System and method for preparing immobilized microbial inoculum

Abstract

The invention relates to the technical field of microorganisms and environmental protection, and discloses a system and method for preparing an immobilized fungicide. The system comprises an immobilized fungicide preparation unit, and a first partition plate is arranged in a fungicide preparation device in a foldable mode; the first partition plate is made to have a folded state enabling the microbial agent preparation device and the cross-linking reactor to be communicated and an unfolded state enabling the microbial agent preparation device and the cross-linking reactor to be separated, and the gel and the bacterial liquid are mixed in the microbial agent preparation device to obtain mixed liquid; the round hole sieve plate is arranged between the first partition plate and the cross-linking reactor, and the vacuumizing device is connected with the cross-linking reactor, so that mixed liquid from the first partition plate passes through the round hole sieve plate to form fungus beads which fall into the cross-linking reactor; the cross-linking reactor is provided with a cross-linking agent inlet, and a cross-linking agent enters the cross-linking reactor through the cross-linking agent inlet. The system can realize the automatic production of the immobilized microbial inoculum, and has the advantages of high productivity, wide applicability and engineering application prospect on the premise of ensuring the performance of the microbial inoculum product.

Description

Technical Field

[0001] The present invention relates to the technical fields of microorganisms and environmental protection, and particularly relates to a system and method for preparing immobilized bacterial agents. Background Art

[0002] During the processes of oil exploration, storage, transportation, processing, and use, due to reasons such as accidents, maintenance, improper operations, and leakage, various organic pollutants enter the soil and groundwater, which will change the physical and chemical properties of the soil and groundwater, causing pollution and damage to the ecological environment; at the same time, through migration and diffusion, the organic pollutants will ultimately enter the biological chain and endanger human life and health.

[0003] Common remediation methods for soil and groundwater pollution include thermal desorption, chemical oxidation, vapor extraction, chemical leaching, etc. However, the above-mentioned remediation technologies have technical limitations to varying degrees, such as high treatment costs, secondary pollution, and low remediation efficiency. The microbial degradation technology has received wide attention due to its advantages of being economical, efficient, simple to operate, green, and free of secondary pollution. However, the engineering application effect of the microbial degradation technology is poor, and the limited factors mainly include that free bacterial agents are difficult to resist high-concentration pollution stress, have poor environmental tolerance, are easily competed by native soil microorganisms, and have a short active period.

[0004] The technology of immobilizing microbial agents has become a research hotspot in recent years due to its advantages such as strong mechanical properties, high biological concentration, strong stress resistance, and good stability. However, at present, the preparation of immobilized bacterial agents mainly relies on manual operation, which is only limited to the small-scale laboratory test level, with low production efficiency and unable to be applied on a large industrial scale. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problems in the prior art that the preparation of immobilized bacterial agents mainly relies on manual operation, with low production efficiency and unable to be applied on a large industrial scale, and to provide a system and method for preparing immobilized bacterial agents.

[0006] To achieve the above purpose, on the one hand, the present invention provides a system for preparing immobilized bacterial agents, which includes an immobilized bacterial agent preparation unit. The immobilized bacterial agent preparation unit includes a bacterial agent preparation device, a first partition board, a round-hole sieve plate, a cross-linking reactor, and a vacuum pumping device. The bacterial agent preparation device has a bacterial liquid inlet and a gelling agent inlet;

[0007] The first partition board is foldably arranged in the bacterial agent preparation device, so that the first partition board has a folded state in which the bacterial agent preparation device and the cross-linking reactor are communicated and an unfolded state in which the bacterial agent preparation device and the cross-linking reactor are separated. When the first partition board is in the unfolded state, the gelling agent and the bacterial liquid of petroleum-degrading bacteria are mixed in the bacterial agent preparation device to obtain a mixed liquid;

[0008] The circular hole sieve plate is arranged between the first partition plate and the crosslinking reactor. The vacuum pumping device is connected to the crosslinking reactor and is used to pump the air in the crosslinking reactor to vacuum. After the mixed liquid from the first partition plate passes through the circular hole sieve plate, bacterial beads are formed and fall into the crosslinking reactor;

[0009] The crosslinking reactor has a crosslinking agent inlet, and the crosslinking agent enters the crosslinking reactor through the crosslinking agent inlet to carry out immobilization treatment on the bacterial beads.

[0010] Preferably, the immobilized bacterium agent preparation unit further includes a first heating device, and the first heating device is used to heat the materials in the bacterium agent preparation device.

[0011] Preferably, the circular hole sieve plate has a plurality of circular holes, and the aperture of the circular holes is 3-8 mm;

[0012] Preferably, the material of the circular hole sieve plate is high boron silicon.

[0013] Preferably, the immobilized bacterium agent preparation unit further includes a filtering device, and the filtering device is arranged in the crosslinking reactor and is used to collect the prepared immobilized bacterium agent.

[0014] Preferably, the immobilized bacterium agent preparation unit further includes a second partition plate, and the second partition plate is movably installed in the crosslinking reactor to have an open state for discharging the mixture in the crosslinking reactor and a closed state for intercepting the mixture in the crosslinking reactor.

[0015] Preferably, a first stirrer is arranged in the bacterium agent preparation device and is used to stir the materials in the bacterium agent preparation device.

[0016] Preferably, the system further includes a bacterium liquid preparation unit, which is used to prepare bacterium liquid and inject the bacterium liquid into the bacterium agent preparation device through the bacterium liquid inlet.

[0017] Preferably, the bacterium liquid preparation unit includes a bacterium liquid preparation device, a second heating device, a second stirrer and an aeration device,

[0018] The bacterium liquid preparation device has a feeding port and a water inlet. The bacterium suspension and the culture medium are injected into the bacterium liquid preparation device through the feeding port, and sterile water is injected into the bacterium liquid preparation device through the water inlet;

[0019] The second heating device is used to heat the materials in the bacterium liquid preparation device;

[0020] The aeration device is used to introduce air into the materials in the bacterium liquid preparation device;

[0021] The second stirrer is used to stir the materials in the bacterium liquid preparation device.

