CN114854795A - Method for producing ethanol by fermenting raw starch with double bacteria - Google Patents
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Abstract
The invention discloses a method for producing ethanol by fermenting raw starch with double bacteria, which adopts phyllospheric PaenibacillusPaenibacillus phyllosphaerae The method has the advantages of low cost, no limitation of conditions such as regions, seasons and the like, suitability for industrial large-scale production and the like. Meanwhile, the raw starch is directly used for alcoholic fermentation, the high-temperature cooking gelatinization and saccharification cooling procedures in the traditional alcoholic fermentation sugar preparation procedure are not needed, the energy consumption is greatly reduced, the procedures are simplified, the production cost is reduced, and the method conforms to the environmental protectionAnd sustainable development requirements.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for producing ethanol by fermenting raw starch with double bacteria.
Background
With the development of society, the problems of exhaustion of energy sources such as coal and petroleum and environmental pollution become more serious, and the production of fuels (fuel ethanol, biodiesel and the like) by using renewable biomass which can replace and protect environment has been widely concerned by people. Among them, fuel ethanol has been developed as the most commonly used liquid biofuel in the world, which can reduce the greenhouse gas emission by 30% -85% compared with fossil fuel, and is helpful to reduce the formation of particulate matters in the atmosphere. Although the fuel ethanol production technology has been rapidly developed, the current ethanol fermentation production mainly uses starch materials such as corn, cassava, sorghum and the like as raw materials. When the fuel ethanol takes starchy crops as raw materials for production, the process flow of the fuel ethanol is generally divided into five stages, namely gelatinization, liquefaction and saccharification, fermentation, distillation and dehydration of the starchy raw materials. However, the traditional fermentation method for producing fuel ethanol has high energy consumption and high cost, and the price cannot compete with gasoline at present. The reason for the high cost of the traditional method is mainly in the boiling and pasting process and the distillation process, and a large amount of energy is consumed. In particular, in the cooking gelatinization process, the required heat energy accounts for 30-40% of the energy consumption required by the whole production, and if the energy consumption of the cooking gelatinization process can be effectively reduced, the competitive capacity of the biomass fuel ethanol can be greatly enhanced.
Raw meal fermentation is one of the best options for solving the above problems. Raw material fermentation is the process of microorganism directly utilizing raw starch which is not gelatinized to grow, reproduce and metabolize. The raw starch is utilized for alcohol fermentation, so that gelatinization, liquefaction and saccharification in the traditional process can be combined into one step for direct saccharification, the high-temperature cooking gelatinization process is omitted, the loss of fermentable sugar under the high-temperature condition is avoided, and meanwhile, cooking and saccharification equipment is not needed, so that the method has the advantages of reducing energy consumption, simplifying operation procedures, reducing production cost and the like, and has huge energy-saving prospect. Under the condition of increasingly competitive conditions of the current alcohol industry, the new technology of non-cooking alcohol fermentation is an effective way for reducing the production cost and enhancing the competitiveness.
