CN105062928A - Zymomonas mobilis resistant to high-concentration acetic acid and high-concentration furfural and application thereof - Google Patents
Zymomonas mobilis resistant to high-concentration acetic acid and high-concentration furfural and application thereof Download PDFInfo
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- CN105062928A CN105062928A CN201510549391.7A CN201510549391A CN105062928A CN 105062928 A CN105062928 A CN 105062928A CN 201510549391 A CN201510549391 A CN 201510549391A CN 105062928 A CN105062928 A CN 105062928A
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- zymomonas mobilis
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Abstract
The invention provides zymomonas mobilis resistant to high-concentration acetic acid and high-concentration furfural and application thereof. The accession number of the zymomonas mobilis is CMGCC No.10946. The zymomonas mobilis can quick grow in a culture solution containing 7.0 g/L of acetic acid and 3.0 g/L of furfural, and alcoholic fermentation can be performed quickly. Therefore, the zymomonas mobilis can directly perform fermentation on cellulose enzymatic hydrolysates on the conditions of high-concentration acetic acid and high-concentration furfural, and fuel alcohol is obtained through preparation.
Description
Technical field
The present invention relates to microbial technology field, particularly, relate to zymomonas mobilis and its application of a kind of resisting high-concentration acetic acid and high density furtural.
Background technology
Utilize the stalk resource of China's abundant for raw material production alcohol fuel and other biological base product, to promotion China agricultural sustainable development, expand Agricultural Function and ensure that national energy security is significant.But in the process of the lignocellulose biomass resources such as stalk at fermentative production of ethanol and bio-based chemical, due to Mierocrystalline cellulose, structure fine and close between hemicellulose and xylogen in raw material, be unfavorable for follow-up cellulase hydrolysis saccharification.Therefore, lignocellulose-like biomass last at enzymolysis as need through the preprocessing means such as physics and chemistry to improve cellulase to cellulosic saccharification efficiency.But in preprocessing process, some Chemical Pretreatment means, as diluted acid etc., are easier to form a series of inhibition microorganism to murder by poisoning left and right such as acetic acid and furtural.Wherein in enzymolysis solution, acetic acid concentration can reach 1-10g/L, and in enzymolysis solution, the concentration of furtural can reach 0.5-11g/L, has had a strong impact on follow-up biological fermentation process.
Zymomonas mobilis (Zymomonasmobilis) is as unique a kind of microorganism by ED pathways metabolism (amphimicrobian) glucose fermentation, there is the advantages such as higher ethanol fermentation efficiency and alcohol resistance, become one of the preferred host building engineered strains for ethanol production and other biological base product (BiotechnologyforBiofuel, 2014,7:101).But in cellulose biomass dilute acid pretreatment technique, there is the inhibitory substance that some have toxic action, still can cause toxic action to existing zymomonas mobilis, cause ethanol fermentation efficiency to reduce.Obvious restraining effect can be had to this bacteria growing when external source interpolation concentration is the acetic acid of 4g/l, and its ethanol production also declines about 10%, and under high-concentration acetic acid (7.0g/L) exists situation, existing zymomonas mobilis then grows more slow, and ethanol production is lower.Obvious restraining effect can be had to this bacteria growing when external source interpolation concentration is the furtural of 1g/L, and its ethanol production also declines 20.54%, and under high density furtural (2.0g/L) exists situation, zymomonas mobilis then can not grow.
The existing zymomonas mobilis of research work to the zymomonas mobilis of enduring high-concentration acetic acid and enduring high-concentration furtural has carried out seed selection respectively, obtain the zymomonas mobilis respectively acetic acid and furtural to tolerance, but, these zymomonas mobilis can not simultaneously enduring high-concentration acetic acid and high density furtural, still can not meet the requirement that stalk fermentation produces ethanol.
Summary of the invention
Being difficult to the defect of simultaneously enduring high-concentration acetic acid and high density furtural in order to overcome existing zymomonas mobilis, the invention provides the Zymomonas mobilis strain that a strain can tolerate the furtural up to 7g/L acetic acid and 3g/L simultaneously.
