Microorganism modified zeolite and preparation and application thereof

Abstract

The invention discloses microorganism modified zeolite, the microorganism modified zeolite is zeolite on which microorganisms grow, and the microorganisms are bacillus, pseudomonas or enterobacter or any combination of bacillus, pseudomonas or enterobacter. The invention also discloses a preparation method and application of the composition. The microorganism modified zeolite provided by the invention can be used for repairing pollution of high-salt underground water, and particularly has an obvious repairing effect on underground water polluted by petroleum and ammonia nitrogen in a landfill of a refinery enterprise.

Description

Technical Field

[0001] The invention relates to a microbial modified zeolite and its preparation and application. Background Art

[0002] Due to the complex and highly toxic nature of pollutants, refining waste is highly susceptible to severe contamination of soil and groundwater in landfills and surrounding areas. Typical contaminants found in refining landfills include petroleum hydrocarbons and ammonia nitrogen. The volume of leachate from landfills is typically substantial, making treatment methods such as reverse osmosis, stripping, and electrochemical methods costly and often lead to secondary pollution. The high salinity, high concentrations of ammonia nitrogen, and petroleum contamination in landfill leachate also restrict the use of many treatment technologies. Summary of the Invention

[0003] To further enrich the options for refining waste pollution control, an embodiment of the present invention provides a microbially modified zeolite that, by coupling microbial degradation and modified zeolite adsorption, effectively treats groundwater containing high-salt, high-concentration ammonia and nitrogen-complex petroleum organic pollutants, with an oil removal rate of up to 72.26% and an ammonia and nitrogen removal rate of up to 90.11%.

[0004] As one aspect of the present invention, it relates to a microbial modified zeolite. The microbial modified zeolite is a zeolite on which microorganisms grow. The microorganisms are Bacillus, Pseudomonas or Enterobacter or any combination thereof.

[0005] In a specific embodiment, the Bacillus is a bacterium with a deposit number of CCTCC M 20231336; the Pseudomonas is a bacterium with a deposit number of CCTCC M 20231335; and the Enterobacter is a bacterium with a deposit number of CCTCC M20231337.

[0006] In a specific embodiment, the ratio of the Bacillus, the Pseudomonas and the Enterobacter is 1-2:1-3:1-2.

[0007] In a specific embodiment, the zeolite is an alkali-modified zeolite, more preferably an alkali-modified natural zeolite from Chifeng, Inner Mongolia. The alkali modification is NaOH modification. The natural zeolite from Chifeng is clinoptilolite with a density of 2.17-2.34 g / cm 3 The cation exchange capacity is 5751-6053 mmol / 100g, the ammonia adsorption value is 144.22-168.84 mmol / 100g, and the porosity is 50.3%-59.3%, making it conducive to microbial adsorption and fixation. Furthermore, the ore from Chifeng, Inner Mongolia, is inexpensive, with 30-60 mesh zeolite granules priced at 260-350 yuan / ton. Zeolite reserves are plentiful and abundant.

[0008] As another aspect of the present invention, a method for preparing the above-mentioned microbial modified zeolite comprises:

[0009] The alkali-modified zeolite and the microbial liquid are placed in sterilized water, kept at a constant temperature of 25° C.-30° C. for 30-36 hours, and then centrifuged to obtain a precipitate.

[0010] In a specific embodiment, the alkali-modified zeolite is obtained by the following method: 30-60 mesh Chifeng zeolite is added to NaOH and the reaction is carried out under the conditions of 120-150 rpm and 25-30 ° C for 4-8 hours. NaOH selectively removes silicon from the zeolite framework, reduces its silicon-aluminum ratio, and the smaller particle size of Na in the solution is reduced. + It will be partially introduced into the zeolite crystal structure, replacing the Ca 2+ Mg 2+ etc., which makes the effective pore size larger and the zeolite shows higher cation exchange performance. Clinoptilolite has a higher NH4 + Selectivity is greater than Na + , thereby promoting the same NH4 + Ion exchange can reduce the ammonia nitrogen concentration in the sewage solution.

