CN105567588A - Microorganism and method for producing butanol by using microorganism - Google Patents
Microorganism and method for producing butanol by using microorganism Download PDFInfo
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- CN105567588A CN105567588A CN201511004392.XA CN201511004392A CN105567588A CN 105567588 A CN105567588 A CN 105567588A CN 201511004392 A CN201511004392 A CN 201511004392A CN 105567588 A CN105567588 A CN 105567588A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
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Abstract
The invention provides a microorganism and a method for producing butanol by using the microorganism. The microorganism is clostridium sp., is preserved on December 02, 2015 in China General Microbiological Culture Collection Center, and has the preservation number of CGMCC NO.11771. The butanol yield of the microorganism is relatively high.
Description
Technical field
The present invention relates to biological chemical field.Particularly, the present invention relates to microorganism and utilize the method for microorganisms producing butanols.
Background technology
Butanols is good oil replacement fuel and important industrial chemicals, is widely used in the aspects such as food, medicine, plastics, printing and dyeing.Compared with ethanol, it is high that butanols has energy density, and corrodibility is low, can arbitrary proportion and gasoline dissolve each other, and has and transform the advantage that directly can utilize butanols without the need to carrying out existing engine and oil pipeline, thus more with potential applications.
But, current microorganism and utilize the method for microorganisms producing butanols still to have much room for improvement.
Summary of the invention
The present invention is intended at least to solve one of technical problem existed in prior art.For this reason, one object of the present invention is a kind of provide microorganism and utilize microorganisms producing butanols method.The butanols output of this microorganism is higher.
In a first aspect of the present invention, the present invention proposes a kind of microorganism.According to embodiments of the invention, described microorganism is clostridium (Clostridiumsp.), China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on December 02nd, 2015, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, deposit number is CGMCCNO.11771.
In a second aspect of the present invention, the present invention proposes a kind of method utilizing microorganisms producing butanols.According to embodiments of the invention, described method comprises: (1) utilizes seed culture medium to carry out activation culture process to previously described microorganism, obtains seed liquor; And (2) utilize fermention medium to carry out fermentation culture process to described seed liquor, to obtain butanols.Utilize higher according to the butanols output utilizing the method for microorganisms producing butanols to obtain of the embodiment of the present invention.
According to embodiments of the invention, the above-mentioned method of microorganisms producing butanols that utilizes can also have following additional technical feature:
According to embodiments of the invention, described seed culture medium is maize powder medium.Thus, utilize according to the butanols output utilizing the method for microorganisms producing butanols to obtain of the embodiment of the present invention higher.
According to embodiments of the invention, based on the cumulative volume of described fermention medium, described fermention medium comprises: the damping fluid of the glucose of 30 ~ 80g/L, the yeast powder of 1g/L, 1 volume % and the nutritive medium of 1 volume %, described damping fluid comprises: dipotassium hydrogen phosphate 50g/L, potassium primary phosphate 50g/L and ammonium acetate 220g/L, and described nutritive medium comprises: 0.1g/L Para-Aminobenzoic, 0.1g/L VitB1,0.001g/L vitamin H, 20g/L magnesium sulfate, 10g/L manganous sulfate, 1g/L ferrous sulfate and 1g/L sodium-chlor.Thus, utilize according to the butanols output utilizing the method for microorganisms producing butanols to obtain of the embodiment of the present invention higher.
According to embodiments of the invention, described microorganism is inoculated in described fermention medium according to 5 ~ 10 volume %.Thus, utilize according to the butanols output utilizing the method for microorganisms producing butanols to obtain of the embodiment of the present invention higher.
According to embodiments of the invention, described activation culture process is 37 DEG C of Anaerobic culturel 24 ~ 48 hours.Thus, utilize according to the butanols output utilizing the method for microorganisms producing butanols to obtain of the embodiment of the present invention higher.