Disclosure of Invention
The invention aims to provide a method for producing ethanol by co-fermenting raw starch through phyllospheric paenibacillus capable of hydrolyzing raw starch and candida huhattaiPaenibacillus phyllosphaeraeThe HYNU-YU148 directly ferments raw starch to prepare glucose, and then produces ethanol by fermentation of Candida shehatae, the process does not need the traditional high-temperature cooking gelatinization and saccharification cooling process, thereby greatly reducing the energy consumption, simplifying the process, reducing the production cost and effectively solving the problems existing in the background technology.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for producing ethanol by fermenting raw starch with two bacteria adopts phyllospheric PaenibacillusPaenibacillus phyllosphaerae HYNU-YU148 fermenting raw starch to obtain glucose, inoculating Candida shehatae in the fermentation liquid, and continuously fermenting to obtain ethanol. The phyllospheric Paenibacillus isPaenibacillus phyllosphaeraeHYNU-YU148, which was deposited at 25.10.20.2021 in GDMCC (GDMCC for short) (Ministry of microorganisms research institute in Guangdong province, Michelia Toxico, Guangzhou), with the deposit number GDMCC NO. 62008. The method comprises the following steps:
s1, mixingPaenibacillus phyllosphaerae Inoculating HYNU-YU148 to a solid plate culture medium, culturing the solid plate culture medium in a constant temperature incubator at 28-32 ℃ for 2-4 days, selecting a single colony from the solid plate culture medium by using an inoculating loop to a liquid seed culture medium, and performing shake culture at 28-32 ℃ and 200rpm for 1-3 days to serve as seed liquid for later use;
s2, inoculating Candida shehatae to a solid plate culture medium, placing the solid plate culture medium in a constant temperature incubator at 28-32 ℃ for culturing for 2-4 days, picking a single colony from the solid plate culture medium by using an inoculating loop to a liquid seed culture medium, and performing shake culture at 28-32 ℃, 100-200rpm for 22-26 hours to serve as seed liquid for later use;
and S3, inoculating the Paenibacillus phyllophilus seed liquid cultured in the S1 into a raw starch fermentation culture medium to obtain a fermentation liquid, wherein the seed liquid in the fermentation liquid accounts for 1-10% by volume.
S4, after the fermentation process in the S3 is carried out for 2-4 days, inoculating Candida shehatae seed liquid cultured in the S2 to obtain fermentation liquid, wherein the seed liquid in the fermentation liquid is 5-10% by volume, continuing to ferment for 2-4 days after the inoculation, and distilling the fermentation liquid to obtain the ethanol.
Further, the components and mass contents of the solid plate culture medium in the S1 are as follows: 9-11g/L of peptone, 4.5-5.5g/L of yeast extract powder, 4.5-5.5g/L of sodium chloride, 9-11g/L of soluble starch and 19-21g/L of agar.
Further, the components and mass contents of the liquid seed culture medium in the S1 are as follows: 9-11g/L of peptone, 4.5-5.5g/L of yeast extract powder, 4.5-5.5g/L of sodium chloride and 9-11g/L of soluble starch.
Further, the components and mass contents of the solid plate culture medium in the S2 are as follows: 9-11g/L of peptone, 4.5-5.5g/L of yeast extract powder, 4.5-5.5g/L of sodium chloride, 9-11g/L of glucose and 19-21g/L of agar.
Further, the components and mass contents of the seed liquid culture medium in the S2 are as follows: 9-11g/L of peptone, 4.5-5.5g/L of yeast extract powder, 4.5-5.5g/L of sodium chloride and 9-11g/L of glucose.
Further, the raw starch fermentation medium in the S3 comprises the following components in percentage by mass: 10-100g/L of raw starch, 1.5-2.5g/L of ammonium sulfate, 2-10g/L of peptone, 1-3g/L of dipotassium phosphate, 0.4-0.6g/L of magnesium sulfate heptahydrate, 0.5-1.5g/L of calcium chloride, 0.008-0.012g/L of ferrous sulfate heptahydrate, and the pH value of the culture medium is 6.5-7.5.
Further, in the fermentation condition of S3, the shaking table speed is 100-200r/min, and the fermentation temperature is 28-32 ℃.
Further, in the fermentation condition of S4, the shaking table speed is 80-120r/min, and the fermentation temperature is 26-30 ℃.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a phyllospheric paenibacillus capable of hydrolyzing raw starch and uses the phyllospheric paenibacillus and candida huhattai to carry out co-fermentation to produce ethanol, and the invention has the advantages of low cost, no limitation of regions, seasons and other conditions, suitability for industrial large-scale production and the like. Meanwhile, the raw starch is directly used for alcoholic fermentation, and the high-temperature cooking gelatinization and saccharification cooling processes in the traditional alcoholic fermentation sugar making process are not needed, so that the energy consumption is greatly reduced, the processes are simplified, the production cost is reduced, and the requirements of environmental protection and sustainable development are met.