[0011] In a specific embodiment, the microbial culture liquid is OD 600 = 0.8-1.5 of enriched activated bacterial solution.

[0012] As another aspect of the present invention, it relates to the application of the above-mentioned microbial modified zeolite in the treatment of refining waste pollution.

[0013] As another aspect of the present invention, it relates to a process for treating refining waste pollution, using the above-mentioned microbial-modified zeolite.

[0014] The modified zeolite obtained using the preparation method provided by the present invention has high adsorption efficiency and rapid adsorption. Its adsorption capacity for screened microorganisms is far higher than that of unmodified zeolite. Furthermore, its preparation cost is low and its operation is simple. The zeolite provided by the present invention has a porous structure. Through modification, the effective pore diameter and pore volume of the zeolite are greatly increased, improving mass transfer performance, making nutrients more accessible to microorganisms, promoting their growth, and significantly increasing microbial degradation efficiency. The microbially modified zeolite provided by the present invention can be used to remediate contaminated high-salinity groundwater, especially groundwater contaminated by petroleum and ammonia nitrogen at refinery landfills. The application scheme provided by the present invention is to enrich salt-tolerant and oil-reducing microorganisms from the contaminated groundwater environment of the original refinery landfill site. As the dominant indigenous bacteria, after being adsorbed and fixed by modified zeolite, the bacteria are not easily lost and are then added back to the original environment. The zeolite can also adsorb ammonia nitrogen to provide nutrients for the bacterial community, overcoming many limitations of traditional bioremediation in the actual application of groundwater remediation. After 7 days of indoor tank experimental simulation operation, the oil removal rate can reach 72.26%, and the ammonia nitrogen removal rate can reach 90.11%. The present invention has the advantages of a wide source of zeolite raw materials, low cost, simple and effective operation, and can be widely used in the remediation of contaminated groundwater in refinery landfill sites.

[0015] Biological Deposits

[0016] The strains screened in the present invention are: YQ2, a strain of Pantoea agglomerans; CQ1, a strain of Bacillus sp. CQ1; PS, a strain of Pseudomonas sp. PS; and WX, a strain of Enterobacter sp. WX. The CQ1, PS, and WX strains from the optimal combination (W7) were deposited in the China Center for Type Culture Collection, Wuhan University, 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China. The deposit date is July 19, 2023, and the deposit numbers are: PS: CCTCC M 20231335; CQ1: CCTCC M 20231336; and WX: CCTCC M20231337. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The oil reduction rate of crude oil under high salt conditions with different composite bacterial communities.

[0018] Figure 2 and Figure 3 SEM images of zeolite before and after modification.

[0019] Figure 4 This is a graph showing the changes in oil concentration and ammonia nitrogen concentration in the effluent after treatment with microbial modified zeolite for different periods of time. DETAILED DESCRIPTION

[0020] The technical solutions and applications of the present invention are described clearly and completely below through various embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0021] Example 1

[0022] This embodiment provides a method for screening salt-tolerant, oil-degrading bacteria from contaminated groundwater at a refinery landfill. The method uses Venezuelan crude oil as a single carbon source and screens the bacteria from the contaminated groundwater at the landfill. The screening process is as follows:

[0023] S1. Obtaining bacterial flora: 5 mL of contaminated groundwater was collected from groundwater monitoring wells in different areas of the refinery landfill and added to an enrichment culture medium. The culture medium was placed in a gyratory shaker at 30°C and 150 rpm for 24 hours to obtain a mixed bacterial solution.

[0024] The enrichment medium was prepared by sequentially adding 10 g of NaCl, 5 g of yeast extract powder, and 10 g of tryptone to 1000 ml of deionized water, adjusting the pH to 7, stirring the mixture to obtain the enrichment medium, and sterilizing the mixture by high pressure.

[0025] S2. Bacterial acclimation: Add crude oil to sterile distilled water to prepare a 10,000 mg / L crude oil culture medium. Pipette 2 ml of the mixed bacterial solution obtained in S1 and incubate on an oscillator at 30°C and 150 rpm for 7 days to complete the first acclimation. Then, prepare 15,000 mg / L and 20,000 mg / L crude oil culture media, and complete the second and third acclimation cycles in the same manner.