[0022] Preferably, the system further includes a gelling agent preparation unit for preparing a gelling agent and injecting the gelling agent into the inoculant preparation device through the gelling agent inlet.

[0023] Preferably, the gelling agent preparation unit includes a gelling agent preparation device, a third heating device, and a third stirrer. The third heating device is used to heat the materials in the gelling agent preparation device, and the third stirrer is used to stir the materials in the gelling agent preparation device.

[0024] The second aspect of the present invention provides a method for preparing an immobilized inoculant, which is carried out in the system as described above. The method includes:

[0025] Set the first partition to the unfolded state. In the inoculant preparation device, the bacterial liquid of petroleum-degrading bacteria and the gelling agent are mixed to obtain a mixed liquid.

[0026] Then set the first partition to the folded state. At the same time, evacuate the crosslinking reactor through the vacuum device. After the mixed liquid from the first partition passes through the round hole sieve plate, it forms bacterial beads and enters the crosslinking reactor. The crosslinking agent in the crosslinking reactor fixes the bacterial beads to obtain the immobilized inoculant.

[0027] Preferably, the weight ratio of the dosage of the bacterial liquid to the gelling agent is 1:8 - 15.

[0028] Preferably, the gelling agent contains sodium alginate and optionally polyvinyl alcohol.

[0029] Preferably, the crosslinking agent contains calcium chloride and optionally boric acid.

[0030] Preferably, the vacuum degree in the crosslinking reactor is 0.06 - 0.1 MPa by evacuation.

[0031] The system provided by the present invention, through the setting of the vacuum device, can provide a constant suction force to the mixed liquid in the inoculant preparation device, making the crushing rate of the prepared spherical inoculant almost 0, and the shape of the inoculant is regular and the size is uniform. Through the design of structures such as the first partition, reactor, round hole sieve plate, and vacuum device, the present invention can realize the automated production of immobilized inoculants. On the premise of ensuring the product performance of the inoculant, it has high productivity (the output is increased by nearly 7 times), wide applicability, is green, has no secondary pollution, and has the prospect of engineering application; the inoculant product can be applied to the harmless biological remediation of organic pollutants. Description of the Drawings

[0032] Figure 1 is a partial structural schematic diagram of an embodiment of the system for preparing an immobilized inoculant provided by the present invention;

[0033] Figure 2 is Figure 1 a schematic structural diagram of the immobilized bacterium agent preparation unit in;

[0034] Figure 3 is Figure 1 a schematic structural diagram of the bacterial liquid preparation unit in;

[0035] Figure 4 is Figure 1 a schematic structural diagram of the gelling agent preparation unit in;

[0036] Figure 5 is a photographed picture of the immobilized bacterium agent prepared in Example 1 of the present invention;

[0037] Figure 6 is a photographed picture of the immobilized bacterium agent prepared in Example 2 of the present invention;

[0038] Figure 7 is a photographed picture of the immobilized bacterium agent prepared in Example 3 of the present invention.

[0039] Description of the reference numerals

[0040] 1 - immobilized bacterium agent preparation unit; 2 - bacterial liquid preparation unit; 3 - gelling agent preparation unit;

[0041] 11 - bacterium agent preparation device; 111 - bacterial liquid inlet; 112 - gelling agent inlet; 12 - first partition; 121 - driving part; 13 - round hole sieve plate; 14 - crosslinking reactor; 141 - crosslinking agent inlet; 15 - vacuum pumping device; 16 - first heating device; 17 - first stirrer; 18 - filtering device; 19 - second partition;

[0042] 21 - bacterial liquid preparation device; 211 - feeding port; 212 - water inlet; 213 - bacterial liquid outlet pipe; 22 - second heating device; 23 - second stirrer; 24 - aeration pipe; 25 - bacterial liquid injection pump; 26 - bacterial liquid flowmeter; 27 - first valve; 28 - first funnel; 29 - first waste liquid pipe;

[0043] 31 - gelling agent preparation device; 311 - gelling agent outlet pipe; 32 - third heating device; 33 - third stirrer; 34 - gelling agent injection pump; 35 - gelling agent flowmeter; 36 - second valve; 37 - second funnel; 38 - second waste liquid pipe. Detailed implementation manners

[0044] The following further describes in detail the specific implementation manners of the present invention with reference to the accompanying drawings. It should be understood that the specific implementation manners described herein are only for the purpose of illustrating and explaining the present invention, and are not intended to limit the present invention.

[0045] The endpoints and any values in the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0046] The present invention provides a system for preparing immobilized bacterial agents. Figures 1 to 4 This is a specific embodiment of the system for preparing immobilized bacterial agents provided by the present invention.

[0047] Please refer to Figure 1 and Figure 2 The system for preparing immobilized bacterial agents according to the present invention includes an immobilized bacterial agent preparation unit 1. The immobilized bacterial agent preparation unit 1 includes a bacterial agent preparation device 11, a first partition 12, a round-hole sieve plate 13, a crosslinking reactor 14, and a vacuum pumping device 15. The bacterial agent preparation device 11 has a bacterial liquid inlet 111 and a gelling agent inlet 112;

[0048] The first partition 12 is foldably arranged in the bacterial agent preparation device 11, so that the first partition 12 has a folded state in which the bacterial agent preparation device 11 and the crosslinking reactor 14 are communicated and an unfolded state in which the bacterial agent preparation device 11 and the crosslinking reactor 14 are separated. When the first partition 12 is in the unfolded state, the gelling agent and the bacterial liquid of petroleum-degrading bacteria are mixed in the bacterial agent preparation device 11 to obtain a mixed liquid;

[0049] The round-hole sieve plate 13 is arranged between the first partition 12 and the crosslinking reactor 14. The vacuum pumping device 15 is connected to the crosslinking reactor 14 and is used to pump vacuum in the crosslinking reactor 14, so that the mixed liquid from the first partition 12 forms bacterial beads and falls into the crosslinking reactor 14 after passing through the round-hole sieve plate 13;

[0050] The crosslinking reactor 14 has a crosslinking agent inlet 141, and the crosslinking agent enters the crosslinking reactor 14 through the crosslinking agent inlet 141 to perform an immobilization treatment on the bacterial beads.