The invention provides a kind of zymomonas mobilis, the deposit number of this zymomonas mobilis (Zymomonasmobilis) is CGMCCNo.10946.
Present invention also offers the purposes of zymomonas mobilis as above in fermentative production of ethanol.
Present invention also offers a kind of method of fermentative production of ethanol, the method comprises the steps: (1) will carry out steam explosion, dilute acid pretreatment and enzymolysis containing cellulosic raw material, obtain enzymolysis solution; (2) to inoculation fermentation bacterial classification in described enzymolysis solution, ferment, obtain fermented liquid; (3) separating alcohol from described fermented liquid; Described fermented bacterium contains zymomonas mobilis as above.
Zymomonas mobilis of the present invention can be used as the good bacterial strain of cellulose alcoholic fermentation and the production of other biological base product, can save enzymolysis solution detoxification treatment step, under higher acetic acid existence condition, directly carry out the fermentation of ethanol and other biological base product.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Biomaterial preservation
A kind of zymomonas mobilis related in comparative example is herein obtained through mutagenesis and screening by laboratory pattern further fermentation single cell bacterium.This strain classification called after zymomonas mobilis (Zymomonasmobilis), its deposit number is CGMCCNo.9987, preservation date is on November 18th, 2014, depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
The zymomonas mobilis of enduring high-concentration acetic acid of the present invention and high density furtural is that the zymomonas mobilis of CGMCCNo.9987 obtains through mutagenesis and screening by deposit number.This strain classification called after zymomonas mobilis (Zymomonasmobilis), its deposit number is CGMCCNo.10946, preservation date is on June 2nd, 2015, depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
The invention provides a strain zymomonas mobilis, wherein, the deposit number of this zymomonas mobilis (Zymomonasmobilis) is CGMCCNo.10946.
Present invention also offers the purposes of zymomonas mobilis as above in fermentative production of ethanol.
Wherein, the special requirement of raw material that fermentative production of ethanol is used can be the various containing cellulosic raw material of routine; Such as can comprise at least one in agricultural crop straw, wood chip, bamboo, rice husk and Pericarppium arachidis hypogaeae.
Wherein, the not special requirement of fermentation condition of described zymomonas mobilis, can be the conventional various fermentation conditions for the preparation of ethanol, because zymomonas mobilis of the present invention has excellent tolerance to acetic acid, preferably, the fermentation condition of described zymomonas mobilis comprises: fermenting process all or part of carries out under the acetic acid concentration being not less than 7g/L and under the furtural concentration being not less than 3g/L.
Wherein, in order to avoid the acetic acid of special high density and the furtural of special high density are on the impact of fermentation, preferably, the fermentation condition of described zymomonas mobilis comprises: fermenting process all or part of under not higher than the acetic acid concentration of 11g/L and not higher than the furtural concentration of 11g/L under carry out.
Present invention also offers a kind of method of fermentative production of ethanol, wherein, the method comprises the steps: (1) will carry out steam explosion, dilute acid pretreatment and enzymolysis containing cellulosic raw material, obtain enzymolysis solution; (2) to inoculation fermentation bacterial classification in described enzymolysis solution, ferment, obtain fermented liquid; (3) separating alcohol from described fermented liquid; Described fermented bacterium contains zymomonas mobilis as above.
Wherein, the described at least one comprised containing cellulosic raw material in agricultural crop straw, wood chip, bamboo, rice husk and Pericarppium arachidis hypogaeae.
Wherein, the condition of carrying out steam explosion, dilute acid pretreatment and enzymolysis containing cellulosic raw material can be conventional dilute acid pretreatment and enzymatic hydrolysis condition, such as, the condition of dilute acid pretreatment can comprise: temperature is 150-190 DEG C, time is 10-100 minute, sulfuric acid concentration is 0.2-3 % by weight, and solid-to-liquid ratio is 1:(5-25); The condition of steam explosion can comprise: temperature is 180-200 DEG C, and pressure is 1.4-2.0 MPa, holding time as 3-7 minute of pressure.The condition of enzymolysis and step can comprise: with the dry weight basis of the raw material of every gram of cellulose, and the consumption of cellulase is 8-20 enzyme activity unit, and temperature is 45-55 DEG C, and the time is 25-48 hour, and pH value is 3.0-7.0.In the process of enzymolysis, the condition of enzymolysis can make described enzymolysis solution contain acetic acid and furtural.
