TWI733269B - Yarrowia lipolytica, use thereof and method for producing diversified fuels using the same - Google Patents

Abstract

The present invention relates to a Yarrowia Lipolytica, a use thereof and a method for producing diversified fuels using the same. By using the Yarrowia LipolyticaINER-W6, agricultural and forestry waste or waste oil can be utilized as raw materials to produce diversified fuels such as alcohol and fuel precursors. Therefore, the Yarrowia LipolyticaINER-W6 and the use thereof are environmentally friendly and have a high industrial utilization value.

Description

Yarrowia lipolytic yeast strain, its use and method for producing multi-element fuel by using it

The present invention relates to a Yeli lipolytic yeast strain and its use, in particular to a Yeli lipolytic yeast strain capable of preparing multiple fuels, and a method for producing multiple fuels using it.

In today's society, human life cannot be separated from fuel, such as the high dependence of transportation on fuel. At present, most fuels are derived from petroleum, but in the process of refining, the pollutants and carbon dioxide produced are the biggest culprits of global warming and climate change. For the sustainable operation of the environment, biofuels have gradually received international attention.

The current development of biofuels includes bioethanol and biofuels. Generally speaking, biomass alcohol can use biomass sources (for example, agricultural products or its waste) as raw materials, and produce biomass alcohol after fermentation by Saccharomyces cerevisiae. The development of biomass fuel oil is the most mature technology of Hydroprocessed Ester and Fatty Acid (HEFA), which can use oil as a raw material and convert it into fuel oil. However, the current domestic oil crops or waste food The remaining amount of oil is not enough to supply the production of biomass fuel. Another oil conversion technology uses algae oil as a raw material. Because microalgae cells contain oil, they have the potential to develop into fuel oil. However, due to the poor growth rate, oil yield and extraction energy consumption of microalgae, microalgae conversion The cost of fuel is too high. Therefore, another technology can use microorganisms to convert sugars to produce biomass fuel oil or its precursors.

Although the current bio-alcohol and bio-fuel respectively have technologies for microbial production, they usually use different strains and are produced separately, and it is impossible to simultaneously produce bio-alcohol and bio-fuel.

Therefore, in order to overcome the deficiencies of the known technologies, the embodiments of the present invention provide a Yeast lipolytic yeast that can use multiple carbon sources (such as oils or agricultural and forestry wastes) as raw materials and simultaneously produce multiple fuels such as alcohol and fuel oil precursors. Strain ( Yarrowia Lipolytica ) INER-W6, its use and the method of using it to produce multiple fuels.

Based on at least one of the foregoing objectives, an embodiment of the present invention provides a Yarrowia Lipolytica strain (Yarrowia Lipolytica) INER-W6, which is deposited at the Biological Resources Preservation and Research Center of the Food Industry Development Institute, and the deposit number is BCRC 920119.

Based on at least one of the foregoing objectives, the embodiments of the present invention provide a use of the aforementioned Yeli lipolytic yeast strain INER-W6, which is used to prepare multi-element fuels.

Optionally, the multi-element fuel is alcohol and fuel oil precursors.

Optionally, the fuel precursor is a fatty acid, and the carbon chain length of the fatty acid is 12, 14, 16, 18, 24 or any combination thereof.

Based on at least one of the foregoing objectives, the present invention provides a multi-fuel production method, including: step A, inoculating the aforementioned Yeli lipolytic yeast strain INER-W6 to a multi-carbon source to form a bacterial solution, and Cultivation and fermentation of the bacterial liquid, wherein the culture temperature is controlled to 18 degrees Celsius to 36 degrees Celsius, and the culture time is controlled to at least 10 hours; step B, the bacterial liquid is separated from liquid to solid to obtain an alcohol-containing liquid, where alcohol is It is secreted by the Yeast lipolytic yeast strain INER-W6; step C, the solid pellet of the bacterial liquid is broken and extracted to obtain the fuel precursor.

Optionally, in the step A, the multi-element carbon source is oil, starch hydrolysate, cellulose hydrolysate, fruit juice, crop juice, or six-carbon sugars derived from oils/sugars. In the step C, the method of bacteriostasis is high-pressure bacteriostasis, vibration bacteriostasis, ultrasonic bacteriostasis, osmotic pressure bacteriostasis, chemical solvent bacteriostasis or freeze-lysis bacteriostasis, and the extraction method is Organic solvent extraction method, physical mechanical extraction method or supercritical extraction method, wherein during extraction, the extraction solvent of organic solvent extraction method is chloroform, alcohol, hexane, n-heptane or any combination thereof

Optionally, in the step A, the concentration of the six-carbon sugar of the multi-carbon source is 2-10%.

