CN108504584A - A kind of culture medium and application for being suitable for improving co-fermentation of glucose and xylose saccharomyces cerevisiae and being resistant to a variety of pretreatment mortifiers - Google Patents
A kind of culture medium and application for being suitable for improving co-fermentation of glucose and xylose saccharomyces cerevisiae and being resistant to a variety of pretreatment mortifiers Download PDFInfo
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Abstract
The present invention discloses a kind of culture medium for being suitable for improving co-fermentation of glucose and xylose saccharomyces cerevisiae and being resistant to a variety of pretreatment mortifiers, the culture medium and is added the yeast powder, peptone and xylose for being suitble to xylose metabolism strain growth and constitutes using containing there are many lignocellulosic pretreatment fluids of mortifier ingredient as matrix.The culture medium of the present invention is suitable for improving tolerance of the co-fermentation of glucose and xylose saccharomyces cerevisiae to a variety of pretreatment mortifiers, conducive to screening to the co-fermentation of glucose and xylose S. cervisiae of pretreatment mortifier tolerance enhancing, with it is existing add a kind of screening and culturing medium of mortifier ingredient merely compared with, culture medium of the invention is greatly improved the screening efficiency of high resistance Saccharomyces cerevisiae.
Description
Technical field
The present invention relates to a kind of screening and culturing medium and its applications, more particularly to one kind to be suitable for improving co-fermentation of glucose and xylose
Saccharomyces cerevisiae is resistant to culture medium and the application of a variety of pretreatment mortifiers, belongs to microorganisms technical field.
Background technology
Agricultural crop straw is arbitrarily abandoned and is burned, and the wasting of resources is caused, and the weight that haze weather is formed in recent years
Want one of reason.Lignocellulosic in stalk, which is converted to clean energy resource such as alcohol fuel, has broad prospect of application.Wooden fibre
The plain raw material of dimension is mainly made of cellulose, hemicellulose and lignin, and stable structure is complicated between each component.Utilize wood fibre
The microbial fermentation of the plain pretreatment for producing the chemicals such as alcohol fuel mainly including raw material, the enzymolysis of cellulose and sugar and etc.
[Sun Y,Cheng J.Hydrolysis of lignocellulosic materials for ethanol
production:a review.Bioresour Technol.2002,83(1):1-11.], bacterium is produced as traditional ethyl alcohol
Accharomyces cerevisiae is unable to the components such as DIRECT UTILIZATION OF CELLULOSE, hemicellulose, it is therefore desirable to be located in advance to lignocellulosic material
Reason and hydrolysis convert it into the monosaccharide that yeast can be utilized directly, and preconditioning technique is the committed step of lignocellulosic conversion.
Efficient cellulose preprocess method includes dilute acid pretreatment at present, (diluted alkaline pretreatment, lime are pre- for alkalescence pretreatment
Processing and alkaline hydrogen peroxide (Alkaline Hydrogen Peroxide, AHP) pretreatment etc.) and ammonia filament expansion
(Ammonia Fiber Expansion, AFEX) pretreatment etc..Fibrin reaction activity increases after dilute acid pretreatment, the degree of polymerization
(DP) decline, follow-up enzymolysis efficiency is provided, the hemicellulose in raw material can be dissolved, increases the accessibility of cellulose.But by
In at high temperature under high pressure, the hemicellulose of removing is degraded into the monosaccharide such as xylose first, and then these pentoses can further occur
Degradation forms furfural etc..Mortifier such as furfural, 5 hydroxymethyl furfural (HMF), acetic acid and the phenols chemical combination that dilute acid pretreatment generates
Object etc. can influence microorganism growth ferment and reduce alcohol getting rate [Almeida JRM, Modig T, Petersson A,B,Lidén G,GorwaGrauslund MF.Increased tolerance and conversion
of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae.J
Chem Technol Biotechnol.2007,82:340–349]；Alkaline hydrogen peroxide (alkaline hydrogen