[0026] S3, isolate single bacteria: pipette 2 ml of the third acclimated bacterial solution in S2 into the enrichment medium and culture for 24 h. Take 1 ml of the enriched bacterial solution and add it to a test tube containing 9 ml of sterile deionized water. Vortex shaker to mix. Then take 1 ml of the bacterial solution from the front test tube to the back test tube. Set the gradient of 10 -3 -10 -9 Finally, take 0.1 mL of the solution and spread it on an agar plate. Place the plate upside down in an incubator and culture it. Select the plate with a single colony and pick a single colony with an inoculation loop for enrichment culture at 30°C for 24 hours.

[0027] S4, obtain salt-tolerant oil-degrading bacteria: add 4 ml of the enriched solution of different single bacteria in S3 to 100 ml of oil-containing water sample with a salinity of 5% and a crude oil concentration of 500 mg / L, and select the oil-degrading bacteria with a relatively significant crude oil degradation rate under high salt conditions. 600= 0.8-1.5 when the enriched activated bacterial solution.

[0028] After 7 days of shaking at 15°C and 120 rpm, crude oil degradation rates were determined by petroleum ether extraction followed by UV spectrophotometry. Each experiment was replicated in triplicate, and the degradation rates were averaged. The results showed that strains YQ2, CQ1, PS, and WX exhibited excellent salt tolerance, remaining viable at salinity levels below 10%. At a salinity of 5%, the four strains exhibited significantly higher degradation efficiencies against an initial crude oil concentration of 500 mg / L, reaching 17.4%, 15.98%, 26.99%, and 19.58%, respectively.

[0029] Example 2

[0030] Through Example 2, the inventors provide a method for optimizing the combination of different oil-reducing bacteria into an optimal composite bacterial population.

[0031] As shown in Example 1, PS bacteria have the best salt tolerance and oil reduction effect. Therefore, it was used as the main component of the bacterial consortium for compounding in different volume ratios. The compounding volumes of different single bacteria are shown in Table 1 below. The types and volumes of each single bacteria added to the compounding of each different bacterial consortium W in the table are different. The oil reduction experimental conditions and process are the same as in Example 1.

[0032] Table 1 Addition amount of each single bacteria in the composite bacterial community

[0033]

[0034]

[0035] The oil reduction rate of each composite bacterial group is shown in the attached Figure 1 It can be seen that the salt-tolerant and oil-reducing effect of the composite bacterial group W7 is the best. Compared with the highest oil-reducing rate of 26.99% of the single bacterial group PS in Example 1, the oil-reducing rate of the bacterial group W7 is increased to 41.10%.

[0036] Comparison with the NCBI molecular biology database revealed that the YQ2 strain was Pantoea agglomerans; the CQ1 strain was Bacillus sp. CQ1; the PS strain was Pseudomonas sp. PS; and the WX strain was Enterobacter sp. WX.

[0037] WX, CQ1 and PS were biologically deposited in the China Center for Type Culture Collection, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the deposit date was July 19, 2023, and the deposit numbers were PS: CCTCC M 20231335; CQ1: CCTCC M 20231336; WX: CCTCC M20231337.

[0038] Example 3

[0039] This embodiment provides a method for preparing a modified zeolite for use as a microorganism immobilization carrier, which comprises the following steps:

[0040] S1. Screening of natural zeolite:

[0041] (1) Purchase natural zeolite from four different mining areas, including Harbin, Heilongjiang, Chaoyang, Liaoning, Shijiazhuang, Hebei, Luoyang, Henan, Shangtianti, Xinyang, Henan, Yantai, Shandong, and Chifeng, Inner Mongolia, and then screen, clean, and dry it for use.