Optionally, in the step A, the culture temperature is better controlled to be 24°C to 30°C, and the culture time is better controlled to be 16-96 hours.

Optionally, in the step B, the concentration of alcohol is not more than 3.5%.

Optionally, in step B, it further includes: step B-2, distilling and purifying the alcohol-containing liquid to obtain alcohol.

In short, the Yersinia lipolytic yeast strain INER-W6 provided by the embodiments of the present invention can simultaneously produce multiple fuels such as alcohol and fuel precursors, so that the utilization of yeast can be improved. In addition, in the method of producing multi-fuel fuels using the Yeast lipolytic yeast strain INER-W6, waste oil or agricultural and forestry wastes can be used as raw materials, which has environmental protection value. Therefore, the implementation cost of the technology of the present invention is not high, and it is highly usable and environmentally friendly, and has a competitive advantage in the fuel market.

In order to make the above and other objects, features and advantages of the present invention more comprehensible, detailed descriptions are made as follows in conjunction with the accompanying drawings.

In order to fully understand the purpose, features and effects of the present invention, the following specific embodiments and test examples are used to illustrate the present invention in detail with the accompanying drawings. The description is as follows.

The embodiment of the present invention provides a Yarrowia lipolytic yeast strain (Yarrowia Lipolytica ) INER-W6, which is produced by the wild-type Yarrowia lipolytic yeast strain BCRC21715 from the Institute of Food Industry after genetic modification, genetic recombination and domestication, wherein , The purpose of genetic improvement is to construct a protein expression system for key genes. The genetically modified Yarrowia lipolytica strain INER-W6 has key genes whose sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 2 in the sequence table. When the key genes are expressed in large quantities, the Yarrowia lipolytic yeast strain INER-W6 can be cultured on multiple carbon sources and fermented, and the multiple carbon sources can be converted into multiple fuels. The multiple fuels are alcohol and fuel precursors. It can be used as environmentally friendly biomass fuel after treatment.

Yarrowia lipolytica strains are translated as Yarrowia lipolytica strains or Yarrowia lipolytica strains. Both wild-type and genetically modified INER-W6 belong to the double type of Dipoascaceae. Yeast yeast, which means that the Yarrowia lipolytic yeast strain can have two cell types at the same time, one is a single cell type, and the other is a hyphae type. Please refer to FIGS. 1A and 1B. FIG. 1A is an image diagram of a single-cell type of Yarrowia lipolytica strain INER-W6 according to an embodiment of the present invention, and FIG. 1B is an image diagram of the hypha type according to an embodiment of the present invention. Image of lipolytic yeast strain INER-W6. As shown in Fig. 1A and Fig. 1B, the single-cell form of Yarrowia lipolytica strain INER-W6 is round or elliptical, and its cell length is about 5 microns (um), but the present invention is not limited thereto. The hyphae of Yarrowia lipolytica strain INER-W6 is filamentous.

Next, please refer to FIG. 1C and FIG. 1D to learn about the method of constructing the Yarrowia lipolytic yeast strain INER-W6. FIG. 1C is a flowchart of the steps for constructing the Yarrowia lipolytic yeast strain INER-W6 according to an embodiment of the present invention, and FIG. 1D It is a schematic diagram of the key genes for constructing the Yarrowia lipolytic yeast strain INER-W6 in an embodiment of the present invention. First, as shown in step S101 of Figure 1C and Figure 1D, the target gene G101 can be synthesized in large quantities by polymerase chain reaction (PCR), where the target gene G101 is derived from the fatB1 gene of SEQ ID NO.1 in the sequence table. Concatenated with the pyrD1 gene of SEQ ID NO. 2 in the sequence table (fatB1-pyrD1). The polymerase chain reaction system adds the target gene G101 as a template, deoxyribonucleic acid (DNA) raw material (not shown), two primers (Primer) and polymerase E101 in a buffer solution, two of which are divided into It is a forward primer (F primer) PR1 and a reverse primer (R primer) PR2, and the sequences are as shown in SEQ ID NO. 3 and SEQ ID NO. 4 in the sequence table, respectively. The sequence of primer PR1 is designed to include restriction site Bam H 1, and the sequence of inverted primer PR2 is designed to include restriction site Hin d III. The polymerase chain reaction includes three main steps, namely denaturation, adhesion and extension. In the present invention, the condition used in the denaturation step is a reaction at 95 degrees Celsius for 5 minutes, but the present invention is not limited to this. The purpose of the denaturation step is to subject the target gene G101 to high temperature denaturation to untie the entangled double-stranded helix into a single strand. In the bonding and elongation steps, 22 temperature-changing cycle steps are required. Each cycle step includes three steps. The order and conditions of the three steps are 95 degrees Celsius for 1 minute/53 degrees Celsius for 1 minute. /72 degrees Celsius for 5 minutes, but the present invention is not limited to this. The purpose of the adhesion and extension step is to attach the pre-primer PR1 and the inverted primer PR2 to the two ends of the target gene G101 that has been untied into a single strand, and to extend the other strand. In each cycle step, twice the amount of " target gene G102 with restriction sites Bam H 1 and Hin d III" can be obtained, and after the polymerase chain reaction is completed, a larger amount of "with restriction enzyme digestion" can be obtained. The target gene G102 of Bam H 1 and Hin d III". Further, the product of the polymerase chain reaction is purified by electrophoresis analysis, but the present invention is not limited thereto.

