CN108220187A - A kind of zymomonas mobilis mutant strain for being resistant to low ph value and its application - Google Patents
A kind of zymomonas mobilis mutant strain for being resistant to low ph value and its application Download PDFInfo
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- CN108220187A CN108220187A CN201711437346.8A CN201711437346A CN108220187A CN 108220187 A CN108220187 A CN 108220187A CN 201711437346 A CN201711437346 A CN 201711437346A CN 108220187 A CN108220187 A CN 108220187A
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
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Abstract
The invention discloses a kind of zymomonas mobilis mutant strain for being resistant to low ph value and its application, which is named as PH1 29, and preserving number is GDMCC 60260；The bacterial strain after normal-temperature plasma mutagenesis, on the culture medium of low pH repeated screening obtain.The bacterial strain not only may insure normal growth under low ph conditions, ensure good fermentation efficiency, and since low ph conditions can eliminate pollution, can also remove the autoclave sterilization of fermentation process from, control production cost, improve fermentation efficiency.
Description
Technical field
The invention belongs to microorganism fields, and in particular to it is a kind of be resistant to low ph value zymomonas mobilis mutant strain and its
Using, can be applied in any not autoclaved open fermentation environment produce alcohol fuel biobased products purposes.
Background technology
In modern biotechnology fermentation industry, high-temperature sterilization is important and joint a link, concerning fermentation costs and production
Product safety, therefore significant.Most of microbe all cannot well be grown under low ph conditions, therefore it is to reduce to reduce pH
The strategy of living contaminants risk.However the pH tolerances of most of fermentation bacterial strains itself are not also high.If utilize sudden change means
The fermentation strain of screening tolerance low ph conditions, not only may insure fermentation efficiency, but also since low ph conditions can eliminate pollution,
One of method of autoclave sterilization and control production cost of fermentation process can also be removed from.
Zymomonas mobilis (Zymomonas mobislis) is the excellent species of producing and ethanol, in recent years in renewable combustion
It is received significant attention in material ethyl alcohol research and production.However it can be given birth in the environment of pH=4 in zymomonas mobilis at present
It is long, but growth poor-performing, fermentation industry can not be normally used for.Therefore for alcohol fuel fermentation system, low ph conditions pair
In the growth of fermentation strain be still a huge challenge.Therefore this patent creates a plant mutant bacterial strain, can be in pH=4
With normal fermentation producing and ethanol in the low ph conditions of pH=3.5, the process is without autoclave sterilization, and in the species still at present
No other reports reach the level.
Invention content
In view of the deficiencies of the prior art, the invention reside in propose a kind of zymomonas mobilis mutant strain for being resistant to low ph value
And its application.
The present invention provides a kind of zymomonas mobilis mutant strain for being resistant to low ph value, strain classification is named as movement
Fermentation single cell bacterium PH1-29, preserving number are：GDMCC 60260 was deposited in Guangdong Province microorganism fungus kind on October 25th, 2017
Collection.
To achieve the above object, the technical solution adopted in the present invention is：
1) irradiation mutagenesis is carried out to zymomonas mobilis thalline by normal-temperature plasma mutation breeding technologies, first will
Starting strain ZM4 carries out activation culture, and cultivation temperature is 30 DEG C, incubation time 16h；
2) overnight culture (10 is taken6~108A cell), 5~10min is centrifuged at 4 DEG C, rotating speed 3000rpm uses physiology
Salt is washed thalline and is resuspended in 1mL physiological saline；It takes 10 μ L that cell is resuspended and is placed in 15~120s of ARTP mutation breeding instrument mutagenesis；
3) renewal cultivation 16h is resuspended in the cell after irradiation mutagenesis, is coated training on the solid medium of pH=3.5 or 4
It supports；
4) all bacterium colonies are selected to be screened on the culture medium of pH=3.5, are repeated at least three times.
