Method for rapidly degrading petroleum hydrocarbon in high-salinity oil-containing wastewater

Abstract

The invention provides a method for rapidly degrading petroleum hydrocarbon in high-salinity oily wastewater, pseudomonas Z1 and achromobacter denitrificans M1 are compounded, an auxiliary liquid is added to obtain a microbial inoculum, the degradation speed of the microbial inoculum to petroleum hydrocarbon and the load tolerance to COD in the wastewater are obviously higher than those of a single strain, and the microbial inoculum has good salinity adaptability. The method has the advantages that in actual wastewater biochemical treatment, flora advantages can be quickly established in a short time under the condition of a small addition amount, the abundance and diversity of microorganisms in a system are effectively improved, and a good petroleum hydrocarbon degradation effect can be achieved within limited hydraulic retention time through continuous operation.

Description

Technical Field

[0001] The invention belongs to the field of oily wastewater treatment, and in particular relates to a method for rapidly degrading petroleum hydrocarbons in high-salinity oily wastewater. Background Art

[0002] With the rapid development of the petroleum industry, a large amount of oily wastewater is inevitably generated during the production, storage, transportation, processing and use of oil, especially the high-salt oily wastewater generated during oil processing and port transportation. These wastewaters have the characteristics of high crude oil content, high salinity, and large fluctuations in batch wastewater, which have a great impact on the continuous operation of the wastewater treatment process, especially the biochemical treatment section in the treatment process, and ultimately result in unsatisfactory wastewater treatment effects and failure to meet treatment requirements.

[0003] At present, the treatment methods for high-salinity oily wastewater include physical method, chemical method and biological method. Biological treatment has the characteristics of high treatment effect, mild conditions, low energy consumption and no secondary pollution. It is usually used in the third stage of oily wastewater treatment, that is, after static oil separation, air flotation oil separation and filtration, the COD of wastewater in this stage is Cr 1000~1500mg / L, the organic matter is mainly long-chain alkanes, difficult-to-degrade hydrocarbon heavy oil, which is emulsified in water, with oil droplet size less than 50μm and salinity of 0.5~1.5%. It is necessary to cultivate special functional microorganisms that are salt-tolerant and have the ability to efficiently degrade long-chain alkanes, introduce them into the biochemical system, and improve the efficiency of organic matter degradation.

[0004] The high efficiency of biological treatment mainly depends on the dominant position of efficient alkane-degrading bacteria and the maintenance of their abundance. Usually in the process of wastewater treatment, low efficiency is mainly reflected in long hydraulic retention time and short microbial retention time.

[0005] At present, in the relevant research and development, patent CN118389348A discloses a strain of Pseudomonas sp. Z1 that can produce surfactants. This strain can transport petroleum hydrocarbons into the cell for degradation by producing surfactants. The degradation rate of alkanes reaches 85% in a suitable salinity and pH environment; patent CN118406583A discloses a strain of Achromobacter denitrificans M1 suitable for the degradation of medium- and long-chain alkanes in saline crude oil wastewater. This strain can produce lipopeptide protein surfactants. The degradation rate of crude oil wastewater after 3 days can reach 90% under suitable salinity and pH. The above strain changes the microemulsification state of petroleum hydrocarbons in water by secreting surfactants or surfactants outside the cell under aerobic conditions, thereby allowing petroleum hydrocarbons to be adsorbed around the cell, and transporting petroleum hydrocarbons into the cell, and converting petroleum hydrocarbons into CO2 through fatty acid metabolism; some petroleum hydrocarbons are adsorbed on the cell surface, and due to the density difference of the bacteria / petroleum hydrocarbon mixture, they are either suspended in the water phase or settled in the sludge.

