阅读说明：本技术 一种由小分子醇制备聚羟基脂肪酸酯的制备方法 (Preparation method for preparing polyhydroxyalkanoate from small molecular alcohol ) 是由 胡超权 邵明远 许雪冰 朱庆山 于 2020-05-07 设计创作，主要内容包括：本发明提供了一种由小分子醇制备聚羟基脂肪酸酯的制备方法,所述的制备方法包括：小分子醇催化重整后得到的混合气作为聚羟基脂肪酸酯生产菌化能自养发酵的碳源和能源。本发明提供的生产工艺使用价格低廉的小分子醇作为发酵过程所需的碳源和能源,而没有使用传统的生物质,例如：纤维素,蔗糖,葡萄糖,废糖蜜等作为发酵过程的碳源和能源。因此本工艺具有更加好的经济效益,相对于传统工艺,产品价格可以降低50％以上,而且没有生物质未反应残渣,更加绿色环保,原料来源广泛且稳定具有工业化扩大生产的前景。(The invention provides a preparation method for preparing polyhydroxyalkanoate by using small molecular alcohol, which comprises the following steps: the mixed gas obtained after the catalytic reforming of the small molecular alcohol is used as a carbon source and an energy source for the chemoautotrophic fermentation of the polyhydroxyalkanoate production bacteria. The production process provided by the invention uses low-cost micromolecule alcohol as a carbon source and an energy source required by the fermentation process, and does not use traditional biomass, such as: cellulose, sucrose, glucose, molasses and the like are used as carbon sources and energy sources in the fermentation process. Therefore, the process has better economic benefit, the product price can be reduced by more than 50% compared with the traditional process, no biomass unreacted residue exists, the process is more green and environment-friendly, the raw material source is wide and stable, and the process has the prospect of industrial expanded production.)

1. A method for preparing polyhydroxyalkanoate from small molecular alcohol is characterized by comprising the following steps:

the mixed gas obtained after the catalytic reforming of the small molecular alcohol is used as a carbon source and an energy source for the chemoautotrophic fermentation of the polyhydroxyalkanoate production bacteria.

2. The preparation method according to claim 1, wherein the preparation method specifically comprises:

carrying out catalytic reforming on micromolecule alcohol to obtain a mixed gas containing carbon dioxide and hydrogen;

(II) inoculating polyhydroxyalkanoate producing bacteria into the culture solution stored in the high-pressure fermentation device;

and (III) introducing the mixed gas obtained in the step (I) into a high-pressure fermentation device, and taking the mixed gas as a carbon source and an energy source to participate in chemoautotrophic fermentation of the polyhydroxyalkanoate production bacteria, and fermenting to generate polyhydroxyalkanoate.

3. The process of claim 2, wherein in step (i), the catalytic reforming reaction process comprises:

the small molecular alcohol and water are mixed and gasified and then are sent into a reaction device by carrier gas, and the mixed gas containing carbon dioxide and hydrogen is generated through the reforming catalytic reaction of the small molecular alcohol and the water;

preferably, the small molecule alcohol comprises methanol and/or ethanol;

preferably, the reaction device is a fixed bed reactor or a fluidized bed reactor;

preferably, the loading amount of the catalyst in the reaction device is 0.2-1 g.

4. The production method according to claim 2 or 3, wherein the catalyst used for the catalytic reforming reaction is a copper-based catalyst;

preferably, the catalyst is an alumina supported copper catalyst;

preferably, the catalyst is a hydrotalcite structure catalyst.

5. The preparation method according to any one of claims 2 to 4, wherein the reaction gas velocity of the reforming catalytic reaction is 50 to 200 mL/min;

preferably, the reaction temperature of the reforming catalytic reaction is 0-300 ℃.

6. The process according to any one of claims 2 to 5, wherein the mixed gas is dried to remove water vapor after the completion of the catalytic reforming reaction.

7. The process according to any one of claims 2 to 6, wherein in step (II), the polyhydroxyalkanoate-producing bacterium comprises one or a combination of at least two of Cupriavidus, Bacillus cereus, Burkholderia, recombinant lysostaphin, and Alcaligenes eutrophus.

8. The process according to any one of claims 2 to 7, wherein in step (III), the chemoautotrophic fermentation process comprises:

introducing oxygen into the mixed gas obtained in the step (I) and introducing the mixed gas into a high-pressure fermentation device to be used as a carbon source and an energy source to participate in chemoautotrophic fermentation of the polyhydroxyalkanoate production bacteria;

preferably, the mixed gas contains carbon dioxide and hydrogen, and the volume ratio of the carbon dioxide to the hydrogen to the oxygen is (10-80): (5-20): (1-5) mixing oxygen into the mixed gas.