Specifically, the medium ion carrier purity of step 2) is 99.999%, gas flow is 10~20 standard throughputs, electric
Press 100~120V, 22 DEG C of plasma radiation temperature；
Specifically, the solid medium used in step 2) and step 3) includes：50g/L glucose, 2g/L biphosphates
Potassium, 10g/L yeast extracts, it is 3.5-4.0 to adjust medium pH using sulfuric acid solution.
The present invention also provides zymomonas mobilis mutant strains as described above not to carry out autoclaved open hair
Producing the purposes of process of alcohol products in ferment environment.
In order to reach object above, the technical solution adopted in the present invention is：
(1) fermentation medium is prepared；
(2) zymomonas mobilis PH1-29 is inoculated on the fermentation medium, carries out fermented and cultured；
(3) ethyl alcohol in separation and fermentation system.
Specifically, the fermentation process of fermentative production of ethanol is all or part of under the yeasting of pH=3.5-4.0
It carries out.
Specifically, the formula of culture medium includes：50g/L glucose, 2g/L potassium dihydrogen phosphates, 10g/L yeast extracts, profit
The pH that culture medium is adjusted with sulfuric acid solution is 3.5-4.0；
Specifically, the inoculum concentration of inoculation fermentation bacterial strain is 10%, fermentation time is 20~90h.
It is worth noting that, under low ph value fermentation system, common bacterial strain has been unable to normal growth, but mutant strain
Can be with normal growth, therefore the pH value for reducing system during the fermentation can both ensure the normal growth of mutant strain PH1-29
And efficient growth ethyl alcohol, while the growth of other bacteriums can also be inhibited, so as to reduce the high pressure in conventional fermentation process
High-temperature sterilization link reduces alcohol fermentation production cost, improves fermentation efficiency.
The beneficial effects of the invention are as follows：
The mutant strain that the present invention obtains can well grow under not autoclaved low ph environment, be 3.5 in pH
Or when 4, the ethanol conversion of mutant strain is far above starting strain；Fermentation process mesohigh sterilizing ring can be reduced in use
Section reduces alcohol fermentation production cost, improves fermentation efficiency.
Biomaterial preservation
The zymomonas mobilis of the enduring high-concentration acetic acid of the present invention is sent out by the movement that deposit number is CICC 41465
Ferment monad is obtained through plasma mutagenesis and screening.The strain classification is named as zymomonas mobilis (Zymomnas
Mobilis) PH1-29, preserving number are：GDMCC 60260, preservation date are on October 25th, 2017.Depositary institution is Guangdong Province
Culture Collection, address are located at three building, the Institute of Micro-biology's laboratory building of Xianlie Middle Road, Guangzhou City 100.
Specific embodiment
Embodiment is set forth below the present invention is further described, but the content being not intended to limit the present invention.
Starting strain in embodiment is zymomonas mobilis ZM4 bacterial strains, purchased from Chinese industrial Microbiological Culture Collection
Center, now number is CICC 41465.
The screening of 1 mutant strain PH1-29 of embodiment
1) irradiation mutagenesis is carried out to zymomonas mobilis thalline by normal-temperature plasma mutation breeding technologies, first will
Starting strain ZM4 carries out activation culture, and cultivation temperature is 30 DEG C, and the activation culture time is 16h；
2) and then overnight culture (10 is taken6-108A cell), 10min is centrifuged at 4 DEG C, and rotating speed 3000rpm uses physiology
Salt is washed thalline and is resuspended in 1mL physiological saline；It takes 10 μ L that cell is resuspended and is placed in ARTP mutation breeding instrument mutagenesis 15-120s；
3) solid medium is prepared：50g/L glucose, 2g/L potassium dihydrogen phosphates, 10g/L yeast extracts utilize sulfuric acid
Solution adjusts medium pH；
4) renewal cultivation 16h is resuspended in the cell after irradiation mutagenesis, is coated cultivating on the solid medium of pH=4；
5) all bacterium colonies are selected to be screened on the culture medium of pH=3.5, screens in triplicate, obtains mutant strain
PH1-29。
2 common fermentation culture medium of embodiment prepares ethyl alcohol
1) preparation of fermentation medium：50g/L glucose, 2g/L potassium dihydrogen phosphates, 10g/L yeast extracts utilize sulphur
Acid solution adjusts medium pH=4；
2) mutant strain PH1-29 and starting strain ZM4 are inoculated in respectively and prepared on culture medium, inoculum concentration 10%,
Carry out fermented and cultured 40h；
3) separating alcohol measures conversion ratio.
