Application of strain and method for producing ethanol by converting crude glycerol with strain
阅读说明：本技术 一种菌株的应用及菌株转化粗甘油生产乙醇的方法 (Application of strain and method for producing ethanol by converting crude glycerol with strain ) 是由 姜莉莉 朱宝伟 刘凤翊 李昌丽 杨薇 修志龙 陈彦昊 于 2020-06-28 设计创作，主要内容包括：本发明适用于生物化学技术领域,提供了一种菌株的应用及菌株转化粗甘油生产乙醇的方法,该菌株为肺炎克雷伯菌E1,其16S rDNA核苷酸序列如序列表SEQ ID NO:1所示；利用该菌株转化粗甘油生产乙醇的方法包括以下步骤：将肺炎克雷伯菌E1进行活化后,再接入种子培养基中进行培养,得到种子培养液；以粗甘油为底物,将上述种子培养液接种到含有粗甘油的发酵培养基中进行发酵培养。该菌株对生物柴油副产物粗甘油的高附加值转化利用,以及提高生物乙醇的市场竞争力均具有重要的应用价值。其中,在批次流加发酵实验中,与纯甘油相比当以粗甘油为底物时,该菌株更有利于乙醇的发酵生产,从而可以降低生物乙醇的生产成本。(The invention is suitable for the technical field of biochemistry, and provides an application of a strain and a method for producing ethanol by converting crude glycerol with the strain, wherein the strain is Klebsiella pneumoniae E1, and the 16S rDNA nucleotide sequence of the strain is shown in a sequence table SEQ ID NO. 1; the method for producing ethanol by converting crude glycerol by using the strain comprises the following steps: activating klebsiella pneumoniae E1, and inoculating into a seed culture medium for culture to obtain a seed culture solution; inoculating the seed culture solution into a fermentation culture medium containing crude glycerol to perform fermentation culture by using the crude glycerol as a substrate. The strain has important application values for high added value conversion and utilization of biodiesel byproduct crude glycerol and improvement of market competitiveness of bioethanol. In batch fed-batch fermentation experiments, compared with pure glycerol, when crude glycerol is used as a substrate, the strain is more beneficial to fermentation production of ethanol, so that the production cost of bioethanol can be reduced.)
1. An application of a bacterial strain in the transformation of crude glycerol is characterized in that the bacterial strain is Klebsiella pneumoniae E1, and the nucleotide sequence of 16SrDNA of the bacterial strain is shown in a sequence table SEQ ID NO. 1.
2. A method for producing ethanol by converting crude glycerol with strains is characterized by comprising the following steps:
activating Klebsiella pneumoniae E1 with 16S rDNA nucleotide sequence shown in SEQ ID NO. 1 of the sequence table, and inoculating into seed culture medium for culture to obtain seed culture solution;
inoculating the seed culture solution into a fermentation culture medium containing crude glycerol to perform fermentation culture by using the crude glycerol as a substrate to obtain fermentation liquor containing ethanol.
3. The method for producing ethanol by transforming crude glycerol with strains according to claim 2, wherein in the step, the fermentation culture mode is a batch fed-batch fermentation method, and the method specifically comprises the following steps:
in the fermentation culture process, the concentration of the crude glycerol is maintained at 15-25 g/L by supplementing the crude glycerol.
4. The method for producing ethanol by transforming crude glycerol with strains according to claim 2, wherein the inoculation amount of the seed culture solution in the step is 8-12%.
5. A method as claimed in claim 2The method for producing ethanol by converting crude glycerol with strains is characterized in that each liter of the seed culture medium comprises the following components: 15-25 g of crude glycerol and KH2PO41～1.5g、K2HP O4·7H2O 4～5g、(NH4)2SO41.5～2.5g、MgSO4·7H2O 0.1～0.3g、CaCO31.5～2.5g、Fe2+0.5-1.5 mL of CaCl solution20.5-1.5 mL of solution, 0.8-1.2 g of yeast powder, 1.5-2.5 mL of trace element A solution and the balance of water.