Next, please refer to step S102 in Figure 1C and Figure 1D to learn how to construct the key gene P101 of Yarrowia lipolytica strain INER-W6, prepare the pYES2 vector V101 and the target genes with restriction sites Bam H 1 and Hin d III G102, the pYES2 carrier system is a yeast expression vector with restriction enzyme sites Bam H 1 and Hin d III, and its commercial number is V82520. Then, Bam H 1 restriction enzyme and Hin d III restriction enzyme were added to cut the vector V101 and the target gene G102, and the reaction was carried out at 37 degrees Celsius for 1-2 hours, but the present invention is not limited by this. Furthermore, electrophoresis analysis is used to confirm the fragment size of the cut vector V101 and the target gene G102 and purify them. Please note that the present invention does not limit the method of purifying gene fragments. Then, take the cut and purified vector fragment and the target gene fragment with a molar ratio of 1:3, and react at 16°C for 16-18 hours for ligation to obtain the recombined key gene P101 The plastid body.

Next, please continue to refer to step S103 of FIG. 1C to learn about the method of amplifying and purifying the key gene P101. Use heat shock treatment to send key genes into E. coli competent cells for transformation experiments, and replicate the key genes by culturing E. coli. Further, the key genes are extracted after bacteriostasis of Escherichia coli. The extraction steps are, for example, but not limited to, breaking the Escherichia coli with an alkaline substance and neutralizing and centrifuging. After obtaining the key genes, run electrophoresis gel and cut gel for purification .

Finally, please refer to step S104 of FIG. 1C to know the construction method of Yarrowia lipolytica strain INER-W6. The steps are, for example, but not limited to electroporation. Using electroporation, the cell membrane of Yarrowia lipolytica competent cell BCRC21715 can be stimulated to produce permeability, so as to further send key genes into Yarrowia lipolysis. Yeast strain INER-W6. The electroporation method is to first mix 1-3 micrograms (ug) of key genes with the Yeli lipolytic yeast competent cell BCRC21715, and perform electric shocks on ice, where the voltage used for the electric shock is 1.5 kilovolts (kV) , The time is 5 milliseconds (msec), and the voltage and time of the electroporation method are not used to limit the present invention. Next, 1 molar concentration (M) of sorbitol (Sorbitol) was used to recover the bacterial cells, and the Yarrowia lipolytic yeast strain INER-W6 was selected through auxotrophy.

