradation.

[0006] Chinese invention patent application CN104403967A discloses a salt- and alkali-tolerant composite microbial active bacterial community, which includes Shewanella, Pseudomonas and Halomonas in a ratio of 4:3:1. The bacterial community has a strong tolerance to pH and salt, and has a wide range of adaptability to drilling waste mud and petroleum-contaminated soil environments. After 14 days of bacterial community treatment, most of the aliphatic hydrocarbons were degraded, with a degradation rate of 92.04%, followed by the degradation rate of aromatic hydrocarbons, which was 79.5%, and the degradation rates of colloid and asphaltene were 10.27% and 16.46%, respectively.

[0007] Chinese invention patent application CN109055261A discloses a strain of Bacillus subtilis that can degrade crude oil at medium and high temperatures. Under the optimal production conditions, the removal rate of 0.3% (V / V) crude oil reaches about 65% after 5 days of degradation reaction. The crude oil degradation rate is high, the reaction rate is fast, and it has the advantages of resistance to medium and high temperatures, alkali and salt. It has high tolerance to surfactants and has great application prospects in microbial oil recovery, bioremediation, oil washing technology and crude oil contaminated water treatment technology.

[0008] At present, the existing technologies are basically aimed at the development and application of microbial strains that are resistant to high and low temperatures and salt and alkali. However, crude oil is petroleum that has not been treated, desulfurized, or purified, and has a high sulfur content. Currently, there are few applications involving microbial degradation of crude oil in produced water that is resistant to high mineralization and high sulfide. It is necessary to develop microbial agents that are suitable for the water quality of produced water, are highly efficient, and grow quickly, to achieve green and efficient treatment of produced water with high mineralization and high sulfide. Summary of the invention

[0009] In view of the problems existing in the prior art, the present invention provides a produced water crude oil degradation microbial agent and a preparation method and application thereof. The microbial agent can be used for crude oil degradation in produced water with high oil content, high salt content and high sulfur content.

[0010] One of the purposes of the present invention is to provide a crude oil degradation microbial composition, comprising: Acinetobacter sp. DQ1, Bacillus sp. X2, Bacillus paramycoides X1 and Enterobacter sp. BR1.

[0011] Preferably, the Acinetobacter sp. DQ1 and Bacillus sp. X2 have the alkane degradation gene alkB; and the Bacillus paramycoides X1 and Enterobacter sp. BR1 have the cycloalkane degradation gene tmoA.

[0012] More preferably, the Acinetobacter sp. DQ1, Bacillus sp. X2, Bacillus paramycoides X1 and Enterobacter sp. BR1 are strains resistant to high salt (salt content 1.0-3.0%) and high sulfur (sulfur content 20m-200mg / L).

[0013] Preferably, the mass ratio of Acinetobacter sp. DQ1, Bacillus sp. X2, Bacillus paramycoides X1, and Enterobacter sp. BR1 is 0.2:0.5:0.8:1.0-0.8:1.0:0.2:0.2.

[0014] More preferably, the mass ratio of Acinetobacter sp. DQ1, Bacillus sp. X2, Bacillus paramycoides X1, and Enterobacter sp. BR1 is 1:1:1:1.

[0015] The second object of the present invention is to provide a method for preparing the above-mentioned microbial composition, which specifically comprises the following steps:

[0016] (1) Acinetobacter sp. DQ1, Bacillus sp. X2, Bacillus paramycoides X1, and Enterobacter sp. BR1 were inoculated into LB medium for culture to prepare corresponding bacterial suspensions;

[0017] (2) Mixing the bacterial suspensions to obtain a microbial composition.

[0018] In some embodiments of the present invention, step (1) uses an inoculation amount of 3-20wt.%, a culture temperature of 20-35°C, a shaker speed of 150-250rpm, a culture time of 10-12h, and centrifugation to collect the bacteria after culturing to the middle of the logarithmic growth phase, and re-adjusting the OD with physiological saline. 600 It is a bacterial suspension of 2.5.

[0019] The third object of the present invention is to provide a crude oil degradation microbial agent, comprising the above-mentioned microbial composition and an inert carrier.

[0020] Preferably, the mass ratio of the microbial composition to the inert carrier is 0.2:1.0-1.0:0.2.

[0021] More preferably, the mass ratio of the microbial composition to the inert carrier is 1:1.

[0022] Preferably, the inert carrier is at least one of a mineral carrier, a plant carrier and a high molecular polymer.

[0023] More preferably, the mineral carrier is at least one of clay, talc, kaolin, montmorillonite, white carbon and zeolite.

[0024] More preferably, the plant carrier is at least one of corn flour, soybean flour and starch.

[0025] More preferably, the high molecular polymer is at least one of polyvinyl alcohol and polyethylene glycol.

