CN110643562A - Culture method for improving capacity of methane oxidation mixed flora for synthesizing poly-beta-hydroxybutyrate - Google Patents
Culture method for improving capacity of methane oxidation mixed flora for synthesizing poly-beta-hydroxybutyrate Download PDFInfo
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Abstract
The invention discloses a culture method for improving the ability of methane oxidation mixed flora to synthesize poly-beta-hydroxybutyrate, belonging to the technical field of organic waste resource utilization and biodegradable plastic synthesis. The methane oxidation mixed flora domesticated from the activated sludge for a long time by taking nitrogen as a nitrogen source is used as a raw material, and the methane oxidation mixed flora is cultured by optimizing a culture mode and circularly utilizing nitrate and nitrogen as nitrogen sources, so that the biological rate, the biomass concentration and the PHB synthetic capacity of the methane oxidation mixed flora are obviously improved. The method provides a technical basis for the industrial application of the PHB technology synthesized by methane oxidation mixed flora by utilizing methane, so as to reduce the pollution caused by petrochemical plastics and the emission of greenhouse gases.
Description
Technical Field
The invention belongs to the technical field of organic waste resource utilization and biodegradable plastic synthesis, and relates to a culture method for improving the capability of synthesizing poly-beta-hydroxybutyrate (PHB) from methane oxidation mixed flora domesticated from activated sludge.
Background
The environmental pollution problem caused by petrochemical plastics and the exhaustion of petroleum resources make the research of sustainable biodegradable plastics become a hot field. Among them, PHB is a high molecular polymer synthesized by microorganisms under the condition of growth restriction, and is a promising substitute for traditional plastics due to good biodegradability and biocompatibility. However, the production cost of PHB is high, which severely limits the wide application of PHB, and studies show that the production cost of PHB can be reduced by about 30-35% by using waste gas containing methane as a substrate. In addition, methane is the second largest greenhouse gas in the world, has the advantage of large quantity, and releases a large amount of methane in the exploitation process of fossil fuels and the anaerobic fermentation process of organic wastes. Therefore, the synthesis of PHB by using the waste gas containing methane as a substrate can reduce the emission of greenhouse gases.
In the previous studies of our group of subjects, it was found that a mixed bacterial group for methane oxidation, which comprises a predominant bacterial group of type II methane-oxidizing bacteria and a large amount of other heterotrophic bacteria having PHB-synthesizing ability, can be acclimatized from activated sludge by using a culture medium containing 5 to 10. mu. mol/LCu2+ under the conditions that methane is used as a sole carbon source and nitrogen is used as a sole nitrogen source and that the headspace gas composition is 10 to 20% oxygen, 25% methane gas and 55 to 65% nitrogen. The process of synthesizing PHB by using the mixed flora does not need aseptic conditions, and the mixed flora has wider substrate range and low requirement on substrate composition and is beneficial to further reducing the production cost of the PHB.
However, nitrogen is used as a nitrogen source, the growth speed of the methane oxidation mixed flora is slow, the biomass concentration is low, large-scale industrial production is not facilitated, and researches show that continuous nitrogen culture can inhibit the activity of pure strains of the methane oxidation bacteria and reduce the PHB synthesis capacity of the pure strains, and the growth rate and the PHB synthesis capacity of the pure strains are obviously improved after nitrate culture. Therefore, by searching for an appropriate culture mode and optimizing the nitrogen source supply method, it is possible to improve the growth rate, biomass concentration and PHB synthesis ability of the methane oxidizing mixed flora cultured in nitrogen for a long period of time.
Disclosure of Invention
The invention aims to provide a culture method for enhancing the PHB synthesis capability of a methane oxidation mixed flora domesticated by taking nitrogen as a nitrogen source by utilizing methane, and provides a technical basis for promoting the industrial application of a PHB synthesis technology by the methane oxidation mixed flora.