Detailed Description
In order to make the technical means and functions realized by the invention easy to understand, the invention is further described by combining the specific embodiments.
First, separation, screening and identification of bacterial strain
Taking a soil sample from the periphery of a starch product factory, preparing the sample into a bacterial suspension by using sterile water, gradiently diluting the bacterial suspension, and coating the bacterial suspension on a separation plate taking starch as a unique carbon source, wherein a plate culture medium comprises 20g/L of raw starch, 2g/L of ammonium sulfate, 5g/L of peptone, 1g/L of dipotassium hydrogen phosphate, 0.5g/L of magnesium sulfate heptahydrate, 1g/L of calcium chloride, 0.01g/L of ferrous sulfate heptahydrate and 20g/L of agar, the raw starch is sterilized by dry heat, and other components are sterilized by wet heat after being prepared. After culturing, selecting the bacterial colony with larger diameter of the transparent ring, and further carrying out streak separation culture to obtain a pure culture, thereby obtaining phyllospheric PaenibacillusPaenibacillus phyllosphaerae HYNU-YU148, preserved on the inclined plane.
Extracting total DNA according to the instruction of the genome DNA extraction kit of the engineering EZ-10 columnar bacteria. PCR amplification was performed using the total DNA extracted above as a template and primers 27F/1492R. The PCR product was electrophoresed in 1% agarose gel, the band of the desired DNA was cut from the electrophoresis result, and purified and recovered according to the instructions of UNIQ-10 column type DNA gel recovery kit. The PCR product was sequenced by Shanghai Bioengineering, Inc. The sequencing results were as follows:
GACTACACCTTCGGGTGTGGTTAGCGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCTGTAAGACCGGGATAACATTCGGAAACGAATGCTAATACCGGATATGCGGTTTGCTCGCATGAGCGAATCGGGAAAGACGGTGCAAGCTGTCACTTACAGATGGACCTGCGGCGCATTAGCTAGTTGGTGGGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGCCAGGGAAGAACGAGTGGGAGAGTAACTGCTCCTGCTATGACGGTACCTGAGAAGAAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTTGTAAGTCAGGTGTTTAAGCTCGGGGCTCAACCCCGATTCGCATCTGAAACTGCAAGACTTGAGTGCAGAAGAGGGAAAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGGCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGGTGTTAGGGGTTTCGATACCCTTGGTGCCGAAGTTAACACATTAAGCATTCCGCCTGGGGAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCTTTGAATCCTCTAGAGATAGAGGCGGCCCTTCGGGGACAGAGGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATTTTAGTTGCCAGCACTTTAAGGTGGGCACTCTAGAATGACTGCCGGTGACAAACCGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTACTACAATGGCCGTTACAACGGGAAGCGAAGGAGCGATCTGGAGCGAATCCTAAAAAGGCGGTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTACAACACCCGAAGCCGGTGGGGT
BLAST analysis is carried out on the 16S rDNA sequence of the detected strain and the sequence in the GenBank database, and the result shows that: the strain of the invention is located in phylogenetic trees and phyllospheric paenibacillus (Paenibacillus phyllosphaerae) The HYNU-YU 14816S rRNA gene sequence has a recent relationship withPaenibacillus phyllosphaerae The homology of strain SM26 is 99%, and the strain is determined to be phyllospheric paenibacillus by combining with a phylogenetic tree: (Paenibacillus phyllosphaerae）。
The invention providesPaenibacillus phyllosphaeraeHYNU-YU148, which is deposited in GDMCC (GDMCC for short) in 2021, 10 months and 25 days (Guangdong province)Mr. zhou 100, guangdong institute for microbiology, guangdong), accession number: GDMCC 62008.