According to embodiments of the invention, described fermentation culture process is 37 DEG C of Anaerobic culturel 48 ~ 96 hours.Thus, utilize according to the butanols output utilizing the method for microorganisms producing butanols to obtain of the embodiment of the present invention higher.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from accompanying drawing below combining to the description of embodiment, wherein:
Fig. 1 shows the schematic diagram of component and content in tunning according to an embodiment of the invention;
Fig. 2 to show in the fermented liquid of initial strains according to an embodiment of the invention component concentration with fermentation time change curve;
Fig. 3 to show in the fermented liquid of G1 according to an embodiment of the invention component concentration with fermentation time change curve;
Fig. 4 to show in the fermented liquid of G2 according to an embodiment of the invention component concentration with fermentation time change curve; And
Fig. 5 to show in the fermented liquid of C1 according to an embodiment of the invention component concentration with fermentation time change curve.
Embodiment
Embodiments of the invention are described below in detail.Embodiment described below is exemplary, only for explaining the present invention, and can not be interpreted as limitation of the present invention.
In a first aspect of the present invention, the present invention proposes a kind of microorganism.According to embodiments of the invention, microorganism is clostridium (Clostridiumsp.), China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on December 02nd, 2015, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, deposit number is CGMCCNO.11771.
For convenience of understanding, be described in detail to the acquisition pattern of microorganism below.
The present invention carries out plasma body mutagenesis process by the clostridium Clostridiumsp. be separated this room as initial strains, obtain mutagenic strain, and carry out four-wheel genome rearrangement by screening the 2 strain forward mutants obtained, finally screening obtains the microorganism of high yield butanols.Wherein, this initial strains clostridium Clostridiumsp. was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 08 26th, 2013, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, deposit number is CGMCCNO.8071.
Particularly,
(1) plasma body mutagenesis process is carried out to initial strains.
(1-1) initial strains is carried out activation culture, obtain seed liquor.
(1-2) seed liquor is carried out fermentation culture, as the OD of bacterium liquid
600when being about 2, collecting thalline and make bacteria suspension.Work as OD
600when being about 2, thalline is just in thalli growth logarithmic phase, and cell viability is vigorous, and Mutagenic Effect is good.
(1-3) plasma body mutagenesis process is carried out to bacteria suspension, and the bacterium liquid obtained is carried out recovery process, then the bacterium liquid after recovery is coated in the neutral red culture containing 1.5 % by weight butanols and grow, select 5 strain bacterial strains as follow-up test bacterial strain, be denoted as G1 ~ G5.
Using the neutral red culture containing high-concentration butanol as screening culture medium, to obtain, to high-concentration butanol, there is resistant strain, with the high-concentration butanol making high-concentration butanol resistant strains can tolerate own metabolism during the fermentation, thus reach the object of high yield butanols.
(1-4) initial strains, G1 ~ G5 are carried out fermentation culture, measure the content of ethanol, acetone and butanols in tunning, compared with initial strains, the butanol content that G1 and G2 obtains is higher, as forward mutant.
(2) genome rearrangement is carried out by screening the forward mutant obtained.
(2-1) thalline of G1 and G2 forward mutant is collected respectively
(2-2) protoplastis of G1 and G2 is prepared respectively
Utilize N,O-Diacetylmuramidase to carry out enzymolysis processing to the thalline that step (2-1) obtains, obtain protoplastis.
(2-3) induced fusion
Get isopyknic G1 and G2 protoplastis to mix, and obtained mixture is carried out induced fusion process.
(2-4) protoplast regeneration
Get induced fusion process product stationary incubation, to make protoplast regeneration, and the mixed solution after regeneration is coated on RCM solid medium, carry out Anaerobic culturel, after growing to single bacterium colony, choose on neutral red culture that single bacterium colony lines containing 2 % by weight butanols, carry out Anaerobic culturel, after growing to bacterium colony, picking individual colonies carries out liquid fermentation and culture, measure butanol content in fermented liquid, obtain the bacterial strain of 5 strain butanols output increased.
(2-5) superior strain repeating step (2-2) ~ (2-4) three times of obtaining of step (2-4), by screening, obtains the clostridium of high yield butanols.
In a second aspect of the present invention, the present invention proposes the method utilizing microorganisms producing butanols.According to embodiments of the invention, the method comprises: (1) utilizes seed culture medium to carry out activation culture process to previously described microorganism, obtains seed liquor; And (2) utilize fermention medium to carry out fermentation culture process to described seed liquor, to obtain butanols.
