CN111518712A - Enterobacter strain and application thereof in synthesis of 2,3-butanediol - Google Patents
Enterobacter strain and application thereof in synthesis of 2,3-butanediol Download PDFInfo
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Abstract
The invention discloses an enterobacter Kosakonia cowanii strain with strain number LT-1, which is preserved in China center for type culture Collection, and the registration number of the registration book is CCTCC No: m2017850, preservation date of 2017, 12 months and 29 days. The invention also discloses application of the enterobacter in synthesizing 2, 3-butanediol. The yield of 2,3-butanediol synthesized by fermentation with glucose as a carbon source and yeast powder as a nitrogen source can reach 42.56-212.49 g/L. The invention has the advantages that the strains used for fermenting and synthesizing the 2,3-butanediol are as follows: the enterobacter K.cowanii LT-1 has the fermentation characteristics that the yield of 2,3-butanediol synthesized by fermentation is high, the byproducts are few, the production cost is reduced, and the purification of the 2,3-butanediol is facilitated. The operation method is simple, the cost is low, the industrial popularization and application prospect is good, and a new process is provided for the biosynthesis of the 2, 3-butanediol.
Description
Technical Field
The invention specifically relates to an enterobacter strain and application thereof in synthesis of 2,3-butanediol, and belongs to the technical fields of microbiology, biological engineering technology and chemical industry.
Background
2,3-Butanediol (2,3-Butanediol) is a valuable liquid fuel, and the combustion value is second only to ethanol and higher than methanol, and reaches 27,200 kJ/kg; 2,3-butanediol is also a very potential chemical raw material and can be used for producing antifreeze agents and high-grade aviation oil; 2,3-butanediol can eliminate free radicals and block peroxidation, so that the compound can be widely applied to industries such as cosmetics, lotion and the like; in addition, 2,3-butanediol is useful as a food additive for high-value perfumes and as a pharmaceutical intermediate.
With the increasing demand of petroleum, petroleum is used as a non-renewable energy source. Therefore, the petroleum product is obtained by the artificial production method, and a new energy development idea is provided for social development. The main production methods of 2,3-butanediol include chemical synthesis and biological fermentation. The chemical synthesis method for producing the 2,3-butanediol is generated by hydrolyzing four-carbon hydrogen compounds generated in petroleum cracking at high temperature and high pressure, and the method has high production cost, complex process, difficult operation and more byproducts, so that the large-scale industrial production is difficult, thereby limiting the development and application of the 2, 3-butanediol. The enterobacter k.cowanii LT-1 can produce 2,3-butanediol by a microbial fermentation technology, so that the difficulties of high chemical production cost and difficult operation are overcome, and the 2,3-butanediol produced by utilizing microbial synthesis fermentation is more economical and has more environmental protection significance.
Through search, no patent report of producing 2,3-butanediol by using Enterobacter K.
Disclosure of Invention
Aiming at the defects in the prior art, the invention solves the problems that: provides an enterobacter k.cowanii LT-1 and application of the enterobacter k.cowanii LT-1 in preparation of 2, 3-butanediol.
The technical problem to be solved by the invention is to provide the application of the enterobacter.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
the inventor screens and obtains a strain of enterobacter from vinasse in 2017, the strain number is Kosakonia cowaniiLT-1, the enterobacter is preserved in China center for type culture Collection (CCTCC for short), and the preservation address is as follows: wuhan city, Hubei province, eight mountainous areas, Wuhan university, mountain type culture collection, zip code: 430072, registration number of registration book is CCTCCNO: m2017850, preservation date of 2017, 12 months and 29 days.
The following all refer to this strain as a production strain.
1. The morphological characteristics and physiological and biochemical characteristics of the colonies are shown in Table 1.
TABLE 1 morphological characteristics and physiological and biochemical characteristics of colonies
2. 16S rDNA sequence analysis:
the length of the nucleic acid sequence of the 16S rDNA gene of the strain is 1388bp, and the gene sequence is shown as SEQID No. 1: as shown. The sequences tested were compared for homology from the Gene Bank database using the BLAST program to construct a phylogenetic tree based on the 16S rDNA full sequence. The results show that: the strain achieves 100% homology with enterobacter HME 8565. According to the results of strain morphology observation and physiological and biochemical experiment analysis, the enterobacter used in the invention is determined, and the enterobacter is specifically Enterobacter K.