[0051] The immobilized bacterial agent product prepared by the system according to the present invention can be used in the bioremediation project for soil and groundwater pollution treatment.

[0052] The system described in the present invention has a wide range of applicability and can prepare various immobilized microbial agent products such as directly embedded microbial beads and biochar-adsorbed embedded microbial beads. The system described in the present invention can realize the automated production of immobilized microbial agents. On the premise of ensuring the performance of the microbial agent products, it has high production capacity (the output is about 7 times higher than that of the natural gravity preparation method in the laboratory), wide applicability, greenness, no secondary pollution, and has the prospect of engineering application.

[0053] During specific implementation, first, the first partition plate 12 is in the unfolded state. The bacterial liquid is injected into the microbial agent preparation device 11 through the bacterial liquid inlet 111, and the gelling agent is injected into the microbial agent preparation device 11 through the gelling agent inlet 112. The gelling agent and the bacterial liquid are mixed in the microbial agent preparation device 11 to obtain a mixed liquid. The crosslinking agent is injected into the crosslinking reactor 14 through the crosslinking agent inlet 141. Then, the first partition plate 12 is in the folded state, and at the same time, the crosslinking reactor 14 is evacuated. In this way, under the action of the vacuum suction force, after the mixed liquid passes through the round hole sieve plate 13, microbial beads are formed and fall into the crosslinking reactor 14, and the microbial beads are immobilized in the crosslinking agent to obtain an immobilized microbial agent product.

[0054] Regarding the specific types and sources of the petroleum-degrading bacteria in the bacterial liquid, the present invention does not make any restrictions as long as they can degrade organic pollutants such as petroleum hydrocarbons. In a specific embodiment, the petroleum-degrading bacteria are Cellulomonas sp. (preservation number: GDMCC No: 62434) and / or Pseudomonas sp. (preservation number: GDMCC No: 62339).

[0055] The present invention does not limit the specific form of the microbial agent preparation device 11 as long as it can provide a space for mixing the microbial agent and the gelling agent. In a preferred embodiment, the microbial agent preparation device 11 is a tank body (i.e., a microbial agent preparation tank).

[0056] The present invention also does not limit the specific manner of driving the first partition plate 12 to switch between the folded state and the unfolded state, which can be manual operation or a driving part 121 can be provided to provide power. In order to achieve automated operation and save labor, in the present invention, the immobilized microbial agent preparation unit 1 further includes a driving part 121, and the driving part 121 is connected to the first partition plate 12 to provide power so that the first partition plate 12 can change from the folded state to the unfolded state or from the unfolded state to the folded state.

[0057] In the present invention, the vacuum pumping device 15 can be a vacuum pump, a micro vacuum pump, a micro air pump, a micro air extraction pump, a micro air extraction and pumping pump and other vacuum pumping devices, preferably a vacuum pump.

[0058] The present invention also does not limit the relationship between the round-hole sieve plate 13 and the microbial agent preparation device 11. The round-hole sieve plate 13 can be arranged inside the microbial agent preparation device 11, or can be arranged between the microbial agent preparation device 11 and the crosslinking reactor 14. In a specific embodiment, the round-hole sieve plate 13 is arranged at the bottom of the microbial agent preparation device 11.

[0059] Further preferably, the round-hole sieve plate 13 has a plurality of round holes, and the aperture of the round holes is 3-8 mm. Among them, the plurality of round holes on the round-hole sieve plate 13 are arranged arbitrarily.

[0060] Further preferably, the material of the round-hole sieve plate 13 is high borosilicate.

[0061] In a preferred embodiment, the immobilized microbial agent preparation unit 1 further includes a first heating device 16, and the first heating device 16 is used to heat the materials in the microbial agent preparation device 11. Thus, dissolution can be promoted, thereby reducing the time required for mixing the microbial liquid and the gelling agent. In a specific embodiment, the first heating device 16 is arranged on the inner wall of the microbial agent preparation device 11 and is arranged along the circumferential direction of the inner wall.

[0062] In a preferred embodiment, the immobilized microbial agent preparation unit 1 further includes a filtering device 18, and the filtering device 18 is arranged in the crosslinking reactor 14 for collecting the prepared immobilized microbial agent. Preferably, the filtering device 18 is a filter mesh.

[0063] In a preferred embodiment, the immobilized microbial agent preparation unit 1 further includes a second partition plate 19, and the second partition plate 19 is movably installed in the crosslinking reactor 14 to have an open state for discharging the mixture in the crosslinking reactor 14 and a closed state for intercepting the mixture in the crosslinking reactor 14.

[0064] In the present invention, the filtering device 18 can be arranged above the second partition plate 19 or below the second partition plate 19. Preferably, it is arranged above the second partition plate 19. Thus, when the crosslinking reaction is completed and the second partition plate 19 is changed from the closed state to the open state, the immobilized microbial agent is not easily damaged. Specifically, during implementation, in the crosslinking reactor 14, when the crosslinking agent solution is required to perform an immobilization treatment on the microbial beads, the second partition plate 19 is in the closed state; when the crosslinking reaction ends and the immobilized microbial agent is obtained, the second partition plate 19 is in the open state. At this time, the immobilized microbial agent is intercepted on the filtering device 18, and the waste liquid is discharged outside the crosslinking reactor 14.

