CN117778479B - Method for preparing 1, 2-propylene glycol by microbial fermentation method - Google Patents
Method for preparing 1, 2-propylene glycol by microbial fermentation method Download PDFInfo
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Abstract
The invention discloses a method for preparing 1, 2-propylene glycol by a microbial fermentation method, which comprises the following steps: mixing klebsiella pneumoniae seed liquid, serratia seed liquid and bacillus aryaensis seed liquid to obtain mixed seed liquid; and (3) inoculating the mixed seed liquid into a fermentation culture medium consisting of beet pulp-bagasse mixed extract and glycerol, and fermenting at 25-30 ℃ for 72-80h to obtain the 1, 2-propylene glycol. The preparation method has the advantages that the raw materials are cheap, the mixed extract of beet pulp and bagasse and the glycerol are used as the culture medium, no additional nutrition source is needed, the fermentation condition is mild, and the production cost is greatly reduced; the invention combines Klebsiella pneumoniae, bacillus aryaensis and Serratia, overcomes the problem of low fermentation yield of the existing single strain or engineering strain, and has synergistic effect in improving the yield of 1, 2-propanediol by compounding the three bacteria.
Description
Technical Field
The invention relates to the technical field of preparation of 1, 2-propylene glycol, in particular to a method for preparing 1, 2-propylene glycol by a microbial fermentation method.
Background
1, 2-Propanediol is an important chemical product and intermediate, and is mainly used in the synthesis process of resin, plasticizer, surfactant and antifreezing agent. Compared with ethylene glycol, the propylene glycol has no toxicity, so the propylene glycol has wide application in the industries of food, cosmetics, medicines and the like, and can be used as a solvent for various products such as food anti-sticking agents, food pigments, food packaging softeners, essence and the like, or used as a humectant, a softener, a solvent and the like in various cosmetics. The traditional production method of 1, 2-propylene glycol mainly comprises a chemical synthesis method, such as a propylene oxide hydration method, an ester exchange method, a1, 2-dichloropropane hydrolysis method and the like, but the chemical synthesis method has the problems of serious environmental pollution, severe reaction conditions, high equipment investment cost, difficult product separation and the like by taking petrochemical resources as raw materials.
With the exacerbation of environmental problems and energy crisis, the utilization of renewable biomass resources to replace traditional fossil raw materials such as propylene oxide, propylene and the like for producing 1, 2-propanediol has great significance. Bagasse is a main byproduct of sugar production of sugarcane, is a renewable biomass resource, and contains abundant cellulose, hemicellulose, soluble sugar and other components; the beet pulp after sugar pressing of beet is taken as industrial residue, the yield is rich, 0.16 to 0.5 ton of beet pulp can be produced per ton of fresh beet, the beet pulp contains rich polysaccharide, mineral elements and vitamins, and no report of directly utilizing the bagasse and the beet pulp for extraction for producing 1, 2-propanediol by fermentation exists at present. Glycerol is a byproduct of biodiesel production, and with the rapid development of the biodiesel industry, glycerol production increases over the years, resulting in over-market. At present, the effective utilization of glycerol for producing 1, 2-propanediol has received a great deal of attention, but the current use of glycerol for producing 1, 2-propanediol requires the use of noble metal catalysts, and the reaction conditions are severe and the process flow is complex.
At present, reports on the synthesis of 1, 2-propylene glycol by fermenting saccharides and glycerol are relatively few, and a microbial fermentation method has the advantages of mild reaction conditions, fewer byproducts, small environmental pollution and the like, and the problems that clostridium contractum, escherichia coli or clostridium pyrolyzicum can ferment the saccharides into the 1, 2-propylene glycol, but the production efficiency is low and the byproducts are more exist by using a single strain are discovered. At present, some researches are carried out to construct engineering bacteria by adopting a genetic engineering method to ferment and produce 1, 2-propanediol, but the construction of the engineering bacteria is very complex, is unfavorable for large-scale popularization and application, and the yield is still very low. Patent CN115261292 a discloses an improved klebsiella bacterium and an application and a method for producing 1, 2-propanediol, which can synthesize 1, 2-propanediol by using substrates such as glycerol and glucose, but the yield of 1, 2-propanediol prepared by fermenting the improved klebsiella pneumoniae is only 6.2g/L at maximum, and the culture medium is prepared from various refined raw materials, so that the cost is high, and the industrial production is difficult to realize. Therefore, how to realize green, economical and efficient preparation of 1, 2-propanediol is a current difficulty.