[0006] Although the strain disclosed in the above patent has a high degradation rate for petroleum hydrocarbons, it is limited by the tolerance and degradation pathway of a single strain in the actual wastewater biochemical treatment process. It usually takes a long time after addition and continues to add a larger dosage to establish a dominant bacterial community in the water body and obtain a higher petroleum hydrocarbon degradation rate. In the actual wastewater treatment process, the hydraulic retention time is usually short, and the relatively slow degradation rate of the strain cannot fully meet the requirements. This problem is particularly prominent in the treatment of wastewater with a high petroleum hydrocarbon content (2000ppm). Considering the actual wastewater treatment capacity and economic cost, it is necessary to develop a method that can quickly and efficiently degrade petroleum hydrocarbons in high-salinity oily wastewater on the existing basis. Summary of the invention

[0007] The specific technical solutions of the present invention are as follows:

[0008] A method for rapidly degrading petroleum hydrocarbons in high-salinity oily wastewater, characterized by comprising the following steps:

[0009] 1) Pseudomonas and Achromobacter denitrificans are cultured in fermentation medium respectively; wherein:

[0010] The culture conditions of Pseudomonas were as follows: medium salinity 1.5%; initial carbon source concentration 5 g / L, feed concentration 0.5 g / L; initial pH 7.5; dissolved oxygen 50%; culture temperature 30°C;

[0011] The culture conditions of Achromobacter denitrificans are as follows: medium salinity 1.0%; initial carbon source concentration 5g / L, feed concentration 0.5g / L; initial pH 7.5; dissolved oxygen 50%; culture temperature 30°C; wherein:

[0012] The Pseudomonas is disclosed in patent CN118389348A, the strain number is Z1, and it is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number: CGMCC No.30070;

[0013] The denitrifying Achromobacter is disclosed in patent CN118406583A, the strain number is M1, and it is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number: CGMCC No.29698;

[0014] 2) The bacterial cultures of the two strains were concentrated to a cell concentration of no less than 8×10 13 CFU; then the concentrated solutions of the two strains were mixed in a ratio of 1:1 in terms of the number of live bacteria, and then one volume of auxiliary liquid was added to the mixed bacterial solution to obtain a bacterial agent; the components of the auxiliary liquid were as follows: Na2HPO4 1.0g / L, KH2PO4 0.5g / L, NH4Cl 1.1g / L, MgSO4 0.2g / L, CaCl20.02g / L, Fe2(SO4)3 0.02g / L, NaCl 30g / L, hexadecane 2000ppm, and the balance was water;

[0015] 3) Add bacterial agents and hydrophilic polyurethane fillers to high-salinity oily wastewater to degrade petroleum hydrocarbons in the wastewater under an aerobic environment.

[0016] Furthermore, the degradation of petroleum hydrocarbons in the wastewater in step 3) is carried out in an aerobic tank, with the amount of wastewater entering the tank being 8 to 10 m3 per day. 3 The corresponding dosage method of the bacterial agent is: the initial dosage is 1-2kg, and then 0.5-1kg is added every day until the COD of the wastewater is no higher than 300mg / L.

[0017] Furthermore, during the degradation of petroleum hydrocarbons in the wastewater in step 3), the pH of the wastewater is maintained at 6.5 to 7.5; and the dissolved oxygen in the wastewater is maintained at 2.5 to 4.5 ppm.

[0018] Furthermore, in the above step 3), after the bacterial agent is added for the first time, an appropriate amount of C, N, and P nutrient sources are added to the wastewater; so that the molar ratio of C, N, and P in the wastewater after the addition is 100:5:1.

[0019] Furthermore, in the above step 3), the dosage of the hydrophilic polyurethane filler is 0.1 to 0.2 times of the total volume of the wastewater by volume.

[0020] Furthermore, the initial COD of the high-salinity oily wastewater is 1000-3000 mg / L, and the salinity is 0.8-2.0%.

[0021] Furthermore, in the step 1), the culture time of Pseudomonas is 40 hours; the culture time of Achromobacter denitrificans is 42 hours.

[0022] The present invention can rapidly degrade petroleum hydrocarbons in high-salinity oily wastewater. The degradation rate of petroleum hydrocarbons and the load tolerance of COD in wastewater by the microbial agent are significantly higher than those of a single bacterial species, and the agent has good salinity adaptability. In actual wastewater biochemical treatment, the bacterial community advantage can be quickly established with a small amount of addition and in a short time, effectively improving the abundance and diversity of microorganisms in the system. Continuous operation can achieve a better degradation effect of petroleum hydrocarbons within a limited hydraulic retention time. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Results of growth antagonism analysis between the two strains.