Wherein, because zymomonas mobilis of the present invention has excellent tolerance to acetic acid, preferably, the condition of enzymolysis makes described enzymolysis solution contain the acetic acid being not less than 7g/L and the furtural being not less than 3g/L.
Further describe the present invention by the following examples:
Preparation embodiment 1
This prepares the preparation of embodiment for illustration of the RM substratum containing acetic acid and furtural, and by preparing the process of enzymolysis solution containing cellulosic raw material (maize straw).
According to 20.0g/L glucose, 10.0g/L yeast powder, 1.0g/LMgCl
2, 1.0g/L (NH
4)
2sO
4and 1.0g/LKH
2pO
4recipe configuration obtain basic RM substratum.Acetic acid and furtural is added in basic RM substratum, obtain 3 kinds of RM and adjust substratum, wherein, one RM adjusts substratum and contains the acetic acid of 6.0g/L and the furtural of 2.0g/L, 2nd RM adjusts substratum and contains the acetic acid of 7.0g/L and the furtural of 3.0g/L, and the 3rd RM adjusts substratum and contains the acetic acid of 7.5g/L and the furtural of 3.5g/L.
Maize straw is cut into the segment of 2 cm, then adds water and regulate water content to be 40 % by weight; Be delivered in heat preservation pressure device, 195 DEG C, carry out steam explosion under 1.6 MPas, and collect steam explosion products, and the water of gained steam explosion products and 70 DEG C is uniformly mixed 30 minutes according to weight ratio 1:3, then add concentration be 72 % by weight sulphuric acid soln make the concentration dilution of sulfuric acid to 2 % by weight, stir 30 minutes at 30 DEG C, obtain dilute acid pretreatment product.Carry out enzymolysis to dilute acid pretreatment product, with the dry weight basis of the raw material of every gram of cellulose, the consumption of cellulase is 18 enzyme activity units, and temperature is 52 DEG C, and the time is 38 hours, and pH value is 4.0; Obtain enzymolysis solution.The content recording acetic acid in enzymolysis solution by liquid phase chromatography is 7.2 % by weight, and the content of furtural is 2.8 % by weight.
Testing example 1
The zymomonas mobilis of to be the zymomonas mobilis of CGMCCNo.9987 and deposit number by deposit number be CGMCCNo.10946 is inoculated in 3 kinds of RM after sterilizing respectively and adjusts in substratum, static gas wave refrigerator 72 hours under 30 DEG C of conditions, ferments and sampled every 12 hours after 24 hours.Utilize ultraviolet spectrophotometer to measure its growing state at 600nm place, result is as shown in table 1.
Table 1
As can be seen from the data of table 1, deposit number of the present invention is the tolerance that the zymomonas mobilis of CGMCCNo.10946 has to high acetic acid concentration and high furtural concentration simultaneously.
Testing example 2
The zymomonas mobilis of to be the zymomonas mobilis of CGMCCNo.9987 and deposit number by deposit number be CGMCCNo.10946 is inoculated in the enzymolysis solution that the preparation embodiment 1 after sterilizing obtains respectively and (is prepared by maize straw, the content of acetic acid is 7.2 % by weight, and the content of furtural is 2.8 % by weight) in.The concentration of inoculation is 10
4cFU/mL, at 30 DEG C in fermentor tank stir culture 40 hours.At 100 DEG C of distillation gained tunnings, gained distillation fraction second distillation at 78.3 DEG C can obtain ethanol, calculates alcohol yied according to the following formula.
