CIRCULATING FLUIDIZED TYPE RAPID SCREENING AND ACCLIMATION DEVICE FOR FUNCTIONAL MICROORGANISMS OF SOIL

Abstract

Circulating fluidized type rapid screening and acclimation device for functional microorganisms of soil. The screening and acclimation device includes a fluidized bed body, an air pump and a peristaltic pump. An upper part and a lower part of the fluidized bed body are respectively provided with a liquid outlet device and a liquid inlet device. A porous sieve plate is provided between the liquid outlet device and the fluidized bed body as well as between the liquid inlet device and the fluidized bed body. The liquid outlet device is provided with a liquid outlet and an exhaust port/sampling port. The liquid inlet device is provided with two connectors which are respectively connected to the air pump and the peristaltic pump, and an inlet of the peristaltic pump is connected to the liquid outlet.

Claims

1. A circulating fluidized type rapid screening and acclimation device for functional microorganisms of soil, comprising: a fluidized bed body, an air pump, and a peristaltic pump; an upper part and a lower part of the fluidized bed body are respectively provided with a liquid outlet device and a liquid inlet device, and a porous sieve plate is provided between the liquid outlet device and the fluidized bed body as well as between the liquid inlet device and the fluidized bed body; the liquid outlet device is provided with a liquid outlet and an exhaust port/sampling port; the liquid inlet device is provided with two connectors which are respectively connected to the air pump and the peristaltic pump, and an inlet of the peristaltic pump is connected to the liquid outlet.

2. The screening and acclimation device according to claim 1, wherein the fluidized bed is cylindrical.

3. The screening and acclimation device according to claim 1, wherein the fluidized bed is made of transparent organic glass.

4. The screening and acclimation device according to claim 2, wherein the fluidized bed is made of transparent organic glass.

5. The screening and acclimation device according to claim 1, wherein the liquid outlet device and the liquid inlet device are both cylindrical.

6. The screening and acclimation device according to claim 2, wherein the liquid outlet device and the liquid inlet device are both cylindrical.

7. The screening and acclimation device according to claim 3, wherein the liquid outlet device and the liquid inlet device are both cylindrical.

8. The screening and acclimation device according to claim 4, wherein the liquid outlet device and the liquid inlet device are both cylindrical.

9. The screening and acclimation device according to claim 1 wherein the porous sieve plate is provided with a plurality of round holes, and the round hole has an aperture of 3-5 mm.

10. The screening and acclimation device according to claim 2 wherein the porous sieve plate is provided with a plurality of round holes, and the round hole has an aperture of 3-5 mm.

11. The screening and acclimation device according to claim 3, wherein the porous sieve plate is provided with a plurality of round holes, and the round hole has an aperture of 3-5 mm.

12. The screening and acclimation device according to claim 4, wherein the porous sieve plate is provided with a plurality of round holes, and the round hole has an aperture of 3-5 mm.

13. The screening and acclimation device according to claim 5, wherein the porous sieve plate is provided with a plurality of round holes, and the round hole has an aperture of 3-5 mm.

14. The screening and acclimation device according to claim 6, wherein the porous sieve plate is provided with a plurality of round holes, and the round hole has an aperture of 3-5 mm.

15. The screening and acclimation device according to claim 7, wherein the porous sieve plate is provided with a plurality of round holes, and the round hole has an aperture of 3-5 mm.

16. The screening and acclimation device according to claim 8, wherein the porous sieve plate is provided with a plurality of round holes, and the round hole has an aperture of 3-5 mm.

17. The screening and acclimation device according to claim 1, wherein the sealing gaskets are provided between the liquid outlet device and the porous sieve plate, between the porous sieve plate and the fluidized bed body, and between the porous sieve plate and the liquid inlet device.

18. The screening and acclimation device according to claim 2, wherein sealing gaskets are provided between the liquid outlet device and the porous sieve plate, between the porous sieve plate and the fluidized bed body, and between the porous sieve plate and the liquid inlet device.

19. The screening and acclimation device according to claim 3, wherein sealing gaskets are provided between the liquid outlet device and the porous sieve plate, between the porous sieve plate and the fluidized bed body, and between the porous sieve plate and the liquid inlet device.