The technical scheme of the invention is as follows:
a culture method for improving the ability of methane oxidation mixed flora to synthesize poly-beta-hydroxybutyrate comprises the following steps:
step 1: the method comprises the following steps of taking methane oxidation mixed flora enriched and domesticated from activated sludge by using nitrogen as a unique nitrogen source and methane as a unique carbon source as a raw material;
step 2: preparation of culture Medium
(1) Preparing a mineral salt NFMS culture medium without nitrogen elements, wherein the mineral salt NFMS culture medium comprises the following components: KH of 0.272 g/L2PO42.868 g/L of Na2HPO4·H2O, 0.1 g/L MgSO4·7H2O, 0.20 g/L CaCl2·6H2O and 1 mL/L of trace elements;
(2) preparing a nitrate-containing mineral salt NMS culture medium, which comprises the following components: 0.1 g/L KNO3KH of 0.272 g/L2PO42.868 g/L of Na2HPO4·H2O, 0.1 g/L MgSO4·7H2O, 0.20 g/L CaCl2·6H2O and 1 mL/L of trace elements;
(3) preparing a trace element solution: 250 mg/L Na-EDTA, 500 mg/L FeSO4·7H2O, 380 mg/L Fe-EDTA, 400 mg/L ZnSO4·7H2O, 15mg/L of H3BO320 mg/L of MnCl2·4H2O, 260 mg/L of Na2MoO4·2H2O, 10 mg/L NiCl2·6H2O, 50 mg/L CoCl2·6H2O；
(4) Preparing CuSO4·5H2O stock solution;
(5) CuSO was added to NFMS and NMS media4·5H2Stock solution of O, CuSO4The molar concentration of (a) is 5-10 mu mol/L;
and step 3: nitrate culture process of methane oxidation mixed flora
(1) Adding NMS culture medium obtained in the step 2 (5) into the system, adding the methane oxidation mixed flora in the step 1 according to the volume ratio of 20-30% to ensure that the volume ratio of the headspace of the system to the liquid phase is 2:1-5:1, and sealing the system;
(2) replacing the headspace gas to make the composition of the headspace gas be 40-60% of oxygen and 40-60% of methane gas; replacing headspace gas every 12-24 hours to ensure sufficient gas substrate; performing nitrate culture on the methane oxidation mixed flora for 36-48 hours;
and 4, step 4: nitrogen cultivation process of methane oxidation mixed flora
(1) Adding the NFMS culture medium obtained in the step 2 (5) into the system, adding the methane oxidation mixed flora cultured by the nitrate obtained in the step 3 according to the volume ratio of 4-6% to ensure that the volume ratio of the headspace of the system to the liquid phase is 2:1-5:1, and sealing the system;
(2) replacing the headspace gas to make the headspace gas consist of 10-20% oxygen, 25% methane and 55-65% nitrogen; replacing headspace gas every 24-72 hours to ensure sufficient gas substrate; carrying out nitrogen culture on the methane oxidation mixed flora for 5-6 days;
and 5: circulating culture of nitrate and nitrogen of methane oxidation mixed flora
Performing circulating culture of nitrate and nitrogen on the methane oxidation mixed flora according to the steps 3 and 4;
step 6: PHB Synthesis Process
(1) Collecting thalli for PHB synthesis after the end of each nitrate and nitrogen culture;
(2) adding the collected thalli and the NFMS culture medium obtained in the step (2) and (5) into the system to ensure that the volume ratio of the headspace of the system to the liquid phase is 5:1-11:1, and sealing the system;
(3) replacing headspace gas to make the composition of headspace gas be 50-60% methane and 40-50% oxygen, and making PHB synthesis under the condition of nitrogen deficiency. In order to ensure sufficient gas substrate, the headspace gas is replaced every 24 to 36 hours, and the synthesis process of the PHB needs about 72 hours;
and 7: after 7-11 times of circulating culture, the PHB synthetic capacity of the methane oxidation mixed flora tends to be stable. In the circulating culture process, after nitrogen culture and nitrate culture, the maximum PHB content of the mixed flora is equivalent to that of continuous nitrogen culture, and the maximum PHB concentration is improved by 0.64-1.27 times after nitrate culture.
Drawings
FIG. 1 shows the oxidation of methane by the mixed bacteria of (a) N2PHB synthesis after cyclic culture for different time, FIG. 2 methane oxidation mixed flora via (b) NO3 -Circulation ofPHB synthesis after different times of culture.
Detailed Description
The following further describes a specific embodiment of the present invention with reference to the drawings and technical solutions. In order to improve the PHB synthesis capacity of the methane oxidation mixed flora domesticated by taking nitrogen as a nitrogen source for a long time, nitrate and nitrogen are used as nitrogen sources to perform circulating culture on the mixed flora.
