Salt-tolerant composite bacterial population, preparation method thereof and application thereof in high-salt oil sludge remediation

Abstract

The application discloses a salt-tolerant composite bacterial community, a preparation method thereof and application of the composite bacterial community in high-salt oil sludge remediation. Halomonas venusta The composite bacterial community comprises salt bacillus Acinetobacter calcoaceticus C. acnes CCTCC KB 20081402, and pseudomonas aeruginosa Pseudomonas aeruginosa CCTCC AB 2010174. The application utilizes H. venusta The Ectoine secretion characteristic of the DSM 4743 provides osmotic pressure protection for non-salt-tolerant strains in the composite bacterial community, a Ectoine secretion-transmission-degradation synergistic mechanism is constructed, and the degradation efficiency of petroleum hydrocarbon in a high-salt environment is greatly improved. Experiments show that the petroleum hydrocarbon degradation rate of the composite bacterial community in high-salt oil sludge with a salt content of 35 g / L can reach 43.12% within 7 days. The application is suitable for oil sludge remediation in high-salt environments such as offshore oil fields and saline-alkali soil petroleum contaminated sites, and has important environmental and economic benefits.

Description

Technical Field

[0001] This invention relates to the field of petroleum hydrocarbon pollution control technology, and in particular to a salt-tolerant composite microbial community, its preparation method, and its application in the remediation of high-salt oil sludge. Background Technology

[0002] Oil sludge is a waste generated during oilfield development, petroleum refining, transportation, and storage. Its main components are petroleum hydrocarbons, water, and solid particles such as soil. This type of material is listed in the "National Hazardous Waste List (2021)" (HW08). If not properly treated, it can cause serious harm to human health and the ecological environment. Therefore, the development of efficient oil sludge degradation technologies has significant economic and social benefits.

[0003] Currently, oil sludge treatment technologies mainly include physical methods (such as mechanical separation, freeze-thaw cycles, air flotation, and ultrasonic treatment), chemical methods (such as hot washing, incineration, pyrolysis, stabilization / solidification, and chemical oxidation), and biological methods. Among these, biological methods have attracted much attention due to their significant advantages such as low cost, thorough degradation, and environmental friendliness. Biological methods primarily employ land cultivation, composting, and bioreactor methods. Bioreactor methods, due to their short treatment cycle, involve mixing oil sludge with water in a reactor, where microorganisms degrade petroleum hydrocarbons into low-toxicity intermediate products or ultimately mineralize them into carbon dioxide and water. The degradation capacity of microorganisms directly determines the reactor's treatment efficiency; therefore, optimizing the microbial community structure and function is crucial for improving system performance. Against this backdrop, bioaugmentation methods (adding exogenous single strains or complex microbial communities) have emerged, enhancing system treatment performance by introducing highly efficient degrading bacteria. Practice has shown that the selection of highly efficient petroleum hydrocarbon-degrading bacteria is key, and compared to single strains, complex microbial communities exhibit superior performance in terms of functional complementarity and environmental adaptability.

[0004] Many of my country's oil fields are located in saline-alkali flats or inland saline-alkali areas. During the oil extraction, processing and transportation process, spilled oil, pipeline leaks or accidents can cause the saline-alkali soil around the oil fields to be polluted, forming high-salt oil sludge (referred to as high-salt oil sludge). Meanwhile, oil and gas extraction water contains petroleum components and salt (salinity can be as high as 200 g / L). When this extraction water leaks into the ground or surface due to leaks from storage tanks or pipelines, it will also produce sludge with both petroleum pollution and high salinity characteristics [Quartaroli, L., Silva, CM, de Paula, SO, da Silva, CC, de Souza, RS, Bassin, JP, 2017. Nitrification of Petroleum Extraction Produced Water: Salt Concentrations and Nitrifying Activity. Environmental EngineeringScience 34(4), 258-264.][Fathepure Babu Z. Recent studies in microbialdegradation of petroleum hydrocarbons in hypersaline environments. Frontiersin Microbiology, 2014. 5.]. Marine oil spills will also produce a large amount of oil-bearing sediments in high-salinity environments. Taking the Penglai 19-3 oil spill in the Bohai Sea in June 2011 as an example, the accident resulted in the leakage of approximately 115 m³ of crude oil and the deposition of 420 m³ of oil-based mud, polluting an area of ​​1600 km² [Guo J, Xie Q, Liu X. Observation of the Penglai 19-3 oil leak and its impact on the sea area ecosystem. IEEE International Geoscience & Remote Sensing Symposium, 2012. 919-922.]. Traditional methods for treating such high-salinity oil sludge, such as dilution and desalination or pretreatment desalination (reverse osmosis, ion exchange, and electrodialysis, etc.), are often costly and have limited efficiency. In contrast, microbial remediation technology can directly degrade oil sludge under high-salinity conditions, avoiding additional desalination treatment. Therefore, conducting research on the degradation of oil sludge by complex microbial communities in high-salinity environments not only has important scientific value but also significant economic benefits and environmental significance.