3 common fermentation culture medium of embodiment prepares ethyl alcohol
1) preparation of fermentation medium：50g/L glucose, 2g/L potassium dihydrogen phosphates, 10g/L yeast extracts utilize sulphur
Acid solution adjusts medium pH=3.5
2) mutant strain PH1-29 and starting strain ZM4 are inoculated in respectively and prepared on culture medium, inoculum concentration 10%,
Carry out fermented and cultured 90h；
3) separating alcohol measures conversion ratio.
Common fermentation culture medium prepares ethyl alcohol under the normal pH of comparative example
1) preparation of fermentation medium：50g/L glucose, 2g/L potassium dihydrogen phosphates, 10g/L yeast extracts utilize sulphur
Acid solution adjusts medium pH=6.6, and culture medium is through 115 DEG C of sterilizing 20min；
2) mutant strain PH1-29 and starting strain ZM4 are inoculated in respectively and prepared on culture medium, inoculum concentration 10%,
Carry out fermented and cultured 32h；
3) separating alcohol measures conversion ratio.
The fermentability of mutant strain PH1-29 to filtering out carries out contrast test with starting strain ZM4, adjusts culture
Two kinds of bacterial strains are inoculated on culture medium and carry out fermented and cultured by base pH value respectively, corresponding fermentation time and conversion ratio such as 1 institute of table
Show：
Fermentability compares 1 mutant strain of table at various ph values with starting strain
Note：PH in culture medium value adjusts gained with sulfuric acid solution, and culture medium does not sterilize under the conditions of pH=4 and pH=3.5,
Under pH=6.6 normal conditions, culture medium sterilizes 20 minutes through 115 DEG C.
As shown in table 1, under the conditions of normal neutral pH (pH=6.6), mutant strain PH1-29 and starting strain ZM4 is inoculated with
In carrying out fermented and cultured on culture medium, as a result, it has been found that mutant strain can all run out of 50g/L glucose after 24 hours, second
Alcohol conversion reaches the 98% of theoretical yield, and the ethyl alcohol after starting strain ZM4 ferments 32 hours under equal conditions just reaches
The 90% of ethanol conversion.Illustrate that performance of the mutant strain under the conditions of normal fermentation also improves.
Under unsterilised environment, the pH to 4.0 of culture medium is adjusted with sulfuric acid, living contaminants are effectively eliminated under the environment.
Mutant strain PH1-29 and starting strain ZM4 are inoculated on culture medium and carry out fermented and cultured, as a result, it has been found that mutant strain fermentation 28 is small
When 50g/L glucose can be fully converted to ethyl alcohol, conversion ratio reaches the 99% of theoretical yield, and control strain growth receives
It is serious to inhibit, ethanol conversion only 0.09 g/g glucose, only up to the 18% of theoretical yield after inoculation 40 hours.
Fermentation pH value is continued to be down to 3.5, it theoretically can normal growth without bacterium.In the environment, mutant strain is although raw
Long rate is also affected, but still can sugar be fully converted to ethyl alcohol after fermenting 88 hours.And under the conditions of this open fermentation,
Control strain ZM4 is unable to normal growth completely.