Peroxide, AHP) mainly using the property of alkaline solution dissolved lignin, destruction lignin structure cuts off carbon water for pretreatment
Connection between compound and lignin causes the dissolution and removing of lignin and hemicellulose, this method to be more advantageous to then
The release of enzymolysis process sugar, and furans aldehydes mortifier [Saha BC, Cotta MA.Comparison of are not generated
pretreatment strategies for enzymatic saccharification and fermentation of
barley straw to ethanol.New Biotechnol.2010,27:10-15.], obtain higher sugar fermentation yield
[Banerjee G,Car S,Scott-Craig JS,Borrusch MS,Walton JD.Rapid optimization of
enzyme mixtures for deconstruction of diverse pretreatment/biomass feedstock
combinations.Biotechnol Biofuels.2010,3:22.], but will produce ferulic acid, p-Coumaric Acid, acetic acid and Na+Equal mortifiers [Banerjee G, Car S, Liu T, Williams DL, Meza SL, Walton JD, Hodge
DB.Scale-up and integration of alkaline hydrogen peroxide pretreatment,
enzymatic hydrolysis,and ethanolic fermentation.Biotechnol Bioeng.2012,109
(4):922-931.]；AFEX pretreatments are to decompose plant cellulose and hemicellulose using ammonia, and biomass is carried out with dense ammonia
It heating (about 100 DEG C), maintains unexpected valve opening release of pressure after a certain period of time, politics and law enter in biomass ammonia due to pressure reduces suddenly,
To make cellulose be swollen, a variety of damage saccharomyces cerevisiae xyloses such as acetamide, ferulic amide, tonka-bean amide are also will produce
Inhibition small molecule [Chundawat SP, Vismeh R, Sharma LN, Humpula JF, the da Costa Sousa of metabolism
L,Chambliss CK,Jones AD,Balan V,Dale BE.Multifaceted characterization of cell
wall decomposition products formed during ammonia fiber expansion(AFEX)and
dilute acid based pretreatments.Bioresour Technol.2010,101(21):8429–8438.].This
A little mortifiers may inhibit the growth of microorganism in fermentation process, so influence downstream metabolic [van der Pol EC,
Bakker RR,Baets P,Eggink G.By-products resulting from lignocellulose
pretreatment and their inhibitory effect on fermentations for(bio)chemicals
and fuels.Appl Microbiol Biotechnol.2014,98(23):9579-9593.], this is also limitation fiber second
One of the reason of alcohol industrialized production [Palmqvist E, Hahn- B.Fermentation of
lignocellulosic hydrolysates.I:inhibition and
detoxification.Bioresour.Technol.2000,74(1):17–24.]。
Saccharomyces cerevisiae is traditional alcohol fermentation bacterial strain, (such as Anaerobic compared with other potential ethyl alcohol production bacterial strains
bacteria、Escherichia coli、Zymomonas mobilis、Scheffersomyces stipitis、
Filamentous fungi etc.), there is higher glucose fermentation alcohol getting rate and to being deposited under a variety of ethanol industry working conditions
Restraining factors there is preferable tolerance, therefore be also more suitable for two Replacing fuels using lignocellulose biomass as raw material
The production of ethyl alcohol.However, with using starchy material produce ethyl alcohol compared with, production two Replacing fuel ethyl alcohol saccharomyces cerevisiae face it is all
Mostly new difficulty.Saccharomyces cerevisiae natural first is unable to fermenting xylose production ethyl alcohol, is secondly deposited in lignocellulosic material hydrolyzate
In the compound of inhibition effect that largely saccharomyces cerevisiae is grown and fermented.Therefore, it is built by means such as genetic engineerings high
It is the production of two Replacing fuel ethyl alcohol economic scales to imitate common fermentation glucose and xylose and the bacterial strain with higher mortifier tolerance
Prerequisite.But a variety of mortifiers are extremely complex to the mechanism of action of Wine brewing yeast strain and at present to yeast in hydrolyzate
The response mechanism understanding of mortifier tolerance is not thorough enough, resistance to effectively improve its mortifier by the method for rationality genetic engineering
Difficulty is brought by property.Therefore, obtain the industrial yeast of the energy common fermentation glucose and xylose that high mortifier tolerance improves for
The development and popularization of cellulosic ethanol are most important, and being utilized to lignocellulosic Efficient Conversion has good industrial applications latent
Power and development prospect.