Further, the method of activating and cultivating Yarrowia lipolytica strain INER-W6 will be explained. First, prepare and sterilize YPD culture solution. YPD culture solution is a culture solution that can be used to cultivate yeast. Its formula consists of 0.85% Yeast extract, 0.3% Peptone and 2% glucose. (Dextrose). First, put the sterilized YPD culture solution into the culture flask. In addition, take out the ^{freezing tube containing the Yeast lipolytic yeast strain INER-W6 stored in the -80 o} C refrigerator, and take out the bacterial liquid containing the Yeast lipolytic yeast strain INER-W6. Next, inoculate the YPD culture fluid of the Yersinia lipolytic yeast strain INER-W6 into the YPD culture medium in the culture flask to cultivate and activate the Yersinia lipolytic yeast strain INER-W6, wherein the inoculated YPD yeast The volume of the bacterial liquid of the strain INER-W6 is 1% of the volume of the YPD culture liquid, but the present invention is not limited to this. The conditioned medium is placed in flask culture incubator for 16 to 24 hours, wherein the temperature control of the incubator at 18 ^o C ~ 36 ^o C, and a speed control 50 revolutions per minute (rpm) ~ 350 rpm. More preferably, the temperature of the incubator is controlled at 24 ^o C to 30 ^o C, and the rotation speed is controlled at 150 to 250 revolutions per minute. After culturing under the above conditions, an activated Lipolytic yeast strain INER-W6 can be obtained, and most of the bacterial system of the Lipolytic yeast strain INER-W6 cultured in the shake flask presents a single cell type. , And the mycelial type of the bacteria occupies a small part, but no matter what the type of the bacteria is, the content of the embodiments provided by the present invention can be realized. Please note here that the present invention is not limited to the culture method, and the culture method may be the above-mentioned shake flask culture method (ie, shaking culture method), static culture method or fermentation tank culture method.

Next, the method of cultivating and fermenting the Yarrowia lipolytic yeast strain INER-W6 to produce multiple fuels will be explained. Please refer to FIG. 2, which is a flowchart of steps for preparing multi-element fuels according to an embodiment of the present invention. As shown in step S201, another culture bottle is prepared, and the sterilized YPD culture solution or the sterilized multi-element carbon source is placed. The multi-element carbon source can be oil, starch hydrolyzate (for example, it can be made of corn, cassava). , Rice and other starch crops are produced by high-temperature cooking or enzyme hydrolysis), cellulose hydrolysate, fruit juice juice, crop juice or six-carbon sugars derived from oils/sugars (for example, glucose). Furthermore, the activated YPD lipolytic yeast strain INER-W6 is inoculated into the sterilized YPD culture medium or the multi-carbon source in the culture bottle, wherein the activated YPD lipolytic yeast strain INER-W6 The volume of the liquid is 1-15% of the volume of the YPD culture liquid or the multi-element carbon source, but the present invention is not limited thereto. When the concentration of the YPD culture fluid or the six-carbon sugar of the multi-carbon source falls within the range of 2-10%, the YPD lipolytic yeast strain INER-W6 can be fermented. The time for cultivating Yarrowia lipolytic yeast strain INER-W6 is at least 10 hours, the culture temperature is controlled at 18 ^o C ~ 36 ^o C, and the rotation speed of the incubator is controlled at 50 to 350 revolutions per minute. More preferably, the culture time is 16 to 96 hours, the culture temperature is controlled at 24 ^o C to 30 ^o C, and the rotation speed of the incubator is controlled at 150 to 250 revolutions per minute. Please note here that the present invention is not limited to the culture method, such as the above-mentioned shake flask culture method, static culture method or fermentation tank culture method.