Embodiment 4 simulates industrial fermentation environment and measures strain fermentation ability
In order to verify mutant strain can be widely used in the raw material of industry fermentation, by the use of 100g/L glucose as carbon source,
Corn leaching juice 3% prepares culture medium as N sources, 2g/L potassium dihydrogen phosphates, 10g/L yeast extracts.Using sulfuric acid solution by pH
It adjusts to 3.7 to effectively prevent living contaminants, mutant strain PH1-29 and starting strain ZM4 is inoculated on culture medium respectively,
Inoculum concentration is 10%, fermented and cultured 40h.As a result, it has been found that the alcohol fermentation efficiency of mutant strain is also significantly better than starting strain, turn
Change high 4 times of speed ratio control strain or more.
Embodiment 5 simulates industrial fermentation environment and measures strain fermentation ability
Carbon source, corn leaching juice are used glucose as N sources, preparation culture medium, by culture medium sugar concentration improve to
200g/L, 2g/L potassium dihydrogen phosphate, 10g/L yeast extracts adjust pH=3.7 using sulfuric acid solution, by mutant strain and go out
Bacterium germination strain is inoculated on culture medium respectively, inoculum concentration 10%, finds that the alcohol fermentation efficiency of mutant strain is bright after fermented and cultured 50h
It is aobvious to be better than control group.Although the biomass accumulation amount of the two does not have difference, the ethyl alcohol conversion rate of mutant strain compares control group
Fast 24 hours or more, alcohol getting rate reached more than 95% theoretical value.
Basic principle, main feature and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (6)
1. a kind of zymomonas mobilis mutant strain for being resistant to low ph value, which is characterized in that strain classification is named as movement hair
Ferment monad PH1-29, preserving number are：GDMCC 60260.
2. a kind of zymomonas mobilis mutant strain for being resistant to low ph value as described in claim 1 do not carry out it is autoclaved
Producing the purposes of process of alcohol products in open fermentation environment.
3. purposes as claimed in claim 2, which is characterized in that the fermentation process of the monad fermentative production of ethanol it is complete
Portion or part carry out in the environment of pH=3.5~4.
4. a kind of method of zymomonas mobilis PH1-29 production ethyl alcohol using described in claim 1, which is characterized in that
This method includes the following steps：
(1) fermentation medium is prepared；
(2) zymomonas mobilis PH1-29 is inoculated on the fermentation medium, carries out fermented and cultured；
(3) ethyl alcohol in separation and fermentation system.
5. a kind of method for producing ethyl alcohol as claimed in claim 5, which is characterized in that the culture medium described in step (1) is matched
Side includes：50-200g/L glucose, 2g/L potassium dihydrogen phosphates, 10g/L yeast extracts adjust culture medium using sulfuric acid solution
PH is 3.5-4.0.
A kind of 6. method for producing ethyl alcohol as claimed in claim 5, which is characterized in that the inoculation campaign described in step (2)
The inoculum concentration of fermentation single cell bacterium PH1-29 is 10%, and fermentation time is 20~90h；Culture environment is adjusted to pH=3.5-4.0, is gone out
Bacterium unsterilised can ferment.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182185A (en) * | 2018-09-18 | 2019-01-11 | 湖北大学 | The ethyl alcohol production Zymomonas mobilis strain and its separating screening method of the low pH of one plant of tolerance and application |
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| CN86107547A (en) * | 1985-10-25 | 1987-10-21 | 昆士兰大学 | Utilize zymomonas mobilis to make starch hydrolyzates change into alcohol |
| CN1928067A (en) * | 2005-09-05 | 2007-03-14 | 福建农林大学 | Motion fermentation single cell bacterium acid-resistant strain |
| US20120329112A1 (en) * | 2009-06-08 | 2012-12-27 | Oak Ridge Associated Universities | Microorganisms Having Enhanced Tolerance To Inhibitors and Stress |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182185A (en) * | 2018-09-18 | 2019-01-11 | 湖北大学 | The ethyl alcohol production Zymomonas mobilis strain and its separating screening method of the low pH of one plant of tolerance and application |
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