[0024] Figure 2 Comparison of short-term degradation efficiency of single strain and mixed bacteria in different compound ratios.

[0025] Figure 3 Comparison of alkane degradation efficiency of mixed bacteria under different salinities.

[0026] Figure 4 Effect of different initial alkane concentrations on the degradation ability of mixed bacteria.

[0027] Figure 5 Growth curve of Pseudomonas Z1 during fermentation.

[0028] Figure 6 Growth curve of Achromobacter denitrifyingus M1 during fermentation.

[0029] Figure 7 The trend of dissolved oxygen change in the system when bacterial agents are used in A / O wastewater treatment system to treat port oily wastewater.

[0030] Figure 8 The trend of system pH change when bacterial agents are used in A / O wastewater treatment system to treat port oily wastewater.

[0031] Fig. 9 COD variation trend when bacterial agent is used to treat port oily wastewater in A / O wastewater treatment system. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below in conjunction with specific embodiments. The materials and methods used in the present invention all belong to the conventional technical field of the present invention.

[0033] Example 1

[0034] Analysis of strain antagonism: The agar diffusion Oxford cup method was used to analyze the growth antagonism of different strains. The specific operation is as follows: Prepare LB solid plates with a salinity of 1% according to the conventional method. The components (g / L) of solid LB culture medium are tryptone 10, yeast extract 5, NaCl 10, and agar 20. Spread the bacterial liquid of strains denitrifying colorless bacteria M1 and Pseudomonas Z1 on the solid plate, and let it stand for 5 minutes with the front side facing up to allow the bacterial liquid to absorb. Then, gently place two sterile Oxford cups vertically on the left and right sides of the solid plate coated with bacterial liquid, and press them lightly with tweezers to make them in close contact with the surface of the culture medium. Then add 200μL of the corresponding bacterial inoculation liquid to the left Oxford cup, and add the same volume of sterile water to the right Oxford cup as a control. Finally, after standing at room temperature for 1h, place it in the incubator and observe whether there is an antagonistic circle after culturing at 30℃ for 12-18h.

[0035] The experimental results show that there is no antagonism between the growth of Achromobacter denitrificans M1 and Pseudomonas alcaligenes Z1. Figure 1 As shown in A, there is no antagonistic zone between the plate coated with denitrifying Achromobacter M1 and the Oxford cup on the right with the inoculum of Pseudomonas. Similarly, there is no antagonistic zone between the plate coated with Pseudomonas Z1 and the Oxford cup on the right with the inoculum of denitrifying Achromobacter M1 ( Figure 1 B).

[0036] Example 2

[0037] Comparison of short-term degradation efficiency of single strains and mixed bacteria with different compound ratios: Hexadecane was used as the sole carbon source to activate the two strains, Achromobacter denitrificans M1 and Pseudomonas Z1, and cultured them to the logarithmic growth phase. Then the bacterial solution was adjusted to OD 600 nmThe values ​​were the same, and the compound ratios were prepared into mixed inoculum according to the ratio of the live bacteria count of denitrifying Achromobacter M1 and Pseudomonas Z1 of 1:1, 1:2, and 2:1, respectively. The mixed inoculum was inoculated into a culture medium containing alkane concentration of 1000ppm at an inoculum volume of 8% (v / v), and the samples were taken after shaking culture at 30℃ and 160r / min for 24h in a shaker to determine the alkane degradation rate. At the same time, a single strain control group was set for comparison. There were 3 parallels for both the control group and the experimental group. The above alkane culture medium components are as follows (g / L): Na2HPO4 0.6, KH2PO4 0.3, NH4Cl 0.66, MgSO4 0.12, CaCl2 0.012, Fe2(SO4)3 0.012, hexadecane 145μL, pH 7.5, salinity 1%, distilled water to 1L, and sterilized at 121℃ for 20min. For details on the alkane extraction steps and the calculation method of the alkane degradation rate, please refer to the "Determination of Petroleum in Water Quality by Ultraviolet Spectrophotometry (Trial)" (HJ 970-2018). First, the supernatant of different bacterial liquids was extracted by n-hexane extraction method, the absorbance of the extract at 225 nm was measured, and then the alkane degradation rate was calculated.

