CN109251869B - Klebsiella for efficiently producing 1, 3-propylene glycol by using crude glycerol - Google Patents

Klebsiella for efficiently producing 1, 3-propylene glycol by using crude glycerol Download PDF

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CN109251869B
CN109251869B CN201810640923.1A CN201810640923A CN109251869B CN 109251869 B CN109251869 B CN 109251869B CN 201810640923 A CN201810640923 A CN 201810640923A CN 109251869 B CN109251869 B CN 109251869B
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glycerol
crude glycerol
klebsiella
propanediol
propylene glycol
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马江山
李昌珠
肖志红
刘汝宽
张良波
李培旺
陈景震
张轩
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Hunan Academy of Forestry
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Abstract

The invention discloses a Klebsiella for efficiently utilizing crude glycerol to produce 1, 3-propanediol. Is named asKlebsiellasp.2e, deposited in Chinese microorganismsThe preservation number of the general microbiological center of the culture preservation management committee is CGMCC No. 15520. The invention makes up the defects of difficult microbial growth, low related enzyme activity, low 1, 3-propylene glycol yield and the like caused by the inhibition of crude glycerol impurities in the process of producing the 1, 3-propylene glycol by using crude glycerol by conventional microorganisms. The strain of the inventionKlebsiellasp.2e in a culture medium containing 36g of crude glycerol (the content of glycerol is 69%), after culturing for 12h at 37 ℃ and 170rpm, the consumed glycerol amount reaches 19.44 +/-0.61 g/L, 1, 3-propanediol of 9.83 +/-0.75 g/L can be produced, and the yield reaches 0.62mol of 1, 3-propanediol/mol of glycerol.