Currently, using metabolic engineering and evolution engineering, grape can efficiently be utilized altogether by having been obtained by genetic engineering means
The bacterial strain of sugar and xylose production ethyl alcohol, (the deposit number CGMCC of the starting strain as used in the embodiment of the present invention
No.11331), fermentation mixed sugar (80g L-1Glucose and 40g L-1Xylose), whole sugars, sugar alcohol can be exhausted in 16h
Conversion ratio is 0.475g g-1, reach 93% [Li H, Shen, Wu M, Hou J, Jiao C, Li Z, Liu X, Bao of theoretical value
X.Engineering a wild-type diploid Saccharomyces cerevisiae strain for second-
generation bioethanol production.Bioresour Bioprocess.2016,3(1):51.].But it is right
The tolerance of mortifier is still to be improved in hydrolyzate.Currently, most researchs for improving yeast tolerance mainly utilize and contain
The yeast culture medium of single component mortifier is screened, and the bacterial strain that final acquired tolerance improves is past in non-detoxification hydrolyzate
Toward or cannot preferably grow.The existing saccharomyces cerevisiae improved to mortifier tolerance is often only to a kind of or certain is a kind of
Mortifier shows certain tolerance, it is difficult to obtain while being resistant to the saccharomyces cerevisiae of a few class mortifiers.In consideration of it, exploitation is a kind of
The efficient raising co-fermentation of glucose and xylose saccharomyces cerevisiae that is suitable for is resistant to the culture mediums of a variety of pretreatment mortifiers with important meaning
Justice contributes to the screening efficiency for quickly improving high resistance Saccharomyces cerevisiae.It is also rarely seen related efficient both at home and abroad at present through retrieval
The report of a variety of pretreatment mortifier culture mediums is resistant to suitable for improving co-fermentation of glucose and xylose saccharomyces cerevisiae.
Invention content
In view of the deficiencies of the prior art, the problem to be solved in the present invention is to provide one kind and is sent out altogether suitable for improving glucose and xylose
Ferment saccharomyces cerevisiae is resistant to culture medium and the application of a variety of pretreatment mortifiers.
The culture of the present invention for being suitable for improving co-fermentation of glucose and xylose saccharomyces cerevisiae and being resistant to a variety of pretreatment mortifiers
Base, the culture medium and are added and are suitble to xylose generation using containing there are many lignocellulosic pretreatment fluids of mortifier ingredient as matrix
The yeast powder, peptone and xylose for thanking to strain growth are constituted；
It is characterized in that：
The lignocellulosic pretreatment fluid is that dilute acid pretreatment lignocellulosic or alkaline hydrogen peroxide (AHP) are located in advance
Manage the lignocellulosic pretreatment fluid that pH is 4.5~5.5 made from lignocellulosic；The addition yeast powder, peptone and wood
Sugar amount be：8~12g/L yeast powders, 18~22g/L peptones, 15~30g/L xyloses.
The above-mentioned co-fermentation of glucose and xylose saccharomyces cerevisiae that is suitable for improving is resistant in the culture medium of a variety of pretreatment mortifiers：
Preferably culture medium prescription is：Using lignocellulosic pretreatment fluid as matrix, the amount of addition yeast powder, peptone and xylose is：
10g/L yeast powders, 20g/L peptones, 20g/L xyloses.
Wherein,
The method that lignocellulosic pretreatment fluid is made in the dilute acid pretreatment lignocellulosic is：Preparing mass fraction is
Lignocellulosic is crushed to 20~80 mesh and and cooking liquor by 1.0% dilution heat of sulfuric acid as the cooking liquor of dilute acid pretreatment
With feed liquid weight ratio 1:10 are uniformly mixed, and are then placed in cylinder of steel and seal, then cylinder of steel is put into digester, and into digester
Add water, water is made the cylinder of steel upper end not being put in digester, insulation reaction at a temperature of digester sealing is placed on 170 ± 5 DEG C
25~30min after reaction immediately takes out cylinder of steel, and being put into makes the temperature of cylinder of steel drop to room temperature in cold water, conventional method of uncapping
It is separated by solid-liquid separation, gained liquid is lignocellulosic pretreatment fluid made from dilute acid pretreatment lignocellulosic；
The method that lignocellulosic pretreatment fluid is made in alkaline hydrogen peroxide (AHP) preprocessing lignocellulose is：
Lignocellulosic is crushed to 20~80 mesh, is added in NaOH solution, stirs evenly, H is then added2O2It stirs evenly, makes system
NaOH dosages are 0.1g/g lignocellulosics, H in the mixed reaction solution obtained2O2Addition is 0.0999g/g lignocellulosics, material
Liquor ratio is 0.15g/ml, adjusts pH to 11.5~11.7, is subsequently placed under the conditions of 30 ± 2 DEG C of temperature, 200 ± 20rpm of rotating speed, is protected
Temperature reaction 24~30 hours, conventional method is separated by solid-liquid separation after reaction, and gained liquid is alkaline peroxide impregnation wood fibre
Lignocellulosic pretreatment fluid made from element.