The application of the enterobacter k.cowanii LT-1 in the fermentative synthesis of 2,3-butanediol is also within the scope of the present invention.
The specific application method is that the enterobacter LT-1 is inoculated in a slant solid culture medium, transferred to a seed culture medium and finally inoculated in a fermentation culture medium for aerobic culture, and the fermentation liquid is rich in 2, 3-butanediol.
The enterobacter k.cowanii LT-1 and the application of the enterobacter k.cowanii LT-1 in the preparation of 2,3-butanediol sequentially comprise the following steps:
1. preparation of a culture medium:
(1) the slant culture medium comprises the following components: 20g/L of glucose, 20g/L of peptone, 5g/L of yeast powder, 10g/L of sodium chloride, 20g/L of agar powder and tap water, adjusting the pH value to 7.0-7.2, and sterilizing at 115 ℃ for 20 min;
(2) the liquid seed culture medium is as follows: 10-30 g/L of carbon source, 4-8 g/L of nitrogen source, 1.0-2.5 g/L of metal salt, preparing with tap water, adjusting the pH value to 7.0-7.2, and sterilizing at 115/121 ℃ for 20 min;
(3) the enrichment liquid culture medium comprises: 30g/L of glucose, 8g/L of yeast powder, 3g/L of sodium nitrate, 1.5g/L of monopotassium phosphate, 0.2g/L of calcium chloride, 0.4g/L of magnesium sulfate, 0.05g/L of manganese sulfate, 20g/L of agar powder, preparing tap water, adjusting the pH value to 7.0-7.2, and sterilizing at 115 ℃ for 20 min;
(4) the enrichment solid culture medium comprises: enriching a liquid culture medium, 20g/L agar powder, preparing tap water, adjusting the pH value to 7.0-7.2, and sterilizing at 115 ℃ for 20 min;
(5) the solid seed culture medium is as follows: preparing a liquid seed culture medium, 20g/L agar powder and tap water, adjusting the pH value to 7.0-7.2, and sterilizing at 115/121 ℃ for 20 min;
(6) fermentation medium: 140g/L of glucose, 6g/L of yeast powder, 6g/L of sodium nitrate, 0.3g/L of monopotassium phosphate, 0.3g/L of calcium chloride, 0.4g/L of magnesium sulfate, 0.04g/L of manganese sulfate, preparation of tap water, adjustment of the pH value to 7.0-7.2, and sterilization at 115 ℃ for 20 min;
2. selecting strains:
selecting deposited strain Enterobacter K
3. Activating strains:
inoculating an enterobacter K.cowanii LT-1 strain to a slant culture medium, performing static culture at 24-36 ℃ for 24-36 h, selecting a single colony again, streaking the single colony onto a common solid culture medium, and performing culture at 24-36 ℃ for 24-36 h to obtain an activated strain for later use;
4. shake flask fermentation seed liquid culture:
taking the activated strain in the step 3, inoculating 2-4 rings of the strain into a shake flask of the seed liquid under an aseptic condition, placing the shake flask on a shaking table with the rotating speed of 220rpm, and culturing at the temperature of 28-38 ℃ for 24 hours to obtain a fermented seed liquid;
5. fermentation culture:
and (3) shake flask fermentation culture: inoculating the seed liquid into a shake flask of a fermentation culture medium under aseptic conditions in an inoculation amount of 4-10% by volume, placing the shake flask on a shaking table with the rotation speed of 220rpm, and culturing for 36-48 h at 28-38 ℃; stopping fermentation when the concentration of butanediol in the fermentation liquor does not rise any more;
6.2, 3-butanediol extraction:
taking the fermentation liquor obtained in the step 5, performing centrifugal sterilization, performing reduced pressure distillation on the clear liquid to obtain 30-55% concentrated solution, and adding 45% of K with the same volume2HPO4A/ethanol system to form a two-phase system, and distilling the solution in the upper phase to obtain the 2, 3-butanediol.