6. The method for producing ethanol by transforming crude glycerol with strains according to claim 5, wherein each liter of Fe2+The solution comprises the following components: 3-5 mL of saturated hydrochloric acid and FeSO4·7H24-6 g of O and the balance of water; the CaCl is2The mass concentration of the solution is 15-25 g/L.
7. The method for producing ethanol by transforming crude glycerol with strains as claimed in claim 5, wherein each liter of the trace element A solution comprises the following components: 0.8-1 mL of saturated hydrochloric acid and CuCl2·2H2O 15～25mg、ZnCl260～80mg、MnCl2·4H2O 80～120mg、H3BO350～70mg、CaCl2·6H2O 180～220mg、NiCl2·6H2O 20～30mg、NaMoO4·2H230-40 mg of O and the balance of water.
8. The method for producing ethanol by transforming crude glycerol with strains according to claim 2, wherein each liter of the fermentation medium comprises the following components: 35-45 g of crude glycerol and KH2PO41～1.5g、(NH4)2SO46～7g、MgCl2·6H2O0.2～0.3g、CaCl20.2-0.4 g, 0.3-0.5 g of citric acid, 1-3 g of yeast powder, 4-6 mL of trace element B solution and the balance of water.
9. According toThe method for producing ethanol by transforming crude glycerol with strains as claimed in claim 8, wherein each liter of the trace element B solution comprises the following components: ZnCl 20.6～0.8g、MnCl2·4H2O 0.1～0.3g、H3BO350～70mg、CuCl2·2H2O 0.4～0.6g、NaMoO4·2H2O 4～6mg、FeCl2·4H2O 3～5g、CoCl2·6H20.4-0.6 g of O, 8-12 mL of saturated hydrochloric acid and the balance of water.
10. The method for producing ethanol by converting crude glycerol with strains as claimed in any one of claims 2 to 9, wherein the glycerol content in the crude glycerol is 80-85%, the ash content is 3-4%, and the moisture content is 10-15%.
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to application of a strain and a method for producing ethanol by converting crude glycerol with the strain.
Background
The excessive dependence of human beings on fossil energy causes a series of problems of increased contradiction between energy supply and demand, global continuous warming, increased energy expenditure burden and the like, and the global demand for biofuel is continuously increased. Currently, bio-energy sources such as bioethanol and biodiesel account for 10% of the total energy supply worldwide, and are expected to increase from the current 2% market share to 27% as transportation fuels in 2050. In recent years, fuel ethanol has become one of the most important biological energy sources in the world due to the characteristics of high energy density, easy transportation, good reproducibility and the like. The global fuel ethanol yield and consumption are rapidly increased, more than 40 countries and regions promote the biofuel ethanol and the vehicle ethanol gasoline at present, and the annual consumption of the ethanol gasoline is about 6 hundred million tons, which accounts for about 60 percent of the total consumption of the global gasoline. The main raw materials for producing the bioethanol comprise: the first is sugar-containing crops such as sugarcane, sugar beet, sweet sorghum, and the like; the second type is starchy crops, such as corn, wheat, sorghum, cassava, sweet potato, jerusalem artichoke and the like; the third category is wood fiber raw materials, such as straws, sawdust, crop shells, urban and rural solid garbage and the like. At present, the production of bioethanol has the problems of grain competition with people, high production cost and the like, and people continuously try to reduce the production cost of bioethanol by using other raw materials in order to expand the source of raw materials, protect the environment and further improve the market competitiveness of bioethanol.
In recent years, biodiesel production has increased exponentially. According to the 2.3 million tons of biodiesel production in europe in 2014 reported by the european biodiesel commission, about 260 million tons of glycerin are produced, and the global glycerin production is predicted to reach 419 million tons in 2020. Although the biodiesel industry is just started, the problem of excessive crude glycerol which is a byproduct generated by the biodiesel industry not only causes the market price of refined glycerol to be greatly reduced, but also causes pollution and burden to the environment, so that the reasonable utilization of the crude glycerol to convert the crude glycerol into a high-value-added product has very important significance to the sustainable development of the biodiesel industry.
In order to utilize crude glycerol and reduce the production cost of bioethanol, microorganisms are used for converting glycerol to produce ethanol in the prior art, but microorganisms used for converting glycerol to produce ethanol in the prior art have the problems of low ethanol yield and the like.