Next, please continue to refer to step S202 in FIG. 2, FIG. 3A and FIG. 3B to learn about the alcohol production process and method. The cultured and fermented Yersinia lipolytic yeast strain INER-W6 can decompose the six-carbon sugar glucose in YPD culture fluid or multiple carbon sources, and can produce alcohol (ethanol) and secrete it into YPD culture fluid or multiple carbon sources. In the carbon source. Please refer to Figure 3A and Figure 3B. Figure 3A is a graph showing the glucose concentration of wild-type yeast strain and Yarrowia lipolytica strain INER-W6 in an embodiment of the present invention, and Figure 3B is an embodiment of the present invention. Line graph of ethanol concentration produced by yeast strain and Yarrowia lipolytica strain INER-W6 in culture. As shown in Figure 3A, the longer the culture time of the bacterial solution, the yeast strain can consume glucose over time, so the concentration of glucose begins to decrease over time. The wild-type Yarrowia lipolytic yeast strain is the control group, and its culture conditions are no different from those of the Yarrowia lipolytic yeast strain INER-W6. The result of Fig. 3A shows that the Yarrowia lipolytica strain INER-W6 can rapidly decompose glucose within about 10 hours of culture, and can continue to decompose glucose during 16-96 hours of culture. Among them, about the 16th hour of culture, The lipolytic yeast strain INER-W6 can decompose most glucose. Compared with the wild-type Yarrowia lipolytic yeast strain, the Yarrowia lipolytic yeast strain INER-W6 consumes glucose at a significantly higher rate. As shown in Figure 3B, the longer the culture time of the bacterial solution, the Yarrowia lipolytica strain INER-W6 can produce ethanol. The wild-type Yarrowia lipolytic yeast strain is the control group, and its culture conditions are no different from those of the Yarrowia lipolytic yeast strain INER-W6. The results in Fig. 3B show that the Yarrowia lipolytica strain INER-W6 can rapidly produce ethanol within about 10 hours of culture, and the ethanol concentration can still be detected at 16-96 hours of culture, which is about the 16th hour of culture. , Yersinia lipolytica strain INER-W6 can produce the highest concentration of ethanol (approximately 33.1 g/liter). The concentration of ethanol will begin to decrease slowly after the 16th hour. The reason is that the ethanol may volatilize, and the higher concentration of ethanol will have the effect of killing the yeast, so when the Yeli lipolytic yeast strain INER-W6 When the bacterial count is slightly affected and decreased, the amount of ethanol produced will also decrease. Among them, the concentration of ethanol is preferably no more than 3.5%. The wild-type Yarrowia lipolytica strain is unable to produce ethanol at any time of cultivation. The results of Fig. 3A and Fig. 3B show that the Yarrowia lipolytica strain INER-W6 can produce and secrete ethanol after decomposing glucose. Please continue to refer to step S202. When ethanol is to be collected, the bacteria liquid needs to be separated from the solid and liquid, and the liquid part containing the alcohol should be collected. The method of separation is centrifugation of the bacterial liquid, wherein the rotation speed of the centrifuge is 4000 to 6000 revolutions per minute, and the centrifugation time is 2-15 minutes. After centrifugation, the bacterial liquid is separated into liquid and precipitated solid. The liquid contains the alcohol secreted by the yeast, and the solid is the pellet with fuel precursor in it. After obtaining the alcohol-containing liquid, it can be purified by distillation to obtain high-concentration alcohol as biomass alcohol.

Please continue to refer to step S203 in FIG. 2 to further understand the production process and method of the fuel precursor. Collect the solid cells of the bacteria liquid after centrifugation, and perform bacteriostasis and extraction to obtain fuel precursors. There are no restrictions on the method of bacteriostasis, which can be high-pressure bacteriostasis, vibration bacteriostasis, and ultrasound. There are no restrictions on bacteriostasis, osmotic pressure bacteriostasis, chemical solvent bacteriostasis, freeze-lysis bacteriostasis, or any method that can be bactericidal, and extraction method, which can be organic solvent extraction method, physical mechanical extraction method , Supercritical extraction method or any method that can extract bacteria. In the extraction, if the organic solvent extraction method is used, the extraction solvent used can be chloroform, alcohol, hexane, n-heptane or any combination thereof. For example, only chloroform or hexane/alcohol can be used to solve the problem. The bacterial body of lipoyeast strain INER-W6 is extracted to obtain fuel precursor, wherein the fuel precursor is fatty acid. Please refer to FIG. 4, which is a bar graph of the content of fatty acids with different carbon chain lengths produced by the Yarrowia lipolytica strain INER-W6 according to an embodiment of the present invention. As shown in Figure 4, the Yarrowia lipolytica strain INER-W6 can produce fatty acids with carbon chain lengths of 12, 14, 16, 18, 24 or any combination thereof.

The Yeli lipolytic yeast strain, its use, and the method for producing multiple fuels using it can assist in the treatment of agricultural and forestry wastes or waste oil, so as to achieve the goals of environmental protection and energy regeneration. Furthermore, the method is a mild microbial fermentation process, which can reduce industrial accidents and risks (such as high temperature and high pressure) associated with traditional fuel production.

In summary, compared with the prior art, the technical effects of the carrier unit and the manufacturing method described in the embodiments and test examples of the present invention are described as follows.

Among the known technologies, bioethanol and biofuels each have the technology of using microorganisms to produce, but the two use different strains and can only be produced separately, and cannot produce bioethanol and biofuels at the same time. In contrast, the Yeli lipolytic yeast strain of the present invention, its use and the method for producing multiple fuels by using it, the Yeli lipolytic yeast strain INER-W6 can be used to simultaneously prepare multiple fuels such as alcohol and fuel precursors, and can be used Oils or agricultural and forestry wastes with sufficient sources are used as raw materials. It is an integrated process with high availability, low cost and environmental protection. Such highly applicable strains and production methods have sufficient competition in the fuel market It can also respond to the international carbon reduction trend.