7.2, 3-butanediol content determination:
centrifuging the fermentation liquid obtained in step 5 at 12,000rpm for 20min to remove thallus, filtering the supernatant with 0.22 μ M filter membrane, and placing in a sample bottle for use, wherein the sample is detected by differential detector, and the chromatographic column contains Aminex HPX-87H (300 × 7.8.8 mm) and mobile phase contains 0.05M H2SO4(ii) a Flow rate: 0.6 mL/min; sample introduction amount: 20 mu L of the solution; column temperature: at 60 ℃.
Has the advantages that: the invention has the following advantages:
(1) the strain for producing the 2,3-butanediol is obtained by screening, can be used for fermenting and synthesizing the 2,3-butanediol by taking glucose as a carbon source and a yeast powder nitrogen source, has few byproducts, is beneficial to purification of the 2,3-butanediol, and has remarkable social and economic benefits.
(2) The yield of 2,3-butanediol synthesized by the strain through fermentation can reach 42.56-212.49 g/L, the glucose conversion rate can reach 62.50% at most, the production cost is greatly reduced, the operation is simple, and the method has very important significance on the production and the expanded application of the 2, 3-butanediol.
Drawings
FIG. 1 shows a 16S rDNA PCR purification agarose gel electrophoresis of Enterobacter LT-1 (A) and a phylogenetic tree of Enterobacter LT-1 (B).
FIG. 2 is a high performance liquid chromatogram of 2,3-butanediol produced by Enterobacter LT-1.
FIG. 3 is an infrared spectrum of 2,3-butanediol produced by Enterobacter LT-1.
FIG. 4 is a graph showing the progress of the horizontal synthesis of 2,3-butanediol by the Enterobacter LT-1 in a shake flask.
FIG. 5 is a graph showing the progress of the fed-batch synthesis of 2,3-butanediol in a 50L fermenter.
FIG. 6 is a graph showing the progress of fed batch synthesis of 2,3-butanediol in a 1t fermenter.
The specific implementation mode is as follows:
the present invention can be better understood from the following examples, however, those skilled in the art will readily appreciate that the descriptions of the examples are only for illustrating the present invention and should not be construed as limiting the invention as detailed in the claims.
Example 1: isolation and screening of Enterobacter K.cowanii LT-1.
The composition of the medium used in this example is as follows:
liquid screening culture medium: 20g/L of glucose, 3g/L of sodium nitrate, 0.2g/L of ferric chloride, 2g/L of dipotassium phosphate, 0.1g/L of calcium chloride, 2g/L of magnesium sulfate, 0.05g/L of manganese sulfate, preparation of tap water, adjustment of the pH value to 7.0-7.2, and sterilization at 115 ℃ for 20 min;
solid screening culture medium: adjusting the value of 20g/L, pH of the liquid screening culture medium and agar powder to 7.0-7.2, and sterilizing at 115 ℃ for 20 min;
fermentation medium: 140g/L of glucose, 6g/L of yeast powder, 6g/L of sodium nitrate, 0.3g/L of monopotassium phosphate, 0.3g/L of calcium chloride, 0.5g/L of magnesium sulfate, 0.04g/L of manganese sulfate, preparation of tap water, adjustment of the pH value to 7.0-7.2, and sterilization at 115 ℃ for 20 min;
the specific operation process of this embodiment is as follows:
screening 2,3-butanediol producing bacteria from 60 parts of distiller's grains from all over the country, and adding 1g of the bacteria into a triangular flask filled with enrichment mediumThe liquid loading amount is 50mL/250mL, and the enrichment culture is carried out for 48h at 37 ℃ in a shaking table at 220 rpm. Taking 1mL of culture solution, transferring the culture solution to the same liquid enrichment medium under the same condition, and carrying out secondary enrichment culture for 48 h. Under aseptic conditions, the culture broth was diluted to 10 deg.C-8And 10-9Respectively coating 0.2mL of the strain on a solid screening culture medium, culturing at 37 ℃ for 24h, after single colonies grow out, selecting a larger single colony, inoculating the single colony into a seed culture medium, culturing to a logarithmic phase, inoculating the single colony into a fermentation culture medium according to the inoculation amount of 5%, culturing at 37 ℃ for 48h on a shaking table at the rotating speed of 220rpm, collecting fermentation liquor, centrifuging, taking supernatant, filtering the supernatant through a 0.22 mu m filter membrane, and measuring the yield of the prescreened strain 2,3-butanediol by using liquid chromatography to obtain the strain with the highest yield.