Secondly, utilizing phyllospheric PaenibacillusPaenibacillus phyllosphaeraeThe production of ethanol by co-fermenting raw starch with HYNU-YU148 and Candida shehatae (Candida shehatae is available from the Guangdong province culture Collection, accession number GDMCC 2.175) includes the following examples:
example 1
The method for producing ethanol by co-fermenting phyllospheric Paenibacillus and Candida shehatae comprises the following steps:
s1, Paenibacillus cereusPaenibacillus phyllosphaerae HYNU-YU148 is inoculated on a solid plate culture medium, and the components and the mass content of the solid plate culture medium are 9g/L of peptone, 4.5g/L of yeast extract powder, 4.5g/L of sodium chloride, 9g/L of soluble starch and 19g/L of agar. The solid plate culture medium is placed in a constant temperature incubator at 28 ℃ for 2 days, a single colony is picked from the solid plate culture medium by using an inoculating loop and is placed in a liquid seed culture medium, and the components and the mass content of the liquid seed culture medium are 9g/L of peptone, 4.5g/L of yeast extract powder, 4.5g/L of sodium chloride and 9g/L of soluble starch. Shaking-culturing at 28 deg.C and 150rpm for 1 day as seed liquid;
s2, inoculating Candida shehatae to a solid plate culture medium, wherein the components and mass content of the solid plate culture medium are peptone 9g/L, yeast extract powder is 4.5g/L, sodium chloride is 4.5g/L, glucose is 9g/L, agar is 19g/L, the solid plate culture medium is placed in a constant-temperature incubator at 28 ℃ for culture for 2 days, inoculating loops are used for picking a single colony from the solid plate culture medium to a liquid seed culture medium, the components and mass content of the liquid seed culture medium are peptone 9g/L, yeast extract powder is 4.5g/L, sodium chloride is 4.5g/L, and glucose is 9g/L, and the culture is shake-cultured for 22 hours at 28 ℃ and 150rpm to serve as seed liquid for later use;
s3, inoculating the cultured phyllospheric paenibacillus seed liquid in the S1 into a raw starch fermentation culture medium according to the volume ratio of 1% for fermentation, wherein the raw starch fermentation culture medium comprises the following components in percentage by mass: 10g/L of raw starch, 1.5g/L of ammonium sulfate, 2g/L of peptone, 1g/L of dipotassium hydrogen phosphate, 0.4g/L of magnesium sulfate heptahydrate, 0.5g/L of calcium chloride and 0.008g/L of ferrous sulfate heptahydrate. The pH of the medium was 6.5. The shaking table speed is 150r/min, the fermentation temperature is 28 ℃, and the fermentation time is 2 days.
S4, inoculating the Candida shehatae seed liquid cultured in the S2 into the fermentation liquid obtained in the S3 according to the volume ratio of 5%, wherein the speed of a shaking table after inoculation is 100r/min, the fermentation temperature is 28 ℃, the fermentation time is 2 days, the ethanol content in the fermentation liquid is 3.6g/L, and the finally obtained fermentation liquid is distilled to obtain the ethanol.
