CN108220187B - Low-pH-value-tolerant zymomonas mobilis mutant strain and application thereof - Google Patents
Low-pH-value-tolerant zymomonas mobilis mutant strain and application thereof Download PDFInfo
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Abstract
The invention discloses a Zymomonas mobilis mutant strain tolerant to low pH value and application thereof, wherein the strain is classified and named as PH1-29 with the accession number of GDMCC 60260; the strain is obtained by repeatedly screening on a culture medium with low pH after being mutagenized by plasma at normal temperature. The strain can not only ensure normal growth in a low pH environment and ensure good fermentation efficiency, but also eliminate pollution in the low pH environment, avoid high-temperature and high-pressure sterilization in the fermentation process, control production cost and improve fermentation efficiency.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a low pH value tolerant zymomonas mobilis mutant strain and application thereof, which can be applied to the production of fuel ethanol bio-based products in any non-autoclaved open fermentation environment.
Background
In the modern biological fermentation industry, high-temperature sterilization is an important and articulated link, and the fermentation cost and the product safety are concerned, so that the attention is worthy. Most microorganisms do not grow well in low pH environments, so lowering pH is a strategy to reduce the risk of microbial contamination. However, most fermentation strains are not highly pH tolerant themselves. If the fermentation strain which is tolerant to the low pH environment is screened by a mutation means, the fermentation efficiency can be ensured, and because the low pH environment can eliminate pollution, high-temperature high-pressure sterilization in the fermentation process can be avoided, and the method is also one of the methods for controlling the production cost.
Zymomonas mobilis (Zymomonas mobilis) is an excellent species for ethanol production and has recently received much attention in the research and production of ethanol as a renewable fuel. However, at present, zymomonas mobilis can grow under the environment of pH 4, but the growth performance is poor, and the zymomonas mobilis cannot be normally used in the fermentation industry. Thus, for ethanol fuel fermentation systems, the low pH environment remains a significant challenge for the growth of the fermenting strain. Therefore, the patent creates a mutant strain which can produce ethanol through normal fermentation in a low pH environment with pH 4 and pH 3.5, and the process does not need high-temperature and high-pressure sterilization, and no other reports exist in the species at present to achieve the level.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a low pH value tolerant zymomonas mobilis mutant strain and application thereof.
The invention provides a Zymomonas mobilis mutant strain tolerant to low pH value, which is classified and named as Zymomonas mobilis PH1-29 with the preservation number as follows: GDMCC 60260, deposited at the guangdong province collection of microorganisms in 2017, 10 and 25 months.
In order to achieve the purpose, the invention adopts the technical scheme that:
1) carrying out irradiation mutagenesis on the zymomonas mobilis by a normal-temperature plasma mutagenesis breeding technology, firstly, carrying out activation culture on a starting strain ZM4, wherein the culture temperature is 30 ℃, and the culture time is 16 h;
2) overnight cultures were taken (10)6～108Cell), centrifuging for 5-10 min at 4 ℃ at the rotating speed of 3000rpm, washing the thalli with physiological saline and suspending in 1mL of physiological saline; putting 10 mu L of the heavy suspension cells into an ARTP mutation breeding instrument for mutation for 15-120 s;
3) resuspending and restoring the cells after the irradiation mutagenesis for 16h, and then coating the cells on a solid culture medium with the pH value of 3.5 or 4 for culture;
4) all colonies were selected for screening on medium pH 3.5 and repeated at least three times.
Specifically, in the step 2), the purity of the plasma carrier gas is 99.999%, the gas flow is 10-20 standard gas flow, the voltage is 100-120V, and the plasma irradiation temperature is 22 ℃;
specifically, the solid medium used in step 2) and step 3) comprises: 50g/L glucose, 2g/L potassium dihydrogen phosphate, and 10g/L yeast extract, and adjusting pH of the culture medium to 3.5-4.0 with sulfuric acid solution.
The invention also provides the use of the zymomonas mobilis mutant strain as described above in an open fermentation environment without autoclaving to produce a fuel ethanol product.
In order to achieve the above purposes, the technical scheme adopted by the invention is as follows:
(1) preparing a fermentation culture medium;
(2) inoculating zymomonas mobilis PH1-29 to the fermentation culture medium for fermentation culture;
(3) separating the ethanol in the fermentation system.
Specifically, all or part of the fermentation process for producing ethanol by fermentation is carried out in a fermentation environment with pH 3.5-4.0.
Specifically, the formula of the culture medium comprises: adjusting the pH of the culture medium to 3.5-4.0 by using a sulfuric acid solution, wherein the glucose is 50g/L, the potassium dihydrogen phosphate is 2g/L, and the yeast extract is 10 g/L;
specifically, the inoculation amount of the inoculated fermentation strain is 10%, and the fermentation time is 20-90 h.