Disclosure of Invention
The aim of the invention is to provide a method for preparing 1, 2-propanediol by a microbial fermentation method.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a method for preparing 1, 2-propanediol by microbial fermentation, comprising the steps of:
(1) Mixing klebsiella pneumoniae seed liquid, serratia seed liquid and bacillus aryaensis seed liquid to obtain mixed seed liquid;
(2) And (3) inoculating the mixed seed liquid into a fermentation culture medium consisting of beet pulp-bagasse mixed extract and glycerol, and fermenting at 25-30 ℃ for 72-80h to obtain the 1, 2-propylene glycol.
Preferably, the strain number of Klebsiella pneumoniae is CGMCC 1.10617; the Serratia strain is CICC 10689; the strain number of Bacillus aryasis was CICC 24574.
Preferably, the inoculation amount of the mixed seed liquid is 5% -10% of the volume of the fermentation medium; in the mixed seed liquid, the volume ratio of Klebsiella pneumoniae seed liquid, bacillus aryabhattai seed liquid and Serratia seed liquid is (3-5): (2-4): (4-6).
Preferably, in the mixed seed solution, the viable count of Klebsiella pneumoniae is more than or equal to 6 multiplied by 10 8 cfu/ml; the viable count of the bacillus arvensis is more than or equal to 4 multiplied by 10 8 cfu/ml; the viable count of Serratia is more than or equal to 7 multiplied by 10 8 cfu/ml.
Preferably, in the step (2), the fermentation medium is prepared by the following method:
(a) Bagasse, beet pulp and water are mixed according to the feed liquid ratio (3-5) g: (2-4) g:50ml of the mixed fermentation liquid is obtained;
(b) Inoculating Trichoderma reesei seed solution and Pseudomonas putida seed solution into the mixed fermentation liquid, fermenting at 25-30deg.C for 36-48 hr, centrifuging to collect supernatant, filtering, and sterilizing to obtain beet pulp-bagasse mixed extract;
(c) Adding 40-60g glycerin into each liter of beet pulp-bagasse mixed extract, and mixing uniformly to obtain the fermentation medium.
Preferably, the strain number of the trichoderma reesei is CGMCC 3.13232; the preservation number of the pseudomonas putida is CGMCC 1.4533.
Preferably, the inoculation amount of the Trichoderma reesei seed liquid and the Pseudomonas putida seed liquid is 3-5% of the volume of the mixed fermentation liquid.
Preferably, the bacterial concentration of the Trichoderma reesei seed solution and the Pseudomonas putida seed solution is more than or equal to 2 multiplied by 10 8 cfu/ml.
The invention has the beneficial effects that:
The invention takes the mixed fermentation extracting solution of beet pulp and bagasse as a fermentation medium by mixing with glycerol, and takes Klebsiella pneumoniae, bacillus aryabhattai and Serratia as a fermentation inoculant. On one hand, the preparation method has cheap raw materials, takes the mixed extract of beet pulp and bagasse and glycerol as a culture medium, does not need to additionally add nutrition sources, has mild fermentation conditions, and greatly reduces the production cost. On the other hand, the invention combines Klebsiella pneumoniae, serratia and Bacillus aryaensis, overcomes the problem of low fermentation yield of the existing single strain or engineering strain, and has synergistic effect in improving the yield of 1, 2-propanediol by compounding the three bacteria.
Drawings
FIG. 1 is a graph showing the number of viable bacteria obtained by culturing Klebsiella pneumoniae, serratia and Bacillus aryaensis alone, culturing Klebsiella pneumoniae and Serratia together, and culturing Klebsiella pneumoniae, serratia and Bacillus aryaensis together for 24 hours.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background art, in the prior art, a chemical synthesis method is used for synthesizing 1, 2-propanediol, but the chemical synthesis method uses petrochemical resources as raw materials, so that the environmental pollution is serious; there is an increasing trend to replace traditional fossil raw materials with renewable biomass resources for the production of 1, 2-propanediol. The saccharide and the glycerol are used as main raw materials for synthesizing the 1, 2-propylene glycol by biomass, reports on the synthesis of the 1, 2-propylene glycol by fermenting the saccharide and the glycerol are relatively few, and in the prior art, the problem of low production efficiency and more byproducts exists when a single strain is used. The construction of engineering bacteria is complex, is unfavorable for large-scale popularization and application, and the yield is still low.