Example 2
The method for producing ethanol by co-fermentation of phyllospheric Paenibacillus and Candida shehatae comprises the following steps:
s1, mixingPaenibacillus phyllosphaerae HYNU-YU148 is inoculated on a solid plate culture medium, and the solid plate culture medium comprises the following components in percentage by mass: 11g/L of peptone, 5.5g/L of yeast extract powder, 5.5g/L of sodium chloride, 11g/L of soluble starch and 21g/L of agar. Placing the solid plate culture medium in a constant temperature incubator at 32 ℃ for culturing for 4 days, and selecting a single colony from the solid plate culture medium into a liquid seed culture medium by using an inoculating loop, wherein the liquid seed culture medium comprises the following components in percentage by mass: 11g/L of peptone, 5.5g/L of yeast extract powder, 5.5g/L of sodium chloride and 11g/L of soluble starch. Shaking-culturing at 32 deg.C and 150rpm for 3 days to obtain seed liquid;
s2, inoculating Candida shehatae to a solid plate culture medium, wherein the solid plate culture medium comprises the following components in percentage by mass: 11g/L of peptone, 5.5g/L of yeast extract powder, 5.5g/L of sodium chloride, 11g/L of glucose and 21g/L of agar, putting a solid plate culture medium into a constant-temperature incubator at 32 ℃ for culturing for 4 days, and selecting a single colony from the solid plate culture medium into a liquid seed culture medium by using an inoculating loop, wherein the liquid seed culture medium comprises the following components in percentage by mass: 11g/L of peptone, 5.5g/L of yeast extract powder, 5.5g/L of sodium chloride and 11g/L of glucose, and performing shake culture at 32 ℃ and 150rpm for 26 hours to serve as seed liquid for later use;
s3, inoculating the cultured seed liquid into a raw starch fermentation culture medium according to the volume ratio of 10% for fermentation, wherein the raw starch fermentation culture medium comprises the following components in percentage by mass: 100g/L of raw starch, 2.5g/L of ammonium sulfate, 10g/L of peptone, 3g/L of dipotassium phosphate, 0.6g/L of magnesium sulfate heptahydrate, 1.5g/L of calcium chloride and 0.012g/L of ferrous sulfate heptahydrate. The pH of the medium was 7.5. The shaking table speed is 150r/min, the fermentation temperature is 32 ℃, and the fermentation time is 4 days.
S4, inoculating the Candida shehatae seed liquid cultured in the S2 into the fermentation liquid obtained in the S3 according to the volume ratio of 10%, wherein the shaking table speed is 100r/min after inoculation, the fermentation temperature is 30 ℃, the fermentation time is 4 days, the ethanol content in the fermentation liquid is 31.3g/L, and the finally obtained fermentation liquid is distilled to obtain the ethanol.
Example 3
The method for producing ethanol by co-fermenting phyllospheric Paenibacillus and Candida shehatae comprises the following steps:
s1, mixingPaenibacillus phyllosphaerae HYNU-YU148 is inoculated on a solid plate culture medium, and the solid plate culture medium comprises the following components in percentage by mass: 10g/L of peptone, 5g/L of yeast extract powder, 5g/L of sodium chloride, 10g/L of soluble starch and 20g/L of agar. Placing the solid plate culture medium in a constant temperature incubator at 30 ℃ for 3 days, and selecting a single colony from the solid plate culture medium into a liquid seed culture medium by using an inoculating loop, wherein the liquid seed culture medium comprises the following components in percentage by mass: 10g/L of peptone, 5g/L of yeast extract powder, 5g/L of sodium chloride and 10g/L of soluble starch. Shaking-culturing at 30 deg.C and 150rpm for 2 days to obtain seed liquid;
s2, inoculating Candida shehatae to a solid plate culture medium, wherein the solid plate culture medium comprises the following components in percentage by mass: 10g/L of peptone, 5g/L of yeast extract powder, 5g/L of sodium chloride, 10g/L of glucose and 20g/L of agar, putting a solid plate culture medium into a 30 ℃ constant-temperature incubator for culturing for 3 days, and picking a single colony from the solid plate culture medium into a liquid seed culture medium by using an inoculating loop, wherein the liquid seed culture medium comprises the following components in percentage by mass: 10g/L of peptone, 5g/L of yeast extract, 5g/L of sodium chloride and 10g/L of glucose, and performing shake culture at 30 ℃ and 150rpm for 24 hours to serve as seed liquid for later use;
s3, inoculating the cultured seed liquid into a raw starch fermentation culture medium according to the volume ratio of 5% for fermentation, wherein the raw starch fermentation culture medium comprises the following components in percentage by mass: 50g/L of raw starch, 2g/L of ammonium sulfate, 10g/L of peptone, 1g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate heptahydrate, 1g/L of calcium chloride and 0.01g/L of ferrous sulfate heptahydrate. The pH of the medium was 7.0. The shaking table speed is 150r/min, the fermentation temperature is 30 ℃, and the fermentation time is 3 days.