Above-mentioned lignocellulosic material is preferably：Corncob, maize straw, wheat stalk, cotton stalk, sweet tea after crushing
Broomcorn straw, rice straw, rape stalk, bagasse, sweet potato dregs, sawdust, waste paper and/or switchgrass.
At least contain mortifier in lignocellulosic pretreatment fluid made from above-mentioned dilute acid pretreatment lignocellulosic ---
Furfural, hydroxymethylfurfural and acetic acid.
At least contain suppression in lignocellulosic pretreatment fluid made from above-mentioned alkaline peroxide impregnation lignocellulosic
Object processed --- ferulic acid and p-Coumaric Acid.
The culture of the present invention for being suitable for improving co-fermentation of glucose and xylose saccharomyces cerevisiae and being resistant to a variety of pretreatment mortifiers
Base is being screened to the application in the co-fermentation of glucose and xylose S. cervisiae of pretreatment mortifier tolerance enhancing.
The culture of the present invention for being suitable for improving co-fermentation of glucose and xylose saccharomyces cerevisiae and being resistant to a variety of pretreatment mortifiers
Base is improving co-fermentation of glucose and xylose saccharomyces cerevisiae to the application in a variety of pretreatment mortifier tolerances.
Invention additionally discloses above-mentioned culture mediums to be highly suitable as the tolerance pretreatment of co-fermentation of glucose and xylose saccharomyces cerevisiae
The screening and culturing medium of mortifier is greatly improved the screening efficiency of high resistance Saccharomyces cerevisiae.
Existing screening and culturing medium generally comprises single component mortifier, and the bacterial strain that final acquired tolerance improves is in non-detoxification
It often still cannot preferably be grown in hydrolyzate.The existing saccharomyces cerevisiae improved to mortifier tolerance is often only to one
Kind or certain a kind of mortifier show certain tolerance, it is difficult to the saccharomyces cerevisiae of a few class mortifiers is obtained while being resistant to, in view of
This prepares the culture that co-fermentation of glucose and xylose bacterial strain can be grown directly using non-detoxification pretreatment hydrolyzate as matrix
Base is the key that the screening efficiency for improving high patience co-fermentation of glucose and xylose saccharomyces cerevisiae.
The present invention carries out change appropriate by component to conventional screening and culturing medium and content, by experiment screening and tests
Card, the final each component and optimum content for determining culture medium, so as to complete the present invention.The experimental results showed that training of the invention
Foster base is suitable as improving screening training of the co-fermentation of glucose and xylose saccharomyces cerevisiae to the tolerance of a variety of pretreatment mortifiers
Base is supported, the screening efficiency of high resistance Saccharomyces cerevisiae is greatly improved.
Description of the drawings
Fig. 1 is starting strain in embodiment 2 on YPX tablets, (YPX+AHP) tablet and (pretreatment of YPX+ acidity) tablet
Growing state.
Wherein：A：Growing state of the starting strain on YPX tablets and (YPX+AHP) tablet；B：Starting strain in YPX and
Growing state on (pretreatment of YPX+ acidity) tablet.
Fig. 2 is growth correlation curve of the starting strain in YPX and (YPX+AHP) fluid nutrient medium in embodiment 2.
Fig. 3 is starting strain in embodiment 3 and utilizes atmospheric pressure at room plasma (Atmospheric and Room
Temperature Plasma, ARTP) gradient life of each number bacterial strain of device mutagenesis gained on YPX and (YPX+AHP) tablet
Long experiment.