[0038] The results showed that Figure 2 As shown in the figure, when the compounding ratio is 1:1, the alkane degradation efficiency in the culture system is the best, and the alkane degradation rate in the 24h system can reach more than 80%, which is significantly higher than that of a single strain and mixed bacteria of other compounding ratios. The mixed bacteria or bacterial agents in the subsequent embodiments are all implemented according to the compounding ratio of two strains of bacteria of 1:1.

[0039] Example 3

[0040] Comparison of alkane degradation efficiency of mixed bacteria under different salinities: Alkane selective culture medium with salinities of 0.5%, 1%, 1.5%, 2%, 2.5%, and 3% was prepared (the culture medium contained 1000 ppm of n-hexadecane). The mixed bacteria were inoculated into the sterilized alkane selective culture medium with different salinities at an inoculum volume of 8% (v / v). The initial concentration of the bacterial solution after inoculation was OD 600 The value was 0.24. Single strains M1 and Z1 were used as blank control groups.

[0041] After 24 hours of shaking culture at 30°C, the supernatants of different bacterial cultures were extracted by n-hexane extraction, and the absorbance of the extract at 225 nm was measured to calculate the alkane degradation rate. The extraction steps and the alkane degradation rate calculation method are referred to Example 2.

[0042] The results are as follows Figure 3 As shown: the mixed bacteria had the highest alkane degradation rate under salinity conditions of 1-1.5%, exceeding 80%; when the salinity reached 2.5%, the 24-hour alkane degradation rate still remained at around 55%, and the alkane degradation rate in the salinity range of 1-2.5 was significantly higher than that of single strains M1 and Z1.

[0043] Example 4

[0044] Effect of different alkane initial concentrations on the degradation ability of mixed bacteria: Alkane selective culture medium with alkane initial concentrations (ppm) of 3000, 2000, 1000, and 500 was prepared, with a salinity of 1% and a pH of 7.5. The mixed bacteria were inoculated into the sterilized selective culture medium with different alkane initial concentrations at an inoculation rate of 8% (v / v). The initial concentration of the bacterial solution after inoculation was OD 600 The value was 0.24. Single strains M1 and Z1 were used as blank control groups.

[0045] After 24 h of shaking culture at 30°C, the supernatants of different bacterial cultures were extracted by n-hexane extraction, and the absorbance of the extract at 225 nm was measured to calculate the alkane content. The extraction steps and alkane content calculation method refer to the "Water Quality Determination of Petroleum-based Ultraviolet Spectrophotometry (Trial)" (HJ 970-2018).

[0046] The results are as follows Figure 4 As shown in the figure: When the alkane concentration is <1000, there is no significant difference in the alkane degradation effect between the control group and the test group. When the initial alkane concentration is >1000, as the alkane concentration increases, the alkane degradation amount of the test group is significantly higher than that of the control group, indicating that the tolerance of mixed bacteria to high concentrations of alkanes is much higher than that of single strains. Under the same conditions, after 24 hours of cultivation, the amount of residual alkanes in the mixed bacterial solution is much lower than that of the single strain control group.

[0047] Example 5

[0048] Application of bacterial agents in A / O wastewater treatment process:

[0049] 1) Pseudomonas Z1 and Achromobacter denitrificans M1 were cultured in 5L fermenters respectively:

[0050] The culture conditions of strain Z1 were as follows: medium salinity 1.5%; initial carbon source concentration 5 g / L, feed concentration 0.5 g / L; initial pH 7.5, regulated by feed ammonia water; dissolved oxygen 50%, regulated by adjusting stirring speed; culture temperature 30°C, regulated by cooling water;

[0051] The growth of strain Z1 during the fermentation process was as follows Figure 5 As shown: the strain has a short growth lag phase (0-16h), a long exponential growth phase of 20h, and enters the growth stable phase at 36h. The biomass accumulation rate is fast, and the OD600 value is as high as 18. During the fermentation process, the strain produces a large amount of foam due to the production of surfactants, and an appropriate amount of defoaming agent needs to be added. The fermentation time of Pseudomonas alcaligenes Z1 is 40h, and the fermentation broth is used for subsequent bacterial agent preparation.