[0065] In a preferred embodiment, a first stirrer 17 is provided inside the microbial agent preparation device 11 for stirring the materials (i.e., microbial liquid and gelling agent) inside the microbial agent preparation device. Specifically, the first stirrer 17 includes a rotating rod, stirring blades, and a driving motor. Among them, the rotating rod is rotatably connected to the top of the microbial agent preparation device 11, the stirring blades include a plurality of blades arranged at intervals along the circumference of the rotating rod, and the output shaft of the driving motor is connected to the rotating rod to drive the rotating rod to rotate. In a specific embodiment, the first stirrer 17 is arranged at the central position inside the microbial agent preparation device 11.

[0066] In the present invention, the system further includes a microbial liquid preparation unit 2 for preparing microbial liquid and injecting the microbial liquid into the microbial agent preparation device 11 through the microbial liquid inlet 111.

[0067] Please refer to Figure 3 , in a preferred embodiment, the microbial liquid preparation unit 2 includes a microbial liquid preparation device 21, a second heating device 22, a second stirrer 23, and an aeration device. The microbial liquid preparation device 21 has a feeding port 211 and a water inlet 212. A microbial suspension and a culture medium are injected into the microbial liquid preparation device 21 through the feeding port 211, and sterile water is injected into the microbial liquid preparation device 21 through the water inlet 212. The second heating device 22 is used to heat the materials inside the microbial liquid preparation device 21. The aeration device is used to introduce air into the materials inside the microbial liquid preparation device 21. The second stirrer 23 is used to stir the materials inside the microbial liquid preparation device 21.

[0068] In a specific embodiment, the aeration device includes an aeration pipe 24 and an aeration disc (not shown in the figure). Among them, the aeration disc is connected to the aeration pipe 24, and the aeration pipe 24 extends to the bottom of the microbial liquid preparation device 21.

[0069] In a specific embodiment, the second heating device 22 is arranged on the inner wall of the microbial liquid preparation device 21 and is arranged along the circumference of the inner wall.

[0070] For the convenience of uniform stirring, in a specific embodiment, the second stirrer 23 is arranged at the central position inside the microbial liquid preparation device 21.

[0071] In a preferred embodiment, the bacterial liquid inlet 111 of the bacterial agent preparation device 11 is connected to the bacterial liquid outlet pipe 213 of the bacterial liquid preparation device 21. A bacterial liquid injection pump 25, a bacterial liquid flowmeter 26, and a first valve 27 are arranged between the bacterial liquid inlet 111 of the bacterial agent preparation device 11 and the bacterial liquid outlet pipe 213 of the bacterial liquid preparation device 21. By providing the bacterial liquid injection pump 25, power can be provided for the transportation of the bacterial liquid. Through the design of the bacterial liquid flowmeter 26 and the first valve 27, the amount of the bacterial liquid transported to the bacterial agent preparation device 11 can be controlled.

[0072] In the present invention, the bacterial liquid preparation unit 2 further includes a first funnel 28 and a first waste liquid pipe 29. Among them, the first funnel 28 is arranged at the bottom of the bacterial liquid preparation device 21 for discharging waste liquid; the first funnel 28 is connected to the first waste liquid pipe 29, and the waste liquid from the first funnel 28 is discharged through the first waste liquid pipe 29.

[0073] In a preferred embodiment, the system further includes a gel agent preparation unit 3 for preparing a gel agent and injecting the gel agent into the bacterial agent preparation device 11 through the gel agent inlet 112.

[0074] Please refer to Figure 4 In a preferred embodiment, the gel agent preparation unit 3 includes a gel agent preparation device 31, a third heating device 32, and a third stirrer 33. The third heating device 32 is used for heating the materials in the gel agent preparation device 31, and the third stirrer 33 is used for stirring the materials in the gel agent preparation device 31.

[0075] In the present invention, the gel agent inlet 112 of the bacterial agent preparation device 11 is connected to the gel agent outlet pipe 311 of the gel agent preparation device 31. A gel agent injection pump 34, a gel agent flowmeter 35, and a second valve 36 are arranged between the gel agent inlet 112 and the gel agent outlet pipe 311. By providing the gel agent injection pump 34, power can be provided for the transportation of the gel agent. Through the design of the gel agent flowmeter 35 and the second valve 36, the amount of the gel agent transported to the bacterial agent preparation device 11 can be controlled.

[0076] In the present invention, the gel agent preparation unit 3 further includes a second funnel 37 and a second waste liquid pipe 38. The second funnel 37 is arranged at the bottom of the gel agent preparation device 31 for discharging waste liquid; the second funnel 37 is connected to the second waste liquid pipe 38, and the waste liquid from the funnel is discharged through the second waste liquid pipe 38.

[0077] In a preferred embodiment, liquid level gauges are provided in both the gel agent preparation device 31 and the bacterial liquid preparation device 21. The liquid level gauges are designed as transparent windows and are embedded on the side walls of the devices.

[0078] It is understood that the gel preparation device 31 also has an inlet for adding water and an inlet for adding materials.

[0079] In a preferred embodiment, the bacterial liquid preparation device 21 is a tank body (i.e., a bacterial liquid preparation tank). The gel preparation device 31 is a tank body (i.e., a gel preparation tank).

[0080] In a preferred embodiment, the structures of the second stirrer 23 and the third stirrer 33 are the same as that of the first stirrer 17, which will not be elaborated here.

[0081] The present invention also provides a method for preparing an immobilized bacterial agent, which is carried out in the system as described above. The method includes:

[0082] Set the first partition 12 to the unfolded state. In the bacterial agent preparation device 11, the bacterial liquid of petroleum-degrading bacteria and the gel are mixed to obtain a mixed liquid;

[0083] Then set the first partition 12 to the folded state. At the same time, evacuate the inside of the crosslinking reactor 14 through the vacuum device 15. After the mixed liquid from the first partition 12 passes through the round hole sieve plate 13, it forms bacterial beads and enters the crosslinking reactor 14. The crosslinking agent in the crosslinking reactor 14 performs immobilization treatment on the bacterial beads to obtain the immobilized bacterial agent.

[0084] In a preferred embodiment, the weight ratio of the dosages of the bacterial liquid and the gel is 1:8 - 15.