Example 2: identification of Enterobacter K.cowanii LT-1 and fermentation product 2, 3-butanediol.
Identification of Enterobacter K.cowanii LT-1
Extracting genome DNA of the strain LT-1 by using a bacterial genome DNA extraction kit, amplifying a 16S rDNA sequence by using an upstream primer 27F and a downstream primer 1492R in a PCR way as shown in figure 1A, performing gel recovery and purification on a product obtained after the PCR amplification, and sending the gel recovery and purification product to Suzhou Jinzhi Biotech limited for sequencing. The length of the nucleotide sequence of the 16S rDNA gene of the strain obtained by sequencing is 1388bp, and the gene sequence is shown as SEQID No. 1. BLAST alignment of the sequencing results with known 16S rDNA sequences in the Gene Bank database and homology comparison using the BLAST program construct a phylogenetic tree based on the full sequence of 16 SrDNA. The results show that: this strain reached 100% homology with enterobacter k.cowanii HME8565 (fig. 1B). According to the results of strain morphological observation and physiological and biochemical experiment analysis, the enterobacter is identified to be used in the invention, and is specifically named as enterobacter K.
② identification of Enterobacter K.cowanii LT-1 fermentation product
Liquid Chromatography (HPLC) and infrared spectroscopy are used to identify whether the fermentation broth contains 2, 3-butanediol. Centrifuging Enterobacter K.cowanii LT-1 fermentation broth at 12,000rpm for 20min, filtering the supernatant with 0.22 μm filter membrane, and placing in sample bottleThe sample is detected by a differential detector, and the chromatographic column comprises Aminex HPX-87H (300 × 7.8.8 mm) and a mobile phase of 0.05M H2SO4(ii) a Flow rate: 0.6 mL/min; sample introduction amount: 20 mu L of the solution; column temperature: at 60 ℃. The liquid chromatogram of the purified product is shown in FIG. 2, and under the same chromatographic conditions, the substance has absorption peaks at the same positions compared with the 2,3-butanediol standard substance, which indicates that the fermentation liquor contains 2, 3-butanediol.
Measuring an infrared spectrum of a K.cowanii LT-1 fermentation 2,3-butanediol purification product by using an infrared spectrometer, measuring an infrared spectrum of a sample by using the infrared spectrometer after the sample is prepared, and scanning the range of 4000-400 cm-1. The results are shown in FIG. 3 and correspond to the structural features of 2, 3-butanediol.
Example 3: optimization of enterobacter LT-1 fermentation synthesis of 2,3-butanediol carbon source variety
This example illustrates the effect of different carbon sources on the preparation of 2,3-butanediol by fermentation of strains, inoculating seed culture solution with an inoculum size of 4% (v/v) into a fermentation medium containing 100g/L of glycerol, glucose, lactose and citrate, respectively, with an initial pH of 7.2, performing shaking culture at 26-34 ℃ and 220rpm, loading the fermentation medium into a 50mL/250mL triangular flask, performing fermentation culture for 42h, taking various fermentation solutions with different carbon sources, performing the operation of step 7, and calculating the content of 2, 3-butanediol. The content of the 2,3-butanediol obtained by taking the glucose as the carbon source is highest, so that the glucose is selected as the optimal carbon source. The yield of the 2,3-butanediol reaches 42.56g/L, the glucose conversion rate is 42.56%, the maximum production rate is as high as 1.01g/L/h, and the concentration of the acetoin in the fermentation liquid is only 0.83 g/L.