S4, inoculating the Candida shehatae seed liquid cultured in the S2 into the fermentation liquid obtained in the S3 according to the volume ratio of 8%, wherein the shaking table speed is 100r/min after inoculation, the fermentation temperature is 28 ℃, the fermentation time is 4 days, the ethanol content in the fermentation liquid is 14.9g/L, and the obtained fermentation liquid is distilled to obtain the ethanol.
Example 4
S3, inoculating the cultured seed liquid into a raw starch fermentation culture medium according to the volume ratio of 10% for fermentation, wherein the fermentation culture medium comprises the following components in percentage by mass: 100g/L of raw starch, 2g/L of ammonium sulfate, 10g/L of peptone, 1g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate heptahydrate, 1g/L of calcium chloride and 0.01g/L of ferrous sulfate heptahydrate. The pH of the medium was 7.0. The shaking table speed is 150r/min, the culture temperature is 30 ℃, and the culture time is 3 days.
S4, inoculating the Candida shehatae seed liquid cultured in the S2 into the S3 fermentation liquid according to the volume ratio of 8%, wherein the shaking table speed is 100r/min after inoculation, the culture temperature is 28 ℃, the culture time is 3 days, the ethanol content in the fermentation liquid is 32.1g/L, and the obtained fermentation liquid is distilled to obtain the ethanol.
The rest of the example was carried out as in example 3.
Example 5
S3, inoculating the cultured seed liquid into a culture medium according to the volume ratio of 5% for fermentation, wherein the fermentation medium comprises the following components in percentage by mass: 50g/L of raw starch, 2g/L of ammonium sulfate, 10g/L of peptone, 1g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate heptahydrate, 1g/L of calcium chloride and 0.01g/L of ferrous sulfate heptahydrate. The pH of the medium was 7.0. The shaking table speed is 150r/min, the culture temperature is 30 ℃, and the culture time is 3 days.
S4, inoculating the Candida shehatae seed liquid cultured in the S2 into the S3 fermentation liquid according to the volume ratio of 8%, wherein the shaking table speed is 100r/min after inoculation, the culture temperature is 28 ℃, the culture time is 4 days, the ethanol content in the fermentation liquid is 33.8g/L, and the obtained fermentation liquid is distilled to obtain the ethanol.
The rest of the example was carried out as in example 3. .
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> Huaiyin college of learning professions
<120> method for producing ethanol by fermenting raw starch with double bacteria
<130> 2022
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1381
<212> DNA
<213> Paenibacillus phyllosphaera HYNU-YU148
<400> 2
gactacacct tcgggtgtgg ttagcggcgg acgggtgagt aacacgtagg taacctgcct 60
gtaagaccgg gataacattc ggaaacgaat gctaataccg gatatgcggt ttgctcgcat 120
gagcgaatcg ggaaagacgg tgcaagctgt cacttacaga tggacctgcg gcgcattagc 180
tagttggtgg ggtaacggct caccaaggcg acgatgcgta gccgacctga gagggtgatc 240
ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt agggaatctt 300
ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag tgatgaaggt tttcggatcg 360
taaagctctg ttgccaggga agaacgagtg ggagagtaac tgctcctgct atgacggtac 420
ctgagaagaa agccccggct aactacgtgc cagcagccgc ggtaatacgt agggggcaag 480
cgttgtccgg aattattggg cgtaaagcgc gcgcaggcgg ttttgtaagt caggtgttta 540
agctcggggc tcaaccccga ttcgcatctg aaactgcaag acttgagtgc agaagaggga 600
aagtggaatt ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca ccagtggcga 660
aggcgacttt ctgggctgta actgacgctg aggcgcgaaa gcgtggggag caaacaggat 720
tagataccct ggtagtccac gccgtaaacg atgaatgcta ggtgttaggg gtttcgatac 780
ccttggtgcc gaagttaaca cattaagcat tccgcctggg gagtacgctc gcaagagtga 840
aactcaaagg aattgacggg gacccgcaca agcagtggag tatgtggttt aattcgaagc 900
aacgcgaaga accttaccag gtcttgacat ccctttgaat cctctagaga tagaggcggc 960
ccttcgggga cagaggagac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt 1020
tgggttaagt cccgcaacga gcgcaaccct tgattttagt tgccagcact ttaaggtggg 1080
cactctagaa tgactgccgg tgacaaaccg gaggaaggcg gggatgacgt caaatcatca 1140
tgccccttat gacctgggct acacacgtac tacaatggcc gttacaacgg gaagcgaagg 1200
agcgatctgg agcgaatcct aaaaaggcgg tctcagttcg gattgcaggc tgcaactcgc 1260
ctgcatgaag tcggaattgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc 1320
gggtcttgta cacaccgccc gtcacaccac gagagtttac aacacccgaa gccggtgggg 1380
t 1381
Claims (10)
1. A method for producing ethanol by fermenting raw starch with double bacteria is characterized in that: using phyllospheric PaenibacillusPaenibacillus phyllosphaerae HYNU-YU148 fermenting raw starch to obtain glucose, inoculating Candida shehatae in the fermentation liquid, and continuously fermenting to obtain ethanol.