20. The screening and acclimation device according to claim 1, wherein an upper part of the porous sieve provided between the fluidized bed and the liquid inlet device is provided with gause.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 is a structure diagram of a circulating fluidized type rapid screening and acclimation device for functional microorganisms of soil of the present disclosure.

[0021] FIG. 2A is a front view of a liquid inlet device/a liquid outlet device in the screening and acclimation device of the present disclosure, FIG. 2B is a left sectional view of the liquid inlet device/the liquid outlet device in the screening and acclimation device of the present disclosure, and FIG. 2C is a top view of the liquid inlet device/the liquid outlet device in the screening and acclimation device of the present disclosure.

[0022] FIG. 3 is a structure diagram of a porous sieve plate in the screening and acclimation device of the present disclosure, the outside denotes a fixed round hole, and the center of the sieve plate is provided with a plurality of round holes.

[0023] FIG. 4 is a structure diagram of a sealing gasket in the screening and acclimation device of the present disclosure.

[0024] FIG. 5A is a front view of a fluidized bed body in the screening and acclimation device of the present disclosure, FIG. 5B is a left sectional view of the fluidized bed body in the screening and acclimation device of the present disclosure, and FIG. 5C is a top view of the fluidized bed body in the screening and acclimation device of the present disclosure.

[0025] FIG. 6 is a degradation effect of a strain obtained through the screening of a screening and acclimation device of the present disclosure on chlorothalonil.

[0026] In the drawings:

[0027] 1—exhaust port/sampling port; 2—porous sieve plate; 3—sealing gasket; 4—fluidized bed body; 5—air pump; 6—peristaltic pump; 7—liquid outlet device; 8—liquid inlet device.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The present disclosure is further described below with reference to the accompanying drawings, but the present disclosure is not limited to the following embodiments.

[0029] FIG. 1 is a structure diagram of a circulating fluidized type screening and acclimation device, which comprises a fluidized bed body 4, an air pump 5, and a peristaltic pump 6. To facilitate the observation of fluidization, the fluidized bed body 4 is made of transparent organic glass to guarantee the lighting, and can be covered with a cover for shading if light-avoidance culture is required. The liquidized bed is cylindrical, thus the internal space utilization is high, and the placement space can be saved, as shown in FIGS. 5A to 5C. An upper part and a lower part of the fluidized bed body 4 can be respectively provided with a liquid outlet device 7 and a liquid outlet device 8, as shown in FIGS. 2A to 2C, the liquid outlet device 7 is provided with a liquid outlet (not shown in figure) and an exhaust port/sampling port 1, and the liquid outlet is connected to the peristaltic pump 6. The liquid inlet device 8 is provided with two connectors which are respectively connected to the air pump 5 and the peristaltic pump 6. The fine solid soil particles and liquid in the fluidized bed body form a slurry state, the air is introduced into the fluidized bed body through the air pump 5 to make a gas velocity greater than the free settling velocity of the particles, then the particles are taken out by the gas, and after the gas is separated from solids, the solid particles of the soil circularly flow in a fluidized state through the peristaltic pump 6, thus forming a slurry-state fluidized bed.

[0030] A porous sieve plate 2 is provided between the liquid outlet device 7 and the fluidized bed body 4 as well as between the liquid outlet device 8 and the fluidized bed body 4, and sealing gaskets 3 are provided between the liquid outlet device 7 and the porous sieve plate, between the porous sieve plate 2 and the fluidized bed body 4, and between the porous sieve plate 2 and the liquid inlet device 8 to strengthen the fit between the porous sieve plate 2 and the fluidized bed body 4. The structure diagrams of the porous sieve plate 2 and the sealing gasket 3 are respectively shown in FIG. 3 and FIG. 4. Wherein an upper part of the porous sieve plate 2 provided between the fluidized bed body 4 and the liquid inlet device 8 is provided gauze (not shown in figure) for preventing fine sand from flowing into the bottom to block a small pipe orifice to affect the circulation and peristalsis.