According to embodiments of the invention, seed culture medium is maize powder medium.Thus, the butanols output utilizing the method for the microorganisms producing butanols of the embodiment of the present invention to obtain is higher.
According to embodiments of the invention, based on the cumulative volume of described fermention medium, described fermention medium comprises the nutritive medium of the glucose of 30 ~ 80g/L, the yeast powder of 1g/L, the damping fluid of 1 volume % and 1 volume %, described damping fluid comprises dipotassium hydrogen phosphate 50g/L, potassium primary phosphate 50g/L and ammonium acetate 220g/L, and described nutritive medium comprises 0.1g/L Para-Aminobenzoic, 0.1g/L VitB1,0.001g/L vitamin H, 20g/L magnesium sulfate, 10g/L manganous sulfate, 1g/L ferrous sulfate and 1g/L sodium-chlor.Thus, utilize according to the butanols output utilizing the method for microorganisms producing butanols to obtain of the embodiment of the present invention higher.
According to embodiments of the invention, microorganism is inoculated in described fermention medium according to 5 ~ 10 volume %.Thus, utilize according to the butanols output utilizing the method for microorganisms producing butanols to obtain of the embodiment of the present invention higher.
According to embodiments of the invention, activation culture process is 37 DEG C of Anaerobic culturel 24 ~ 48 hours.Thus, utilize according to the butanols output utilizing the method for microorganisms producing butanols to obtain of the embodiment of the present invention higher.
According to embodiments of the invention, fermentation culture process is 37 DEG C of Anaerobic culturel 48 ~ 96 hours.Thus, utilize according to the butanols output utilizing the method for microorganisms producing butanols to obtain of the embodiment of the present invention higher.
Below in conjunction with embodiment, the solution of the present invention is made an explanation.It will be understood to those of skill in the art that the following examples only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
The ARTP plasma body mutagenesis instrument of chemical industry system of Tsing-Hua University is adopted to carry out mutagenic treatment.
Seed culture medium: 6g Semen Maydis powder is mixed with 80mL water and boils 15min, and obtained mixture water is settled to 100ml, 115 DEG C of sterilizing 15min, cooling.
RCM substratum: yeast leaching powder 3g/L, Tryptones 10g/L, extractum carnis 10g/L, Zulkovsky starch 1g/L, glucose 5g/L, NaCl5g/L, NaAc3g/L, Cys 0.5g/L, 12.5mMMgCl
2, 12.5mMCaCl
2, 10g/L caseinhydrolysate and water.
Neutral red culture: comprise glucose 40g/L, Tryptones 6g/L, yeast extract paste 2g/L, extractum carnis 2g/L, ammonium acetate 3g/L, KH
2pO
40.5g/L, toluylene red 0.2g/L, MgSO
47H
2o0.2g/L, FeSO
47H
2o0.02g/L and agar powder 25g/L, pH6.2.
CPB substratum: get yeast leaching powder 3g/L, Tryptones 10g/L, extractum carnis 10g/L, Zulkovsky starch 1g/L, glucose 5g/L, NaCl5g/L, NaAc3g/L, Cys 0.5g/L, 500mM sucrose, 25mMMgCl
2and 25mMCaCl
2, pH8.5.
Fermention medium: comprise the glucose of 30 ~ 80g/L, the yeast powder of 1g/L, the damping fluid of 1 volume %, the nutritive medium of 1 volume %.Damping fluid comprises: dipotassium hydrogen phosphate 50g/L, potassium primary phosphate 50g/L and ammonium acetate 220g/L, and nutritive medium comprises: 0.1g/L Para-Aminobenzoic, 0.1g/L VitB1,0.001g/L vitamin H, 20g/L magnesium sulfate, 10g/L manganous sulfate, 1g/L ferrous sulfate and 1g/L sodium-chlor.
Utilize the content of conventional gas phase chromatography determination butanols, ethanol and acetone.
Embodiment 1
Initial strains: clostridium Clostridiumsp. (be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 08 26th, 2013, deposit number is CGMCCNO.8071), is separated by this room and preserves.