Fig. 4 is starting strain in embodiment 3 and utilizes atmospheric pressure at room plasma (Atmospheric and Room
Temperature Plasma, ARTP) growth of device mutagenesis No. 1 bacterial strain of gained in YPX and (YPX+AHP) fluid nutrient medium
Situation correlation curve.
Fig. 5 is starting strain in embodiment 4 and utilizes atmospheric pressure at room plasma (Atmospheric and Room
Temperature Plasma, ARTP) life of device mutagenesis No. 1 bacterial strain of gained on YPX and (YPX+ dilute acid pretreatments) tablet
Long situation.
Specific implementation mode
Technical scheme of the present invention is described further with reference to example, it should be understood that although listed embodiment is enumerated
The preferred embodiment of the present invention, but listed specific embodiment is merely to preferably illustrate the present invention and list, but this hair
Bright protected range is not limited within the scope of embodiments.
Embodiment 1：Containing there are many screening and culturing medium preparations of pretreatment mortifier
It is as follows：
(1) maize straw pretreatment fluid is made in dilute acid pretreatment maize straw：With Sulphuric acid mass fraction be 1.0% it is dilute
Sulfuric acid solution, as the cooking liquor of dilute acid pretreatment, by maize straw (being crushed to 20~80 mesh) and cooking liquor with solid-liquid ratio 1:
10 are uniformly mixed and are put into the small cylinder of steel of 1.5L, and canister is put into digester after sealing, and is added into digester a certain amount of
Water, so that water the canister upper end not being put in digester, 170 DEG C of required reaction temperature be set after digester is sealed and is opened
Begin to heat, reaches insulation reaction 25min after temperature.Canister is taken out immediately after reaction, is put into cold water and makes canister
Temperature drops to room temperature.Pretreatment fluid in canister is separated by solid-liquid separation, liquid is maize straw pretreatment fluid, wherein there are many containing
Mortifier, such as furfural, hydroxymethylfurfural, acetic acid etc..
(2) maize straw pretreatment fluid is made in alkaline hydrogen peroxide (AHP) pretreatment maize straw：By maize straw (powder
It is broken to 20~80 mesh) it is added in NaOH solution, it stirs evenly, H is then added2O2It stirs evenly, makes mixed reaction solution obtained
Middle NaOH dosages are 0.1g/g maize straws, H2O2Addition be 0.0999g/g maize straws, solid-liquid ratio 0.15g/ml, adjust
PH to 11.5~11.7 is saved, then under the conditions of 30 DEG C of temperature, rotating speed 200rpm, insulation reaction 24 hours, solid-liquid point after reaction
From liquid is maize straw pretreatment fluid, wherein containing there are many mortifiers, such as ferulic acid, p-Coumaric Acid.
(3) by step (1) and (2) two kinds of maize straw pretreatment fluids, pH is measured, NaOH or sulfuric acid tune are then utilized respectively
PH to 4.5~5.5 is prepared using the pretreatment hydrolyzate for mixing up pH value containing the two kinds of pretreating process generations of diluted acid and AHP
Mortifier, culture medium for screening patience yeast, culture medium prescription are：Yeast powder 10g/L, peptone 20g/L, xylose
Agar powder 20g/L is added in 20g/L, solid medium.
Embodiment 2：Influence of the culture medium of hydrolyzate mortifier to Yeast Growth is pre-processed containing diluted acid and AHP
It is as follows：
(1) dilution spread is tested：It will be by metabolic engineering, the efficient Co metabolism glucose and xylose of energy wine brewing ferment
Starting strain of female industrial strain (deposit number is CGMCC No.11331) as this experiment, is connected to 5mL YPD (YPD
Culture medium prescription：Yeast powder 10g/L, peptone 20g/L, glucose 20g/L.) in, it is incubated overnight, takes 1ml bacteria suspensions,
8000r/min is centrifuged, and removes supernatant, and with 1mL sterile water wash suspension thallines, supernatant is removed in centrifugation, adjusts OD600=1.0 or so, it is dilute
1000 times are released, 100 μ L is taken to be applied to YPX tablets, (YPX+ dilute acid pretreatments) tablet and (YPX+AHP) tablet, 30 DEG C of cultures 3~
4 days, strain growth situation is observed, is taken pictures, the results showed that (as shown in Figure 1A and B), after measured, starting strain is on YPX tablets
Well-grown, on (YPX+ dilute acid pretreatments) tablet growing poor, only fragmentary bacterium colony grows, in (YPX+AHP) tablet
Upper growing state is also obviously poorer than on YPX tablet.