Description

Klebsiella for efficiently producing 1, 3-propylene glycol by using crude glycerol
Technical Field
The invention belongs to the technical field of microbial engineering, and particularly relates to a Klebsiella for efficiently producing 1, 3-propanediol by using crude glycerol.
Background
Crude glycerol is a major by-product in the biodiesel industry, with about 1kg of crude glycerol being produced per 10kg of biodiesel produced on average, and about 67% of the crude glycerol globally being derived from biodiesel production statistically about 67% of the crude glycerol globally being derived from biodiesel industrial production. The content of glycerin in the crude glycerin is only 35-50%, and the crude glycerin also contains a large amount of impurities such as alcohols, salts, soaps, heavy metals, pigments, residual fatty acids and the like. The crude glycerol has huge yield and high refining cost, and if the crude glycerol is not timely treated and utilized, the production cost of the biodiesel is not reduced, and the crude glycerol can become a new pollution source. The method directly converts crude glycerol into high-value chemicals such as 1, 3-propylene glycol through microbial fermentation, is a green and efficient utilization way with great prospect at present, and has important practical significance for reducing production cost and environmental pollution in large-scale development of biodiesel industry. However, the most important problem in the process of fermenting crude glycerol by microorganisms is that complex components in the crude glycerol influence secretion expression and action activity of key enzymes, so that the yield of fermentation products is too low, the production intensity is not high, and the effective utilization of the crude glycerol is severely restricted.
At present, crude glycerol is mainly refined into pure glycerol, however, the cost in the refining process is high, and the market price of the glycerol is continuously lowered due to supply and demand, so that a large part of the crude glycerol is discarded. The screening and the utilization of the microorganisms to convert the crude glycerol into the 1, 3-propylene glycol are beneficial to solving the problem of environmental pollution of the crude glycerol, prolonging the industrial chain of the biodiesel and promoting the development of the 1, 3-propylene glycol production industry. At present, little microbial resources capable of efficiently utilizing the crude glycerol to ferment into the 1, 3-propylene glycol are available, and strains with industrial application potential are rarely reported.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the Klebsiella capable of efficiently utilizing the crude glycerol to produce the 1, 3-propanediol, and the Klebsiella can efficiently convert the crude glycerol into the 1, 3-propanediol product, and has great potential in the aspect of industrial application.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
the Klebsiella sp.2e for producing the 1, 3-propanediol by efficiently utilizing the crude glycerol is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 15520.
The strain is preserved by China general microbiological culture Collection center (CGMCC) (the preservation address is No. 3 of Xilu No.1 of Beijing Kogyo-Yang district), the preservation number is CGMCC No.15520, the classification name is Klebsiella sp, and the strain survives after detection in 2018, 3 months and 26 days.
The strain is derived from the soil polluted by the waste residue generated in the production of biodiesel, and is identified as Klebsiella sp, named 2e, by combining the physiological and biochemical characteristics of the strain and the phylogenetic analysis based on the 16S rDNA gene sequence. The conversion rate of the strain Klebsiella sp.2e to crude glycerol and the yield of 1, 3-propanediol are superior to those of other most Klebsiella strains reported at home and abroad, and the invention provides an excellent microbial resource for producing 1, 3-propanediol by utilizing crude glycerol.
The separation, purification and screening processes of the strain Klebsiella sp.2e comprise the following steps: a soil sample near a biodiesel production plant area of the great famous biotechnology available company in North Hunan is suspended in sterile water, is static and is diluted in a gradient manner, and then a proper amount of diluent is inoculated on a separation enrichment medium plate by a coating method and is cultured in a constant temperature incubator at 37 ℃. Then, the grown colonies are picked out and singly inoculated to a new separation enrichment medium plate by a streaking method, and the streaking inoculation culture is repeated until a pure culture of the microorganism is obtained. Inoculating the separated pure bacterial colony into a fermentation medium containing crude glycerol, culturing for 24h at 37 ℃ at 170rpm in a shaking table, and detecting the content of 1, 3-propanediol in a culture solution by adopting gas chromatography, wherein the strain with the highest concentration of 1, 3-propanediol is the bacterial strain for efficiently utilizing the crude glycerol to produce the 1, 3-propanediol.