Based on this, the present invention provides a method for preparing 1, 2-propanediol by microbial fermentation, and firstly, in terms of raw material selection, in order to replace the conventional petrochemical resources, the inventors first considered saccharides, the sources of which are selected from bagasse and beet pulp, in order to reduce costs and achieve reuse of agricultural byproducts, but found that the yield of 1, 2-propanediol is not high by using a single bagasse-bagasse mixed extract as a fermentation substrate. The inventor tries to ferment and produce 1, 2-propylene glycol by using single glycerol as a fermentation substrate, but experiments show that the yield of 1, 2-propylene glycol is not high by using single glycerol as a fermentation substrate, and experiments show that the yield of 1, 2-propylene glycol can be greatly improved by compounding beet pulp-bagasse mixed extract and glycerol according to a certain proportion.
For the selection of the strain, firstly, the existing reported strains capable of converting glucose or glycerol, including klebsiella, clostridium wedge, anaerobic bacillus pyrolyticus, serratia freundii and the like, are selected, and are combined to verify the effect of the strains on producing 1, 2-propanediol, and finally, the compound of klebsiella pneumoniae and serratia can improve the yield of 1, 2-propanediol, but the yield is still not as high as that of the modified klebsiella. Then, the inventors separately and co-culturing Klebsiella pneumoniae and Serratia respectively, found that the number of living bacteria of Klebsiella pneumoniae and Serratia is high when the number of living bacteria of Serratia is not separately cultured in a co-fermentation medium, and hypothesized that the number of living bacteria of Klebsiella pneumoniae and Serratia is lower than that of living bacteria when separately cultured due to a certain competition relationship between Klebsiella pneumoniae and Serratia in the culture process, and the two compete for nutrients together and generate metabolic substances inhibiting the growth of the other party, so that the number of living bacteria of Klebsiella pneumoniae and Serratia in the co-culture medium is lower than that of living bacteria when separately cultured. Therefore, in order to solve this problem, the inventors thought that if the third bacterium can be added, this bacterium can produce a metabolic substance favorable for Klebsiella pneumoniae and Serratia, and thus can increase the activities of Klebsiella pneumoniae and Serratia, and thus can contribute to an increase in the yield of 1, 2-propanediol. Considering that klebsiella pneumoniae and serratia are gram-negative bacteria, the inventors selected bacillus animalis as a gram-positive bacterium, which has remarkable physiological and biochemical differences from klebsiella pneumoniae and serratia, and bacillus animalis can produce various enzymes such as protease, amylase, lipase, etc., mainly used for decomposing macromolecules such as proteins, starch, cellulose, and fat, converting these macromolecules into small molecules that can be utilized by bacteria, thereby being able to promote the growth of klebsiella pneumoniae and serratia; in addition, it can produce extracellular polysaccharide, which is a viscous substance capable of forming a protective layer, which contributes to the survival of klebsiella pneumoniae and serratia pneumoniae in the environment.
Therefore, the inventor compounds Klebsiella pneumoniae, serratia and Bacillus aryaensis, and discovers that a synergistic effect exists among the three, so that the number of living bacteria of three bacteria in a co-culture medium is higher than that of living bacteria in single culture, and the synergistic effect is achieved in the aspect of improving the yield of 1, 2-propanediol by compounding the three bacteria.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present application, the technical scheme of the present application will be described in detail with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and are commercially available.
Wherein, the strain number of the klebsiella pneumoniae (Klebsiella pneumoniae) used in the invention is CGMCC 1.10617, and the klebsiella pneumoniae is purchased from the common microorganism center of China Committee for culture Collection of microorganisms; serratia sp. With strain number CICC 10689, available from China industry microbiological culture Collection center; the strain of bacillus arvensis (Bacillus aryabhattai) was designated as cic 24574 and was purchased from the chinese industrial microbiological bacterial collection center.