[0026] A fourth object of the present invention is to provide a method for using the above-mentioned microorganisms or microbial agents.

[0027] The specific method of use is: inoculating the microbial composition or microbial agent into produced water, and degrading the crude oil in the produced water through shaking table culture or biological contact oxidation reaction HRT.

[0028] Furthermore, the inoculation amount of the microbial composition is 3-20wt.%; the inoculation amount of the microbial agent is 0.05-2g / L.

[0029] Furthermore, the shaker culture degradation conditions are: shaker speed 150-250rpm, temperature 20-35°C, and culture time 4-24h.

[0030] Furthermore, the biological contact oxidation reaction degradation conditions are: the dissolved oxygen in the reactor is controlled at 2-4 mg / L; the temperature is 20-50° C., and the HRT time of the biological contact oxidation reaction is 4-20 h.

[0031] A fifth object of the present invention is to provide an application of the above-mentioned microbial agent in the degradation of crude oil in produced water.

[0032] Preferably, the produced water has an oil content of 10-500 mg / L, a sulfur content of 20-200 mg / L, and a salt content of 1.0-3.0%.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The microbial composition and microbial agent provided by the present invention, the synergistic compatibility of the microbial composition and the agent in Acinetobacter sp. DQ1, Bacillus sp. X2, Bacillus paramycoides X1 and Enterobacter sp. BR1 greatly improves the degradation rate of crude oil in produced water;

[0035] 2. Acinetobacter sp. DQ1 and Bacillus sp. X2 in the microbial composition and bacterial agent of the present invention have the alkane degradation gene alkB; Bacillus paramycoides X1 and Enterobacter sp. BR1 have the cycloalkane degradation gene tmoA; and they are resistant to high salt and high sulfur. For produced water with an oil content of 10-500 mg / L, a sulfur content of 20-200 mg / L, and a salt content of 1.0-3.0%, the crude oil removal rate can reach more than 90%;

[0036] 3. The microbial composition and microbial agent of the present invention can be used to degrade crude oil by shaking culture or biological contact oxidation reactor operation, and the latter can reach a crude oil degradation rate of more than 90% in just 4 hours, greatly improving the degradation efficiency;

[0037] 4. The preparation method of the microbial composition and microbial agent of the present invention is simple and efficient, has no secondary pollution, does not require special treatment, has high production efficiency, and is suitable for industrial application. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.

[0039] The sources of the strains used in the present invention are as follows:

[0040] Acinetobacter sp.DQ1 was deposited in China Center for Type Culture Collection on June 5, 2023, address: Wuhan University, Wuhan, China, with the deposit number: CCTCC No: M2023937;

[0041] Bacillus sp. X2 was deposited in China Center for Type Culture Collection on June 5, 2023, address: Wuhan University, Wuhan, China, with the deposit number: CCTCC No: M2023936;

[0042] Bacillus paramycoides X1 was deposited in China Center for Type Culture Collection on June 5, 2023, address: Wuhan University, Wuhan, China, with the deposit number: CCTCCNo: M2023939;

[0043] Enterobacter sp. BR1 was deposited in the China Center for Type Culture Collection on June 5, 2023, address: Wuhan University, Wuhan, China, with the deposit number: CCTCC No: M2023938.

[0044] Embodiment 1:

[0045] (1) Preparation of bacterial suspension:

[0046] Acinetobacter sp. DQ1, Bacillus sp. X2, Bacillus paramycoides X1 and Enterobacter sp. BR1 were inoculated into 100 mL LB liquid medium with an inoculum size of 5 wt.%, respectively, and cultured on a shaker at 150 rpm for 12 h at 30 ° C until the middle of the logarithmic growth phase, centrifuged at 4000 rpm for 7 min, and the obtained cells were collected and washed twice with saline; then the OD 600 The bacterial suspension reaching 2.5 is ready for use;

[0047] (2) Preparation of microbial composition:

[0048] Mixing bacterial suspensions of Acinetobacter sp. DQ1, Bacillus sp. X2, Bacillus paramycoides X1, and Enterobacter sp. BR1 at a mass ratio of 1:1:1:1 to obtain a microbial composition;

[0049] (3) A microbial composition was inoculated at a 10 wt.% inoculum into produced water containing 100 mg / L oil, 100 mg / L sulfur and 3.0% salt, and cultured at 25°C and a shaking speed of 150 rpm for 24 h.