[0085] In a preferred embodiment, the gel contains sodium alginate and optionally polyvinyl alcohol. Further preferably, the gel contains sodium alginate and polyvinyl alcohol.

[0086] In a specific embodiment, the gel is a mixture of sodium alginate, polyvinyl alcohol and water. Preferably, the weight ratio of water, sodium alginate and polyvinyl alcohol is 100:2 - 5:10 - 15.

[0087] In another specific embodiment, the gel is a mixture of sodium alginate and water. Preferably, the weight ratio of water and sodium alginate is 100:2 - 5.

[0088] The present invention does not limit the specific type of the crosslinking agent, which can be a conventional crosslinking agent in the art. In the present invention, the crosslinking agent contains calcium chloride and optionally boric acid. Preferably, the crosslinking agent contains calcium chloride and boric acid.

[0089] In a specific embodiment, the crosslinking agent is a mixture of calcium chloride, boric acid and water. Preferably, the weight ratio of water, calcium chloride and boric acid is 100:3 - 5:3 - 5.

[0090] In another specific embodiment, the crosslinking agent is a mixture of calcium chloride and water. Preferably, the weight ratio of water to calcium chloride is 100:3 to 5.

[0091] The present invention does not limit the specific dosage of the crosslinking agent, as long as the dosage of the crosslinking agent can submerge the bacterial beads so that the crosslinking agent can immobilize all the bacterial beads.

[0092] In a preferred embodiment, the vacuum degree in the crosslinking reactor 14 is 0.06 to 0.1 MPa by evacuating. It can be understood that the "vacuum degree" here refers to the absolute vacuum degree. In specific implementation, the vacuum pumping device 15 is a vacuum pump, and the load flow rate of the vacuum pump is 10 to 20 L / min.

[0093] In the method of the present invention, it also includes preparing a bacterial liquid according to the following process: mixing a bacterial suspension of petroleum-degrading bacteria with sterile water and a culture medium, and then fermenting for 20 to 24 h under the conditions of a temperature of 30 to 37 °C, a rotation speed of 100 to 150 rpm, and an aeration rate of 0.3 to 0.7 L / min to obtain the bacterial liquid.

[0094] The present invention will be described in detail below through examples, but the protection scope of the present invention is not limited thereto.

[0095] Adopt as Figures 1 - 4 shown in the system to perform the operations of the following examples. The system includes an immobilized bactericide preparation unit 1, a bacterial liquid preparation unit 2, and a gelling agent preparation unit 3;

[0096] Among them, the immobilized bactericide preparation unit 1 includes a bactericide preparation tank, a first partition plate 12, a round hole sieve plate 13, a crosslinking reactor 14, and a vacuum pump. The bactericide preparation tank has a bacterial liquid inlet 111 and a gelling agent inlet 112; the first partition plate 12 is foldably arranged in the bactericide preparation tank so that the first partition plate 12 has a folded state in which the bactericide preparation tank and the crosslinking reactor 14 are communicated and an unfolded state in which the bactericide preparation tank and the crosslinking reactor 14 are separated. The round hole sieve plate 13 is arranged between the first partition plate 12 and the crosslinking reactor 14, and the vacuum pump is connected to the crosslinking reactor 14 for evacuating the inside of the crosslinking reactor 14; the crosslinking reactor 14 has a crosslinking agent inlet 141, and the crosslinking agent enters the crosslinking reactor 14 through the crosslinking agent inlet 141;

[0097] The immobilized bacterium agent preparation unit 1 further includes a first heating device 16, a filtering device 18, a second partition plate 19, and a first stirrer 17. The second partition plate 19 is movably installed in the crosslinking reactor 14 to have an open state for discharging the mixture in the crosslinking reactor 14 and a closed state for intercepting the mixture in the crosslinking reactor 14.

[0098] The bacterial liquid preparation unit 2 includes a bacterial liquid preparation tank, a second heating device 22, a second stirrer 23, and an aeration device. The bacterial liquid outlet pipe 213 of the bacterial liquid preparation tank is connected to the bacterial liquid inlet 111 of the bacterium agent preparation tank.

[0099] The gelling agent preparation unit 3 includes a gelling agent preparation tank, a third heating device 32, and a third stirrer 33. The gelling agent outlet pipe 311 of the gelling agent preparation tank is connected to the gelling agent inlet 112 of the bacterium agent preparation tank.

[0100] Example 1

[0101] This example is used to illustrate the system and method for preparing the immobilized bacterium agent according to the present invention, specifically, the method for preparing the petroleum hydrocarbon immobilized bacterium agent by the direct embedding method of a single strain.

[0102] (1) Cellulomonas sp. A (preservation date: April 6, 2022, preservation number: GDMCC No: 62434) is selected as the petroleum-degrading bacterium, and its degradation applicable range is temperature 15 - 40°C, pH = 6.5 - 10.5. 10 L of sterile water is added into the bacterial liquid preparation tank through the water inlet 212, and then the bacterial suspension of Cellulomonas sp. A (OD 600 = 1.0) and the culture medium powder (5 g / L of yeast extract, 10 g / L of tryptone, 10 g / L of sodium chloride) are added through the feeding port 211 according to an inoculation amount of 5%. The parameters of the second heating device 22, the second stirrer 23, and the aeration device are set, so that the bacterial suspension ferments under the conditions of a temperature of 32°C, a rotation speed of 135 rpm, and an aeration volume of 0.5 L / min. After stirring and fermenting for 20 h, the bacterial liquid is obtained.

[0103] (2) 30 L of sterile water is added into the gelling agent preparation tank, and then sodium alginate and polyvinyl alcohol are added. Among them, the addition amount of sodium alginate is 4% of the mass of the sterile water, and the addition amount of polyvinyl alcohol is 12% of the mass of the sterile water. The parameters of the third heating device 32 and the third stirrer 33 are set, so that the temperature for preparing the gelling agent is 30°C and the rotation speed is 80 rpm. After stirring for 3 h, the gelling agent is obtained.