The strain number of Trichoderma reesei (Trichoderma reesei) used in the invention is CGMCC 3.13232, and the Trichoderma reesei is purchased from the common microorganism center of China Committee for culture Collection of microorganisms; pseudomonas putida (Pseudomonas putida) has a strain number of CGMCC 1.4533 and is purchased from the China general microbiological culture Collection center.
Example 1:
(1) Preparation of fermentation medium:
Sugar cane bagasse, beet pulp and water are mixed according to 3g of feed liquid: 2g:50ml of the mixed fermentation liquid is obtained;
The method comprises the steps of (1) inoculating Trichoderma reesei seed liquid and Pseudomonas putida seed liquid into a mixed fermentation liquid, wherein the inoculum size of the Trichoderma reesei seed liquid and the Pseudomonas putida seed liquid is 3% of the volume of the mixed fermentation liquid, and the bacterial concentration of the Trichoderma reesei seed liquid and the Pseudomonas putida seed liquid is 2X 10 8 cfu/ml;
fermenting at 28deg.C for 42 hr, centrifuging to collect supernatant, filtering, and sterilizing to obtain beet pulp-bagasse mixed extractive solution;
adding 40g of glycerol into each liter of beet pulp-bagasse mixed extract, and uniformly mixing to prepare a fermentation medium;
(2) Mixing klebsiella pneumoniae seed liquid, bacillus aryaensis seed liquid and Serratia seed liquid to obtain mixed seed liquid;
In the mixed seed liquid, the volume ratio of Klebsiella pneumoniae seed liquid to Bacillus aryabhattai seed liquid to Serratia seed liquid is 3:2:4, a step of;
In the mixed seed solution, the viable count of Klebsiella pneumoniae is 6 multiplied by 10 8 cfu/ml; the viable count of the bacillus arvensis is 4 multiplied by 10 8 cfu/ml; the viable count of Serratia is 8×10 8 cfu/ml;
(3) Inoculating the mixed seed liquid into a fermentation medium, and fermenting for 72 hours at 28 ℃ to obtain 1, 2-propylene glycol; wherein, the inoculation amount of the mixed seed liquid is 7 percent of the volume of the fermentation medium.
Example 2
(1) Preparation of fermentation medium:
Bagasse, beet pulp and water were mixed according to feed liquid 4g:3g:50ml of the mixed fermentation liquid is obtained;
the method comprises the steps of (1) inoculating trichoderma reesei seed liquid and pseudomonas putida seed liquid into a mixed fermentation liquid, wherein the inoculum size of the trichoderma reesei seed liquid and the pseudomonas putida seed liquid is 4% of the volume of the mixed fermentation liquid, and the bacterial concentration of the trichoderma reesei seed liquid and the pseudomonas putida seed liquid is 2 multiplied by 10 8 cfu/ml;
fermenting at 28deg.C for 42 hr, centrifuging to collect supernatant, filtering, and sterilizing to obtain beet pulp-bagasse mixed extractive solution;
Adding 50g of glycerin into each liter of beet pulp-bagasse mixed extract, and uniformly mixing to prepare a fermentation medium;
(2) Mixing klebsiella pneumoniae seed liquid, bacillus aryaensis seed liquid and Serratia seed liquid to obtain mixed seed liquid;
in the mixed seed liquid, the volume ratio of Klebsiella pneumoniae seed liquid to Bacillus aryabhattai seed liquid to Serratia seed liquid is 4:3:5, a step of;
In the mixed seed solution, the viable count of Klebsiella pneumoniae is 6 multiplied by 10 8 cfu/ml; the viable count of the bacillus arvensis is 4.5X10 8 cfu/ml; the viable count of Serratia is 7.5X10 8 cfu/ml;
(3) Inoculating the mixed seed liquid into a fermentation medium, and fermenting for 72 hours at 28 ℃ to obtain 1, 2-propylene glycol; wherein, the inoculation amount of the mixed seed liquid is 7 percent of the volume of the fermentation medium.