(1) plasma body mutagenesis process
(1-1) initial strains be stored in glycerine pipe is seeded in seed culture medium, Anaerobic culturel 48h in 37 DEG C of thermostat containers.
(1-2) the bacterium liquid that step (1-1) obtains being forwarded to glucose concn is in the fermention medium of 30g/L, and Anaerobic culturel in 37 DEG C of thermostat containers, as the OD of bacterium liquid
600when being about 2, be just in thalli growth logarithmic phase, cell viability is vigorous.Get 5mL bacterium liquid, after centrifugal, abandon supernatant, with stroke-physiological saline solution somatic cells is washed, resuspended, make bacteria suspension.
(1-3) draw 10 μ L bacteria suspensions, drip metal slide glass central authorities after sterilization, then metal slide glass is placed in ARTP plasma body mutagenesis instrument and carries out plasma body mutagenesis, mutation time is 10s, and mutagenesis power is 100w.
(1-4) 1 milliliter of RCM substratum is added in the bacterium liquid after the mutagenesis obtained to step (1-3), be placed in 37 DEG C of water bath with thermostatic control recovery 2h, the bacterium liquid got after 20 μ L recoveries is coated on RCM substratum, is placed in 37 DEG C of anaerobic box constant temperature culture 4d, to recover.Treat substratum to grow new single bacterium colony, picking individual colonies coats the neutral red culture containing 1.5 % by weight butanols, to obtain, to high-concentration butanol, there is resistant strain, thus make high-concentration butanol resistant strains can tolerate the high-concentration butanol of own metabolism during the fermentation, thus reach the object of high yield butanols.Through screening, obtain the higher and enduring high-concentration butanols bacterial strain of 5 strain butanols output, called after G1-G5, as subsequent experimental bacterial strain.
(1-5) initial strains, G1 ~ G5 being inoculated in respectively glucose concn is in the fermention medium of 80g/L, 37 DEG C of Anaerobic culturel 96 hours, measure the content of ethanol, acetone and butanols in tunning, detected result as shown in figures 1-4, can find out, the butanol concentration that the butanol concentration that initial strains obtains is 12.59g/L, G1, G2 obtains is respectively 13.68,13.42g/L, the two comparatively initial strains improve 8.6%, 6.5% respectively.Compared with initial strains, the butanol content that G1 and G2 obtains is higher, as forward mutant.
(2) genome rearrangement
(2-1) thalline of G1 and G2 forward mutant is collected respectively
Be seeded to by forward mutant in seed culture medium, 37 DEG C of Anaerobic culturel 24 hours, then get 5mL bacterium liquid, the centrifugal 10min of 3000r/min, collect thalline.Thalline is suspended in CPB, centrifugal, collect thalline.So resuspended 2 times, after centrifugal, thalline is resuspended in 0.5mLCPB.
(2-2) protoplastis of G1 and G2 is prepared respectively
In the bacterium liquid that step (2-1) obtains, add 20mg/L N,O-Diacetylmuramidase, 35 DEG C of water bath heat preservation process, when more than 90% cell englobement protoplastis, by bacterium liquid in the centrifugal 5min of 12000r/min, abandon supernatant liquor, obtain protoplastis.Wash protoplastis with CPB to dezymotize, then the protoplastis obtained is suspended in 0.5mLCPB solution.
(2-3) induced fusion
Get isopyknic G1 and G2 protoplastis to mix, centrifugal and be resuspended in the CPB of 0.1mL.Add the 50%PEG6000 of 0.9mL, incubated at room 30min induced fusion.
(2-4) protoplast regeneration
5mlCPB is added in induced fusion product, centrifugal, collecting precipitation thing, and by the resuspended precipitation of CPB containing 0.1mol/LN-acetylglucosamine, hatch 4.5h for 34 DEG C, protoplastis is recovered.Then obtained mixture is coated on RCM solid medium, 37 DEG C of Anaerobic culturel 4d, after growing single bacterium colony, choose on neutral red culture that single bacterium colony lines containing 2 % by weight butanols, 37 DEG C of Anaerobic culturel 4d, after growing to bacterium colony, picking individual colonies is inoculated in fermention medium, 37 DEG C of Anaerobic culturel 96 hours, measure the concentration of butanols, acetone and ethanol, obtain the bacterial strain that 5 plant heights produce butanols.