[0052] The culture conditions of strain M1 were as follows: medium salinity 1.0%; initial carbon source concentration 5 g / L, fed concentration 0.5 g / L; initial pH 7.5, regulated by fed ammonia water; dissolved oxygen 50%, regulated by adjusting stirring speed; culture temperature 30°C, regulated by cooling water.

[0053] The growth of strain M1 during fermentation was as follows Figure 6 As shown: the strain has a long growth lag phase (0-24h), an exponential growth phase of 12h, and enters a stable growth phase at 36h. The biomass accumulates quickly, with an OD600 value of up to 22. During the fermentation process, less foam is produced. The fermentation time of Achromobacter denitrificans M1 is 42h, and the fermentation broth is used for subsequent bacterial agent preparation.

[0054] 2) The fermentation broths of the two strains were placed at 4°C for 8-10 hours, the bacteria at the bottom were collected, and about 4 / 3 of the supernatant of the fermentation broth was discarded to make the bacterial cell concentration of each strain after concentration to be 8×10 13 CFU; the concentrates of the two strains were mixed in a 1:1 ratio according to the number of live bacteria, and then one volume of auxiliary liquid was added to the mixed bacterial solution to obtain a bacterial agent.

[0055] The composition of the auxiliary liquid is as follows: Na2HPO4 1.0 g / L, KH2PO4 0.5 g / L, NH4Cl 1.1 g / L, MgSO4 0.2 g / L, CaCl2 0.02 g / L, Fe2(SO4)3 0.02 g / L, NaCl 30 g / L, hexadecane 2000 ppm, and the balance is water.

[0056] 3) Apply the bacterial agent to the A / O wastewater treatment process system, which has a total volume of 20m 3 The special bacteria continuous expansion biochemical system has a water treatment capacity of 10m 3 / d, which can realize real-time intelligent monitoring of dissolved oxygen, pH and COD. The influent of the expansion system is port oily sewage (COD 1000-1500mg / L), hydrophilic polyurethane filler is added to the aerobic pool, and low-concentration batch operation is started. Its process mainly includes three stages: enrichment of microbial populations in the system, establishment of alkane-degrading bacterial community and treatment of oily wastewater. The enrichment of microbial populations in the system is to pump the sewage and sludge of the aerobic pool of the biochemical system into the aerobic zone of the special bacteria expansion equipment, and allow aerobic bacteria to fully proliferate in a saturated aerated environment; then, a small amount of bacterial agent is added to the raw water of the aerobic pool every day to establish the alkane-degrading bacterial community; after entering the oily wastewater treatment stage, bacterial agents are added in multiple batches. After multiple cycles of operation, while efficiently degrading petroleum hydrocarbons, an aerobic pool biochemical colony environment with alkane-degrading bacteria as the dominant bacterial community is formed.

[0057] Taking the first operation cycle (3 days) in the oily wastewater treatment stage as an example, the specific operations are as follows:

[0058] On the first day, the aerobic pool was filled with 10m 3 , add 1.5kg of bacterial agent, and then add appropriate amount of C, N, P nutrient sources; make the molar ratio of C, N, P in the wastewater after addition to be 100:5:1; among which, the amount of C nutrient source is the sum of COD in the wastewater and the added carbon source. On the second and third days, add 0.5kg of bacterial agent every day, and measure the dissolved oxygen, pH and COD of the aerobic pool water at 9:00, 14:00 and 19:00 every day.

[0059] Wastewater treatment and monitoring results Figures 7 to 9 As shown in Figure 2. Among them, the dissolved oxygen changes as shown in Figure 2. Figure 7 As shown in the figure: During the operation of this cycle, the dissolved oxygen dropped rapidly to 0.9 after the carbon source and bacterial agent were added, and dropped to 1.6 after the second bacterial agent supplementation. This shows that the abundance of the bacterial community increased after the carbon source or bacterial agent was added, and the increased metabolic aerobicity led to a rapid drop in dissolved oxygen. The dissolved oxygen was maintained between 3 and 4.5 during other time periods. The pH changes are shown in the figure. Figure 8 As shown in the figure, except for the pH value dropping to 6.8 at the time point with low dissolved oxygen after carbon source supplementation, the pH value remained stable at around 7.3 during the rest of the time period. Fig. 9 As shown: Under the condition that the dissolved oxygen and pH value of the whole system are basically stable, the COD of the wastewater decreases at a fast rate. On the third day, it has dropped from the initial 730 to 289, and the COD removal rate has reached 60.41%.