Example 1
(1) The method is characterized in that a methane oxidation mixed flora domesticated from activated sludge collected from a secondary sedimentation tank of a municipal sewage treatment plant of Daizhuang of Dalian city, Liaoning province by using nitrogen as a unique nitrogen source is used as a raw material, and the mixed flora has stable PHB synthesis capacity;
(2) preparing NFMS and NMS culture medium, adding CuSO4Stock solution of CuSO4The concentration of (b) is 5-10. mu. mol/L. Then, the pH value of the NMFS culture medium is adjusted to 6.8 by using 1 mol/L sodium hydroxide solution;
(3) taking 10-15 mL of the methane oxidation mixed flora, centrifugally collecting, using NMS culture medium for resuspension, diluting to 50 mL by using NMS culture medium, inoculating into a 300 mL serum bottle, and sealing by using a rubber plug and a screw cap;
(4) communicating the 300 mL serum bottle inoculated with the methane oxidation mixed flora with the atmosphere by using a fine needle with a filter, and then enabling the headspace gas of the serum bottle to consist of 40-60% of oxygen and 40-60% of methane by using a method of vacuumizing and gas refilling. The serum bottle is placed in a shaker at 30 ℃ and 150 r/min for nitrate culture of methane-oxidizing bacteria mixed flora for 36-48 hours. Periodically monitoring the gas composition of the headspace by using a gas chromatograph with a thermal conductivity detector, and replacing the headspace gas every 12-24 hours to ensure sufficient gas substrate;
(5) after the end of the nitrate culture, 2-3 mL of the bacterial solution was inoculated into a 300 mL serum bottle containing 50 mL of NFMS medium and sealed with a rubber stopper and a screw cap. The blood serum bottle is communicated with the atmosphere by a fine needle head with a filter, and then the composition of the headspace gas of the blood serum bottle is 10-20% of oxygen, 25% of methane and 55-65% of nitrogen by the methods of vacuumizing and gas refilling. Placing the serum bottle in a shaker at 30 deg.C and 150 r/min, and culturing with nitrogen gas for 5-6 days. Periodically monitoring the gas composition of the headspace by using a gas chromatograph with a thermal conductivity detector, and replacing the headspace gas every 24-72 hours to ensure sufficient gas substrate;
(6) performing nitrate and nitrogen circulation culture on the mixed flora according to the steps (4) and (5), and performing circulation culture for about 11 times;
(7) after each culture, respectively centrifuging for 5 minutes under 10000 r/min by using a centrifuge to collect thalli after nitrate culture and nitrogen culture, and then re-suspending the collected thalli to the condition that the collected thalli contains 5 mu mol/L CuSO4In the NFMS culture medium, the total volume of the bacterial liquid after heavy suspension is the same as that of the collected bacterial liquid, and PHB synthesis is carried out;
(8) the resuspended methanogenic mixed bacterial population was inoculated into a series of 120mL sera, each serum bottle containing 10mL of bacterial solution, and sealed with a rubber stopper and a screw cap. A120 mL serum bottle containing bacterial liquid is communicated with the atmosphere by using a fine needle with a filter, and then the composition of headspace gas of the serum bottle is 50-60% of methane and 40-50% of oxygen by using a method of vacuumizing and gas refilling. Periodically monitoring the gas composition of the headspace by using a gas chromatograph with a thermal conductivity detector, and replacing the headspace gas every 24 hours to ensure sufficient gas substrate;
(9) the 120mL serum bottle containing the bacterial solution after the NFMS heavy suspension was placed in a shaker at 30 ℃ and 150 r/min for PHB synthesis. After 72 hours, the synthesis process of PHB of the methane oxidation mixed flora is finished, and two 120mL serum bottles are taken periodically each time during the process, and the change of the PHB content in the mixed flora cells is detected;
(10) after the methane oxidation mixed flora is circularly cultured by nitrate and nitrogen for about 7 times, the capability of synthesizing PHB by using methane is stable, the maximum PHB content of the mixed flora is equivalent to that of continuous nitrogen culture after the nitrogen culture and the nitrate culture, and the maximum PHB concentration is improved by 0.95-1.27 times after the nitrate culture. FIG. 1 shows PHB synthesis curves of the mixed bacterial flora after 1, 3, 7 and 11 circulations.
Example 2
Nitrate and nitrogen circulation culture was performed using a methane-oxidizing mixed bacterial group having a stabilized PHB synthesis ability, which was acclimatized from activated sludge collected from a secondary sedimentation tank of Ling river municipal sewage treatment plant, Dalian city, Liaoning, using nitrogen as the only nitrogen source, and the other embodiments were the same as in example 1.
Claims (6)
1. A culture method for improving the ability of methane oxidation mixed flora to synthesize poly-beta-hydroxybutyrate is characterized by comprising the following steps:
the methane oxidation mixed flora enriched and domesticated from the activated sludge by using nitrogen as a unique nitrogen source and methane as a unique carbon source is used as a raw material.