9. The preparation method according to any one of claims 2 to 8, wherein the fermentation pressure of the chemoautotrophic fermentation is 0.1 to 5 MPa;

preferably, the fermentation temperature of the chemoautotrophic fermentation is 20-30 ℃.

10. The method according to any one of claims 1 to 9, wherein the method comprises the following steps:

(1) mixing and gasifying the micromolecule alcohol and water, and then sending the mixture into a reaction device by carrier gas, wherein the loading amount of a catalyst in the reaction device is 0.2-1 g, and generating a mixed gas containing carbon dioxide and hydrogen through the reforming catalytic reaction of the micromolecule alcohol and water; the catalyst adopted in the reforming reaction is a copper-based catalyst, the reaction gas speed is 50-200 mL/min, and the reaction temperature is 0-300 ℃; after the reforming reaction is finished, drying the mixed gas to remove water vapor in the mixed gas;

(2) inoculating polyhydroxyalkanoate producing bacteria into a culture solution stored in a high-pressure fermentation device;

(3) and (2) introducing oxygen into the mixed gas obtained in the step (I), introducing the mixed gas into a high-pressure fermentation device, taking the mixed gas as a carbon source and an energy source to participate in chemoautotrophic fermentation of the polyhydroxyalkanoate production bacteria, and fermenting to generate polyhydroxyalkanoate, wherein the volume ratio of carbon dioxide to hydrogen to oxygen is (10-80): (5-20): (1-5), the fermentation pressure is 0.1-5 MPa, and the fermentation temperature is 20-30 ℃.

Technical Field

The invention belongs to the technical field of production of polyhydroxyalkanoate, relates to a preparation method of polyhydroxyalkanoate, and particularly relates to a preparation method of polyhydroxyalkanoate by using small molecular alcohol.

Background

Polyhydroxyalkanoates are a generic name for a class of biodegradable thermoplastic polyesters synthesized by microorganisms. Under conditions of excess carbon source and deficiency of certain nutrients (e.g., nitrogen limitation, phosphorus limitation), many microorganisms disrupt their normal metabolic pathways, and accumulate certain structures of PHA in their cytoplasm as a carbon and energy storage material. When there is a lack of energy in the environment, microorganisms can re-catabolize these PHA stores to provide the energy needed for life activities. Thus, PHAs are completely biodegradable and biocompatible. PHA can be obtained by fermenting various microorganisms with various substances as a carbon source, and has the advantages of low carbon source price, high molecular weight of the obtained polymer up to more than 105, complete degradation of PHA into water and carbon dioxide in natural ecological environment, and small environmental pollution, so that the PHA has attracted extensive research interest.

CN101892271A discloses a fermentation method for producing polyhydroxyalkanoate. The main technical characteristics are as follows: the cell suspension containing PHA producing bacteria is seed cultured to obtain activated cell, which is used as seed for large scale culture to obtain high density cell. Then, the starch is converted into PHA to be accumulated in the bacterial cells in a large amount through metabolism regulation and fermentation. After the fermentation process is finished, separating out thalli by suction filtration, mechanically breaking the cell walls of the obtained thalli, then removing cell fragments by centrifugal separation, adding an organic solvent into the PHA-containing supernatant for extraction, adding methanol, and refining the precipitated PHA to obtain a PHA product.

CN100448911C discloses a method for extracting intracellular polyhydroxyalkanoate of a microorganism, which comprises the following steps: 1) after fermenting the microbial strains for producing the polyhydroxyalkanoate, collecting thallus cells in fermentation liquor; 2) adding an ester organic solvent with a structure similar to that of a polyhydroxyalkanoate monomer into the wet thalli collected in the step 1) or thalli powder prepared by drying the wet thalli, uniformly mixing, and heating at 80-120 ℃ for 1-4 hours to extract polyhydroxyalkanoate; 3) separating and removing thallus residue, and reserving an organic phase; 4) precipitating the polyhydroxyalkanoate from the organic phase obtained in step 3) with an organic solvent miscible with the ester extraction solvent used in step 2); 5) separating and washing the precipitate; 6) drying the precipitate to obtain the polyhydroxyalkanoate.

CN103571894A discloses a method for preparing medium-long-chain Polyhydroxyalkanoate (PHAMCL) by fermentation with cellulose as a carbon source, which is mainly technically characterized by comprising the following steps: an escherichia coli recombinant engineering bacterium containing a polyhydroxyalkanoate synthase gene phaC1 is constructed and fermented in a culture medium containing cellulose to obtain medium-long chain polyhydroxyalkanoate. The invention further relates to a preparation method of long-chain hydroxy fatty acid methyl ester (3HAME) in the biofuel, which is obtained by alcoholysis of medium-long-chain polyhydroxy fatty acid ester under the catalysis of hydrolase.