[0104] (3) Keep the first partition plate 12 in the bacterium agent preparation tank in the unfolded state to separate the bacterium agent preparation tank from the crosslinking reactor 14. Open the bacterium liquid injection pump 25 and the first valve 27 respectively, and transport the bacterium liquid prepared in the bacterium liquid preparation tank to the bacterium agent preparation tank through the bacterium liquid inlet 111. Open the gelling agent injection pump 34 and the second valve 36, and transport the gelling agent prepared in the gelling agent preparation tank to the bacterium agent preparation tank through the gelling agent inlet 112. Control the weight ratio of the bacterium liquid to the gelling agent in the bacterium agent preparation tank to be 1:10 through the bacterium liquid flowmeter 26 and the gelling agent flowmeter 35. By setting the parameters of the first heating device 16 and the first stirrer 17, stir the bacterium liquid and the gelling agent at 30°C and 80 rpm. After stirring for 0.5 h, a mixed liquid is obtained.

[0105] Inject the crosslinking agent into the crosslinking reactor 14 through the crosslinking agent inlet 141. Among them, the crosslinking agent is a mixed liquid of calcium chloride, boric acid and water, and the weight ratio of calcium chloride, boric acid and water is 4:4:100. Make the first partition plate 12 become the folded state through the driving part 121 to connect the bacterium agent preparation tank and the crosslinking reactor 14. At the same time, turn on the vacuum pump (set parameters: vacuum degree 0.08 MPa; load flow rate 15 L / min). Under the action of the vacuum suction force, after the mixed liquid passes through the round hole sieve plate 13, bacterium beads are formed and fall into the crosslinking reactor 14. After the bacterium beads are fixed in the crosslinking agent solution for 4 h, an immobilized bacterium agent is obtained. Open the second partition plate 19 to make it in the open state to let the waste liquid flow out, and then open the filter screen to collect the immobilized bacterium agent product (as Figure 5 shown).

[0106] Example 2

[0107] This example is used to illustrate the system and method for preparing the immobilized bacterium agent of the present invention, specifically the method for preparing the petroleum hydrocarbon immobilized bacterium agent by the direct embedding method of compound strains.

[0108] (1) Select Cellulomonas sp. A (preservation number: GDMCC No: 62434) and Pseudomonas sp. E (preservation number: GDMCC No: 62339), compound Cellulomonas sp. A and Pseudomonas sp. E, and the ratio of the number of colonies of the two is 1:1 to obtain a mixed bacterial suspension, and its degradation applicable range is temperature 15-40°C, pH = 6.5-10.5; Add 10 L of sterile water to the bacterium liquid preparation tank through the water inlet 212, and then add the mixed bacterial suspension (OD 600 = 1.0), the culture medium powder (5 g / L of yeast extract, 10 g / L of tryptone, 10 g / L of sodium chloride) through the feeding port 211 according to an inoculation amount of 5%. Set the parameters of the second heating device 22, the second stirrer 23 and the aeration device, and ferment the bacterial suspension at a temperature of 32°C, a rotation speed of 135 rpm, and an aeration volume of 0.5 L / min. After stirring and fermenting for 20 h, a bacterium liquid is obtained.

[0109] (2) Add 30 L of sterile water into the gelling agent preparation tank, and then add sodium alginate and polyvinyl alcohol. Among them, the addition amount of sodium alginate is 4% of the mass of the sterile water, and the addition amount of polyvinyl alcohol is 12% of the mass of the sterile water. Set the parameters of the third heating device 32 and the third stirrer 33 to make the temperature of gelling agent preparation 30 °C and the rotation speed 80 rpm. After stirring for 3 h, a gelling agent is obtained.

[0110] (3) Make the first partition plate 12 in the inoculum preparation tank in the unfolded state to separate the inoculum preparation tank from the crosslinking reactor 14. Open the inoculum injection pump 25 and the first valve 27 respectively, and transport the inoculum prepared in the inoculum preparation tank to the inoculum preparation tank through the inoculum inlet 111. Open the gelling agent injection pump 34 and the second valve 36, and transport the gelling agent prepared in the gelling agent preparation tank to the inoculum preparation tank through the gelling agent inlet 112. Control the weight ratio of the inoculum to the gelling agent in the inoculum preparation tank to be 1:10 through the inoculum flowmeter 26 and the gelling agent flowmeter 35. By setting the parameters of the first heating device 16 and the first stirrer 17, stir the inoculum and the gelling agent at 30 °C and 80 rpm. After stirring for 0.5 h, a mixed solution is obtained;

[0111] Inject the crosslinking agent into the crosslinking reactor 14 through the crosslinking agent inlet 141. Among them, the crosslinking agent is a mixed solution of calcium chloride, boric acid and water, and the weight ratio of calcium chloride, boric acid and water is 4:4:100. Make the first partition plate 12 become the folded state through the driving part 121 to connect the inoculum preparation tank and the crosslinking reactor 14. At the same time, turn on the vacuum pump (set parameters: vacuum degree 0.08 MPa; load flow rate 15 L / min). Under the action of the vacuum suction force, the mixed solution passes through the round hole sieve plate 13 and then forms bacteria beads falling into the crosslinking reactor 14. After the bacteria beads are fixed in the crosslinking agent solution for 4 h, an immobilized inoculum is obtained. Open the second partition plate 19 to make the waste liquid flow out, and then open the filter screen to collect the immobilized inoculum product (as Figure 6 shown).

[0112] Example 3

[0113] This example is used to illustrate the system and method for preparing the immobilized inoculum of the present invention, specifically the method for preparing the petroleum hydrocarbon immobilized inoculum by the compound strain adsorption and embedding method.