[0005] In the bioremediation of oil sludge in high-salt environments, a complex microbial community is typically constructed by isolating multiple salt-tolerant or halophilic bacterial strains from oil fields, industrial wastewater, and saline environments. Common strains include *Halomonas* spp. Halomonas ), Bacillus spp. ( Bacillus ), Rhodococcus spp. ( Rhodococcus Marine bacteria () Marinobacter )wait. Halomonas It is a typical representative of moderately halophilic bacteria (salt concentration range 30-150 g / L), and taxonomically belongs to the class Gamma-Proteobacteria. γ - Proteobacteria ), Marine Spirospirales ( Oceanospirillales ), Halomonas family ( Halomonadaceae To cope with high osmotic pressure stress, Halomonas Cellular activity is maintained by accumulating large amounts of osmotic-compensating solutes to balance osmotic pressure inside and outside the cell. Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is a typical example of an osmotic-compensating solute and is a cyclic derivative of aspartic acid. Based on the metabolic characteristics of ectoine, strains can be divided into two categories: non-secretory types (such as...) H. elongata DSM 2581T accumulates only intracellularly; secretory types (such as...) H. salinaDSM 5928T can secrete ectoine into the extracellular environment and accumulate it in the culture medium under conditions of relatively constant environmental osmotic pressure [Zhang LH, Lang YJ, Nagata S. Efficient production of Ectoine using Ectoine-excreting strain. Extremophiles, 2009. 13(4):717-724.]. The "Kunte model" explains the mechanism by which non-secreting strains trigger negative feedback inhibition through an ectoine concentration threshold. However, secreting strains relieve this inhibition due to continuous efflux (release rate > absorption rate), forming a "generalized Kunte model" [Gao S., et al., Comparison of ectoine synthesis regulation in secreting and non-secreting strains of Halomonas. Annals of Microbiology, 2014. 64(3): 1357-1361.]. Under hyperosmolar stress, non-halophilic bacteria (such as *Escherichiacoli*) take up extracellular ectoine via the ProP (constitutive low-affinity transport system) and ProU (protein-dependent high-affinity transport system). The amount absorbed is positively correlated with osmotic pressure and accumulates intracellularly without being metabolized [Jebbar M, Talibart R, Gloux K, et al. Osmoprotection of *Escherichia coli* by ectoine: uptake and accumulation characteristics. Journal of Bacteriology, 1992. 174(15):5027-5035.]. Ectoine absorbed by non-halophilic bacteria has stabilizing effects on enzymes, proteins, nucleic acids, and cells.Scholars have offered several explanations, including the "water replacement hypothesis" [Clegg JS, Seitz P, Seitz W et al. Cellular responses to extreme water loss: the water-replacement hypothesis. Cryobiology, 1982. 19:306-316.], the "preferential exclusion model" [Arakawa T, Timasheff SN. The stabilization of proteins by osmolytes. Biophysical Journal, 1985. 47(3):411-414.], and the "molecular crowding theory" [Zimmerman SB, Minton A P. Macromolecular crowding: biochemical, biophysical and physiological consequences. Annual Review of Biophysics and Biomolecular Structure, 1993. 22:27-65.]. This synergistic "secretion-uptake" mechanism enables the complex microbial community to acquire overall stress resistance.