Example 3:
(1) Preparation of fermentation medium:
mixing bagasse, beet pulp and water according to 5g of feed liquid: 4g:50ml of the mixed fermentation liquid is obtained;
The method comprises the steps of (1) inoculating trichoderma reesei seed liquid and pseudomonas putida seed liquid into a mixed fermentation liquid, wherein the inoculum size of the trichoderma reesei seed liquid and the pseudomonas putida seed liquid is 5% of the volume of the mixed fermentation liquid, and the bacterial concentration of the trichoderma reesei seed liquid and the pseudomonas putida seed liquid is 2 multiplied by 10 8 cfu/ml;
fermenting at 28deg.C for 42 hr, centrifuging to collect supernatant, filtering, and sterilizing to obtain beet pulp-bagasse mixed extractive solution;
Adding 50g of glycerin into each liter of beet pulp-bagasse mixed extract, and uniformly mixing to prepare a fermentation medium;
(2) Mixing klebsiella pneumoniae seed liquid, bacillus aryaensis seed liquid and Serratia seed liquid to obtain mixed seed liquid;
in the mixed seed liquid, the volume ratio of Klebsiella pneumoniae seed liquid to Bacillus aryabhattai seed liquid to Serratia seed liquid is 5:4:6, preparing a base material;
In the mixed seed solution, the viable count of Klebsiella pneumoniae is 6 multiplied by 10 8 cfu/ml; the viable count of the bacillus arvensis is 4.8X10 8 cfu/ml; the viable count of Serratia is 7.2X10 8 cfu/ml;
(3) Inoculating the mixed seed liquid into a fermentation medium, and fermenting for 72 hours at 28 ℃ to obtain 1, 2-propylene glycol; wherein, the inoculation amount of the mixed seed liquid is 7 percent of the volume of the fermentation medium.
Comparative example 1:
(1) Preparation of fermentation medium:
Sugar cane bagasse, beet pulp and water are mixed according to 3g of feed liquid: 2g:50ml of the mixed fermentation liquid is obtained;
The method comprises the steps of (1) inoculating Trichoderma reesei seed liquid and Pseudomonas putida seed liquid into a mixed fermentation liquid, wherein the inoculum size of the Trichoderma reesei seed liquid and the Pseudomonas putida seed liquid is 3% of the volume of the mixed fermentation liquid, and the bacterial concentration of the Trichoderma reesei seed liquid and the Pseudomonas putida seed liquid is 2X 10 8 cfu/ml;
Fermenting at 28deg.C for 42 hr, centrifuging to collect supernatant, filtering, and sterilizing to obtain mixed extract of glycerol beet pulp and bagasse as fermentation medium;
(2) Mixing klebsiella pneumoniae seed liquid, bacillus aryaensis seed liquid and Serratia seed liquid to obtain mixed seed liquid;
In the mixed seed liquid, the volume ratio of Klebsiella pneumoniae seed liquid to Bacillus aryabhattai seed liquid to Serratia seed liquid is 3:2:4, a step of;
In the mixed seed solution, the viable count of Klebsiella pneumoniae is 6 multiplied by 10 8 cfu/ml; the viable count of the bacillus arvensis is 4 multiplied by 10 8 cfu/ml; the viable count of Serratia is 8×10 8 cfu/ml;
(3) Inoculating the mixed seed liquid into a fermentation medium, and fermenting for 72 hours at 28 ℃ to obtain 1, 2-propylene glycol; wherein, the inoculation amount of the mixed seed liquid is 7 percent of the volume of the fermentation medium.
The difference between this comparative example and example 1 is that: glycerol was omitted from the fermentation medium.
Comparative example 2:
(1) Adding 40g of glycerol into each liter of water, and uniformly mixing to obtain a fermentation medium;
(2) Mixing klebsiella pneumoniae seed liquid, bacillus aryaensis seed liquid and Serratia seed liquid to obtain mixed seed liquid;
In the mixed seed liquid, the volume ratio of Klebsiella pneumoniae seed liquid to Bacillus aryabhattai seed liquid to Serratia seed liquid is 3:2:4, a step of;
In the mixed seed solution, the viable count of Klebsiella pneumoniae is 6 multiplied by 10 8 cfu/ml; the viable count of the bacillus arvensis is 4 multiplied by 10 8 cfu/ml; the viable count of Serratia is 8×10 8 cfu/ml;
(3) Inoculating the mixed seed liquid into a fermentation medium, and fermenting for 72 hours at 28 ℃ to obtain 1, 2-propylene glycol; wherein, the inoculation amount of the mixed seed liquid is 7 percent of the volume of the fermentation medium.