[0114] (1) Select Cellulomonas sp. A (preservation number: GDMCC No: 62434) and Pseudomonas sp. E (preservation number: GDMCC No: 62339), and compound Cellulomonas sp. A and Pseudomonas sp. E with the ratio of the number of colonies of the two being 1:1 to obtain a mixed bacterial suspension, the applicable degradation range of which is a temperature of 15 - 40 °C and pH = 6.5 - 10.5; add 10 L of sterile water into the bacterial liquid preparation tank through the water inlet 212, and then add the mixed bacterial suspension (OD 600 = 1.0), the culture medium powder (5 g / L of yeast extract, 10 g / L of tryptone, 10 g / L of sodium chloride) and biochar particles (particle size ≤ 0.075 mm; pH = 7.5, specific surface area is 13 m 2 / g, average pore diameter is 2 nm) through the feeding port 211 according to an inoculation amount of 5%, set the parameters of the second heating device 22, the second stirrer 23 and the aeration device, and ferment the bacterial suspension at a temperature of 32 °C, a rotation speed of 135 rpm and an aeration rate of 0.5 L / min for 20 h to obtain the bacterial liquid.

[0115] (2) Add 30 L of sterile water into the gel preparation tank, and then add sodium alginate and polyvinyl alcohol. Among them, the addition amount of sodium alginate is 4% of the mass of the sterile water, and the addition amount of polyvinyl alcohol is 12% of the mass of the sterile water. Set the parameters of the third heating device 32 and the third stirrer 33, and make the temperature for gel preparation be 30 °C and the rotation speed be 80 rpm. After stirring for 3 h, obtain the gel.

[0116] (3) Make the first partition 12 in the bacterial agent preparation tank in the unfolded state to separate the bacterial agent preparation tank from the cross-linking reactor 14. Open the bacterial liquid injection pump 25 and the first valve 27 respectively, and transport the bacterial liquid prepared in the bacterial liquid preparation tank to the bacterial agent preparation tank through the bacterial liquid inlet 111. Open the gel injection pump 34 and the second valve 36, and transport the gel prepared in the gel preparation tank to the bacterial agent preparation tank through the gel inlet 112. Control the weight ratio of the bacterial liquid to the gel in the bacterial agent preparation tank to be 1:10 through the bacterial liquid flowmeter 26 and the gel flowmeter 35. By setting the parameters of the first heating device 16 and the first stirrer 17, stir the bacterial liquid and the gel at 30 °C and 80 rpm. After stirring for 0.5 h, obtain the mixed liquid;

[0117] Inject the crosslinking agent into the crosslinking reactor 14 through the crosslinking agent inlet 141. The crosslinking agent is a mixed solution of calcium chloride, boric acid and water, and the weight ratio of calcium chloride, boric acid and water is 4:4:100. Drive the first partition 12 into a folded state through the driving part 121 to connect the inoculant preparation tank and the crosslinking reactor 14. At the same time, turn on the vacuum pump (set parameters: vacuum degree 0.08 MPa; load flow rate 15 L / min). Under the action of the vacuum suction force, after the mixed solution passes through the round hole sieve plate 13, bacterial beads are formed and fall into the crosslinking reactor 14. After the bacterial beads are fixed in the crosslinking agent solution for 4 h, the immobilized inoculant is obtained. Open the second partition 19 to let the waste liquid flow out, and then open the filter screen to collect the immobilized inoculant product (as Figure 7 shown).

[0118] Comparative Example 1

[0119] Implement according to the method described in Example 1. The difference is that the immobilized inoculant is prepared by a laboratory method (manual operation).

[0120] Specifically, after mixing the bacterial liquid and the gelling agent, store them in a separatory funnel, and drop the bacterial beads into the crosslinking agent by the method of natural gravity for crosslinking.

[0121] Comparative Example 2

[0122] Implement according to the method described in Example 2. The difference is that the immobilized inoculant is prepared by a laboratory method (manual operation).

[0123] Specifically, after mixing the bacterial liquid and the gelling agent, store them in a separatory funnel, and drop the bacterial beads into the crosslinking agent by the method of natural gravity for crosslinking.

[0124] Comparative Example 3

[0125] Implement according to the method described in Example 3. The difference is that the immobilized inoculant is prepared by a laboratory method (manual operation).

[0126] Specifically, after mixing the bacterial liquid and the gelling agent, store them in a separatory funnel, and drop the bacterial beads into the crosslinking agent by the method of natural gravity for crosslinking.

[0127] Comparative Example 4

[0128] Implement according to the method described in Example 1. The difference is that the immobilized inoculant is prepared by a laboratory method (manual operation).

[0129] Specifically, after mixing the bacterial liquid and the gelling agent, store them in a syringe, and drop the bacterial beads into the crosslinking agent by the method of manual extrusion for crosslinking.

[0130] The immobilized microbial agent prepared in this comparative example has poor uniformity, which may be because it is difficult to ensure constant pressure during manual extrusion.

[0131] Test Example 1

[0132] Perform performance measurements on the immobilized microbial agent products prepared in the examples and comparative examples, including shape, crushing rate, mechanical strength, bacteria loading, and yield.

[0133] The test method for mechanical strength is as follows: Take 40 g of immobilized microbial agent pellets, dry the moisture with filter paper, place them on an electronic balance, zero it, and slowly squeeze the pellets with a glass plate. Use the reading on the electronic balance when the pellets are crushed as the index of the immobilization mechanical strength.

[0134] The test method for bacteria loading Log(CFU / g) is as follows: Take 3 portions (3 g / portion) of the immobilized microbial agent, add 20 ml of sterile water to each portion, shake well by ultrasonic oscillation (power 100 w, 10 s / 10 s, frequency 20 kHz, 5 min), and perform gradient dilution coating at 10 -2 、10 -4 、10 -6 、10 -8 Then incubate at a constant temperature for 24 h and record the number of colonies. The calculation formula is as follows:

[0135]

[0136] Where y is the number of viable bacteria after dilution;

[0137] n is the dilution factor;

[0138] v is the total liquid volume;

[0139] m is the mass of the immobilized microbial agent.