Example 4: optimization of concentration of 2,3-butanediol glucose synthesized by enterobacter LT-1 fermentation
This example illustrates the effect of different glucose concentrations on the fermentative production of 2,3-butanediol by strains, inoculating seed culture fluid with an inoculum size of 4% (v/v) into fermentation media with glucose contents of 100g/L, 110g/L, 120g/L, 130g/L, 140g/L, 150g/L and 160g/L, respectively, with an initial pH of 7.2, shaking and culturing at 32 ℃, 220rpm for 42h, with the volume of fermentation media being 50mL/250mL, taking fermentation broth with each content of glucose, performing step 7, calculating the content of 2,3-butanediol, and determining that when the glucose concentration is 140g/L, the content of 2,3-butanediol is the highest, so the glucose concentration is 140g/L for fermentation. The yield of the 2,3-butanediol reaches 60.04g/L, the glucose conversion rate is 42.89%, the maximum production rate is as high as 1.43g/L/h, and the concentration of the acetoin in the fermentation liquor is only 1.03 g/L.
Example 5: optimization of nitrogen source variety for fermenting and synthesizing 2,3-butanediol by enterobacter LT-1
In this example, which illustrates the influence of different nitrogen sources on the preparation of 2,3-butanediol by strain fermentation, a seed culture solution is inoculated into a fermentation medium containing 5g/L beef extract, tryptone, yeast extract, corn steep liquor, bean cake powder, peanut powder, soybean powder, wheat germ powder and yeast powder in an inoculation amount of 4% (v/v), the initial pH value is 7.2, the mixture is subjected to shaking culture at 32 ℃ and 220rpm, the liquid loading amount of the fermentation medium is 50mL/250mL of a triangular flask, the fermentation culture is performed for 42 hours, various nitrogen source fermentation liquids are taken to perform the operation of step 7, the content of 2,3-butanediol is calculated, and when the yeast powder is used as a nitrogen source, the content of 2,3-butanediol is the highest, so that the yeast powder is selected as the optimal nitrogen source. The yield of the 2,3-butanediol reaches 62.24g/L, the glucose conversion rate is 44.46%, the maximum production rate is as high as 1.48g/L/h, and the concentration of the acetoin in the fermentation liquor is only 1.12 g/L.
Example 6: optimization of concentration of yeast powder for synthesizing 2,3-butanediol by virtue of enterobacter LT-1 fermentation
This example illustrates the effect of different concentrations of yeast powder on the fermentation of strains to produce 2,3-butanediol, inoculating a seed culture solution at an inoculum size of 4% (v/v) into a fermentation medium containing 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, and 10g/L of yeast powder, respectively, at an initial pH of 7.2, performing shaking culture at 32 ℃, 220rpm, at a fermentation medium loading capacity of 50mL/250mL triangular flask, performing fermentation culture for 42h, taking the fermentation broth of the yeast powder at each concentration, performing the operation of step 7, and calculating the content of 2, 3-butanediol. When the concentration of the yeast powder is 6g/L, the content of the 2,3-butanediol is highest, so that 6g/L of the yeast powder is selected for fermentation. The yield of the 2,3-butanediol reaches 64.21g/L, the glucose conversion rate is 45.86%, the maximum production rate is as high as 1.53g/L/h, and the concentration of the acetoin in the fermentation liquor is only 1.38 g/L.
Example 7: optimization of temperature for synthesizing 2,3-butanediol by virtue of enterobacter LT-1 fermentation
This example illustrates the effect of different temperatures on the yield of 2,3-butanediol produced by microbial fermentation, inoculating a seed culture solution 4% (v/v) in a fermentation medium at an initial pH of 7.2, performing shaking culture at 220rpm in a flask containing 50mL/250mL of the medium, performing fermentation culture for 42h at different temperatures of 24 deg.C, 26 deg.C, 28 deg.C, 30 deg.C, 32 deg.C, 34 deg.C, and 36 deg.C, taking the fermentation broth at each temperature, performing the operation of step 7, calculating the content of 2,3-butanediol, and selecting 32 deg.C as the optimum fermentation temperature, where the highest content of 2,3-butanediol is obtained. The yield of the 2,3-butanediol reaches 65.24g/L, the glucose conversion rate is 46.60%, the maximum production rate is as high as 1.55g/L/h, and the concentration of acetoin in the fermentation liquor is only 1.41 g/L.