2. The culture method for improving the ability of methane-oxidizing mixed bacteria to synthesize poly-beta-hydroxybutyrate according to claim 1, wherein the culture medium is prepared from:
(1) preparing a mineral salt NFMS culture medium without nitrogen elements, wherein the mineral salt NFMS culture medium comprises the following components: KH of 0.272 g/L2PO42.868 g/L of Na2HPO4·H2O, 0.1 g/L MgSO4·7H2O, 0.20 g/L CaCl2·6H2O and 1 mL/L of trace elements;
(2) preparing a nitrate-containing mineral salt NMS culture medium, which comprises the following components: 0.1 g/L KNO3KH of 0.272 g/L2PO42.868 g/L of Na2HPO4·H2O, 0.1 g/L MgSO4·7H2O, 0.20 g/L CaCl2·6H2O and 1 mL/L of trace elements;
(3) preparing a trace element solution: 250 mg/L Na-EDTA, 500 mg/L FeSO4·7H2O, 380 mg/L Fe-EDTA, 400 mg/L ZnSO4·7H2O, 15mg/L of H3BO320 mg/L of MnCl2·4H2O, 260 mg/L of Na2MoO4·2H2O, 10 mg/L NiCl2·6H2O, 50 mg/L CoCl2·6H2O；
(4) Preparing CuSO4·5H2O stock solution;
(5) CuSO was added to NFMS and NMS media4·5H2Stock solution of O, CuSO4The molar concentration of (b) is 5-10 [ mu ] mol/L.
3. The culture method for improving the ability of the mixed methane-oxidizing bacteria to synthesize the poly-beta-hydroxybutyrate according to claim 1, which is characterized in that the nitrate culture process of the mixed methane-oxidizing bacteria comprises the following steps:
(1) adding the methane oxidation mixed flora in the step 1 into the NMS culture medium obtained in the step 2 (5) according to the volume ratio of 20-30% to ensure that the volume ratio of the headspace of the system to the liquid phase is 2:1-5:1, and sealing the system;
(2) replacing the headspace gas to make the composition of the headspace gas be 40-60% of oxygen and 40-60% of methane gas; replacing headspace gas every 12-24 hours to ensure sufficient gas substrate; and carrying out nitrate culture on the methane oxidation mixed flora for 36-48 hours.
4. The culture method for improving the ability of methane-oxidizing mixed flora to synthesize poly-beta-hydroxybutyrate according to claim 1, wherein the nitrogen culture process of the methane-oxidizing mixed flora comprises the following steps:
(1) adding the mixed flora cultured by the nitrate in the step 3 into the NFMS culture medium obtained in the step 2 (5) according to the volume ratio of 4-6% to ensure that the volume ratio of the headspace of the system to the liquid phase is 2:1-5:1, and sealing the system;
(2) replacing the headspace gas to make the headspace gas consist of 10-20% oxygen, 25% methane and 55-65% nitrogen; replacing headspace gas every 24-72 hours to ensure sufficient gas substrate; the methane oxidation mixed flora is cultured for 5 to 6 days in nitrogen.
5. The culture method for improving the capability of the mixed methane-oxidizing bacteria to synthesize the poly-beta-hydroxybutyrate according to claim 1, which is characterized in that the mixed methane-oxidizing bacteria is cultured in a circulating mode by nitrate and nitrogen:
the mixed bacterial colonies for methane oxidation were subjected to circulating culture of nitrate and nitrogen as described in 3 and 4.
6. The culture method for improving the ability of methane-oxidizing mixed bacteria to synthesize poly-beta-hydroxybutyrate according to claim 1, wherein PHB synthesis process comprises:
(1) at the end of each nitrate and nitrogen culture, cells were collected for PHB synthesis.
(2) Adding the collected thalli and the NFMS culture medium obtained in the step (2) and (5) into the system to ensure that the volume ratio of the headspace of the system to the liquid phase is 5:1-11:1, and sealing the system;
(3) replacing headspace gas to make the composition of headspace gas be 50-60% methane and 40-50% oxygen, and making PHB synthesis under the condition of nitrogen deficiency. In order to ensure sufficient gas substrate, the headspace gas is replaced every 24-36 hours, and the synthesis process of PHB takes about 72 hours.
(4) After 7-11 times of circulating culture, the PHB synthetic capacity of the methane oxidation mixed flora tends to be stable. In the circulating culture process, after nitrogen culture and nitrate culture, the maximum PHB content of the mixed flora is equivalent to that of continuous nitrogen culture, and the maximum PHB concentration is improved by 0.64-1.27 times after nitrate culture.
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