(2) culture monitoring growing state：Starting strain is connected in 5mLYPD, is incubated overnight, 1ml bacteria suspensions are taken,
8000r/min is centrifuged, and removes supernatant, with fresh culture medium adjustment starting OD600=1.0 or so, it is inoculated in YPX and (YPX+ respectively
AHP) in fluid nutrient medium, 10 times is diluted, starting OD is made600=0.1 or so, 30 DEG C of continuous cultures 30 hours or so, using it is complete from
Dynamic growth curve instrument monitors growing state (as shown in Figure 2) automatically.
Embodiment 3：Saccharomyces cerevisiae tolerance AHP, which is improved, using ARTP induced-mutation techniques pre-processes corn stalk hydrolysis mortifier
Ability
It is as follows：
(1) starting strain activates：Starting strain is seeded to YPD tablets, 30 DEG C are inverted culture 2~3 days, are then seeded to
In YPD liquid, shaken cultivation 12h~24 hour, control cell concentration OD in bacterium solution to fresh YPD of transferring600=0.1,30 DEG C
It cultivates shake culture a few hours, control thalline final concentration OD600Within=1.0, as ARTP mutagenesis starting strains.
(2) ARTP mutagenesis：1mL bacterium solutions are taken, suspension is in 1.5mL EP pipes, 8000r/min, centrifuges 2min, discards supernatant
Liquid.After brine 2 times, cell concentration is made 10 in dilution6~107Between bacteria suspension, take 10 μ L bacteria suspensions uniform
It is coated on sterile slide surface.Then slide glass is placed on the microscope carrier of ARTP mutation breeding systems, is placed in ARTP mutation breedings
Mutagenic treatment is carried out in instrument, mutagenesis instrument working gas is 99.99% high-purity helium, and radio-frequency power 100W, helium gas flow are
10SLM, processing time are set as 80s and 120s, and the distance between sample and plasma emission source are 3mm.
(3) it is cultivated after mutagenesis：It takes 1mL sterile waters to wash down the saccharomycete after mutagenesis, is coated on containing highly concentrated after appropriateness dilution
It spends on (YPX+AHP) tablet of AHP pretreatment hydrolyzate mortifiers, 30 DEG C are inverted culture 3~4 days, and mutation time is the flat of 80s
There are 4 bacterium colony growths on plate, there are 29 bacterium colonies to grow on the tablet that mutation time is 120s, in general, mutagenesis 120s ratios 80s
Effect is good, and the bacterium colony grown is more, and bacterium colony is larger.
(4) the bacterial strain primary dcreening operation of resistance to AHP pretreatments hydrolyzate mortifier：It selects well-grown on (YPX+AHP) tablet
Starting strain difference before bacterium colony (selecting 2 on the tablet of mutagenesis 80s, 7 are selected on the tablet of mutagenesis 120s) and mutagenesis
It is inoculated into the YPD of 5mL and cultivates, 30 DEG C are incubated overnight to exponential phase, and thalline were collected by centrifugation, with 0.5mL sterile water wash
Sterile water suspension thalline is used in suspension thalline, centrifugation again, is placed it in 30 DEG C of 10~12h of placement, is prepared resting cell.It adjusts
Bacterial concentration makes bacteria suspension OD600=1.0 or so, 10 times of gradient dilutions are carried out, take 1~4 μ L bacteria suspensions drops flat in YPX successively
Plate and (YPX+AHP) tablet, 30 DEG C are cultivated 3~4 days, are observed strain growth situation, are taken pictures, gradient growth experiment the result shows that
The growth on YPX tablets and (YPX+AHP) tablet of (as shown in Figure 3), only No. 1 bacterial strain shows clear superiority.