2. The method for producing ethanol by fermenting raw starch with two bacteria according to claim 1, wherein: the method comprises the following steps:
s1, Paenibacillus cereusPaenibacillus phyllosphaerae Inoculating HYNU-YU148 to a solid plate culture medium, culturing the solid plate culture medium in a constant temperature incubator at 28-32 ℃ for 2-4 days, selecting a single colony from the solid plate culture medium by using an inoculating loop to a liquid seed culture medium, and performing shake culture at 28-32 ℃ and 200rpm for 1-3 days to serve as seed liquid for later use;
s2, inoculating Candida shehatae to a solid plate culture medium, placing the solid plate culture medium in a constant temperature incubator at 28-32 ℃ for culturing for 2-4 days, picking a single colony from the solid plate culture medium by using an inoculating loop to a liquid seed culture medium, and performing shake culture at 28-32 ℃, 100-200rpm for 22-26 hours to serve as seed liquid for later use;
and S3, inoculating the Paenibacillus phyllophilus seed liquid cultured in the S1 into a raw starch fermentation culture medium to obtain a fermentation liquid, wherein the seed liquid in the fermentation liquid accounts for 1-10% by volume.
And 3, S4, S3 is fermented for 2 to 4 days, and then inoculated into the Candida shehatae seed liquid cultured in S2 to obtain fermentation liquid, the seed liquid in the fermentation liquid is 5 to 10 percent by volume, and after 2 to 4 days of fermentation, the fermentation liquid is distilled to obtain the ethanol.
4. The method for producing ethanol by fermenting raw starch with two bacteria according to claim 2, wherein: the components and mass contents of the solid plate culture medium in the S1 are 9-11g/L of peptone, 4.5-5.5g/L of yeast extract powder, 4.5-5.5g/L of sodium chloride, 9-11g/L of soluble starch and 19-21g/L of agar.
5. The method for producing ethanol by fermenting raw starch with two bacteria according to claim 2, wherein: the components and mass contents of the liquid seed culture medium in the S1 are 9-11g/L of peptone, 4.5-5.5g/L of yeast extract powder, 4.5-5.5g/L of sodium chloride and 9-11g/L of soluble starch.
6. The method for producing ethanol by fermenting raw starch with two bacteria according to claim 2, wherein: the components and mass contents of the solid plate culture medium in the S2 are 9-11g/L of peptone, 4.5-5.5g/L of yeast extract powder, 4.5-5.5g/L of sodium chloride, 9-11g/L of glucose and 19-21g/L of agar.
7. The method for producing ethanol by fermenting raw starch with two bacteria according to claim 2, wherein: the components and mass contents of the liquid seed culture medium in the S2 are 9-11g/L of peptone, 4.5-5.5g/L of yeast extract powder, 4.5-5.5g/L of sodium chloride and 9-11g/L of glucose.