[0050] Embodiment 2:

[0051] (1) Preparation of bacterial suspension:

[0052] Acinetobacter sp. DQ1, Bacillus sp. X2, Bacillus paramycoides X1 and Enterobacter sp. BR1 were inoculated into 100 mL LB liquid medium with an inoculum size of 10 wt.%, respectively, and cultured on a shaker at 250 rpm for 10 h at 30 ° C until the middle of the logarithmic growth phase, centrifuged at 4000 rpm for 7 min, and the obtained cells were collected and washed twice with saline; then the OD 600 The bacterial suspension reaching 2.5 is ready for use;

[0053] (2) Preparation of microbial agents:

[0054] The bacterial suspensions of Acinetobacter sp.DQ1, Bacillus sp.X2, Bacillus paramycoides X1 and Enterobacter sp.BR1 are mixed at a mass ratio of 1:1:1:1 to obtain a liquid bacterial agent; the liquid bacterial agent is then mixed with a kaolin carrier at a mass ratio of 1:1, and the mixture is shaken evenly; the shaken sample is passed into a spray dryer, the air inlet temperature is set to 120°C, and the air outlet temperature is set to 60°C, to obtain a microbial bacterial agent;

[0055] (3) Take 0.05 g / L of microbial agent and inoculate it into produced water with an oil content of 500 mg / L, a sulfur content of 20 mg / L, and a salt content of 2.0%, and culture it at 25°C and a shaking speed of 200 rpm for 24 hours.

[0056] Embodiment 3:

[0057] (1) Preparation of bacterial suspension: same as in Example 1;

[0058] (2) Preparation of microbial composition: same as in Example 1;

[0059] (3) The microbial composition was transferred to a biological contact oxidation reactor at an inoculum amount of 10 wt.%, and inoculated into produced water with an oil content of 100 mg / L, a sulfur content of 100 mg / L, and a salt content of 3%; the dissolved oxygen in the reactor was controlled at 4 mg / L, the temperature was controlled at 20°C, and the HRT treatment time was 6 h.

[0060] Embodiment 4:

[0061] (1) Preparation of bacterial suspension: same as in Example 2;

[0062] (2) Preparation of microbial agent: same as in Example 2;

[0063] (3) The microbial agent was transferred to the biological contact oxidation reactor at an inoculum rate of 1 g / L and inoculated into produced water with an oil content of 200 mg / L, a sulfur content of 50 mg / L, and a salt content of 2.5%. The dissolved oxygen in the reactor was controlled at 4 mg / L, the temperature was controlled at 20°C, and the HRT treatment time was 6 h.

[0064] Embodiment 5:

[0065] (1) Preparation of bacterial suspension: same as in Example 2;

[0066] (2) Preparation of solid bacterial agent: same as in Example 2;

[0067] (3) The microbial agent was transferred to the biological contact oxidation reactor at an inoculation rate of 1.5 g / L and inoculated into produced water with an oil content of 100 mg / L, a sulfur content of 50 mg / L, and a salt content of 2.5%. The dissolved oxygen in the reactor was controlled at 4 mg / L, the temperature was controlled at 20°C, and the HRT treatment time was 4 h.

[0068] Embodiment 6:

[0069] Compared with Example 1, the only difference is that the bacterial suspensions of Acinetobacter sp. DQ1, Bacillus sp. X2, Bacillus paramycoides X1 and Enterobacter sp. BR1 are in a mass ratio of 0.2:0.5:0.8:1.0.

[0070] Embodiment 7:

[0071] Compared with Example 1, the only difference is that the bacterial suspensions of Acinetobacter sp. DQ1, Bacillus sp. X2, Bacillus paramycoides X1 and Enterobacter sp. BR1 are in a mass ratio of 0.8:1.0:0.2:0.2.

[0072] Comparative Example 1:

[0073] The only difference from Example 1 is that the microbial composition is a bacterial suspension of Acinetobacter sp. DQ1, Bacillus sp. X2 and Enterobacter sp. BR1 mixed in a mass ratio of 1:1:1.

[0074] Comparative Example 2:

[0075] The only difference from Example 1 is that the microbial composition is replaced by a single Bacillus paramycoides X1.

[0076] Comparative Example 3:

[0077] The only difference from Example 1 is that the microbial composition is a bacterial suspension of Acinetobacter sp. DQ1, Bacillus sp. X2 and Bacillus paramycoides X1 mixed in a mass ratio of 1:1:1.

[0078] Comparative Example 4:

[0079] The only difference from Example 1 is that the microbial composition is a bacterial suspension of Bacillus sp. X2, Bacillus paramycoides X1 and Enterobacter sp. BR1 mixed in a mass ratio of 1:1:1.

[0080] Comparative Example 5:

[0081] The only difference from Example 1 is that the microbial composition is a bacterial suspension of Acinetobacter sp. DQ1, Bacillus paramycoides X1 and Enterobacter sp. BR1 mixed in a mass ratio of 1:1:1.

[0082] Comparative Example 6:

[0083] The only difference from Example 1 is that the bacterial suspensions of Acinetobacter sp. DQ1, Bacillus sp. X2, Bacillus paramycoides X1 and Enterobacter sp. BR1 are in a mass ratio of 0.8:1.0:0.1:0.2.