(2-5) the superior strain repeating step (2-2) step (2-4) obtained ~ (2-4) three times, obtain the Microbial biomass C 1 of high yield butanols, result is as shown in table 1.Can find out, the butanols output that fermentation initial strains obtains is the butanols output of 12.09g/L, C1 bacterial strain is 16.32g/L.C1 bacterial strain is clostridium (Clostridiumsp.), and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 02nd, 2015, deposit number is CGMCCNO.11771.
The output of table 1 butanols, ethanol and acetone
Embodiment 2
In this embodiment, butanols is produced according to the following step:
(1) by C1 inoculation in seed culture medium, in 37 DEG C of constant incubators, Anaerobic culturel 24h, obtains seed liquor;
(2) seed liquor being inoculated in glucose content according to the inoculum size of 7% (v/v) is in the fermention medium of 70g/L, and in 37 DEG C of constant incubators, Anaerobic culturel 60 hours, obtains tunning.
As shown in Figure 5, the output of butanols is 16.32g/L to the growth curve of C1 bacterial strain.
In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not must for be identical embodiment or example.And the specific features of description, structure, material or feature can combine in one or more embodiment in office or example in an appropriate manner.In addition, when not conflicting, the feature of the different embodiment described in this specification sheets or example and different embodiment or example can carry out combining and combining by those skilled in the art.
Although illustrate and describe embodiments of the invention above, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, and those of ordinary skill in the art can change above-described embodiment within the scope of the invention, revises, replace and modification.
Claims (7)
1. a microorganism, it is clostridium (Clostridiumsp.), and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 02nd, 2015, deposit number is CGMCCNO.11771.
2. utilize a method for the microorganisms producing butanols described in claim 1, it is characterized in that, comprising:
(1) utilize seed culture medium to carry out activation culture process to microorganism according to claim 1, obtain seed liquor; And
(2) fermention medium is utilized to carry out fermentation culture process to described seed liquor, to obtain butanols.
3. method according to claim 2, is characterized in that, described seed culture medium is maize powder medium.
4. method according to claim 2, is characterized in that, based on the cumulative volume of described fermention medium,
Described fermention medium comprises: the damping fluid of the glucose of 30 ~ 80g/L, the yeast powder of 1g/L, 1 volume % and the nutritive medium of 1 volume %,
Described damping fluid comprises: dipotassium hydrogen phosphate 50g/L, potassium primary phosphate 50g/L and ammonium acetate 220g/L,
Described nutritive medium comprises: 0.1g/L Para-Aminobenzoic, 0.1g/L VitB1,0.001g/L vitamin H, 20g/L magnesium sulfate, 10g/L manganous sulfate, 1g/L ferrous sulfate and 1g/L sodium-chlor.
5. method according to claim 2, is characterized in that, described microorganism is inoculated in described fermention medium according to 5 ~ 10 volume %.
6. method according to claim 2, is characterized in that, described activation culture process is 37 DEG C of Anaerobic culturel 24 ~ 48 hours.
7. method according to claim 2, is characterized in that, described fermentation culture process is 37 DEG C of Anaerobic culturel 48 ~ 96 hours.
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| CN112626062A (en) * | 2020-12-25 | 2021-04-09 | 成都雅途生物技术有限公司 | Gene recombination iterative composite mutation breeding method for high-yield A40926 strain |
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| 麦帅等: "等离子体诱变技术筛选高产丁醇菌株的研究", 《2015年中国化工学会年会论文集》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112626062A (en) * | 2020-12-25 | 2021-04-09 | 成都雅途生物技术有限公司 | Gene recombination iterative composite mutation breeding method for high-yield A40926 strain |
| CN112626062B (en) * | 2020-12-25 | 2023-01-31 | 成都雅途生物技术有限公司 | Gene recombination iterative composite mutation breeding method for high-yield A40926 strain |
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