[0031] The working process of the circulating fluidized type screening and acclimation device for soil functional microorganisms is as follows:

[0032] placing about 200 g of specific soil into the fluidized bed body 4 which is provided with four layers of gauze at the bottom to prevent fine sand from flowing into the bottom to block a small pipe orifice to affect the circulation and peristalsis; and then injecting a prepared liquid nutrient solution to make the soil and the liquid form a slurry state; providing oxygen for the slurry-state fluid through the air pump 5, and enabling the fluid to flow out along with the gas, wherein the pollutant circularly flows in a fluidized state through the peristaltic pump, thus making the water content in the soil be constant and be uniformly distributed in the soil in the screening and acclimation process, and finally forming three-phase equilibrium of gas, liquid, and solid to achieve the acclimation process of the soil.

[0033] In the specific acclimation process, the gas displacement of the air pump 5 can be changed, the greater the air pump 5 is, the more sufficient the oxygen supply of the slurry-state fluid is, the activation of the soil microorganisms is facilitated, and the acclimation and bacterial screening process is accelerated. However, over-sized air pump can make the liquid in the liquid outlet device splash, resulting in the increase of the liquid supplementation frequency. Therefore, the optimal gas displacement range is 2-3.2 L/min.

[0034] In the specific acclimation process, a rotational speed of the peristaltic pump 6 can be changed, the larger the peristaltic pump is, the faster the circulating velocity of the slurry-state fluid is. However, the circulation effect of the pipeline may be effected due to local lack of oxygen if the peristaltic pump is over-sized, thus the optimal rotational speed range is 170-200 r/min.

[0035] The process of screening using the device of the present disclosure is illustrated below by taking the separation of chlorothalonil-degrading bacteria as an example:

[0036] 1. Culture Medium

[0037] inorganic salt liquid culture medium: 1.5 g of NH.sub.4NO.sub.3, 0.5 g of KH.sub.2PO.sub.4, 1.5 g of K.sub.2HPO.sub.4, 1.0 g of NaCl, 0.2 g of MgSO.sub.4.7H.sub.2O, 1000 mL of deionized water, sterilizing at 121° C. for 15 min.

[0038] LB solid culture medium: 10 g of peptone, 5 g of yeast, 10 g of NaCl, 25 g of agar, 1000 mL of deionized water, sterilizing at 121° C. for 15 min.

[0039] inorganic salt solid culture medium: 1.5 g of NH.sub.4NO.sub.3, 0.5 g of KH.sub.2PO.sub.4, 1.5 g of K.sub.2HPO.sub.4, 1.0 g of NaCl, 0.2 g of MgSO.sub.4.7H.sub.2O, 25 g of agar, 1000 mL of deionized water, sterilizing at 121° C. for 15 min.

[0040] PDA culture medium: taking 38 g of potato dextrose agar culture medium and 1000 mL of deionized water, oscillating and mixing, and then sterilizing at 115° C. for 20 min.

[0041] Chlorothalonil-containing culture medium: adding a chlorothalonil solution with a certain concentration dissolved in acetone into the liquid or the solid culture medium, and sterilizing at 121° C. for 15 min.

[0042] 2. Test Soil

[0043] harvesting normal soil (15-30 cm) without chlorothalonil applied from the garden of the Science and Technology Park of Xuancheng in Anhui province; and picking out impurities such as straw fragments, broken stones, plastic and roots for later use.

[0044] 3. Screening of Soil Through Circulating Acclimation Method

[0045] sterilizing the fluidized bed body 4, the pipe and the connector at 115° C. for 30 min, then adding the test soil into the fluidized bed body 4, with the soil layer thickness of 5-6 cm; starting the air pump at first, wherein the gas displacement of the air pump is 3.2 L/min, then adding 2 L of sterilized inorganic salt liquid culture medium with the chlorothalonil content of 300 mg/L into the device through the peristaltic pump, wherein the culture medium starts circulation as the peristaltic pump is immersed into the soil layer, and the rotational speed of the peristaltic pump is 300 rpm; and supplementing the circulating liquid at fixed period according to the evaporation condition of the circulating liquid of the inorganic salt culture medium; respectively taking muddy water from the lower connector and the liquid from the upper connector after 2 d, 4 d, 6 d, and 10 d, sequentially diluting by 10, 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6 and 10.sup.7 times; respectively applying 200 μL of liquid onto the LB solid culture medium, the inorganic salt solid culture medium and the PDA culture medium with the chlorothalonil contents of 100 mg/L, 200 mg/L, 400 mg/L, 500 mg/L, and 600 mg/L, placing the culture mediums in an incubator for inverted culture at 30° C. for 7 d, observing the growth condition of the bacterial colonies, and the picking out strains with different forms to respectively separate and purify, conducting streak separation for multiple times until single colonies are obtained, and refrigerating and storing well-grown pure strains in a refrigerator at 4° C. for later use.