Claims

1. A method for rapidly degrading petroleum hydrocarbons in high-salinity oily wastewater, characterized in that The steps include: 1) Pseudomonas and Achromobacter denitrificans are cultured in fermentation medium respectively; wherein: The culture conditions of Pseudomonas were as follows: medium salinity 1.5%; initial carbon source concentration 5 g / L, feed concentration 0.5 g / L; initial pH 7.5; dissolved oxygen 50%; culture temperature 30°C; The culture conditions of Achromobacter denitrificans are as follows: medium salinity 1.0%; initial carbon source concentration 5g / L, feed concentration 0.5g / L; initial pH 7.5; dissolved oxygen 50%; culture temperature 30°C; wherein: The Pseudomonas is disclosed in patent CN118389348A, the strain number is Z1, and it is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number: CGMCC No.30070; The denitrifying Achromobacter is disclosed in patent CN118406583A, the strain number is M1, and it is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number: CGMCC No.29698; 2) The culture solutions of the two strains were concentrated to a cell concentration of no less than 8×10 13 CFU; then the concentrated solutions of the two strains were mixed in a ratio of 1:1 in terms of the number of live bacteria, and then one volume of auxiliary liquid was added to the mixed bacterial solution to obtain a bacterial agent; the components of the auxiliary liquid were as follows: Na2HPO4 1.0g / L, KH2PO4 0.5g / L, NH4Cl 1.1g / L, MgSO4 0.2g / L, CaCl20.02g / L, Fe2(SO4)3 0.02g / L, NaCl 30g / L, hexadecane 2000ppm, and the balance was water; 3) Add bacterial agents and hydrophilic polyurethane fillers to high-salinity oily wastewater to degrade petroleum hydrocarbons in the wastewater under an aerobic environment.

2. The method for rapidly degrading petroleum hydrocarbons in high-salinity oily wastewater according to claim 1, characterized in that: The degradation of petroleum hydrocarbons in the wastewater in step 3) is carried out in an aerobic tank, with the amount of wastewater entering the tank being 8 to 10 m3 per day. 3 The corresponding dosage method of the bacterial agent is: the initial dosage is 1-2kg, and then 0.5-1kg is added every day until the COD of the wastewater is no higher than 300mg / L.

3. The method for rapidly degrading petroleum hydrocarbons in high-salinity oily wastewater according to claim 1, characterized in that: During the degradation of petroleum hydrocarbons in the wastewater in step 3), the pH of the wastewater is maintained at 6.5 to 7.5; and the dissolved oxygen in the wastewater is maintained at 2.5 to 4.5 ppm.

4. The method for rapidly degrading petroleum hydrocarbons in high-salinity oily wastewater according to claim 2, characterized in that: In the step 3), after the bacterial agent is added for the first time, an appropriate amount of C, N, and P nutrient sources are added to the wastewater; so that the molar ratio of C, N, and P in the wastewater after the addition is 100:5:

1.

5. The method for rapidly degrading petroleum hydrocarbons in high-salinity oily wastewater according to claim 1, characterized in that: The dosage of the hydrophilic polyurethane filler is 0.1 to 0.2 times of the total volume of the wastewater by volume.

6. The method for rapidly degrading petroleum hydrocarbons in high-salinity oily wastewater according to claim 1, characterized in that: The initial COD of the high-salinity oily wastewater is 1000-3000 mg / L, and the salinity is 0.8-2.0%.

7. The method for rapidly degrading petroleum hydrocarbons in high-salinity oily wastewater according to claim 1, characterized in that: In the step 1), the culture time of Pseudomonas is 40 hours; the culture time of Achromobacter denitrificans is 42 hours.

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