[0140] The performance parameters of the immobilized microbial agent products prepared in Example 1 and Comparative Example 1 are compared as shown in Table 1.

[0141] Table 1

[0142]

[0143] The performance parameters of the immobilized microbial agent products prepared in Example 2 and Comparative Example 2 are compared as shown in Table 2.

[0144] Table 2

[0145]

[0146]

[0147] The performance parameters of the immobilized microbial agent products prepared in Example 3 and Comparative Example 3 are compared as shown in Table 3.

[0148] Table 3

[0149]

[0150] As can be seen from the results in Table 1-3, the performance of the immobilized microbial agent product prepared in the embodiments of the present invention is the same as or even better than that in the laboratory (manual operation), and its yield has increased significantly (by about 7 times).

[0151] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A system for preparing immobilized microbial agents, characterized in that, The system includes an immobilized bacteria agent preparation unit (1), and the immobilized bacteria agent preparation unit includes a bacteria agent preparation device (11), a first partition plate (12), a round hole sieve plate (13), a crosslinking reactor (14) and a vacuum pumping device (15). The bacteria agent preparation device (11) has a bacteria liquid inlet (111) and a gelling agent inlet (112). The first partition plate (12) is foldably arranged in the bacteria agent preparation device (11), so that the first partition plate (12) has a folded state in which the bacteria agent preparation device (11) communicates with the crosslinking reactor (14) and an unfolded state in which the bacteria agent preparation device (11) is separated from the crosslinking reactor (14). When the first partition plate (12) is in the unfolded state, the gelling agent and the bacteria liquid of the petroleum-degrading bacteria are mixed in the bacteria agent preparation device (11) to obtain a mixed liquid. The round hole sieve plate (13) is arranged between the first partition plate (12) and the crosslinking reactor (14), and the vacuum pumping device (15) is connected to the crosslinking reactor (14) for pumping the air in the crosslinking reactor (14). After the mixed liquid from the first partition plate (12) passes through the round hole sieve plate (13), bacteria beads are formed and fall into the crosslinking reactor (14). The crosslinking reactor (14) has a crosslinking agent inlet (141), and the crosslinking agent enters the crosslinking reactor (14) through the crosslinking agent inlet (141) to perform an immobilization treatment on the bacteria beads.

2. The system according to claim 1, wherein The immobilized bacteria agent preparation unit (1) further includes a first heating device (16), and the first heating device (16) is used for heating the materials in the bacteria agent preparation device (11).

3. The system according to claim 1, wherein The round hole sieve plate (13) has a plurality of round holes, and the aperture of the round holes is 3-8 mm. Preferably, the material of the round hole sieve plate is high borosilicate.

4. The system according to claim 1, wherein The immobilized bacteria agent preparation unit (1) further includes a filtering device (18), and the filtering device (18) is arranged in the crosslinking reactor (14) for collecting the prepared immobilized bacteria agent.

5. The system according to claim 1, characterized in that The immobilized bacteria agent preparation unit (1) further includes a second partition plate (19), and the second partition plate (19) is movably installed in the crosslinking reactor (14) to have an open state for discharging the mixture in the crosslinking reactor (14) and a closed state for intercepting the mixture in the crosslinking reactor (14).

6. The system according to claim 1, wherein A first stirrer (17) is arranged in the bacteria agent preparation device for stirring the materials in the bacteria agent preparation device (11).

7. The system according to claim 1, wherein The system further includes a bacteria liquid preparation unit (2) for preparing bacteria liquid and injecting the bacteria liquid into the bacteria agent preparation device (11) through the bacteria liquid inlet (111).

8. The system according to claim 7, wherein The bacteria liquid preparation unit (2) includes a bacteria liquid preparation device (21), a second heating device (22), a second stirrer (23) and an aeration device. The bacterial liquid preparation device (21) has a feeding port (211) and a water inlet (212). A bacterial suspension and a culture medium are injected into the bacterial liquid preparation device (21) through the feeding port, and sterile water is injected into the bacterial liquid preparation device (21) through the water inlet. The second heating device (22) is used to heat the materials in the bacterial liquid preparation device (21). The aeration device is used to introduce air into the materials in the bacterial liquid preparation device (21). The second stirrer (23) is used to stir the materials in the bacterial liquid preparation device (21).

9. The system according to claim 1, wherein The system further includes a gelling agent preparation unit (3) for preparing a gelling agent and injecting the gelling agent into the bacterial agent preparation device (11) through the gelling agent inlet (112).

10. The system according to claim 1, wherein, The gelling agent preparation unit (3) includes a gelling agent preparation device (31), a third heating device (32), and a third stirrer (33). The third heating device (32) is used to heat the materials in the gelling agent preparation device (31), and the third stirrer (33) is used to stir the materials in the gelling agent preparation device.

11. A method for preparing an immobilized microbial agent, characterized in that, This method is carried out in the system according to any one of claims 1-10, and the method includes: Setting the first partition (12) to an unfolded state. In the bacterial agent preparation device (11), the bacterial liquid of petroleum-degrading bacteria and the gelling agent are mixed to obtain a mixed liquid. Then setting the first partition (12) to a folded state, and at the same time evacuating the inside of the crosslinking reactor (14) through a vacuum device (15). After the mixed liquid from the first partition (12) passes through the round hole sieve plate (13), it forms bacterial beads and enters the crosslinking reactor (14). The crosslinking agent in the crosslinking reactor (14) performs an immobilization treatment on the bacterial beads to obtain an immobilized bacterial agent.

12. The method according to claim 11, wherein The weight ratio of the dosages of the bacterial liquid and the gelling agent is 1:8-15. Preferably, the gelling agent contains sodium alginate and optionally polyvinyl alcohol.

13. The method according to claim 11, characterized in that, The crosslinking agent contains calcium chloride and optionally boric acid. Preferably, the degree of vacuum inside the crosslinking reactor (14) is 0.06-0.1 MPa by evacuation.

Images