In the conventional process, the main source of carbon source required for the fermentation process of polyhydroxyalkanoate is biomass such as cellulose, sucrose, glucose, molasses and the like. Biomass unreacted residue is generated during fermentation, and biomass is expensive, resulting in an increase in production cost of polyhydroxyalkanoate.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide a preparation method for preparing polyhydroxyalkanoate by using small molecular alcohol, and the production process provided by the invention uses low-cost small molecular alcohol as a carbon source and an energy source required by a fermentation process, and does not use traditional biomass, such as: cellulose, sucrose, glucose, molasses and the like are used as carbon sources and energy sources in the fermentation process. Therefore, the process has better economic benefit, the product price can be reduced by more than 50% compared with the traditional process, no biomass unreacted residue exists, the process is more green and environment-friendly, the raw material source is wide and stable, and the process has the prospect of industrial expanded production.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a preparation method for preparing polyhydroxyalkanoate by using small molecular alcohol, which comprises the following steps:

the mixed gas obtained after the catalytic reforming of the small molecular alcohol is used as a carbon source and an energy source for the chemoautotrophic fermentation of the polyhydroxyalkanoate production bacteria.

The production process provided by the invention uses low-cost micromolecule alcohol as a carbon source and an energy source required by the fermentation process, and does not use traditional biomass, such as: cellulose, sucrose, glucose, molasses and the like are used as carbon sources and energy sources in the fermentation process. Therefore, the process has better economic benefit, the product price can be reduced by more than 50% compared with the traditional process, no biomass unreacted residue exists, the process is more green and environment-friendly, the raw material source is wide and stable, and the process has the prospect of industrial expanded production.

As a preferred technical scheme of the present invention, the preparation method specifically comprises:

carrying out catalytic reforming on micromolecule alcohol to obtain a mixed gas containing carbon dioxide and hydrogen;

(II) inoculating polyhydroxyalkanoate producing bacteria into the culture solution stored in the high-pressure fermentation device;

and (III) introducing the mixed gas obtained in the step (I) into a high-pressure fermentation device, and taking the mixed gas as a carbon source and an energy source to participate in chemoautotrophic fermentation of the polyhydroxyalkanoate production bacteria, and fermenting to generate polyhydroxyalkanoate.

In a preferred embodiment of the present invention, in step (i), the catalytic reforming reaction process comprises:

the small molecular alcohol and water are mixed and gasified, and then are sent into a reaction device by carrier gas, and the mixed gas containing carbon dioxide and hydrogen is generated through the reforming catalytic reaction of the small molecular alcohol and the water.

Preferably, the small molecule alcohol comprises methanol and/or ethanol.

Preferably, the reaction device is a fixed bed reactor or a fluidized bed reactor;

preferably, the loading of the catalyst in the reactor is 0.2 to 1g, for example 0.2g, 0.3g, 0.4g, 0.5g, 0.6g, 0.7g, 0.8g, 0.9g or 1.0g, but is not limited to the recited values, and other values not recited in the range of values are also applicable.

As a preferable technical scheme of the invention, the catalyst adopted by the catalytic reforming reaction is a copper-based catalyst.

Preferably, the catalyst is an alumina supported copper catalyst.

Preferably, the catalyst is a hydrotalcite structure catalyst.

In the process of preparing carbon dioxide and hydrogen by reforming micromolecule alcohol, the selection of the catalyst is very important, the copper-based catalyst is selected in the process, and the reforming reaction temperature of the micromolecule alcohol can be effectively controlled to be carried out in a low-temperature normal-pressure environment by preparing the catalyst with the copper-zinc-aluminum hydrotalcite structure.

In a preferred embodiment of the present invention, the reaction gas flow rate of the reforming catalyst reaction is 50 to 200mL/min, for example, 50mL/min, 60mL/min, 70mL/min, 80mL/min, 90mL/min, 100mL/min, 110mL/min, 120mL/min, 130mL/min, 140mL/min, 150mL/min, 160mL/min, 170mL/min, 180mL/min, 190mL/min, or 200mL/min, but is not limited to the above-mentioned values, and other values in the above-mentioned range are also applicable.