The present invention has been disclosed in preferred embodiments above, but those skilled in the art should understand that the above-mentioned embodiments are only used to describe the present invention and should not be construed as limiting the scope of the present invention. It should be noted that all changes and substitutions equivalent to the foregoing test examples should be included in the scope of the present invention.

E101: polymerase G101: target gene G102: target gene with restriction sites Bam H 1 and Hin d III P101: key gene PR1: pre-primer PR2: reverse primer S101~S104, 201~S203: Step V101: Carrier

Fig. 1A is an image diagram of a single-cell type of Yarrowia lipolytica strain INER-W6 according to an example of the present invention.

Fig. 1B is an image diagram of the Yarrowia lipolytica strain INER-W6 in the form of hyphae in an example of the present invention.

Fig. 1C is a flow chart of the steps for constructing the Yarrowia lipolytic yeast strain INER-W6 according to an embodiment of the present invention.

Fig. 1D is a schematic diagram of the key genes for constructing the Yarrowia lipolytic yeast strain INER-W6 according to an embodiment of the present invention.

Figure 2 is a flow chart of the steps for preparing multi-element fuels according to an embodiment of the present invention.

Figure 3A is a graph showing the glucose concentration of wild-type yeast strain and Yarrowia lipolytica strain INER-W6 in an embodiment of the present invention.

Fig. 3B is a broken line graph of the ethanol concentration produced by the wild-type yeast strain and the Yarrowia lipolytica strain INER-W6 in the embodiment of the present invention.

Fig. 4 is a bar graph showing the content of fatty acids with different carbon chain lengths produced by P. lipolytica strain INER-W6 according to an embodiment of the present invention.

Biological Resources Conservation and Research Center of Food Industry Development Institute, BCRC 920119, November 18, 2019.

S201~S203: Steps

Claims (10)

A Yarrowia lipolytic yeast strain ( Yarrowia Lipolytica ) INER-W6, which is deposited in the Biological Resources Preservation and Research Center of the Food Industry Development Institute, and the deposit number is BCRC 920119. A use of the Yeast lipolytic yeast strain INER-W6 as described in claim 1 is to prepare a multi-element fuel. The use according to claim 2, wherein the multi-element fuel is an alcohol and a fuel precursor. The use according to claim 3, wherein the fuel precursor is a fatty acid, and the carbon chain length of the fatty acid is 12, 14, 16, 18, 24 or any combination thereof. A method for producing multi-element fuels, comprising: step A, inoculating the Yarrowia lipolytic yeast strain INER-W6 as described in claim 1 to multi-element carbon sources to form a bacterial liquid, and culturing and fermenting the bacterial liquid , Wherein the culture temperature is controlled to 18 degrees Celsius to 36 degrees Celsius, and the culture time is controlled to at least 10 hours; step B, solid-liquid separation of the bacteria liquid is carried out to obtain a liquid containing alcohol, wherein the alcohol is derived from the Yeah solution Lipomyces cerevisiae strain INER-W6 is secreted; in step C, a solid pellet of the bacterial liquid is broken and extracted to obtain a fuel precursor. The method for producing a multi-element fuel according to claim 5, wherein in step A: the multi-element carbon source is a fat, a starch hydrolyzate, a cellulose hydrolysate, a fruit juice, a crop juice or a fat / A six-carbon sugar derived from sugar; and Yubu Step C: The method of bacteriostasis is a high-pressure bacteriostasis, an oscillating bacteriostasis method, an ultrasonic bacteriostasis method, an osmotic bacteriostasis method, a chemical solvent bacteriostasis method or a freeze lysis bacteriostasis method, and The extraction method is an organic solvent extraction method, a physical mechanical extraction method or a supercritical extraction method, wherein during extraction, the extraction solvent of the organic solvent extraction method is chloroform, alcohol, hexane, n-heptane or any of them combination. The method for producing multi-element fuels according to claim 6, wherein in step A: the concentration of the hexa-sugar of the multi-element carbon source is 2-10%. The multi-fuel production method according to claim 5, wherein in step A: the cultivation temperature is more preferably controlled to be 24°C to 30°C, and the cultivation time is more preferably controlled to be 16-96 hours. The method for producing multi-element fuels according to claim 5, wherein in step B: the concentration of the alcohol is not more than 3.5%. The method for producing multi-element fuels according to claim 5, wherein in step B, it further includes: step B-2, distilling and purifying the liquid containing alcohol to obtain the alcohol.

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