[0006] Currently, biological treatment of oil sludge mostly employs methods such as field cultivation and composting, but these methods are unsuitable for high-salt environments with large fluctuations in osmotic pressure, and the microbial agents are easily deactivated. In addition, there are methods that utilize microorganisms to degrade oil sludge in high-salt environments, but their efficiency remains low. The main reason is that high salt inhibits common non-salt-tolerant petroleum hydrocarbon-degrading strains (such as Acinetobacter calcareae)... Acinetobacter calcoaceticus Pseudomonas aeruginosa Pseudomonas aeruginosa The growth and metabolic activity of high-salt oil sludge are limited; many naturally salt-tolerant or halophilic strains have limited ability to degrade petroleum hydrocarbons, making it impossible to effectively treat high-salt oil sludge.

[0007] Therefore, developing an efficient method for constructing sludge-degrading microbial communities suitable for high-salt environments has significant economic and social value. Summary of the Invention

[0008] This invention provides a salt-tolerant composite microbial community, its preparation method, and its application in the remediation of high-salt oily sludge, in order to overcome the above-mentioned problems.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows: This invention provides a salt-tolerant complex bacterial group including halomonas. Halomonas venusta DSM4743, Acinetobacter calciacetate Acinetobacter calcoaceticus CCTCC KB 20081402, Pseudomonas aeruginosa Pseudomonas aeruginosa CCTCC AB 2010174; The ratio of viable counts of Acinetobacter calciacetate, Pseudomonas aeruginosa, and Halomonas is (0.5-2):(0.5-2):(0.5-2).

[0010] Furthermore, the ratio of viable counts of Acinetobacter calcareae, Pseudomonas aeruginosa, and Halomonas is 1:1:1.

[0011] This invention provides a method for preparing the aforementioned salt-tolerant complex bacterial community, characterized by comprising the following steps: (1) Acinetobacter calcitrinum, Pseudomonas aeruginosa, and Halomonas were inoculated into activation medium and cultured in shake flasks to obtain seed culture of each strain; (2) The seed liquid of each strain obtained in step (1) is added to the degradation substrate at an inoculation rate of 1% for scale-up fermentation culture to obtain salt-tolerant complex bacteria.

[0012] Further, in step (1), the activated culture medium comprises: 10 g / L peptone, 5 g / L yeast extract, 10-30 g / L NaCl, and pH 7.2; wherein the NaCl concentration used for Halomonas is 30 g / L, and the NaCl concentration used for Pseudomonas aeruginosa and Acinetobacter calcium acetate is 10 g / L.

[0013] Further, the degradation matrix components described in step (2) include: 20 g / L glucose, 20 g / L monosodium glutamate, 10 g / L trisodium citrate, 10 g / L ammonium sulfate, 1 g / L yeast extract, 3 g / L KH2PO4, 9 g / L K2HPO4·3H2O, 0.4 g / L MgSO4·7H2O, 0.01 g / L MnSO4·H2O, 0.01 g / L FePO4, 25.625 g / L NaCl, and pH 8.0.

[0014] This invention provides, in one aspect, the application of the salt-tolerant composite microbial community in the remediation of high-salt oily sludge.

[0015] Furthermore, the application method includes the following steps: (1) After mechanically crushing the high-salt oil sludge, it was soaked in a degradation matrix; (2) Add salt-tolerant complex bacteria to the system of step (1) at a concentration of 3% of the degradation substrate; (3) Degraded for 7 days at 30℃, 120 rpm and aerobic conditions.

[0016] Furthermore, the amount of high-salt oily sludge added is 1 / 8 of the mass of the degradation matrix.

[0017] The beneficial effects of this invention are: (1) The composite species of the present invention introduces Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) secretory strains. Halomonas venusta DSM 4743, together with two highly efficient degrading bacteria, established a unique "Ectoine secretion-transfer-degradation" synergistic mechanism. Halomonas venusta DSM 4743 can secrete Ectoine into the extracellular environment. *Acinetobacter calciacetate* and *Pseudomonas aeruginosa* can effectively absorb Ectoine, thus significantly improving the survival and degradation rates of non-salt-tolerant strains in high-salt environments. This fundamentally solves the core bottleneck of limited activity of non-salt-tolerant, highly efficient degrading bacteria in high-salt oil sludge. Experiments show that after treating oil sludge with a salinity of 35 g / L for 7 days using the method of this invention, the petroleum hydrocarbon degradation rate can reach 43.12%, far exceeding that of using only the other two degrading bacteria (5.18%) or using only one of them. Halomonas venusta The control group (12.41%) fully demonstrates the effectiveness and advantages of this synergistic mechanism. The technology based on this synergistic mechanism is particularly suitable for the remediation of oil sludge in high-salt environments such as offshore oilfields, oil-contaminated sites in saline-alkali land, and petrochemical wastewater treatment systems.