8. The method for producing ethanol by fermenting raw starch with two bacteria according to claim 2, wherein: the raw starch fermentation medium in the S3 comprises, by mass, 10-100g/L of raw starch, 1.5-2.5g/L of ammonium sulfate, 2-10g/L of peptone, 1-3g/L of dipotassium phosphate, 0.4-0.6g/L of magnesium sulfate heptahydrate, 0.5-1.5g/L of calcium chloride, 0.008-0.012g/L of ferrous sulfate heptahydrate, and the pH value of the medium is 6.5-7.5.
9. The method for producing ethanol by fermenting raw starch with two bacteria according to claim 2, wherein: the fermentation conditions in the S3 are that the shaking table speed is 100-200r/min and the fermentation temperature is 28-32 ℃.
10. The method for producing ethanol by fermenting raw starch with two bacteria according to claim 2, wherein: the fermentation conditions in the S4 are that the shaking table speed is 80-120r/min and the fermentation temperature is 26-30 ℃.
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|---|---|---|---|---|
| CN114874941A (en) * | 2022-05-11 | 2022-08-09 | 淮阴师范学院 | Paenibacillus phyllophilus with raw starch hydrolysis capacity and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1629298A (en) * | 2003-12-19 | 2005-06-22 | 首都师范大学 | Process for preparing alcohol through utilizing xylose and glucose by microzyme |
| US20110151532A1 (en) * | 2008-05-23 | 2011-06-23 | United of Georgia Research Foundation, Inc. | Paenibacillus spp. and methods for fermentation of lignocellulosic materials |
| CN107129948A (en) * | 2017-05-22 | 2017-09-05 | 东莞市保得生物工程有限公司 | A kind of fermentation medium and its optimization method of fixed nitrogen series bacillus 1 49 |
| CN114874941A (en) * | 2022-05-11 | 2022-08-09 | 淮阴师范学院 | Paenibacillus phyllophilus with raw starch hydrolysis capacity and application thereof |
-
2022
- 2022-05-11 CN CN202210510252.3A patent/CN114854795B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1629298A (en) * | 2003-12-19 | 2005-06-22 | 首都师范大学 | Process for preparing alcohol through utilizing xylose and glucose by microzyme |
| US20110151532A1 (en) * | 2008-05-23 | 2011-06-23 | United of Georgia Research Foundation, Inc. | Paenibacillus spp. and methods for fermentation of lignocellulosic materials |
| CN107129948A (en) * | 2017-05-22 | 2017-09-05 | 东莞市保得生物工程有限公司 | A kind of fermentation medium and its optimization method of fixed nitrogen series bacillus 1 49 |
| CN114874941A (en) * | 2022-05-11 | 2022-08-09 | 淮阴师范学院 | Paenibacillus phyllophilus with raw starch hydrolysis capacity and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| KAYLEEN NAIDU等: "Purification and characterization of α-amylase from Paenibacillus sp. D9 and Escherichia coli recombinants", BIOCATALYSIS AND BIOTRANSFORMATION, pages 1 - 11 * |
| YAN, G.: "Paenibacillus phyllosphaerae strain CS-148 16S ribosomal RNA gene, partial sequence", GENBANK DATABASE, pages 608826 * |
| 曾丽娟: "类芽孢杆菌属菌株产生淀粉酶的分离纯化及对淀粉结合域CBM25的初步研究", 万方学位论文数据库 * |
| 王磊;陈宇飞;杨柳;: "产淀粉酶芽孢杆菌的分离和鉴定", 食品研究与开发, no. 06, pages 175 - 178 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114874941A (en) * | 2022-05-11 | 2022-08-09 | 淮阴师范学院 | Paenibacillus phyllophilus with raw starch hydrolysis capacity and application thereof |
| CN114874941B (en) * | 2022-05-11 | 2023-08-11 | 淮阴师范学院 | Paenibacillus phylloides with starch hydrolysis capability and application thereof |
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