[0084] Comparative Example 7:

[0085] The only difference from Example 1 is that the bacterial suspensions of Acinetobacter sp. DQ1, Bacillus sp. X2, Bacillus paramycoides X1 and Enterobacter sp. BR1 are in a mass ratio of 0.2:0.5:1.0:1.0.

[0086] Table 1 Crude oil degradation results

[0087]

[0088]

[0089] It can be seen from the crude oil degradation rate results of the above embodiments and comparative examples that the microbial agent of the present invention can effectively treat high-oil, high-sulfur and high-salt produced water under the synergistic effect of Acinetobacter sp. DQ1, Bacillus sp. X2, Bacillus paramycoides X1 and Enterobacter sp. BR1, and the crude oil degradation rate in produced water with an oil content of 10-500 mg / L, a sulfur content of 20-150 mg / L and a salt content of 1.0-3.0% reaches more than 90%; and the use method of the microbial agent is simple and diverse, and can achieve a good effect of degrading crude oil regardless of direct shaking table treatment or biological contact oxidation reaction treatment. In addition, the biological contact oxidation reaction treatment efficiency is higher.

[0090] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A crude oil degrading microbial composition, characterized in that: include: Acinetobacter sp. DQ1, Bacillus sp. X2, Bacillus paramycoides X1 and Enterobacter sp. BR1.

2. The microbial composition according to claim 1, characterized in that Acinetobacter sp. DQ1 and Bacillus sp. X2 have the alkane degradation gene alkB.

3. The microbial composition according to claim 1, characterized in that Bacillus paramycoides X1 and Enterobacter sp. BR1 have the cycloalkane degradation gene tmoA.

4. The microbial composition according to claim 1, characterized in that The mass ratio of Acinetobacter sp. DQ1, Bacillus sp. X2, Bacillus paramycoides X1 and Enterobacter sp. BR1 is 0.2:0.5:0.8:1.0-0.8:1.0:0.2:0.

2.

5. The method for preparing the microbial composition according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Acinetobacter sp. DQ1, Bacillus sp. X2, Bacillus paramycoides X1, and Enterobacter sp. BR1 were inoculated into LB medium for culture to prepare corresponding bacterial suspensions; (2) Mixing the bacterial suspensions to obtain a microbial composition.

6. The preparation method according to claim 5, characterized in that: In step (1), the inoculation amount is 3-20wt.%, the culture temperature is 20-35°C, the shaking speed is 150-250rpm, the culture time is 10-12h, and the cells are collected by centrifugation after culturing to the middle of the logarithmic growth phase and re-adjusted to OD 600 It is a bacterial suspension of 2.

5.

7. A crude oil degradation microbial agent, characterized in that: The invention comprises the microbial composition according to any one of claims 1 to 4 and an inert carrier.

8. The microbial agent according to claim 7, characterized in that: The mass ratio of the microbial composition to the inert carrier is 0.2:1.0-1.0:0.

2.

9. The microbial agent according to claim 7 or 8, characterized in that: The inert carrier is at least one of a mineral carrier, a plant carrier and a high molecular polymer.

10. The microbial agent according to claim 9, characterized in that: The mineral carrier is at least one of clay, talc, kaolin, montmorillonite, white carbon and zeolite; the plant carrier is at least one of corn flour, bean flour and starch; and the high molecular polymer is at least one of polyvinyl alcohol and polyethylene glycol.

11. A method for using the microbial composition according to any one of claims 1 to 4 or the microbial agent according to any one of claims 7 to 10, characterized in that: The microbial composition or microbial agent is inoculated into produced water, and crude oil in the produced water is degraded by shaking table culture or biological contact oxidation reaction HRT.

12. The method of use according to claim 11, characterized in that: The inoculation amount of the microbial composition is 3-20wt.%; the inoculation amount of the microbial agent is 0.05-2g / L.

13. The method of use according to any one of claims 11-12, characterized in that: The shaker culture degradation conditions are: shaker speed 150-250 rpm, temperature 20-35° C., and culture time 4-24 h.

14. The method of use according to any one of claims 11-12, characterized in that: The degradation conditions of the biological contact oxidation reaction are: the dissolved oxygen in the reactor is controlled at 2-4 mg / L; the temperature is 20-50° C., and the HRT time of the biological contact oxidation reaction is 4-20 h.

15. Use of the microbial composition according to any one of claims 1 to 4 and the microbial agent according to any one of claims 7 to 10 in the degradation of crude oil in produced water.

16. The use according to claim 15, characterized in that The produced water has an oil content of 10-500 mg / L, a sulfur content of 20-200 mg / L, and a salt content of 1.0-3.0%.

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