Preferably, the reforming catalyst reaction temperature is 0 to 300 ℃, for example, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 100 ℃, 110 ℃, 120 ℃, 130 ℃, 140 ℃, 150 ℃, 160 ℃, 170 ℃, 180 ℃, 190 ℃, 200 ℃, 210 ℃, 220 ℃, 230 ℃, 240 ℃, 250 ℃, 260 ℃, 270 ℃, 280 ℃, 290 ℃ or 300 ℃, but not limited to the values, and other values not listed in the range of the values are also applicable.

In a preferred embodiment of the present invention, after the catalytic reforming reaction is completed, the mixed gas is dried to remove water vapor therein.

In a preferred embodiment of the present invention, in step (ii), the polyhydroxyalkanoate-producing bacteria include one or a combination of at least two of cuprinophilus, bacillus cereus, burkholderia, recombinant lysostaphin, and alcaligenes eutrophus.

As a preferred technical solution of the present invention, in step (iii), the chemoautotrophic fermentation process specifically includes:

and (3) introducing oxygen into the mixed gas obtained in the step (I) and then introducing the mixed gas into a high-pressure fermentation device to be used as a carbon source and an energy source to participate in the chemoautotrophic fermentation of the polyhydroxyalkanoate production bacteria.

In the invention, the chemoautotrophic fermentation process of the polyhydroxyalkanoate producing bacteria needs to be carried out in an aerobic environment, and the mixed gas of carbon dioxide, hydrogen and oxygen is mixed according to a certain proportion to be used as a carbon source and energy to be supplied to the bacteria for fermentation so as to produce polyhydroxyalkanoate. The reaction in the high-pressure fermentation device is a reaction after process strengthening, can effectively solve the problem of low efficiency of the traditional method, and is beneficial to industrial large-scale production.

Preferably, the mixed gas contains carbon dioxide and hydrogen, and the volume ratio of the carbon dioxide to the hydrogen to the oxygen is (10-80): (5-20): (1-5) mixing oxygen into the mixed gas.

As a preferable technical scheme, the fermentation pressure of the chemoautotrophic fermentation is 0.1-5 MPa; for example, the pressure may be 0.1MPa, 1MPa, 1.5MPa, 2MPa, 2.5MPa, 3MPa, 3.5MPa, 4MPa, 4.5MPa or 5MPa, but the pressure is not limited to the values listed, and other values not listed in the numerical range are also applicable.

Preferably, the fermentation temperature of the chemoautotrophic fermentation is 20 to 30 ℃, and may be, for example, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃ or 30 ℃, but is not limited to the recited values, and other values not recited in the range of the values are also applicable.

As a preferred technical scheme of the present invention, the preparation method specifically comprises the following steps:

(1) mixing and gasifying the micromolecule alcohol and water, and then sending the mixture into a reaction device by carrier gas, wherein the loading amount of a catalyst in the reaction device is 0.2-1 g, and generating a mixed gas containing carbon dioxide and hydrogen through the reforming catalytic reaction of the micromolecule alcohol and water; the catalyst adopted in the chemical reforming reaction is a copper-based catalyst, the reaction gas speed is 50-200 mL/min, and the reaction temperature is 0-300 ℃; after the reforming reaction is finished, drying the mixed gas to remove water vapor in the mixed gas;

(2) inoculating polyhydroxyalkanoate producing bacteria into a culture solution stored in a high-pressure fermentation device;

(3) and (2) introducing oxygen into the mixed gas obtained in the step (I), introducing the mixed gas into a high-pressure fermentation device, taking the mixed gas as a carbon source and an energy source to participate in chemoautotrophic fermentation of the polyhydroxyalkanoate production bacteria, and fermenting to generate polyhydroxyalkanoate, wherein the volume ratio of carbon dioxide to hydrogen to oxygen is (10-80): (5-20): (1-5), the fermentation pressure is 0.1-5 MPa, and the fermentation temperature is 20-30 ℃.

But not limited to, the recited values and other values not recited within the range of values are equally applicable.

Compared with the prior art, the invention has the beneficial effects that:

the production process provided by the invention uses low-cost micromolecule alcohol as a carbon source and an energy source required by the fermentation process, and does not use traditional biomass, such as: cellulose, sucrose, glucose, molasses and the like are used as carbon sources and energy sources in the fermentation process. Therefore, the process has better economic benefit, the product price can be reduced by more than 50% compared with the traditional process, no biomass unreacted residue exists, the process is more green and environment-friendly, the raw material source is wide and stable, and the process has the prospect of industrial expanded production.

Drawings

FIG. 1 is a graph showing the relationship between the reaction pressure and the yield of polyhydroxyalkanoate provided in example 1 of the present invention.

Detailed Description

The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings.