CN105002128B - A kind of zymomonas mobilis of resisting high-concentration acetic acid and its application - Google Patents
A kind of zymomonas mobilis of resisting high-concentration acetic acid and its application Download PDFInfo
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- CN105002128B CN105002128B CN201510549342.3A CN201510549342A CN105002128B CN 105002128 B CN105002128 B CN 105002128B CN 201510549342 A CN201510549342 A CN 201510549342A CN 105002128 B CN105002128 B CN 105002128B
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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
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
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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 present invention provides a kind of zymomonas mobilis of enduring high-concentration acetic acid (Zymomonas mobilis), the deposit number of the zymomonas mobilis is CGMCC No.10945.The zymomonas mobilis of the present invention can mushroom out in the culture solution of the acetic acid containing 8.0g/L or more, and can quickly carry out alcohol fermentation.This makes the zymomonas mobilis of the present invention can be under the conditions of higher acetic acid concentration directly to cellulase hydrolysis product fermentations, and alcohol fuel is prepared.
Description
Technical field
The present invention relates to microorganisms technical fields, and in particular, to a kind of zymomonas mobilis of resisting high-concentration acetic acid
With its application.
Background technology
It is that raw material produces alcohol fuel and other biological base product using the abundant stalk resource in China, to promoting China's agriculture
Industry sustainable development expands Agricultural Function and ensures that national energy security is of great significance.But in lignocellulosics such as stalks
Biomass resource is during fermentative production of ethanol and biological-based chemicals, due to cellulose, hemicellulose and wood in raw material
Fine and close structure between quality is unfavorable for subsequent cellulase hydrolysis saccharification.Therefore, lignocellulose-like biomass is before enzymolysis
The preprocessing means such as physics and chemistry need to generally be passed through to improve saccharification efficiency of the cellulase to cellulose.But pretreated
Cheng Zhong, some chemical pretreatment means, such as diluted acid, be relatively easy to form acetic acid etc. it is a series of to microorganism have poison left and right
Mortifier.Acetic acid concentration has seriously affected subsequent biological fermentation process up to 1-10g/L wherein in enzymolysis liquid.
Zymomonas mobilis (Zymomonas mobilis) passing through ED metabolic pathways (amphimicrobian) as unique one kind
The microorganism of glucose fermentation has the advantages such as higher alcohol fermentation efficiency and alcohol resistance, it has also become structure producing and ethanol
One of preferred host of engineering bacteria and other biological base product (Biotechnology for Biofuel, 2014,7:101).So
And in cellulose biomass dilute acid pretreatment technique, there are some inhibiting substances with toxic effect, still can be to having
Zymomonas mobilis cause toxic action, cause alcohol fermentation efficiency reduction.The acetic acid of a concentration of 4g/l is added in external source
In the case of the bacterium can be grown there is obvious inhibiting effect, and its ethanol production also declines 10% or so, and highly concentrated
In the case of degree acetic acid (7.0g/L) exists, existing zymomonas mobilis then grows more slowly, and ethanol production is lower.
Have research work and selection and breeding have been carried out to the zymomonas mobilis of enduring high-concentration acetic acid, has obtained having acetic acid
There is the zymomonas mobilis of tolerance, still, under the acetic acid concentration of 8.0g/L, the growth of these zymomonas mobilis is still
More slowly, ethanol production is still relatively low.
Invention content
In order to overcome the existing lower defect of zymomonas mobilis high-concentration acetic acid tolerance, the present invention provides one
Strain is resistant to the Zymomonas mobilis strain of up to 8g/L acetic acid.
The present invention provides a kind of zymomonas mobilis, the zymomonas mobilis (Zymomonas mobilis)
Deposit number is CGMCC No.10945.
The present invention also provides purposes of the zymomonas mobilis as described above in fermentative production of ethanol.
The present invention also provides a kind of methods of fermentative production of ethanol, and this method comprises the following steps：(1) fiber will be contained
The raw material of element carries out steam blasting, dilute acid pretreatment and enzymolysis, obtains enzymolysis liquid；(2) the inoculation fermentation bacterium into the enzymolysis liquid
Kind, it ferments, obtains zymotic fluid；(3) separating alcohol from the zymotic fluid；The fermenting microbe contains fortune as described above
Dynamic fermentation single cell bacterium.
The present invention zymomonas mobilis can be used as cellulose alcoholic fermentation and other biological base product production it is good
Bacterial strain can save enzymolysis liquid detoxification treatment step, and ethyl alcohol and the production of other biological base are carried out directly under higher acetic acid existence condition
The fermentation of product.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Biomaterial preservation
A kind of zymomonas mobilis involved in this paper comparative examples is by laboratory pattern further fermentation single cell bacterium through mutagenesis
It is obtained with screening.The strain classification is named as zymomonas mobilis (Zymomonas mobilis), and deposit number is
CGMCC No.9987, the deposit date is on November 18th, 2014, and depositary institution is China Committee for Culture Collection of Microorganisms
Common micro-organisms center, address are located at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica.