It is worth noting that under a low pH value fermentation system, the common strain can not grow normally, but the mutant strain can grow normally, so that the pH value of the system is reduced in the fermentation process, so that the normal growth and the high-efficiency ethanol growth of the mutant strain PH1-29 can be guaranteed, and the growth of other bacteria can be inhibited, thereby reducing the high-pressure high-temperature sterilization link in the conventional fermentation process, reducing the ethanol fermentation production cost and improving the fermentation efficiency.
The invention has the beneficial effects that:
the mutant strain obtained by the invention can grow well in a low pH value environment without autoclaving, and the ethanol conversion rate of the mutant strain is far higher than that of the original strain when the pH is 3.5 or 4; during the use process, the high-pressure sterilization link in the fermentation process can be reduced, the production cost of ethanol fermentation is reduced, and the fermentation efficiency is improved.
Biological material preservation
The Zymomonas mobilis tolerant to high-concentration acetic acid is obtained by carrying out plasma mutagenesis and screening on Zymomonas mobilis with the preservation number of CICC 41465. The strain is classified and named as zymomonas mobilis (Zymomonas mobilis) PH1-29 with the preservation number as follows: GDMCC 60260 with a preservation date of 2017, 10 months and 25 days. The preservation unit is Guangdong province microorganism strain preservation center, and the address is located in the third floor of the experimental building of the first-fierce middle-route 100 # microorganism institute in Guangzhou city.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
The starting strain in the examples is ZM mobilis strain ZM4 which is purchased from China center for culture Collection of Industrial microorganisms and is currently numbered CICC 41465.
EXAMPLE 1 screening of mutant Strain PH1-29
1) Carrying out irradiation mutagenesis on the zymomonas mobilis by a normal-temperature plasma mutagenesis breeding technology, firstly, carrying out activation culture on a starting strain ZM4, wherein the culture temperature is 30 ℃, and the activation culture time is 16 h;
2) the overnight culture was then taken (10)6-108Individual cells) at 4 ℃ for 10min at the rotating speed of 3000rpm, washing the thalli with physiological saline and suspending in 1mL of physiological saline; putting 10 mu L of the resuspended cells into an ARTP mutation breeding instrument for mutation for 15-120 s;
3) preparing a solid culture medium: 50g/L glucose, 2g/L potassium dihydrogen phosphate and 10g/L yeast extract, and adjusting the pH value of the culture medium by using a sulfuric acid solution;
4) resuspending and restoring the cells subjected to the irradiation mutagenesis for 16h, and then coating the cells on a solid culture medium with pH 4 for culture;
5) all colonies were selected for screening on a medium at pH 3.5 and the screening was repeated three times to obtain mutant strain pH 1-29.
EXAMPLE 2 preparation of ethanol from common fermentation Medium
1) Preparation of a fermentation medium: 50g/L glucose, 2g/L potassium dihydrogen phosphate and 10g/L yeast extract, and adjusting the pH value of the culture medium to be 4 by using a sulfuric acid solution;
2) respectively inoculating the mutant strain PH1-29 and the original strain ZM4 on a prepared culture medium, wherein the inoculation amount is 10%, and performing fermentation culture for 40 h;
3) the ethanol was separated and the conversion was determined.
EXAMPLE 3 preparation of ethanol from common fermentation Medium
1) Preparation of a fermentation medium: 50g/L glucose, 2g/L potassium dihydrogen phosphate, 10g/L yeast extract, adjusting pH of culture medium to 3.5 with sulfuric acid solution
2) Respectively inoculating the mutant strain PH1-29 and the original strain ZM4 on a prepared culture medium, wherein the inoculation amount is 10%, and performing fermentation culture for 90 hours;
3) the ethanol was separated and the conversion was determined.
Comparative example preparation of ethanol from common fermentation Medium at Normal pH
1) Preparation of a fermentation medium: adjusting pH of culture medium to 6.6 with sulfuric acid solution, and sterilizing the culture medium at 115 deg.C for 20 min;
2) respectively inoculating the mutant strain PH1-29 and the original strain ZM4 on a prepared culture medium, wherein the inoculation amount is 10%, and performing fermentation culture for 32 hours;
3) the ethanol was separated and the conversion was determined.
The fermentation capacity of the screened mutant strain PH1-29 and the original strain ZM4 are compared and tested, the pH value of a culture medium is adjusted, the two strains are respectively inoculated on the culture medium for fermentation culture, and the corresponding fermentation time and the corresponding conversion rate are shown in the table 1:
TABLE 1 comparison of fermentation Capacity of mutant strains and starting strains at different pH values
Note: the pH of the medium was adjusted with sulfuric acid solution, and the medium was not sterilized at pH 4 and pH 3.5, and was sterilized at 115 ℃ for 20 minutes under normal conditions at pH 6.6.
As shown in table 1, the mutant strain pH1-29 and the starting strain ZM4 were inoculated onto a medium under normal neutral pH (pH 6.6) conditions for fermentation culture, and as a result, it was found that 50g/L glucose was completely consumed after 24 hours of the mutant strain, and the ethanol conversion rate reached 98% of the theoretical conversion rate, whereas the ethanol conversion rate reached 90% of the ethanol conversion rate after 32 hours of fermentation with the starting strain ZM4 under the same conditions. The performance of the mutant strain under normal fermentation conditions is also improved to a certain extent.