The separation enrichment culture medium comprises the following components: 25.0g/L of glycerol, 10.0g/L of peptone, 10.0g/L of yeast extract, 5.0g/L of sodium acetate, K2HPO42.0g/L,MgSO40.2g/L,MnSO40.02g/L,CaCl20.1g/L and pH of 7.0-7.2. The composition of the crude glycerol fermentation medium is as follows: 36.0g/L of crude glycerol (glycerol content 69%), 10.0g/L of yeast extract, (NH)4)2SO42.0g/L,K2HPO42.0g/L,KH2PO42.0g/L,MgSO40.2g/L,CaCl20.1g/L,MnSO40.02g/L,FeSO40.05g/L, pH 7.0-7.2, and nitrogen gas is filled before sterilization to remove oxygen in the culture medium.
Compared with the prior art, the invention has the following beneficial effects: the invention makes up the defects of difficult microbial growth, low related enzyme activity, low 1, 3-propylene glycol yield and the like caused by the inhibition of crude glycerol impurities in the process of producing the 1, 3-propylene glycol by using crude glycerol by conventional microorganisms. After the strain Klebsiella sp.2e is cultured in a culture medium containing 36g of crude glycerol (the content of the glycerol is 69%) for 12 hours at 37 ℃ and 170rpm, the consumed glycerol amount reaches 19.44 +/-0.61 g/L, 1, 3-propanediol of 9.83 +/-0.75 g/L can be generated, and the yield reaches 0.62mol of 1, 3-propanediol/mol of glycerol.
Drawings
FIG. 1: a colony morphology of the strain of the invention;
FIG. 2: the strain of the invention is based on a phylogenetic tree diagram of a 16S rDNA sequence;
FIG. 3: the strain growth chart of the invention in the course of culturing in a crude glycerol culture medium;
FIG. 4: the activity change diagram of the glycerol dehydratase, the 1, 3-propanediol oxidoreductase and the glycerol dehydrogenase of the strain in the process of culturing the crude glycerol culture medium is shown;
FIG. 5: the consumption of the glycerol and the content of the 1, 3-propylene glycol of the strain are shown after the strain is cultured in a crude glycerol culture medium for 12 hours.
Detailed Description
Example 1: species identification of strains
1. Analysis of colony morphology
And (3) streaking and inoculating the single colony of the strain to a nutrient agar culture medium (10 g/L of peptone, 3g/L of beef extract, 5g/L of NaCl, 15g/L of agar and 7.0-7.2 of pH), then inversely placing the plate in a 30 ℃ constant temperature incubator, culturing for 18-24 h, and observing the form of the grown colony. FIG. 1 shows that the bacterial colony of the strain is light yellow, moist, small, round, convex, neat in edge and smooth in surface. Is a gram-negative bacterium.
The characteristics are substantially as follows: colony characteristics: the bacterial colony is yellowish, the surface is rough, wrinkled and opaque, round or irregular, and the edge is irregular; gram staining is carried out on the strain by using a kit, thalli observation is carried out under an oil lens, and a picture is taken, wherein a strain is shown in a figure 2 to be rod-shaped, is bluish purple and is a gram-positive bacterium.
2. Physiological and biochemical experimental analysis of strains
The strain is respectively subjected to physiological and biochemical experiment detection such as citric acid utilization, nitrate utilization, hydrogen sulfide production, arginine hydrolysis, V-P experiment and the like, the strain can utilize citrate and nitrate, produce hydrogen sulfide, hydrolyze arginine, produce indole, ferment sucrose, lactose, mannose and the like, and is negative in the experiment of not producing oxidase and methyl red and the like, the physiological and biochemical characteristics are basically consistent with those of Klebsiella, and the experiment results are shown in Table 1.
TABLE 1 Klebsiellas.2e physiological and biochemical experiment test results
Figure BDA0001702365710000031
3. 16S rDNA analysis of strains
The bacterial genome DNA extraction kit provided by Tiangen Biochemical technology Co., Ltd is adopted to extract the genome DNA of the strain, the 16S rDNA sequence segment of the strain is amplified by utilizing the bacterial 16S rDNA universal primer through PCR reaction by taking the extracted genome DNA as a template, and the amplified segment is sequenced by Shanghai biological engineering Co., Ltd (the result is shown in the nucleotide sequence in the attached table). BLAST comparison analysis of the 16S rDNA sequence of the sequenced strain with the nucleic acid database of NCBI, construction of phylogenetic tree (FIG. 2) by MEGA 5.1 software based on the proximity method
The bacteria is identified as Klebsiella by combining the colony morphological characteristics, physiological and biochemical experiments and 16S rDNA sequence analysis experiment results.