(5) the bacterial strain secondary screening of resistance to AHP pretreatments hydrolyzate mortifier：No. 1 bacterium that will be grown fine on (YPX+AHP) tablet
Strain, the starting strain before mutagenesis are inoculated into the YPD of 5mL and cultivate respectively, and 30 DEG C are incubated overnight to exponential phase, with fresh
Culture medium adjustment starting OD600=1.0 or so, it is inoculated in respectively in YPX and (YPX+AHP) fluid nutrient medium, dilutes 10 times, make
Originate OD600=0.1 or so, 30 DEG C of continuous cultures 16 hours or so, growth feelings are monitored using full-automatic growth curve instrument automatically
Condition, the results showed that (as shown in Figure 4), although No. 1 bacterial strain growing state in (YPX+AHP) fluid nutrient medium is still not so good as
In YPX, but compared with the starting strain before mutagenesis, No. 1 bacterial strain is either in YPX, or in (YPX+AHP) fluid nutrient medium
In, growing to have largely improves, and the time for reaching stationary phase shortens.Genetic stability is tested：Secondary screening is verified and is obtained
The bacterial strains of resistance to AHP pretreatment hydrolyzate mortifiers pass through setting-out repeatedly, detach single bacterium colony, then 8~10 generation of continuous passage, bacterium
The holding of strain is resistant to mortifier ability.
(6) shake flat experiment is tested：By the starting strain before No. 1 bacterial strain, mutagenesis growing fine on (YPX+AHP) tablet point
Be not inoculated into the YPD of 5mL and cultivate, 30 DEG C are incubated overnight to exponential phase, bacterium solution is inoculated in respectively 40mL YPX and
(YPX+AHP) in fluid nutrient medium, adjustment starting OD600=0.1 or so, 30 DEG C of continuous cultures, by amplify volume of culture,
Test vector generation for testing IC under pressure condition is selected, compared with starting strain, the time of No. 1 bacterial strain being grown in YPX into stationary phase carries
Preceding 1.5h, the growth in (YPX+AHP) fluid nutrient medium improve 20% or so.
Embodiment 4：Tolerance feelings of the bacterial strain of resistance to AHP pretreatments hydrolyzate mortifier to dilute acid pretreatment hydrolyzate mortifier
Condition is tested
It is as follows：
No. 1 bacterial strain of the resistance to AHP pretreatments hydrolyzate mortifier of 4 gained of embodiment and starting strain are inoculated into the YPD of 5mL
In, 30 DEG C are incubated overnight, and take 1ml bacteria suspensions, and supernatant is removed in 8000r/min centrifugations, with 1mL sterile water wash suspension thallines, from
The heart removes supernatant, adjusts OD600=1.0 or so, 1000 times are diluted, 100 μ L bacterium solutions is taken to be applied to YPX tablets and (YPX+ diluted acids are pre-
Processing) on tablet, 30 DEG C are cultivated 3~4 days, are observed strain growth situation, are taken pictures, the results showed that (as shown in Figure 5) and goes out bacterium germination
Strain is compared, and growth of No. 1 bacterial strain on (YPX+ dilute acid pretreatments) tablet obviously improves, and illustrates after ARTP mutagenesis, gained 1
Number bacterial strain improves the tolerance of dilute acid pretreatment hydrolyzate mortifier.Prove that the raising glucose and xylose of the present invention that is suitable for is total to
Ferment wine brewing yeast is resistant to the screening efficiency that a variety of culture mediums for pre-processing mortifiers are greatly improved high resistance Saccharomyces cerevisiae.
Claims (8)
1. a kind of culture medium for being suitable for improving co-fermentation of glucose and xylose saccharomyces cerevisiae and being resistant to a variety of pretreatment mortifiers, the training
Foster base and is added and is suitble to xylose metabolism bacterial strain life using containing there are many lignocellulosic pretreatment fluids of mortifier ingredient as matrix
Long yeast powder, peptone and xylose is constituted；
It is characterized in that：
The lignocellulosic pretreatment fluid is dilute acid pretreatment lignocellulosic or alkaline hydrogen peroxide (AHP) pretreatment wood
The lignocellulosic pretreatment fluid that pH made from matter cellulose is 4.5~5.5；The addition yeast powder, peptone and xylose
Amount is：8~12g/L yeast powders, 18~22g/L peptones, 15~30g/L xyloses.