[0018] (2) The composite microbial community constructed in this invention possesses three functions: secreting ectoine, degrading petroleum hydrocarbons, and producing biosurfactants. Among them, H. venusta DSM 4743 secretes Ectoine to maintain the overall stress resistance of the bacterial community, and also has the function of degrading petroleum hydrocarbons. Acinetobacter calciacetate can degrade petroleum hydrocarbons, and Pseudomonas aeruginosa can degrade petroleum hydrocarbons and produce bioactive substances.

[0019] (3) The strains in the composite microbial community constructed in this invention do not exhibit growth inhibition or resource competition, thus achieving long-term stable coexistence. This characteristic enables the microbial community to adapt to complex environments and maintain continuous degradation capabilities in field applications. (4) The process of this method is clear. It does not require expensive desalination pretreatment of high-salt oil sludge. It directly utilizes the composite microbial community for biodegradation, which reduces the treatment cost. Attached Figure Description

[0020] 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.

[0021] Figure 1 Results of microbial diversity analysis for the degradation of high-salt landed oil sludge by a complex microbial community. Detailed Implementation

[0022] 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.

[0023] 1. Strains and materials: In this invention, moderately halophilic monoclonal bacteria Halomonas venusta DSM 4743, purchased from DSMZ (German Collection of Microorganisms and Cell Cultures), Germany; Pseudomonas aeruginosa ( Pseudomonas aeruginosa CCTCC AB 2010174, this strain was purchased from the China Center for Type Culture Collection (CCTCC); Acinetobacter calciacetate ( Acinetobacter calcoaceticus CCTCC KB 20081402, this strain was purchased from the China Center for Type Culture Collection (CCTCC).

[0024] Oil sludge sample: taken from Daqing Oilfield, with a salt content of about 35 g / L and an oil content of 8.38%.

[0025] 2. Culture medium: Activation medium (g / L): peptone 10, yeast extract 5, NaCl 10-30, pH 7.2, sterilized at 121℃ for 20 min. Halomonas venusta The activation medium used for DSM 4743 contains 30 g / L of NaCl. Pseudomonas. aeruginosa CCTCC AB 2010174 and ( A. calcoaceticus The activation medium used in CCTCC KB 20081402 contains 10 g / L of NaCl.

[0026] Degradation matrix: 20 g / L glucose, 20 g / L monosodium glutamate, 10 g / L trisodium citrate, 10 g / L ammonium sulfate, 1 g / L yeast extract, 3 g / L KH₂PO₄, 9 g / L K₂HPO₄·3H₂O, 0.4 g / L MgSO₄·7H₂O, 0.01 g / L MnSO₄·H₂O, 0.01 g / L FePO₄, 25.625 g / L NaCl, pH 8.0, autoclaved at 121 °C for 20 min. Glucose was added separately after sterilization (autoclaved at 105 °C for 20 min). Adding NaCl to the matrix to bring the system salt concentration to a specific value (e.g., 30 g / L) is to simulate the real environment of the target treatment site (the salt content of high-salt sludge in Daqing Oilfield is about 35 g / L), ensuring the activity and stability of the microbial community in subsequent field applications (such as composting or biomass treatment of high-salt sludge).

[0027] Ectoine induction medium: 37.4 g / L sodium glutamate, 3 g / L KH₂PO₄, 11.8 g / L K₂HPO₄·3H₂O, 0.4 g / L MgSO₄·7H₂O, 0.01 g / L MnSO₄·H₂O, 58.44 g / L NaCl. pH 7.0, autoclaved at 121 ℃ for 15 min.

[0028] Ectoine concentration determination method. Extracellular Ectoine concentration determination: Centrifuge the sample at 14000×g, and use the supernatant for HPLC determination (e.g., Ectoine secreted by cells). Intracellular Ectoine concentration determination: Centrifuge as described above, collect the precipitate, wash with NaCl-Kpi buffer (100 mM, pH 7.2, NaCl concentration the same as the culture medium concentration), add an equal volume of 80% ethanol (V / V) to the centrifuged precipitate for extraction, resuspend, and incubate overnight at room temperature. Centrifuge the suspension again, and use the supernatant for HPLC analysis (e.g., intracellular Ectoine). The total Ectoine concentration is the sum of the extracellular and intracellular Ectoine concentrations. Ectoine concentration is determined by HPLC. The chromatographic column is a TSK-GEL reversed phase column (TOSOH corporation, Japan). A UV detector with a detection wavelength of 210 nm is used.