The zymomonas mobilis of the enduring high-concentration acetic acid of the present invention is by the movement that deposit number is CGMCC No.9987
Fermentation single cell bacterium is obtained through mutagenesis and screening.The strain classification is named as zymomonas mobilis (Zymomonas mobilis),
Its deposit number is CGMCC No.10945, and the deposit date is on June 2nd, 2015, depositary institution was protected for Chinese microorganism strain
Administration committee's common micro-organisms center is hidden, address is located at the micro- life of the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences
Object research institute.
Specific implementation mode
The specific implementation mode of the present invention is described in detail below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The present invention provides one plant of zymomonas mobilis, wherein the zymomonas mobilis (Zymomonas
Mobilis deposit number) is CGMCC No.9987.
The present invention also provides purposes of the zymomonas mobilis as described above in fermentative production of ethanol.
Wherein, the raw material used in fermentative production of ethanol does not require particularly, can be that the various of routine contain cellulose
Raw material；Such as may include at least one of agricultural crop straw, sawdust, bamboo, rice husk and peanut shell.
Wherein, the fermentation condition of the zymomonas mobilis does not require particularly, can be that the various of routine are used for
The fermentation condition for preparing ethyl alcohol, since the zymomonas mobilis of the present invention has excellent tolerance to acetic acid, it is preferable that institute
The fermentation condition for stating zymomonas mobilis includes：Under all or part of acetic acid concentration not less than 8g/L of fermentation process
It carries out.
Wherein, the influence in order to avoid the acetic acid of special high concentration to fermentation, it is preferable that the zymomonas mobilis
Fermentation condition includes：It is carried out under all or part of acetic acid concentration not higher than 11g/L of fermentation process.
The present invention also provides a kind of methods of fermentative production of ethanol, wherein this method comprises the following steps：(1) will contain
There is the raw material of cellulose to carry out steam blasting, dilute acid pretreatment and enzymolysis, obtains enzymolysis liquid；(2) it is inoculated with into the enzymolysis liquid
Fermenting microbe ferments, and obtains zymotic fluid；(3) separating alcohol from the zymotic fluid；The fermenting microbe contains institute as above
The zymomonas mobilis stated.
Wherein, the raw material containing cellulose include in agricultural crop straw, sawdust, bamboo, rice husk and peanut shell extremely
Few one kind.
Wherein, the condition that the raw material containing cellulose carries out steam blasting, dilute acid pretreatment and enzymolysis can be conventional
Dilute acid pretreatment and enzymatic hydrolysis condition, for example, the condition of dilute acid pretreatment may include：Temperature is 150-190 DEG C, time 10-
100 minutes, sulfuric acid concentration was 0.2-3 weight %, solid-to-liquid ratio 1:(5-25)；The condition of steam blasting may include：Temperature is
180-200 DEG C, pressure is 1.4-2.0 megapascal, and holding time for pressure is 3-7 minutes.The condition and step of enzymolysis may include：
In terms of the dry weight of every gram of cellulose-containing raw material, the dosage of cellulase is 8-20 enzyme activity units, and temperature is 45-55 DEG C, when
Between be 25-48 hours, pH value 3.0-7.0.During enzymolysis, the condition of enzymolysis can make the enzymolysis liquid contain second
Acid.
Wherein, since the zymomonas mobilis of the present invention has excellent tolerance to acetic acid, it is preferable that the item of enzymolysis
Part makes the enzymolysis liquid contain the acetic acid not less than 8g/L.
Present invention be described in more detail by the following examples：
Prepare embodiment 1
This preparation embodiment is used to illustrate the preparation of the RM culture mediums containing acetic acid, and by the raw material system containing cellulose
The process of standby enzymolysis liquid.
According to 20.0g/L glucose, 10.0g/L yeast powders, 1.0g/L MgCl2、1.0g/L(NH4)2SO4And 1.0g/L
KH2PO4Recipe configuration obtain basic RM culture mediums.Acetic acid is added in basic RM culture mediums so that acetic acid concentration is respectively
7.5g/L, 8.0g/L and 8.5g/L.
Wheat stalk is cut into 2 centimetres or so of segment, then plus water adjusts water content as 30 weight %；It is delivered to heat preservation
In pressure apparatus, steam blasting is carried out under 200 DEG C, 1.8 megapascal, and collect steam explosion products, and by gained steam blasting
Product and 80 DEG C of water are according to weight ratio 1：3 are stirred 30 minutes, and the sulfuric acid solution of a concentration of 72 weight % is then added and makes
The concentration dilution of sulfuric acid is stirred 30 minutes at 30 DEG C, obtains dilute acid pretreatment product to 3 weight %.Dilute acid pretreatment product is carried out
Enzymolysis, in terms of the dry weight of every gram of cellulose-containing raw material, the dosage of cellulase is 20 enzyme activity units, and temperature is 52 DEG C, when
Between be 38 hours, pH value 4.0；Obtain enzymolysis liquid.The content that acetic acid in enzymolysis liquid is measured with liquid chromatography is 8.3 weights
Measure %.