[0046] 4. Liquid Phase Enrichment Method

[0047] taking 10 g of test soil sample to add in a 300 mL conical flask filled with 90 mL of inorganic salt culture medium, and adding an appropriate amount of glass beads; oscillating the conical flask at 30° C. and 170 rpm for 5 h; and centrifuging a soil mixed solution at 3000 rpm for 5 min; inoculating the centrifuged supernatant into the inorganic salt culture medium with the chlorothalonil content of 200 mg/L, and oscillating and culturing at 30° C. and 170 rpm for 3 d; transferring 2 mL of liquid into another conical flask for enrichment culture using a sterile pipette; transferring the liquid for three times to apply the liquid on the LB solid culture medium, the inorganic salt solid culture medium and the PAD culture medium respectively, placing the culture mediums in the incubator for inverted culture at 30° C. for 7 d, observing the growth condition of the bacterial colonies, and then picking out the strains with different forms to respectively separate and purify, conducting streak separation for multiple times until single colonies are obtained, and refrigerating and storing well-grown pure strains in a refrigerator at 4° C. for later use.

[0048] 5, Screening Result of Chlorothalonil Degradation Strain

[0049] screening the chlorothalonil degradation strain from the same source using two methods (the method of the present disclosure and the liquid phase enrichment method), wherein the results show that 17 strains of bacteria and 3 strains of fungi obtained through the screening of a soil circulating acclimation method; and only two strains of bacteria and no fungi are obtained through the screening of the liquid phase enrichment method. Through subsequent strain identification, the result is shown in the following table 1, confirming that the two strains of bacteria obtained through the screening of the liquid-phase enrichment method can also be obtained through the screening of circulating acclimation. Therefore, the strains obtained through the screening of the liquid-phase enrichment method are few in variety and single in bacterial colony; and chlorothalonil degradation strains with rich varieties can be screened by applying a soil circulating acclimation method, the bacterial colonies have rich diversity, and an important foundation is laid for development of efficient chlorothalonil degradation bacteria agent.

TABLE-US-00001 TABLE 1 Chlorothalonil degradation bacteria obtained using different methods Screening method Bacteria Fungi Soil circulating Enterobacter cloacae, Cunninghamella acclimation Burkholderia Symbiont of bertholletiae, method Cavelerius saccharivorus, TUAH-f2 Burkholderia cepacian, TUAH-f3 Bacillus nanhaiensis, Serratia marcescens, Pseudomonas putida, Burkholderia Zhejiangensis, Pseudomonas geniculate, Pseudomonas monteilii, Pseudomonas aeruginosa, TUAH-11, Stenotrophomonas maltophilia, TUAH-13, Pseudomonas dentitrificans, Pseudomonas hibiscicola, Burkholderia anthina, Stenotrophomonas maltophilia Liquid phase Burkholderia Symbiont of — enrichment Cavelerius method Saccharivorus, Burkholderia cepacia

[0050] 6. Determination of Degradation Ability of Screened Strains on the Chlorothalonil

[0051] Respectively naming 17 strains of bacteria obtained through screening as TUAH-1 to TUAH-17, and naming three strains of fungi as TUAH-f1, TUAH-f2, and TUAH-f3, wherein the degradation rate of the strain to 20 mg/L chlorothalonil for two days is as shown in FIG. 6.

[0052] The result shows that the strains obtained through the screening of the soil circulating acclimation method using the device of the present disclosure have degradation effect on Chlorothalonil, and the degradation rate is up to 63.6%, which has laid a good foundation for the subsequent practical degradation of Chlorothalonil in the field.

[0053] The screening and acclimation device of the present disclosure is simple in sampling in later period, the gas introduction in the device is controllable, the acclimation flow rate is controllable, and other gases can be used for creating anaerobic or other special environments; and the circulating liquid can be replaced at any time to meet different experiment requirements.

[0054] In the circulating acclimation process, the advantages of simple operation, energy conservation, emission reduction, low energy consumption and low cost of the device are fully reflected.