[0042] (2) The cation exchange capacity and ammonia absorption values ​​of natural zeolites from different mining areas were measured using barium chloride exchange-atomic absorption spectrophotometry (GB / T 20973-2007) and Nessler's reagent-spectrophotometry (HJ 535-2009). The Chifeng zeolite from Inner Mongolia, which is primarily composed of clinoptilolite, was found to have the best ammonia absorption value and cation exchange capacity. This zeolite was confirmed as a suitable zeolite raw material.

[0043] S2. Modification of Chifeng zeolite from Inner Mongolia using six different methods:

[0044] (1) NaOH modification: Take 10 g of a 30-60 mesh zeolite sample that has been sieved, washed, and dried for pretreatment and add it to 100 mL of a 1 mol / L NaOH solution in a conical flask. Incubate the sample in a shaking incubator at 120-150 rpm and 25-30°C for 4-8 hours. Filter the sample through a 0.45 μm filter membrane, rinse with deionized water until neutral, and dry it in a drying oven at 105°C to obtain the alkali-modified zeolite.

[0045] (2) HCl modification: 10 g of a 30-60 mesh zeolite sample that had been pretreated by sieving, washing, and drying was added to a 100 mL conical flask containing a 0.5 mol / L HCl solution. The mixture was stirred in an incubator at 100 rpm and 25°C for 24 h. After filtration through a 0.45 μm filter, the zeolite surface was rinsed with deionized water until neutral and dried in a drying oven at 105°C for 2 h.

[0046] (3) NaCl Modification: 10 g of a 30-60 mesh zeolite sample, pre-screened, washed, and dried, was added to a 100 mL conical flask containing a 4% NaCl solution. The mixture was shaken in an incubator at 200 rpm and 30°C for 2 h. The mixture was filtered through a 0.45 μm filter, rinsed with deionized water, and dried in a drying oven at 105°C for 2 h.

[0047] (4) CTS modification: 10 g of 30-60 mesh zeolite sample that had been pretreated by sieving, washing, and drying was added to 100 mL of 2% acetic acid and 5 g / L chitosan acetate gel. The mixture was stirred in a magnetic stirrer for 10 h and dried at 60 °C for 12 h.

[0048] (5) Copper loading modification: Take 10 g of 30-60 mesh zeolite sample that has been sieved, washed, and dried, add it to 100 mL of 0.1 mol / L copper sulfate solution, and rotate the sample at 25°C and 200 rpm for 2 h. Rinse with deionized water and dry it in a drying oven at 105°C for 2 h.

[0049] (6) High-temperature modification: 10 g of a 30-60 mesh zeolite sample that had been sieved, washed, and dried was placed in a crucible and calcined in a muffle furnace at 400°C for 3 h. After calcination, the sample was cooled to room temperature to obtain a modified zeolite.

[0050] The zeolite before modification was used as a control and compared with the modified zeolite, and an ammonia nitrogen adsorption experiment was carried out. The treatment of high-concentration ammonia nitrogen-contaminated water body includes the following steps:

[0051] S1: Weigh 3.8190 g of ammonium chloride (NH4Cl, high-grade, dried at 100-105°C for 2 h), dissolve it in water, transfer it into a 1000 mL volumetric flask, and dilute to the mark. This is the contaminated water sample with an ammonia nitrogen concentration of 1000 mg / L.

[0052] S2: Take 100mL of ammonium chloride solution in S1 into a 250mL conical flask, add 5g of zeolite modified by different methods, and react in a shaker at 15℃ and 200rpm. Centrifuge at 0.5, 1, 2, 5, 10, 24, 48, and 72h, and filter the supernatant through a 0.45μm filter membrane to obtain samples. The residual ammonia nitrogen concentration in the sample was determined by the Nessler reagent method. According to the ammonia nitrogen adsorption rate and the ammonia nitrogen equilibrium adsorption capacity, the optimal modification method for Chifeng zeolite in Inner Mongolia was NaOH modification.