[0029] Example 1: H. venusta Ectoine secretion characteristics of DSM 4743 Will H. venustaDSM 4743 was activated in activation medium for 24 h. The cells were then inoculated at a 1% inoculum into Ectoine induction medium and cultured at 30°C and 120 rpm. Samples were taken at 0 h, 48 h, and 72 h, and the supernatant was collected by centrifugation to determine intracellular and extracellular Ectoine levels.

[0030] The results showed that H. venusta After 48 hours of culture in DSM 4743, the total ectoine production reached 1282.5 mg / L. Extracellular ectoine was 1008.99 mg / L, and intracellular ectoine was 273.51 mg / L, with an ectoine secretion rate of 78.67%. After 72 hours, the total ectoine production increased to 3559.84 mg / L, with extracellular ectoine accumulation reaching 3088.92 mg / L and intracellular content at 470.92 mg / L, and the ectoine secretion rate increasing to 86.77%. H. venusta DSM 4743 can synthesize and secrete ectoine extracellularly. Ectoine production and secretion rates both significantly increase with prolonged culture time.

[0031] Example 2: Growth stability test of the composite microbial community in a high-salt oily sludge environment Halomonas Halomonas venusta DSM 4743, Acinetobacter calciacetate Acinetobacter calcoacetic acid CCTCC KB 20081402, Pseudomonas aeruginosa Pseudomonas aeruginosa CCTCC AB2010174 was activated on activation medium for 24 h to obtain seed culture of each strain. The seed culture of the three strains was then co-inoculated into Erlenmeyer flasks containing a degradation substrate at an inoculation rate of 10% (v / v) (viable cell ratio of 1:1:1). The degradation substrate included high-salt sludge from Daqing Oilfield (the salt content of the high-salt sludge was determined to be 35 g / L, with an addition rate of 5 g sludge / 40 mL substrate). The inoculated system was cultured at 30℃ and 120 rpm with shaking for 7 days. After culture, cell growth and microbial diversity were measured. The total viable cell count in the culture medium was determined using the plate count method (plate culture medium). After 7 days of culture, the viable cell concentration of the complex reached 2.83 × 10¹. 0 The CFU / mL level indicates that the complex microbial community can grow well in a high-salt sludge environment.

[0032] After the culture was completed, fermentation broth samples were taken and sent to Shanghai Paisenno Biotechnology Co., Ltd. to analyze the composition of the microbial community at the end of the culture using 16S rRNA gene high-throughput sequencing technology.

[0033] Figure 1 Microbial diversity analysis showed that the microbial community remained stably detectable at the end of the culture. Halomonas , Pseudomonas and Acinetobacter The strains belong to the same genus and their relative abundance is relatively balanced. This result indicates that the strains in this invention can stably coexist during co-culture.

[0034] Example 3 Ectoine-secreting bacteria H. venusta Experiment on DSM 4743 enhancing the ability of complex microbial communities to degrade petroleum hydrocarbons Set up a control group and an experimental group: Control group 1: Inoculated only with petroleum hydrocarbon degrading bacteria Pseudomonas aeruginosa CCTCC AB 2010174 and Acinetobacter calcoaceticus CCTCC KB 20081402.

[0035] Control group 2: Inoculated only with Ectoine-secreting bacteria Halomonas venusta DSM 4743.

[0036] Experimental group: Simultaneous inoculation with three strains of bacteria, namely Halomonas. Halomonas venusta DSM 4743, Acinetobacter calciacetate Acinetobacter calcoaceticus CCTCC KB 20081402, Pseudomonas aeruginosa Pseudomonas aeruginosa CCTCC AB 2010174.

[0037] The above-mentioned test strains were activated and cultured in LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH adjusted to 7.0-7.2) at 30℃ and 120 rpm for 24 hours to obtain seed culture.

[0038] 5.0 g of high-salt oil sludge from Daqing Oilfield (oil content 8.38%, salt content 35 g / L) was added to 40 mL of degradation matrix. Subsequently, the seed culture of each group was inoculated into the corresponding system at an inoculation rate of 10% (v / v).