The difference between this comparative example and example 1 is that: the mixed extract of beet pulp and bagasse is omitted from the fermentation medium.
Comparative example 3:
(1) Preparation of fermentation medium:
Sugar cane bagasse, beet pulp and water are mixed according to 3g of feed liquid: 2g:50ml of the mixed fermentation liquid is obtained;
The method comprises the steps of (1) inoculating Trichoderma reesei seed liquid and Pseudomonas putida seed liquid into a mixed fermentation liquid, wherein the inoculum size of the Trichoderma reesei seed liquid and the Pseudomonas putida seed liquid is 3% of the volume of the mixed fermentation liquid, and the bacterial concentration of the Trichoderma reesei seed liquid and the Pseudomonas putida seed liquid is 2X 10 8 cfu/ml;
fermenting at 28deg.C for 42 hr, centrifuging to collect supernatant, filtering, and sterilizing to obtain beet pulp-bagasse mixed extractive solution;
adding 40g of glycerol into each liter of beet pulp-bagasse mixed extract, and uniformly mixing to prepare a fermentation medium;
(2) Inoculating klebsiella pneumoniae seed liquid into a fermentation medium, and fermenting for 72 hours at 28 ℃ to obtain 1, 2-propanediol; wherein, the inoculation amount of Klebsiella pneumoniae seed liquid is 7% of the volume of the fermentation medium; the viable count of the Klebsiella pneumoniae seed liquid is 1.8X10 9 cfu/ml.
The difference between this comparative example and example 1 is that: the single klebsiella pneumoniae is selected for fermentation to produce the 1, 2-propanediol.
Comparative example 4:
(1) Preparation of fermentation medium:
Sugar cane bagasse, beet pulp and water are mixed according to 3g of feed liquid: 2g:50ml of the mixed fermentation liquid is obtained;
The method comprises the steps of (1) inoculating Trichoderma reesei seed liquid and Pseudomonas putida seed liquid into a mixed fermentation liquid, wherein the inoculum size of the Trichoderma reesei seed liquid and the Pseudomonas putida seed liquid is 3% of the volume of the mixed fermentation liquid, and the bacterial concentration of the Trichoderma reesei seed liquid and the Pseudomonas putida seed liquid is 2X 10 8 cfu/ml;
fermenting at 28deg.C for 42 hr, centrifuging to collect supernatant, filtering, and sterilizing to obtain beet pulp-bagasse mixed extractive solution;
adding 40g of glycerol into each liter of beet pulp-bagasse mixed extract, and uniformly mixing to prepare a fermentation medium;
(2) Inoculating the bacillus arvensis seed solution into a fermentation medium, and fermenting for 72 hours at the temperature of 28 ℃ to obtain 1, 2-propylene glycol; wherein, the inoculation amount of the bacillus arvensis seed liquid is 7 percent of the volume of the fermentation medium; the viable count of the bacillus arvensis seed solution is 1.8X10 9 cfu/ml.
The difference between this comparative example and example 1 is that: and (3) fermenting and producing the 1, 2-propylene glycol by using single bacillus aryabhattai.
Comparative example 5
(1) Preparation of fermentation medium:
Sugar cane bagasse, beet pulp and water are mixed according to 3g of feed liquid: 2g:50ml of the mixed fermentation liquid is obtained;
The method comprises the steps of (1) inoculating Trichoderma reesei seed liquid and Pseudomonas putida seed liquid into a mixed fermentation liquid, wherein the inoculum size of the Trichoderma reesei seed liquid and the Pseudomonas putida seed liquid is 3% of the volume of the mixed fermentation liquid, and the bacterial concentration of the Trichoderma reesei seed liquid and the Pseudomonas putida seed liquid is 2X 10 8 cfu/ml;
fermenting at 28deg.C for 42 hr, centrifuging to collect supernatant, filtering, and sterilizing to obtain beet pulp-bagasse mixed extractive solution;
adding 40g of glycerol into each liter of beet pulp-bagasse mixed extract, and uniformly mixing to prepare a fermentation medium;
(2) Inoculating Serratia seed solution into fermentation medium, fermenting at 28deg.C for 72 hr to obtain 1, 2-propylene glycol; wherein, the inoculation amount of the Serratia seed solution is 7% of the volume of the fermentation medium; the viable count of Serratia is 1.8X10 9 cfu/ml;
The difference between this comparative example and example 1 is that: a single Serratia is selected for fermentation to produce 1, 2-propanediol.