[0053] The SEM images of zeolite before and after modification with NaOH are shown in the attached figure. Figure 2 and Figure 3 It can be seen that the surface of natural zeolite is uneven and scaly. The scaly structure of the surface of zeolite modified by NaOH disappears and irregular pores increase. This is mainly because NaOH selectively removes silicon from the zeolite skeleton, reduces its silicon-aluminum ratio, and the smaller Na particles in the solution + It will be partially introduced into the zeolite crystal structure, replacing the larger particle size Ca in the zeolite framework itself. 2+ Mg 2+The pores are widened, the effective pore size becomes larger, the steric hindrance becomes smaller, the internal diffusion rate is accelerated, and the zeolite exhibits higher cation exchange performance. Clinoptilolite has a higher NH4 + Selectivity is greater than Na + , thereby promoting the same NH4 + Ion exchange can reduce the ammonia nitrogen concentration in the sewage solution.

[0054] Example 4

[0055] This embodiment provides a method for preparing microbial-modified zeolite.

[0056] 20g of dried Chifeng zeolite before and after modification and 50ml of enrichment culture medium were sterilized at 121°C for 20 minutes in a 250ml conical flask. After cooling, 50ml of enriched and activated bacterial culture of composite bacterial strain W7 was added to the culture medium and the mixture was shaken at 120-150rpm at 25-30°C for 30-36 hours. The sample was centrifuged at 1000rpm for 10 minutes, the supernatant was discarded, and the precipitate obtained by centrifugation was rinsed three times with sterile saline. The precipitate was then freeze-dried in a vacuum at -60°C to obtain modified zeolite particles immobilized with composite bacterial strain W7.

[0057] After centrifugation, the discarded supernatant was measured by OD 600 The results show that the effective pore volume of the modified zeolite is significantly increased, and the adsorption capacity of the composite bacterial community is increased from 8.22×10 10 cell / g increased to 1.03×10 11 cell / g.

[0058] Hereinafter, the inventors tested the removal performance of the microbial-modified zeolite provided in the above embodiments on petroleum and ammonia nitrogen in contaminated groundwater of refinery landfills through Examples 5-9. The tests were conducted using a tank device for indoor simulation.

[0059] Example 5

[0060] According to the measurement of tetrachloroethylene extraction and infrared oil measuring instrument, the petroleum concentration of the contaminated groundwater in the landfill of the refinery enterprise is about 500 mg / L. According to the measurement of Nessler's reagent method, the ammonia nitrogen concentration of the inlet water is about 1000 mg / L. According to the measurement of salinity meter, the salinity of the groundwater is about 5%.

[0061] In the laboratory, a tank experiment was conducted to simulate the prevention and control effect of petroleum and ammonia nitrogen pollution in the high-salt groundwater environment of a refinery landfill. The media prepared in different Examples 5-8 were filled as the wall reaction zone to establish a permeable reaction wall. The wall was divided into five parts. The first and second parts were the coarse sand inlet area and the fine sand inlet area, respectively. The coarse sand particle size was selected in the range of 15-30 mesh, and the fine sand particle size was in the range of 60-80 mesh. The third part in the middle was the wall reaction area, and the fourth and fifth parts were the coarse sand and fine sand outlet areas.

[0062] Microbial modified zeolite is added to the wall reaction zone of the permeable reaction wall prepared in this embodiment, that is, the wall reaction zone is composed of: adsorption material modified zeolite and the composite salt-tolerant and oil-reducing bacterial group W7 immobilized on the modified zeolite after freeze-drying.

[0063] Example 6

[0064] The wall reaction zone of the permeable reaction wall prepared in this embodiment is composed of natural zeolite as an adsorption material and the oil-degrading bacteria group W7 immobilized on the natural zeolite after freeze-drying.

[0065] Example 7

[0066] The wall reaction zone of the permeable reaction wall prepared in this embodiment is composed of: free composite bacterial colony W7.

[0067] Example 8

[0068] The wall reaction zone of the permeable reaction wall prepared in this embodiment is composed of: adsorption material natural zeolite.

[0069] Application Example 1

[0070] The permeable reaction wall prepared in Example 5 was used.

[0071] Application Example 2

[0072] The permeable reaction wall prepared in Example 6 was used.