Testing example 1
It is CGMCC No.10945 fortune by zymomonas mobilis that deposit number is CGMCC No.9987 and deposit number
Dynamic fermentation single cell bacterium is inoculated in the RM fluid nutrient mediums that the preparation embodiment 1 after sterilizing obtains respectively, and (acetic acid concentration is respectively
7.5g/L, 8.0g/L and 8.5g/L) in, static gas wave refrigerator 72 hours under the conditions of 30 DEG C, fermentation 24 hours after every 12 hours into
Row sampling.Growth situation is measured at 600nm using ultraviolet specrophotometer, the results are shown in Table 1.
Table 1
The deposit number that can be seen that the present invention from the data of table 1 is the zymomonas mobilis of CGMCC No.10945
With high acetic acid concentration tolerance.
Testing example 2
It is CGMCC No.10945 by zymomonas mobilis that deposit number is CGMCC No.9987 and deposit number
Zymomonas mobilis is inoculated in the obtained enzymolysis liquid of preparations embodiment 1 after sterilizing that (content of acetic acid is 8.3 weights respectively
Measure %).A concentration of the 10 of inoculation4CFU/mL, at 30 DEG C in fermentation tank stir culture 40 hours.At 100 DEG C obtained by distillation
Tunning, gained distillation fraction second distillation at 78.3 DEG C can obtain ethyl alcohol, calculate alcohol yied according to the following formula.
Alcohol yied=100% × ethyl alcohol weight/stalk dry weight
Result of calculation is shown, under identical fermentation condition, deposit number of the invention is the fortune of CGMCC No.10945
The alcohol yied of dynamic fermentation single cell bacterium reaches 27.3 weight %, and the alcohol yied that deposit number is CGMCC No.9987 is only
22.7 weight %, it can thus be seen that under higher acetic acid existence condition, zymomonas mobilis of the invention produces more
Ethyl alcohol.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail can carry out a variety of simple variants to technical scheme of the present invention within the scope of the technical concept of the present invention, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (9)
1. a kind of zymomonas mobilis, it is characterised in that：The zymomonas mobilis (Zymomonas mobilis) preservation
Number is CGMCC No.10945.
2. purposes of the zymomonas mobilis described in claim 1 in fermentative production of ethanol.
3. purposes according to claim 2, wherein the raw material used in fermentative production of ethanol include agricultural crop straw, sawdust,
At least one of bamboo, rice husk and peanut shell.
4. purposes according to claim 2 or 3, wherein the fermentation condition of the zymomonas mobilis includes：It fermented
It is carried out under all or part of acetic acid concentration not less than 8g/L of journey.
5. purposes according to claim 4, wherein the fermentation condition of the zymomonas mobilis includes：Fermentation process
The all or part of acetic acid concentration not higher than 11g/L under carry out.
6. a kind of method of fermentative production of ethanol, which is characterized in that this method comprises the following steps：(1) cellulose will be contained
Raw material carries out steam blasting, dilute acid pretreatment and enzymolysis, obtains enzymolysis liquid；(2) the inoculation fermentation strain into the enzymolysis liquid, into
Row fermentation, obtains zymotic fluid；(3) separating alcohol from the zymotic fluid；The fermenting microbe contains as described in claim 1
Zymomonas mobilis.
7. according to the method described in claim 6, wherein, the raw material containing cellulose includes agricultural crop straw, sawdust, bamboo
At least one of son, rice husk and peanut shell.
8. the method described according to claim 6 or 7, wherein the condition of enzymolysis makes the enzymolysis liquid contain acetic acid.
9. according to the method described in claim 8, wherein, the condition of enzymolysis makes the enzymolysis liquid contain the second not less than 8g/L
Acid.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1472309A (en) * | 2003-06-23 | 2004-02-04 | 华东理工大学 | Acetate proof mutant escherichia coli DH5 alpha and its culturing method and use |
| CN104673737A (en) * | 2015-04-01 | 2015-06-03 | 农业部沼气科学研究所 | Ethanol-tolerant zymomonas mobilis as well as preparation method and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1472309A (en) * | 2003-06-23 | 2004-02-04 | 华东理工大学 | Acetate proof mutant escherichia coli DH5 alpha and its culturing method and use |
| CN104673737A (en) * | 2015-04-01 | 2015-06-03 | 农业部沼气科学研究所 | Ethanol-tolerant zymomonas mobilis as well as preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Adaptative laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors;Zong-xia Shui et al.;《Appl Microbiol Biotechnol》;20150503;第99卷(第13期);摘要，第5742页右栏第2-4段，第5743页右栏第1段，图4 * |
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