Example 8: optimization of pH value in fermentation synthesis of 2,3-butanediol by enterobacter LT-1
This example illustrates the effect of different pH values on the preparation of 2,3-butanediol by fermentation of a strain, inoculating a seed culture solution in an inoculum size of 4% (v/v) into a fermentation medium, culturing at 32 ℃ under shaking at 220rpm in a medium-filled volume of 50mL/250mL triangular flask, culturing in fermentation media with pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, performing the operation of step 7 on the fermentation broth at each pH value, and calculating the content of 2,3-butanediol according to a standard curve to obtain the highest content of 2,3-butanediol when the pH value is 7.0, so that the pH value of 7.0 is selected as the optimal fermentation pH value. The yield of the 2,3-butanediol reaches 66.44g/L, the glucose conversion rate is 47.46%, the maximum production rate is as high as 1.58g/L/h, and the concentration of the acetoin in the fermentation liquor is only 1.45 g/L.
Example 9: optimization of species and concentration of metal salts required for the fermentative synthesis of 2,3-butanediol by enterobacter LT-1
This example illustrates the effect of different metal salts and their concentrations on the fermentative production of 2,3-butanediol by inoculating a seed culture broth with an inoculum size of 4% (v/v) in the following concentrations of the respective metal salts, as a single factor variable experiment:
sodium nitrate: 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0g/L and a control group, wherein the optimal concentration of sodium nitrate is 6.0 g/L;
potassium dihydrogen phosphate: 0.10, 0.20, 0.30, 0.40, 0.50, 0.60g/L and a control group, wherein the optimal concentration of the potassium dihydrogen phosphate is 0.3 g/L;
calcium chloride: 0.10, 0.20, 0.30, 0.40, 0.50, 0.60g/L and a control group, wherein the optimal concentration of calcium chloride is 0.30 g/L;
magnesium sulfate: 0.10, 0.20, 0.30, 0.40, 0.50, 0.60g/L and a control group, wherein the optimal concentration of magnesium sulfate is 0.50 g/L;
manganese sulfate 0.01, 0.02, 0.03, 0.04, 0.05, 0.06g/L and control group, the optimal concentration of manganese sulfate is 0.04 g/L;
for the fermentation culture media of different experimental groups, 3 groups of parallel controls are set for metal salts with different concentrations, the initial pH value is 7.0, shaking culture is carried out at 32 ℃ and 220rpm, the liquid loading capacity of the culture media is 50mL/250mL triangular bottles, the fermentation culture is carried out for 42h, the fermentation liquid under each metal salt concentration is taken to carry out the operation of the step 7 to calculate the content of 2,3-butanediol, when under the conditions, the content of 2,3-butanediol produced by the strain reaches up to 68.41g/L, the glucose conversion rate is 48.86%, the maximum yield is 1.63g/L/h, and the concentration of acetoin in the fermentation liquid is only 1.50g/L (figure 4).
Example 10: fed-batch synthesis of 2,3-butanediol in 50L fermentation tank
Inoculating enterobacter K.cowanii LT-1 seed liquid into a sterile fermentation culture medium according to the inoculation amount of 10% by volume, wherein the total liquid loading of a fermentation tank is 30L, the fermentation temperature is 32 ℃, the stirring speed is 400rpm, and the ventilation volume is 1.2vvm for fermentation; the initial pH value of the fermentation is 7.0, and the pH value is controlled to be 7.0 in the fermentation process; the fermentation time was 84 hours. Sampling every 6 hours to determine the concentration of 2,3-butanediol in the fermentation broth. Determination and analysis: and (3) centrifuging the fermentation liquor at 12,000rpm for 2 minutes, taking the supernatant, diluting the supernatant by a proper time, detecting that the yield of the 2,3-butanediol in the fermentation liquor reaches 201.35g/L, the glucose conversion rate is 59.22%, the maximum production rate is 2.40g/L/h, and the concentration of the acetoin in the fermentation liquor is 3.26g/L (figure 5).