2. according to claim 1 be suitable for improving a variety of pretreatment mortifiers of co-fermentation of glucose and xylose saccharomyces cerevisiae tolerance
Culture medium, it is characterised in that：The culture medium using lignocellulosic pretreatment fluid as matrix, addition yeast powder, peptone and
The amount of xylose is：10g/L yeast powders, 20g/L peptones, 20g/L xyloses.
3. according to claim 1 or 2 be suitable for improving a variety of pretreatments suppressions of co-fermentation of glucose and xylose saccharomyces cerevisiae tolerance
The culture medium of object processed, which is characterized in that
The method that lignocellulosic pretreatment fluid is made in the dilute acid pretreatment lignocellulosic is：Preparing mass fraction is
Lignocellulosic is crushed to 20~80 mesh and and cooking liquor by 1.0% dilution heat of sulfuric acid as the cooking liquor of dilute acid pretreatment
With feed liquid weight ratio 1:10 are uniformly mixed, and are then placed in cylinder of steel and seal, then cylinder of steel is put into digester, and into digester
Add water, water is made the cylinder of steel upper end not being put in digester, insulation reaction at a temperature of digester sealing is placed on 170 ± 5 DEG C
25~30min after reaction immediately takes out cylinder of steel, and being put into makes the temperature of cylinder of steel drop to room temperature in cold water, conventional method of uncapping
It is separated by solid-liquid separation, gained liquid is lignocellulosic pretreatment fluid made from dilute acid pretreatment lignocellulosic；
The method that lignocellulosic pretreatment fluid is made in alkaline hydrogen peroxide (AHP) preprocessing lignocellulose is：It will be wooden
Matter cellulose powder is broken to 20~80 mesh, is added in NaOH solution, stirs evenly, and H is then added2O2It stirs evenly, makes obtained
NaOH dosages are 0.1g/g lignocellulosics, H in mixed reaction solution2O2Addition is 0.0999g/g lignocellulosics, solid-liquid ratio
For 0.15g/ml, pH to 11.5~11.7 is adjusted, is subsequently placed under the conditions of 30 ± 2 DEG C of temperature, 200 ± 20rpm of rotating speed, heat preservation is anti-
It answers 24~30 hours, conventional method is separated by solid-liquid separation after reaction, and gained liquid is alkaline peroxide impregnation lignocellulosic system
The lignocellulosic pretreatment fluid obtained.
4. according to claim 3 be suitable for improving a variety of pretreatment mortifiers of co-fermentation of glucose and xylose saccharomyces cerevisiae tolerance
Culture medium, it is characterised in that：The lignocellulosic material be crush after corncob, maize straw, wheat stalk, cotton
Stalk, sweet sorghum stalk, rice straw, rape stalk, bagasse, sweet potato dregs, sawdust, waste paper and/or switchgrass.
5. according to claim 3 be suitable for improving a variety of pretreatment mortifiers of co-fermentation of glucose and xylose saccharomyces cerevisiae tolerance
Culture medium, it is characterised in that：At least contain in lignocellulosic pretreatment fluid made from the dilute acid pretreatment lignocellulosic
There is mortifier --- furfural, hydroxymethylfurfural and acetic acid.
6. according to claim 3 be suitable for improving a variety of pretreatment mortifiers of co-fermentation of glucose and xylose saccharomyces cerevisiae tolerance
Culture medium, it is characterised in that：Lignocellulosic pretreatment fluid made from the alkaline peroxide impregnation lignocellulosic
In at least contain mortifier --- ferulic acid and p-Coumaric Acid.
7. being suitable for improving co-fermentation of glucose and xylose saccharomyces cerevisiae described in claims 1 or 2 is resistant to a variety of pretreatment mortifiers
Culture medium is being screened to the application in the co-fermentation of glucose and xylose S. cervisiae of pretreatment mortifier tolerance enhancing.
8. being suitable for improving co-fermentation of glucose and xylose saccharomyces cerevisiae described in claims 1 or 2 is resistant to a variety of pretreatment mortifiers
Culture medium is improving co-fermentation of glucose and xylose saccharomyces cerevisiae to the application in a variety of pretreatment mortifier tolerances.
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