Test example 1:
Separately culturing Klebsiella pneumoniae, serratia and Bacillus aryabhattai in LB liquid culture medium; co-culturing Klebsiella pneumoniae and Serratia; and co-culturing Klebsiella pneumoniae, serratia and Bacillus aryaensis, and counting the number of viable bacteria in a fermentation medium at different time points to verify the synergistic effect of the three strains during co-culture, wherein the method comprises the following steps:
Firstly, activating Klebsiella pneumoniae, serratia and Bacillus aryaensis with LB plates and LB liquid culture media in sequence to obtain Klebsiella pneumoniae seed liquid, bacillus aryaensis seed liquid and Serratia seed liquid, and then culturing respectively:
1. klebsiella pneumoniae, serratia and Bacillus aryaensis were cultured alone:
the seed liquid of Klebsiella pneumoniae (number of viable bacteria: 6×10 8), seed liquid of Bacillus aryabhattai (number of viable bacteria: 4×10 8) and seed liquid of Serratia (number of viable bacteria: 7×10 8) were inoculated into LB plain liquid medium at an inoculum size of 5% (volume fraction), cultured at 30℃for 24 hours, and the number of viable bacteria of the three bacteria was measured by plate counting method every four hours, and the results are shown in FIG. 1.
2. Combination culture one: klebsiella pneumoniae and Serratia are co-cultured:
uniformly mixing klebsiella pneumoniae seed liquid and Serratia seed liquid according to a volume ratio of 1:1 to obtain mixed seed liquid (the number of viable bacteria of the klebsiella pneumoniae seed liquid in the mixed seed liquid is 6×10 8; the number of viable bacteria of the Serratia seed liquid is 7×10 8), inoculating into LB flat liquid medium according to an inoculum size of 5% (volume fraction), culturing at 30deg.C for 24 hours, and measuring the number of viable bacteria of three bacteria by adopting a plate counting method every four hours, wherein the result is shown in figure 1.
3. And (3) combined culture: klebsiella pneumoniae, bacillus aryabhattai and Serratia co-culture
Uniformly mixing Klebsiella pneumoniae seed liquid, bacillus aryaensis seed liquid and Serratia seed liquid according to a volume ratio of 1:1:1 to obtain mixed seed liquid (in the mixed seed liquid, the viable count of the Klebsiella pneumoniae seed liquid is 6×10 8, the viable count of the Bacillus aryasis seed liquid is 4×10 8, the viable count of the Serratia seed liquid is 7×10 8), inoculating into LB flat liquid culture medium according to an inoculum size of 5% (volume fraction), culturing at 30 ℃ for 24 hours, and measuring the viable count of three bacteria by adopting a plate counting method every four hours, wherein the result is shown in figure 1.
As can be seen from FIG. 1, the number of live bacteria of Klebsiella pneumoniae and Serratia are both lower than those when both bacteria are cultured alone by co-culturing Klebsiella pneumoniae and Serratia; however, after the Klebsiella pneumoniae, the Serratia and the Bacillus aryaensis are combined together, the number of the living bacteria of the Klebsiella pneumoniae, the Serratia and the Bacillus aryaensis is obviously improved, which proves that the activity of the Klebsiella pneumoniae and the Serratia during co-culture can be obviously improved by adding the Bacillus aryaensis, and the three bacteria have a synergistic effect.