Example 11: fed-batch synthesis of 2,3-butanediol in 1t fermentation tank
Inoculating enterobacter K.cowanii LT-1 seed liquid into a sterile fermentation culture medium according to the inoculation amount of 10% by volume, wherein the total liquid loading of a fermentation tank is 30L, the fermentation temperature is 32 ℃, the stirring speed is 300rpm, and the ventilation volume is 1.2vvm for fermentation; the initial pH value of the fermentation is 7.0, and the pH value is controlled to be 7.0 in the fermentation process; the fermentation time was 84 hours. Sampling every 6 hours to determine the concentration of the 2,3-butanediol in the fermentation liquor. Determination and analysis: and (2) centrifuging the fermentation liquor at 12,000rpm for 2 minutes, taking the supernatant, diluting the supernatant by a proper time, detecting that the yield of the 2,3-butanediol in the fermentation liquor reaches 212.49g/L, the glucose conversion rate is 62.50%, the maximum production rate is 2.53g/L/h, and the concentration of the acetoin in the fermentation liquor is only 3.46g/L (figure 6).
Finally, it should also be noted that the above-mentioned list is only a specific embodiment of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the teachings of the present invention are to be considered within the scope of the present invention.
Sequence listing
<110> institute of technology and technology
<120> Enterobacter strain and application thereof in synthesis of 2,3-butanediol
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accagccaca ctggaactga gacacggtcc agactcctac gggaggcagc agtggggaat 300
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ttgtaaagta ctttcagcgg ggaggaaggc gatgtggtta ataaccgcgt cgattgacgt 420
tacccgcaga agaagcaccg gctaactccg tgccagcagc cgcggtaata cggagggtgc 480
aagcgttaat cggaattact gggcgtaaag cgcacgcagg cggtctgtca agtcggatgt 540
gaaatccccg ggctcaacct gggaactgca tccgaaactg gcaggcttga gtctcgtaga 600
ggggggtaga attccaggtg tagcggtgaa atgcgtagag atctggagga ataccggtgg 660
cgaaggcggc cccctggacg aagactgacg ctcaggtgcg aaagcgtggg gagcaaacag 720
gattagatac cctggtagtc cacgccgtaa acgatgtcga cttggaggtt gtgcccttga 780
ggcgtggctt ccggagctaa cgcgttaagt cgaccgcctg gggagtacgg ccgcaaggtt 840
aaaactcaaa tgaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgat 900
gcaacgcgaa gaaccttacc tggtcttgac atccacagaa cttggcagag atgccttggt 960
gccttcggga actgtgagac aggtgctgca tggctgtcgt cagctcgtgt tgtgaaatgt 1020
tgggttaagt cccgcaacga gcgcaaccct tatcctttgt tgccagcggt ccggccggga 1080
actcaaagga gactgccagt gataaactgg aggaaggtgg ggatgacgtc aagtcatcat 1140
ggcccttacg accagggcta cacacgtgct acaatggcgc atacaaagag aagcaaactc 1200
gcgagagcaa gcggacctca taaagtgcgt cgtagtccgg attggagtct gcaactcgac 1260
tccatgaagt cggaatcgct agtaatcgtg aatcagaatg tcacggtgaa tacgttcccg 1320
ggccttgtac acaccgcccg tcacaccatg ggagtgggtt gcaaaagaag taggtagctt 1380
aaccttcg 1388
Claims (9)
1. An enterobacter strain is named as enterobacter Kosakonia cowanii LT-1, and the strain is preserved in China Center for Type Culture Collection (CCTCC) in 29 months 12 and 2017, and the preservation number is CCTCC No: m2017850.
2. Use of the enterobacterium of claim 1 for the preparation of 2, 3-butanediol.
3. Use according to claim 2, characterized in that it comprises the following steps:
(1) strain activation:
(2) seed culture:
(3) fermentation culture: inoculating the seed liquid in the step (2) to a fermentation medium, and fermenting for 42-84 h to obtain a fermentation liquid containing 2, 3-butanediol;
the fermentation medium comprises the following components: 80-140 g/L of carbon source, 4-8 g/L of nitrogen source, 1-2.5 g/L of metal salt, 7.0-7.2 of pH value and the balance of water;
the carbon source is any one or combination of more of glycerol, glucose and lactose; the nitrogen source is any one or combination of more of beef extract, tryptone, yeast extract, corn steep liquor, bean cake powder, soybean powder, wheat germ powder, peanut cake powder and yeast powder; the metal salt is one or more of magnesium sulfate, potassium dihydrogen phosphate, manganese sulfate, calcium chloride, and sodium nitrate.