The pH value of the culture medium is adjusted to 4.0 by using sulfuric acid in a non-sterile environment, and the mixed bacteria pollution is effectively eliminated in the environment. The mutant strain PH1-29 and the original strain ZM4 are inoculated on a culture medium for fermentation culture, and the result shows that 50g/L of glucose can be completely converted into ethanol by the mutant strain after being fermented for 28 hours, the conversion rate reaches 99% of the theoretical conversion rate, the growth of the control strain is severely inhibited, and the conversion rate of ethanol is only 0.09 g/g of glucose after being inoculated for 40 hours, and only 18% of the theoretical conversion rate is reached.
The fermentation pH was continued to drop to 3.5 and theoretically no bacteria could grow normally. In this environment, the mutant strain can completely convert sugars to ethanol after 88 hours of fermentation, although the growth rate is also affected. In this open fermentation condition, the control strain ZM4 did not grow normally at all.
Example 4 measurement of fermentation Capacity of Strain by simulating Industrial fermentation Environment
In order to verify that the mutant strain can be widely used for industrial raw material fermentation, a culture medium is prepared by using 100g/L glucose as a carbon source, 3% corn steep liquor as an N source, 2g/L potassium dihydrogen phosphate and 10g/L yeast extract. The pH value is adjusted to 3.7 by using a sulfuric acid solution to effectively prevent mixed bacteria pollution, and the mutant strain PH1-29 and the original strain ZM4 are respectively inoculated on a culture medium, the inoculation amount is 10%, and the fermentation culture is carried out for 40 h. The result shows that the ethanol fermentation efficiency of the mutant strain is also obviously better than that of the original strain, and the conversion rate of the mutant strain is higher than that of the control strain by more than 4 times.
Example 5 measurement of fermentation Capacity of Strain by simulating Industrial fermentation Environment
Glucose is used as a carbon source, corn steep liquor is used as an N source, a culture medium is prepared, the sugar concentration of the culture medium is increased to 200g/L, 2g/L potassium dihydrogen phosphate and 10g/L yeast extract are added, the pH value is adjusted to 3.7 by using a sulfuric acid solution, the mutant strain and the original strain are respectively inoculated on the culture medium, the inoculation amount is 10%, and the ethanol fermentation efficiency of the mutant strain is obviously better than that of a control group after fermentation culture for 50 hours. Although the biomass accumulation amount of the mutant strain and the control strain is not different, the ethanol conversion rate of the mutant strain is faster than that of the control group by more than 24 hours, and the ethanol yield reaches more than 95 percent of a theoretical value.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
1. A low pH tolerant Zymomonas mobilis mutant strain PH1-29 is characterized in that the strain taxonomy is named as Zymomonas mobilis(Zymomnas mobilis)The preservation number is: GDMCC No. 60260.
2. Use of a low pH tolerant Zymomonas mobilis mutant strain PH1-29 of claim 1 in an open fermentation environment without autoclaving to produce a fuel ethanol product.
3. The use according to claim 2, wherein the fermentation process of zymomonas mobilis PH1-29 fermentation for ethanol production is wholly or partially carried out in an environment with a PH of 3.5-4.
4. A process for the production of ethanol using zymomonas mobilis PH1-29 as claimed in claim 1, comprising the steps of:
(1) preparing a fermentation culture medium;
(2) inoculating zymomonas mobilis PH1-29 to the fermentation culture medium for fermentation culture;
(3) separating the ethanol in the fermentation system.
5. The method of claim 4, wherein the media formulation of step (1) comprises: 50-200g/L glucose, 2g/L potassium dihydrogen phosphate, and 10g/L yeast extract, and adjusting pH of the culture medium to 3.5-4.0 with sulfuric acid solution.
6. The method for producing ethanol according to claim 4, wherein the amount of inoculated Zymomonas mobilis PH1-29 in the step (2) is 10%, and the fermentation time is 20-90 h; the culture environment is adjusted to pH 3.5-4.0, and fermentation can be carried out with or without sterilization.
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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 |
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| CN86101245A (en) * | 1985-02-22 | 1986-10-08 | 昆士兰大学 | Utilize the zymomonas mobilis bacterium to produce ethanol by sucrose |
| US20100311137A1 (en) * | 2009-06-08 | 2010-12-09 | Ut-Battelle, Llc | Microorganisms Having Enhanced Tolerance To Inhibitors and Stress |
| CN105062928B (en) * | 2015-08-31 | 2018-07-13 | 农业部沼气科学研究所 | A kind of zymomonas mobilis and its application of resisting high-concentration acetic acid and high concentration furtural |
| CN105002128B (en) * | 2015-08-31 | 2018-09-25 | 农业部沼气科学研究所 | A kind of zymomonas mobilis of resisting high-concentration acetic acid and its 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 |
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