METHOD FOR PROMOTING GROWTH OF GAS-FERMENTED MICROORGANISMS

Abstract

The present invention relates to a method for promoting growth of gas-fermented microorganisms, comprising the following steps: S1. ultrasonically blending a surfactant and a culture medium, then adding a fluorine-containing alkyl compound to a mixture, and ultrasonically processing the mixture to obtain a perfluorocarbon nanoemulsion; S2. inoculating a bacterial suspension into the perfluorocarbon nanoemulsion, and introducing simultaneously a mixed gas to obtain a precursor; S3. cultivating the precursor in a shaker, wherein the precursor has a diameter of bubbles of 2.0-4.2 mm, and the bubbles have the total volume of less than 40 ml. Under these conditions, the microorganisms have a high utilization rate of the gas, can grow and metabolize more quickly, and can obtain bacteria and products more quickly. Compared with a traditional culture system, addition of a mass transfer material can promote gas mass transfer. Therefore, the gas consumption is small, and the cost is low.

Claims

1. A method for promoting growth of gas-fermented microorganisms, comprising the following steps: S1. ultrasonically blending a surfactant and a culture medium, then adding a fluorine-containing alkyl compound to a mixture, and ultrasonically processing the mixture to obtain a perfluorocarbon nanoemulsion; S2. inoculating a bacterial suspension into the perfluorocarbon nanoemulsion, and introducing simultaneously a mixed gas to obtain a precursor; S3. cultivating the precursor in a shaker; wherein, bubbles in the precursor have a diameter of 2-4.2 mm, and the total volume of less than 40 ml.

2. The method for promoting the growth of the gas-fermented microorganisms according to claim 1, wherein the mixed gas is selected from one or more of nitrogen, argon, oxygen, carbon dioxide, carbon monoxide and hydrogen.

3. The method for promoting the growth of the gas-fermented microorganisms according to claim 1, wherein the compositions of the culture medium comprise one or more of halogenide, phosphate, hydrophosphate, sulphate, and sulphite, hydrates of the forgoing compositions, and trace elements.

4. The method for promoting the growth of the gas-fermented microorganisms according to claim 1, wherein the bacterial suspension has an OD600 value of 0.05-1.

5. The method for promoting the growth of the gas-fermented microorganisms according to claim 1, wherein the surfactant is selected from a polymer surfactant containing an alkoxy chain segment.

6. The method for promoting the growth of the gas-fermented microorganisms according to claim 1, wherein the fluorine-containing alkyl compound is selected from one or more of perfluorodecalin, tetradecafluorohexane, dodecafluorocyclohexane, perfluoroheptane, dodecafluoropentane, decafluoropentane, and heptafluoropropane.

7. The method for promoting the growth of the gas-fermented microorganisms according to claim 1, wherein the perfluorocarbon nanoemulsion has an average

8. The method for promoting the growth of the gas-fermented microorganisms according to claim 1, wherein a bacterium is cupriavidus necator.

9. The method for promoting the growth of the gas-fermented microorganisms according to claim 1, wherein the culture medium has a temperature of 30-37° C., a rotational speed of 100-300 rpm; time of 48-120 h and pH of 6.5-7.5.

10. The method for promoting the growth of the gas-fermented microorganisms according to claim 2, wherein the mixed gas has a percent by volume of hydrogen of 20-50 vt %.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 shows an average particle size distribution diagram of a perfluorocarbon nanoemulsion of Embodiment 1.

[0033] FIGS. 2A-2C show an appearance of cultured precursors of Embodiment 1 and Comparative Embodiments 1-2, respectively.

[0034] FIG. 3 shows the OD600 value and PHB produced in Embodiment 1, Comparative Embodiments 1-2.

DETAILED DESCRIPTION OF EMBODIMENTS

[0035] In order to illustrate the technical solutions of the present invention more clearly, the following embodiments are given. Unless otherwise stated, the raw materials, reactions and post-processing means in the embodiments are common raw materials on market and technical means well known to those skilled in the art.

[0036] Bacteria described in embodiments of the present invention adopt cupriavidus necator.

[0037] A surfactant described in the embodiments of the present invention is a polymer surfactant containing an alkoxy chain segment, which is Pluronic series products purchased from BASF.

[0038] A method for testing a particle size of a perfluorocarbon nanoemulsion described in the embodiments and comparative embodiments of the present invention uses a nanoparticle size and a surface potential tester for measurement.

[0039] The method for testing a diameter of bubbles described in the embodiments and the comparative embodiment of the present invention adopts ImageJ software to measure a real picture.

[0040] The method for testing a total volume of the bubbles described in the embodiments and the comparative embodiment of the present invention adopts ImageJ software to measure the real picture.

[0041] pH of a culture medium described in Embodiments 1-3 of the present invention is 6.8, and the compositions and concentration of the culture medium are described in Table 1 below.