[0073] Application Example 3

[0074] Use the permeable reaction wall prepared in Example 7.

[0075] Application Example 4

[0076] The permeable reaction wall prepared in Example 8 was used.

[0077] Comparative Application Example 1

[0078] The permeable reaction wall prepared in this comparative example does not have a third part, the wall reaction zone.

[0079] Analysis and Exploration

[0080] Peristaltic pumps were used to control the flow rate of contaminated groundwater at the water inlets of the five devices in Application Examples 1 to 4 and Application Comparative Example 1. The ammonia nitrogen and petroleum pollutant content in the effluent of different tanks was monitored regularly. After 7 days of operation, the effluent ammonia nitrogen and petroleum pollutant content were the final test results. They are recorded in Table 2 below. The changes in the effluent oil concentration and ammonia nitrogen concentration of Application Example 1 treated with microbial modified zeolite during the 7-day operation period are shown in the attached figure. Figure 4 .

[0081] Table 2

[0082]

[0083] The salt-tolerant and oil-reducing microbial flora W7, enriched from the contaminated groundwater environment of the original refining enterprise landfill, was adsorbed and fixed by modified zeolite. The flora survived and proliferated in this environment. While the zeolite adsorbed and fixed the flora, it also adsorbed and utilized ammonia nitrogen to provide nutrients for the flora, optimizing the oil-reduction ability. Under the adsorption coupling of the zeolite and the degradation effect of the composite flora W7, a relatively effective synergistic remediation of the high-salinity groundwater body containing oil and ammonia nitrogen was achieved in 7 days. The oil removal rate and ammonia nitrogen removal rate of the microbial modified zeolite provided in Example 5 can be increased by 41.91% and 60.75% respectively compared with the application control ratio.

[0084] In summary, considering the raw material cost, preparation method and removal rate of petroleum, ammonia and nitrogen compound pollutants of the microbial modified zeolite provided by the present invention in simulated applications, the composite bacteria-immobilized zeolite particles can effectively act in the field of bioremediation of contaminated groundwater in landfills of refineries and chemical enterprises.

[0085] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A microbial modified zeolite, characterized in that: The microorganism-modified zeolite is a zeolite on which microorganisms grow, and the microorganisms are Bacillus, Pseudomonas or Enterobacter or any combination thereof.

2. The microbial modified zeolite according to claim 1, characterized in that: The Bacillus is a bacterium with a preservation number of CCTCC M20231336; the Pseudomonas is a bacterium with a preservation number of CCTCC M 20231335; and the Enterobacter is a bacterium with a preservation number of CCTCC M 20231337.

3. The microbial modified zeolite according to claim 1, characterized in that: The ratio of the Bacillus, the Pseudomonas and the Enterobacter is 1-2:1-3:1-2.

4. The microbial modified zeolite according to claim 1, characterized in that: The zeolite is an alkali-modified zeolite.

5. The microbial modified zeolite according to claim 4, characterized in that: The zeolite is alkali-modified natural zeolite from Chifeng, Inner Mongolia.

6. The microbial modified zeolite according to claim 5, characterized in that: The alkali modification is NaOH modification.

7. A method for preparing the microbial modified zeolite according to any one of claims 1 to 6, characterized in that: include: The alkali-modified zeolite and the microbial liquid are placed in sterilized water, kept at a constant temperature of 25°C-30°C for 30-36 hours, and then centrifuged to obtain a precipitate.

8. The method according to claim 7, characterized in that The alkali-modified zeolite is obtained by the following method: 30-60 mesh Chifeng zeolite is added to NaOH, and the reaction is carried out under the conditions of 120-150 rpm and 25-30° C. for 4-8 hours.

9. The method of claim 7, characterized in that The microbial culture solution is OD 600 =0.8-1.5 of enriched activated bacterial solution.

10. Use of the microbial modified zeolite according to any one of claims 1 to 6 in the treatment of refining waste pollution.

11. A process for treating pollution from refining waste, using the microbial modified zeolite according to any one of claims 1 to 6.

Images