Alcohol yied=100% × ethanol weight/stalk dry weight
Calculation result shows, under identical fermentation condition, deposit number of the present invention is that the alcohol yied of the zymomonas mobilis of the zymomonas mobilis of CGMCCNo.10946 reaches 28.5 % by weight, and deposit number is the alcohol yied only 14.5 % by weight of the zymomonas mobilis of CGMCCNo.9987, this shows, higher acetic acid and higher furtural while under existence condition, deposit number of the present invention is that the zymomonas mobilis of CGMCCNo.10946 can generate more ethanol.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be combined by any suitable mode, in order to avoid unnecessary repetition, the present invention illustrates no longer separately to various possible array mode.
In addition, also can carry out arbitrary combination between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (8)
1. a zymomonas mobilis, is characterized in that: the deposit number of this zymomonas mobilis (Zymomonasmobilis) is CGMCCNo.10946.
2. the purposes of zymomonas mobilis according to claim 1 in fermentative production of ethanol.
3. purposes according to claim 2, wherein, fermentative production of ethanol raw material used comprises at least one in agricultural crop straw, wood chip, bamboo, rice husk and Pericarppium arachidis hypogaeae.
4. the purposes according to Claims 2 or 3, wherein, the fermentation condition of described zymomonas mobilis comprises: fermenting process all or part of carries out under the acetic acid concentration being not less than 7g/L and under the furtural concentration being not less than 3g/L.
5. a method for fermentative production of ethanol, is characterized in that, the method comprises the steps: (1) will carry out steam explosion, dilute acid pretreatment and enzymolysis containing cellulosic raw material, obtain enzymolysis solution; (2) to inoculation fermentation bacterial classification in described enzymolysis solution, ferment, obtain fermented liquid; (3) separating alcohol from described fermented liquid; Described fermented bacterium is containing, for example zymomonas mobilis according to claim 1.
6. method according to claim 5, wherein, the described at least one comprised containing cellulosic raw material in agricultural crop straw, wood chip, bamboo, rice husk and Pericarppium arachidis hypogaeae.
7. the method according to claim 5 or 6, wherein, the condition of enzymolysis makes described enzymolysis solution contain acetic acid and furtural.
8. method according to claim 7, wherein, the condition of enzymolysis makes described enzymolysis solution contain the acetic acid being not less than 7g/L and the furtural being not less than 3g/L.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107893043A (en) * | 2017-12-26 | 2018-04-10 | 农业部沼气科学研究所 | A kind of zymomonas mobilis mutant strain of enduring high-concentration acetic acid and its application |
| CN108220187A (en) * | 2017-12-26 | 2018-06-29 | 农业部沼气科学研究所 | A kind of zymomonas mobilis mutant strain for being resistant to low ph value and its application |
| CN109971671A (en) * | 2019-02-14 | 2019-07-05 | 农业部沼气科学研究所 | Zymomonas mobilis, preparation method and the application of resisting high-concentration acetic acid and furtural simultaneously |
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| 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 |
| CN104845924A (en) * | 2015-04-01 | 2015-08-19 | 农业部沼气科学研究所 | Furfural-resistant zymomonas mobilis and preparation method and application thereof |
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| 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 |
| CN104845924A (en) * | 2015-04-01 | 2015-08-19 | 农业部沼气科学研究所 | Furfural-resistant zymomonas mobilis and preparation method and application thereof |
Non-Patent Citations (1)
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| ZONG-XIA SHUI ET AL.: "Adaptive laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors", 《APPL MICROBIOL BIOTECHNOL》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107893043A (en) * | 2017-12-26 | 2018-04-10 | 农业部沼气科学研究所 | A kind of zymomonas mobilis mutant strain of enduring high-concentration acetic acid and its application |
| CN108220187A (en) * | 2017-12-26 | 2018-06-29 | 农业部沼气科学研究所 | A kind of zymomonas mobilis mutant strain for being resistant to low ph value and its application |
| CN107893043B (en) * | 2017-12-26 | 2020-12-04 | 农业部沼气科学研究所 | Zymomonas mobilis mutant strain tolerant to high-concentration acetic acid and application thereof |
| CN109971671A (en) * | 2019-02-14 | 2019-07-05 | 农业部沼气科学研究所 | Zymomonas mobilis, preparation method and the application of resisting high-concentration acetic acid and furtural simultaneously |
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