Disclosure of Invention
The embodiment of the invention aims to provide application of a strain in transformation of crude glycerol, and aims to solve the problems in the background art.
The embodiment of the invention is realized by applying a strain in the transformation of crude glycerol, wherein the strain is Klebsiella pneumoniae E1, and the nucleotide sequence of 16S rDNA is shown in a sequence table SEQ ID NO. 1.
Another object of an embodiment of the present invention is to provide a method for producing ethanol by transforming crude glycerol with a strain, which includes the following steps:
activating Klebsiella pneumoniae E1 with 16S rDNA nucleotide sequence shown in SEQ ID NO. 1 of the sequence table, and inoculating into seed culture medium for culture to obtain seed culture solution;
inoculating the seed culture solution into a fermentation culture medium containing crude glycerol to perform fermentation culture by using the crude glycerol as a substrate to obtain fermentation liquor containing ethanol.
As a preferable scheme of the embodiment of the present invention, in the step, the fermentation culture mode is a batch fed-batch fermentation method, which specifically comprises the following steps:
in the fermentation culture process, the concentration of the crude glycerol is maintained at 15-25 g/L by supplementing the crude glycerol.
In another preferable embodiment of the present invention, in the step, the inoculation amount of the seed culture solution is 8% to 12%.
As an originalIn another preferred embodiment of the present invention, each liter of the seed culture medium comprises the following components: 15-25 g of crude glycerol and KH2PO41～1.5g、K2HPO4·7H2O 4～5g、(NH4)2SO41.5～2.5g、MgSO4·7H2O 0.1～0.3g、CaCO31.5～2.5g、Fe2+0.5-1.5 mL of CaCl solution20.5-1.5 mL of solution, 0.8-1.2 g of yeast powder, 1.5-2.5 mL of trace element A solution and the balance of water.
As another preferred embodiment of the present invention, Fe is added per liter2+The solution comprises the following components: 3-5 mL of saturated hydrochloric acid and FeSO4·7H24-6 g of O and the balance of water; the CaCl is2The mass concentration of the solution is 15-25 g/L.
As another preferable scheme of the embodiment of the present invention, the trace element a solution comprises the following components per liter: 0.8-1 mL of saturated hydrochloric acid and CuCl2·2H2O 15～25mg、
As another preferred embodiment of the present invention, the fermentation medium comprises the following components per liter: 35-45 g of crude glycerol and KH2PO41～1.5g、(NH4)2
As another preferable scheme of the embodiment of the present invention, each liter of the trace element B solution includes the following components: ZnCl20.6～0.8g、MnCl2·4H2O 0.1～0.3g、H3BO350～70mg、CuCl2·2H2O 0.4～0.6g、NaMoO4·2H2O 4～6mg、FeCl2·4H2O 3～5g、CoCl2·6H20.4-0.6 g of O, 8-12 mL of saturated hydrochloric acid and the balance of water.
In another preferred embodiment of the present invention, the content of glycerin in the crude glycerin is 80% to 85%, the content of ash is 3% to 4%, and the content of water is 10% to 15%.
The application of the strain in the conversion of the crude glycerol provided by the embodiment of the invention is Klebsiella pneumoniae E1(Klebsiella pneumoniae E1), and the strain has important application value in the high added value conversion and utilization of the crude glycerol as a biodiesel byproduct and the improvement of the market competitiveness of bioethanol. In batch fed-batch fermentation experiments, compared with pure glycerol, when crude glycerol is used as a substrate, the strain is more beneficial to fermentation production of ethanol, so that the production cost of bioethanol can be reduced.
Drawings
FIG. 1 is a graph showing the effect of glycerol concentration on the growth of a single bacterium E1 in batch fermentation.
FIG. 2 is a graph showing the effect of glycerol concentration on glycerol consumption time in batch fermentation.
FIG. 3 is a graph showing the metabolism of E1 in a single strain during fermentation in example 4.
FIG. 4 is a graph showing the metabolism of E1, a single bacterium, during the fermentation culture in comparative example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
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