[0039] All treatment groups were incubated in a shaker at 30℃ and 120 rpm for 7 days. After incubation, the mixture of oil sludge and culture medium was collected for determining the residual petroleum hydrocarbon content. The determination of petroleum hydrocarbon content was strictly carried out in accordance with the Environmental Protection Standard of the People's Republic of China, "Determination of Petroleum in Soil - Infrared Spectrophotometry (HJ 1051-2019)," and the petroleum hydrocarbon degradation rate was calculated accordingly. The formula for calculating the petroleum hydrocarbon degradation rate is as follows: Petroleum hydrocarbon degradation rate (%) = [(C0 - C7) / C0] × 100% In the formula: C0: initial content of petroleum hydrocarbons in the sludge before treatment (unit: mg / kg dry soil), C7: residual content of petroleum hydrocarbons in the sludge after 7 days of treatment with compound microbial community (unit: mg / kg dry soil).

[0040] After 7 days of cultivation, the degradation rates of petroleum hydrocarbons in each treatment group are shown in Table 1.

[0041] Table 1. No Addition / Addition Halomonas venusta DSM 4743's complex microbial community degrades petroleum hydrocarbons in oil sludge under saline-alkali conditions.

[0042] The results in Table 1 demonstrate that the addition of Ectoine-secreting bacteria... H. venusta DSM 4743 can produce a significant synergistic effect with petroleum hydrocarbon degrading bacteria, thereby greatly improving the overall degradation efficiency of petroleum hydrocarbons in oil sludge by the composite microbial community under high salinity conditions, verifying the effectiveness of the synergistic Ectoine secretion-transfer-degradation mechanism constructed in this invention.

[0043] 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 salt-tolerant complex bacterial community, characterized in that, Including Halomonas Halomonas venusta DSM 4743, Acinetobacter calciacetate Acinetobacter calcoaceticus CCTCC KB 20081402, Pseudomonas aeruginosa Pseudomonas aeruginosa CCTCC AB 2010174; The ratio of viable counts of Acinetobacter calciacetate, Pseudomonas aeruginosa, and Halomonas is (0.5-2):(0.5-2):(0.5-2).

2. The salt-tolerant complex microbial community according to claim 1, characterized in that, The ratio of viable counts of Acinetobacter calcareae, Pseudomonas aeruginosa, and Halomonas is 1:1:

1.

3. A method for preparing the salt-tolerant complex bacterial flora according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Acinetobacter calcitrinum, Pseudomonas aeruginosa, and Halomonas were inoculated into activation medium and cultured in shake flasks to obtain seed culture of each strain; (2) The seed liquid of each strain obtained in step (1) is added to the degradation substrate at an inoculation rate of 1% for scale-up fermentation culture to obtain the salt-tolerant complex bacterial group.

4. The preparation method according to claim 3, characterized in that, In step (1), the activated culture medium consists of: 10 g / L peptone, 5 g / L yeast extract, 10-30 g / L NaCl, and pH 7.2; wherein the NaCl concentration used for Halomonas is 30 g / L, and the NaCl concentration used for Pseudomonas aeruginosa and Acinetobacter calcium acetate is 10 g / L.

5. The preparation method according to claim 3, characterized in that, The degradation matrix components described in step (2) include: glucose 20 g / L, monosodium glutamate 20 g / L, trisodium citrate 10 g / L, ammonium sulfate 10 g / L, yeast extract 1 g / L, KH2PO4 3 g / L, K2HPO4·3H2O 9 g / L, MgSO4·7H2O 0.4 g / L, MnSO4·H2O 0.01 g / L, FePO4 0.01 g / L, NaCl 25.625 g / L, pH 8.

0.

6. The application of the salt-tolerant composite microbial community according to any one of claims 1-2 in the remediation of high-salt oily sludge.

7. The application according to claim 6, characterized in that, The application method includes the following steps: (1) After mechanically crushing the high-salt oil sludge, it was soaked in a degradation matrix; (2) Add salt-tolerant complex bacteria to the system of step (1) at a concentration of 3% of the degradation substrate; (3) Degraded for 7 days at 30℃, 120 rpm and aerobic conditions.

8. The application according to claim 7, characterized in that, The amount of high-salt oily sludge added is 1 / 8 of the mass of the degradation matrix.

Images