Example 2: growth characteristics of strains in crude glycerol fermentation process
The strain liquid cultured to logarithmic phase is inoculated into a crude glycerol fermentation medium in an inoculation amount of 5% (v/v), cultured for 12h at 37 ℃ and 170rpm, and sampled every 2h for thallus growth analysis. The thallus growth analysis adopts an ultraviolet spectrophotometer to detect the light absorption value (OD600) of the sample solution at 600nm, and the result is shown in figure 3.
Example 3: enzymatic activity characteristics related to 1, 3-propanediol synthesis in crude glycerol fermentation process of strain
Inoculating the strain liquid cultured to logarithmic phase into crude glycerol fermentation medium at 5% (v/v), and culturing at 37 deg.C and 170rpmCulturing for 12h, and sampling every 2h for detecting the activities of glycerol dehydratase, 1, 3-propanediol oxidoreductase and glycerol dehydrogenase. After a sample for enzyme activity detection is centrifuged at 8000rpm for 5min, thalli precipitates are washed twice by dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (0.02M, pH 7.2) and suspended, suspended thalli are crushed by an ultrasonic crusher and then centrifuged at 12000rpm and 4 ℃ for 10min, and supernate is stored for subsequent enzyme activity detection. The glycerol dehydratase was determined as follows: the following reagents were mixed in a final concentration in a total reaction system of 1.0 ml: 0.2 mol/L1, 2-propylene glycol, 0.05mol/L KCl, 0.035mol/L potassium phosphate buffer (pH 8.0), 15. mu. mol/L CoB12 and a proper amount of diluted enzyme solution. The reaction system was mixed well and incubated at 37 ℃ for 10min, then 1.0mL of 0.1M potassium citrate buffer (pH 3.6) and 0.5mL of 0.1% MBTH were added, incubated at 37 ℃ for 15min, then 1.0mL of water was added, and the change in absorbance was detected at 305 nm. Propionaldehyde had a molar absorption coefficient at 305nm of 13.3X 103mol/L-1 cm-1. 1 enzyme activity unit is defined as the amount of enzyme required to produce 1. mu. mol propionaldehyde per minute. The glycerol dehydrogenase was determined as follows: in 1.5ml of the total reaction system, 0.03mol/L (NH) of the following reagent was mixed at the final concentration4)2SO40.2mol/L glycerol, 0.002mol/L NAD, 0.001mmol/L Fe (NH)4)2(SO4)20.1mol/L potassium carbonate buffer solution and a proper amount of diluted enzyme solution. And (3) starting the reaction after adding enzyme liquid into the reaction system, and monitoring and recording the change of the light absorption value at 340nm within 3 min. 1 enzyme activity unit is defined as the amount of enzyme required to reduce 1. mu. mol glycerol per minute. The method for measuring 1, 3-propanediol oxidoreductase is as follows: in 1.5ml of the total reaction system, 0.03mol/L (NH) of the following reagent was mixed at the final concentration4)2SO40.1 mol/L1, 3-propanediol, 0.002mol/L NAD, 0.001mmol/L Fe (NH)4)2(SO4)20.1mol/L potassium carbonate buffer solution and a proper amount of diluted enzyme solution. And (3) starting the reaction after adding enzyme liquid into the reaction system, and monitoring and recording the change of the light absorption value at 340nm within 3 min. 1 enzyme activity unit is defined as the amount of enzyme required to reduce 1. mu. mol of 1, 3-propanediol per minute. The results are shown in FIG. 4.
Example 4: consumption of glycerol and yield of 1, 3-propanediol after crude glycerol fermentation by strain
Inoculating the bacterial liquid cultured to logarithmic phase into a crude glycerol fermentation culture medium in an inoculation amount of 5% (v/v), culturing at 37 ℃ and 170rpm for 12h, centrifuging the culture liquid at 8000rpm for 5min, discarding the precipitate, and filtering the supernatant with a 0.22-micron filter membrane for detecting the concentrations of glycerol and 1, 3-propanediol. The concentration of glycerol and 1, 3-propanediol was measured by HPLC using a differential refractometer equipped with an Aminex HPX-87H column (300 nm. times.7.8 mm) according to the following procedure: the sample loading was 10. mu.L, the mobile phase was 0.005mol/L sulfuric acid, the flow rate was 0.5mL/min, and the column temperature was 60 ℃. The results are shown in FIG. 5. After 12 hours of fermentation, the consumed glycerol amount reaches 19.44 +/-0.61 g/L, 1, 3-propylene glycol of 9.83 +/-0.75 g/L can be produced, and the yield reaches 0.62mol of 1, 3-propylene glycol/mol of glycerol.

Claims (2)

1. Klebsiella for producing 1, 3-propanediol by using crude glycerol, which is named asKlebsiellasp, 2e, preserved in the China general microbiological culture Collection center with the preservation number of CGMCC 15520.
2. Use of the strain according to claim 1 for the production of 1, 3-propanediol from crude glycerol, wherein the glycerol content of said crude glycerol is 69%.
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