4. The use according to claim 3, wherein in step (1), the strain is activated under the following culture conditions:
inoculating enterobacter K.cowanii LT-1 to a slant culture medium, performing static culture at 24-36 ℃ for 24-36 h, selecting a single colony again, streaking the single colony onto a seed culture medium, and performing culture at 24-36 ℃ for 24-36 h to obtain an activated strain for later use;
slant culture medium: 20g/L of peptone, 5g/L of yeast powder, 10g/L of sodium chloride, 20g/L of agar powder and tap water, adjusting the pH value to 7.0-7.2, and sterilizing at 121 ℃ for 20 min;
solid seed culture medium: 10-30 g/L of carbon source, 4-8 g/L of nitrogen source, 1.0-2.5 g/L of metal salt, 20g/L of agar powder and the balance of water, wherein the pH value is 7.0-7.2.
5. The use according to claim 3, wherein in step (2), the seed culture comprises the following steps: inoculating the activated strain into a liquid seed culture medium, and performing shake culture at the temperature of 24-36 ℃ for 24-48 h to obtain a fermented seed liquid;
liquid seed culture medium: 10-30 g/L of carbon source, 4-8 g/L of nitrogen source, 1.0-2.5 g/L of metal salt and the balance of water, wherein the pH value is 7.0-7.2.
6. The use according to claim 3, wherein in step (3), the temperature of the fermentation is 28-38 ℃.
7. The use according to claim 3, wherein in step (3), the fermentation is characterized by a shake flask inoculum size of 1-10%; the inoculation amount of the fermentation tank is 1-30%; the fermentation mode of the fermentation tank is a fed-batch method.
8. The use according to claim 3, wherein the 2,3-butanediol is extracted by a method comprising: taking fermentation liquor, centrifuging to remove thalli, carrying out reduced pressure distillation to obtain a concentrated solution with the volume fraction of 2,3-butanediol of 30-50%, and then adding K which has the same volume as that of concentrated acid2HPO4K with mass fraction of 45%2HPO4Ethanol solution to form a two-phase system, and distilling the solution in the upper phase to obtain the 2, 3-butanediol.
9. The method as claimed in claim 2, wherein the crude extraction method of 2,3-butanediol is salting-out extraction method.
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| CN111518711A (en) * | 2019-02-02 | 2020-08-11 | 常熟理工学院 | Enterobacter strain and application thereof in coproduction of microbial exopolysaccharide and 2,3-butanediol |
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| CN111518710A (en) * | 2019-02-02 | 2020-08-11 | 常熟理工学院 | Enterobacter strain and application thereof in preparation of microbial polysaccharide |
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| CN111518710A (en) * | 2019-02-02 | 2020-08-11 | 常熟理工学院 | Enterobacter strain and application thereof in preparation of microbial polysaccharide |
Non-Patent Citations (3)
| Title |
|---|
| HUA GAO ET AL.: "Production exopolysaccharide from Kosakonia cowanii LT-1 through solid-state fermentation and its application as a plant growth promoter", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 * |
| LIYING WANG ET AL.: "Development of sugarcane resource for efficient fermentation of exopolysaccharide by using a novel strain of Kosakonia cowanii LT-1", 《BIORESOURCE TECHNOLOGY》 * |
| 李媛媛: "基于全基因组序列系统分类Kosakonia属的细菌", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111518711A (en) * | 2019-02-02 | 2020-08-11 | 常熟理工学院 | Enterobacter strain and application thereof in coproduction of microbial exopolysaccharide and 2,3-butanediol |
| CN111518711B (en) * | 2019-02-02 | 2021-12-07 | 常熟理工学院 | Enterobacter strain and application thereof in coproduction of microbial exopolysaccharide and 2,3-butanediol |
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