TABLE-US-00001 TABLE 1 Compositions and Concentration of Culture Medium Compositions Concentration (g/l) NH.sub.4C1 1.0 NaH,PO4•2H.sub.2O 5.2 Na.sub.2HPO.sub.4•12H.sub.2O 11.6 K.sub.2SO.sub.4 0.45 MgSO.sub.4•7H.sub.2O 0.80 CaCl.sub.2•2H.sub.2O 0.08 Trace elements 0.001

[0042] The forgoing trace elements as well as the compositions and concentrations thereof were described in Table 2 below.

TABLE-US-00002 TABLE 2 Composition and Concentration of Trace Elements Compositions Concentration (g/l) HCl 0.1 FeSO.sub.4•7H.sub.2O 15 MnSO.sub.4•H.sub.2O 2.4 ZnSO.sub.4•7H.sub.2O 2.4 CuSO.sub.4•5H.sub.2O 0.48

Embodiment 1

[0043] A method for promoting growth of gas-fermented microorganisms, comprising the following steps:

[0044] S1. ultrasonically blending a surfactant Pluronic F68 and 50 ml of a culture medium (wherein, Pluronic F68 is 2.8 wt % of the culture medium) with an ultrasonic cleaner for 30 min, so that the surfactant was completely dissolved; taking 2250 μl of perfluorodecalin and 2250 μl of tetradecafluorohexane, ultrasonically blending with an ultrasonic cleaner for 30 min to obtain a perfluorocarbon nanoemulsion, the perfluorocarbon nanoemulsion at this time having a measured average particle size of about 254 nm, and obtained particle size test results being shown in FIG. 1;

[0045] S2. culturing a cupriavidus necator with a culture medium for 20 h to obtain an intermediate, then centrifuging, pouring out a supernatant, washing with 25 ×PBS, and then pouring out the supernatant by centrifugation. This step was repeated three times, and bacteria were dispersed in the culture medium to obtain a bacterial suspension.

[0046] Establishing 2 groups of parallel experiments: covering an anaerobic bottle with a rubber stopper and an aluminum seal, sterilizing in a sterilizer at 121° C. for 20 min, adding the perfluorocarbon nanoemulsion to the bacterial suspension, mixing evenly, then inoculating 45 ml of the bacterial suspension containing the perfluorocarbon nanoemulsion into the anaerobic bottle, introducing a mixed gas (a volume ratio of each gas in the mixed gas was N.sub.2:H.sub.2:O.sub.2:CO.sub.2=49:37:7:7) at the same time, making a pressure in the anaerobic bottle be about 0.3 MPa to obtain a precursor, the bubbles in the precursor having the diameter of 2 mm, and the bubbles having the total volume of 30 ml.

[0047] S3. putting the precursor into a shaker, and culturing at a constant temperature of 30° C., at a rotation speed of 100 rpm, and at pH of 6.5 for 72 h.

Embodiment 2

[0048] A method for promoting growth of gas-fermented microorganisms, comprising the following steps:

[0049] S1. ultrasonically blending a surfactant Pluronic F68 and 30 ml of a culture medium (wherein, Pluronic F68 is 2.9 wt % of the culture medium) with an ultrasonic cleaner for 30 min, so that the surfactant was completely dissolved; taking 1350 μl of perfluorodecalin and 1350 μl of tetradecafluorohexane, ultrasonically blending with an ultrasonic cleaner for 20 min to obtain a perfluorocarbon nanoemulsion, the perfluorocarbon nanoemulsion at this time having a measured average particle size of about 280 nm.

[0050] S2. culturing a cupriavidus necator with a culture medium for 22 h to obtain an intermediate, then centrifuging, pouring out a supernatant, washing with 25×PBS, and then pouring out the supernatant by centrifugation. This step was repeated three times, and bacteria were dispersed in the culture medium to obtain a bacterial suspension.

[0051] Establishing 2 groups of parallel experiments: covering an anaerobic bottle with a rubber stopper and an aluminum seal, sterilizing in a sterilizer at 121° C. for 20 min, adding the perfluorocarbon nanoemulsion to the bacterial suspension, mixing evenly, then inoculating 50 ml of the bacterial suspension containing the perfluorocarbon nanoemulsion into the anaerobic bottle, introducing a mixed gas (a volume ratio of each gas in the mixed gas was N.sub.2:H.sub.2:O.sub.2:CO.sub.2=40:40:10:10) at the same time, making a pressure in the anaerobic bottle be about 0.3 MPa to obtain a precursor, the bubbles in the precursor having the diameter of 4.2 mm, and the bubbles having the total volume of 40 ml.

[0052] S3. putting the precursor into a shaker, and culturing at a constant temperature of 32° C., at a rotation speed of 300 rpm, and at pH of 7.5 for 96 h.

Embodiment 3

[0053] A method for promoting growth of gas-fermented microorganisms, comprising the following steps:

[0054] S1. ultrasonically blending a surfactant Pluronic F68 and 20 ml of a culture medium (wherein, Pluronic F68 is 3.0 wt % of the culture medium) with an ultrasonic cleaner for 30 min, so that the surfactant was completely dissolved; taking 900 μl of perfluorodecalin and 900 μl of tetradecafluorohexane, ultrasonically blending with an ultrasonic cleaner for 35 min to obtain a perfluorocarbon nanoemulsion, the perfluorocarbon nanoemulsion at this time having a measured average particle size of about 250 nm.