Test example 2:
The fermentation broths containing 1, 2-propanediol prepared in examples 1 to 3 and comparative examples 1 to 5 were examined for the yield of 1, 2-propanediol by high performance liquid chromatography. The fermentation broth was centrifuged at 14000 r/min for 10: 10 min, and the supernatant was filtered through a 0.22 μm filter and analyzed by HPLC. The column was Rezex ROA 300 mm X7.8 mm (phenomenex Co.), the mobile phase was 5 mmol/L sulfuric acid, the mobile phase flow rate was 0.5 mL/min, the detection instrument was differential detection RID, the temperature of RID was 55deg.C, the column temperature was 60deg.C, and the results are shown in Table 1.
TABLE 1 production of 1, 2-propanediol from different treatment groups
As can be seen from Table 1, the present application uses beet pulp-bagasse mixed extract and glycerin as fermentation substrates, uses Klebsiella pneumoniae, bacillus aryabhattai and Serratia as fermentation strains for preparing 1, 2-propanediol, the yields of 1, 2-propanediol are all above 10g/L, while comparative examples 1-2 are 1, 2-propanediol prepared by fermentation using beet pulp-bagasse mixed extract and glycerin alone as fermentation substrates, the yields of which are significantly lower than those of examples 1-3 of the present application; comparative examples 3 to 5 were prepared by using Klebsiella pneumoniae, bacillus aryabhattai and Serratia alone as fermentation strains for the production of 1, 2-propanediol in a yield of only 2.21g/L at the maximum, which demonstrates that the Klebsiella pneumoniae, bacillus aryabhattai and Serratia combination of the present application have a synergistic effect in improving the yield of 1, 2-propanediol.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (5)
1. A method for preparing 1, 2-propylene glycol by a microbial fermentation method, which is characterized by comprising the following steps:
(1) Mixing klebsiella pneumoniae seed liquid, serratia seed liquid and bacillus aryaensis seed liquid to obtain mixed seed liquid;
(2) The mixed seed liquid is inoculated into a fermentation culture medium consisting of beet pulp-bagasse mixed extract and glycerin, and is fermented for 72 to 80 hours at the temperature of 25 to 30 ℃ to obtain 1, 2-propanediol;
In the step (1), the strain number of the klebsiella pneumoniae is CGMCC 1.10617; the Serratia strain is CICC 10689; the strain number of the bacillus aryasis is CICC 24574;
in the step (2), the fermentation medium is prepared by the following method:
(a) Bagasse, beet pulp and water are mixed according to the feed liquid ratio (3-5) g: (2-4) g:50ml of the mixed fermentation liquid is obtained;
(b) Inoculating Trichoderma reesei seed solution and Pseudomonas putida seed solution into the mixed fermentation liquid, fermenting at 25-30deg.C for 36-48 hr, centrifuging to collect supernatant, filtering, and sterilizing to obtain beet pulp-bagasse mixed extract;
(c) Adding 40-60g of glycerol into each liter of beet pulp-bagasse mixed extract, and uniformly mixing to prepare a fermentation medium;
In the step (b), the strain number of the trichoderma reesei is CGMCC 3.13232; the strain number of the pseudomonas putida is CGMCC 1.4533.
2. The method for preparing 1, 2-propanediol by a microbial fermentation process according to claim 1 wherein the mixed seed liquid is inoculated in an amount of 5% to 10% by volume of fermentation medium; in the mixed seed liquid, the volume ratio of Klebsiella pneumoniae seed liquid, bacillus aryabhattai seed liquid and Serratia seed liquid is (3-5): (2-4): (4-6).
3. The method for preparing 1, 2-propanediol by a microbial fermentation method according to claim 1, wherein the number of viable bacteria of klebsiella pneumoniae is not less than 6 x 10 8 cfu/ml in the mixed seed solution; the viable count of the bacillus arvensis is more than or equal to 4 multiplied by 10 8 cfu/ml; the viable count of Serratia is more than or equal to 7 multiplied by 10 8 cfu/ml.
4. The method for preparing 1, 2-propanediol by microbial fermentation according to claim 1, wherein the seed solution of trichoderma reesei and the seed solution of pseudomonas putida are both inoculated in an amount of 3% -5% by volume of the mixed fermentation broth.
5. The method for preparing 1, 2-propanediol by a microbial fermentation method according to claim 1, wherein the bacterial concentration of the trichoderma reesei seed solution and the pseudomonas putida seed solution is not less than 2x 10 8 cfu/ml.
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