[0055] S2. culturing a cupriavidus necator with a culture medium for 20 h to obtain an intermediate, then centrifuging, pouring out a supernatant, washing with 25×PBS, and then pouring out the supernatant by centrifugation. This step was repeated three times, and bacteria were dispersed in the culture medium to obtain a bacterial suspension.

[0056] Establishing 2 groups of parallel experiments: covering an anaerobic bottle with a rubber stopper and an aluminum seal, sterilizing in a sterilizer at 121° C. for 20 min, adding the perfluorocarbon nanoemulsion to the bacterial suspension, mixing evenly, then inoculating 45 ml of the bacterial suspension containing the perfluorocarbon nanoemulsion into the anaerobic bottle, introducing a mixed gas (a volume ratio of each gas in the mixed gas was N.sub.2:H.sub.2:O.sub.2:CO.sub.2=45:40:8:7) at the same time, making a pressure in the anaerobic bottle be about 0.4 MPa to obtain a precursor, the bubbles in the precursor having the diameter of 3.5 mm, and the bubbles having the total volume of 35 ml.

[0057] S3. putting the precursor into a shaker, and culturing at a constant temperature of 35° C., at a rotation speed of 200 rpm, and at pH of 7.0 for 120 h.

Comparative Embodiment 1

[0058] S1. culturing a cupriavidus necator with a culture medium for 20 h to obtain an intermediate, then centrifuging, pouring out a supernatant, washing with 25×PBS, and then pouring out the supernatant by centrifugation. This step was repeated three times, and bacteria were dispersed in the culture medium to obtain a bacterial suspension.

[0059] Establishing 2 groups of parallel experiments: covering an anaerobic bottle with a rubber stopper and an aluminum seal, sterilizing in a sterilizer at 121° C. for 20 min, then inoculating 45 ml of the bacterial suspension into the anaerobic bottle, introducing a mixed gas (compositions of each gas in the mixed gas was N.sub.2:H.sub.2:O.sub.2:CO.sub.2=49:37:7:7) at the same time, and making a pressure in the anaerobic bottle be about 0.3 MPa to obtain a precursor.

[0060] S2. putting the precursor into a shaker, and culturing at a constant temperature of 30° C., at a rotation speed of 100 rpm, and at pH of 6.5 for 72 h. No air bubbles are provided in the precursor.

Comparative Embodiment 2

[0061] Substances and an operation method used in Comparative Embodiment 2 were the same as those in Embodiment 1. Only difference was that during preparation of the precursor, a shaking force of the perfluorocarbon nanoemulsion was slightly increased, so that the bubbles in the precursor had a diameter of 1.1 mm, and a total volume of 60 ml.

Test Embodiments

[0062] An anaerobic bottle of a cultured precursor of Embodiment 1 and Comparative Embodiments 1-2 was opened. An OD600 value measured by a bacterial liquid was used to characterize growth of microorganisms. 2 ml of the bacterial liquid was centrifuged and dissolved simultaneously. A liquid chromatograph was used to measure PHB to characterize a product yield.

[0063] FIGS. 2A-2C showed an appearance of cultured precursors of Embodiment 1 and Comparative Embodiments 1-2, respectively. As can be seen from FIGS. 2a-c, a diameter and a total volume of bubbles in Embodiment 1 were moderate. In Comparative Embodiment 1, since no surfactant was added, no bubbles were generated. In Comparative Embodiment 2, due to addition of a surfactant, vigorously shaking was performed. Therefore, the bubbles had a smaller diameter and the largest total volume.

[0064] FIG. 3 showed measured OD600 and PHB results of the cultured precursor of Embodiment 1 and Comparative Embodiments 1-2.

[0065] As can be seen from FIG. 3, in Embodiment 1, the surfactant was added. The bubbles were controlled to be moderate in the diameter. Growth of microorganisms and a yield of a product PHB were the best. No surfactant was added in Comparative Embodiment 1. The PHB yield under normal growth of the microorganisms was 12.8 mg/l. However, in Comparative Embodiment 2, due to the small diameter and the excessive total volume of the bubbles, a mass transfer coefficient was small, which greatly hindered the growth of the microorganisms and the synthesis of the product.

[0066] It will be apparent to those skilled in the art that the present invention is not limited to the details of the forgoing exemplary embodiments, but that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments are to be regarded in all respects as illustrative and not restrictive. The scope of the present invention is defined by the appended claims rather than the foregoing description. All changes within the meaning and scope of the equivalents of claims are included in the present invention.

[0067] In addition, it should be understood that although this specification is described in terms of embodiments, not each embodiment only includes an independent technical solution. This description in the specification is only for the sake of clarity. Those skilled in the art should take the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementations that can be understood by those skilled in the art.