CN110358690A - A kind of S. cervisiae of resistance to mortifier and its selection and application - Google Patents
A kind of S. cervisiae of resistance to mortifier and its selection and application Download PDFInfo
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Abstract
The present invention relates to a kind of S. cervisiae of resistance to mortifier and its selection and applications, the saccharomyces cerevisiae of the resistance to mortifier, it is named as ZR/SC-UV-2, classification naming are as follows: saccharomyces cerevisiae (Saccharomyces cerevisiae), China Committee for Culture Collection of Microorganisms's common micro-organisms center was preserved on 06 13rd, 2019, deposit number are as follows: CGMCC No.17924, preservation address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.Its selection are as follows: the S. cervisiae of xylose can be utilized as starting strain, obtain the S. cervisiae of the resistance to mortifier by Uv-induced screening.The condition of the ultraviolet mutagenesis are as follows: under the conditions of being protected from light, 30~600s is irradiated under the irradiation distance of 15~20cm using 15~20W ultraviolet lamp, carries out ultraviolet mutagenesis.Compared with prior art, S. cervisiae of the present invention is strong to the mortifier tolerance in cellulosic hydrolysate, high to the degradation rate of xylose, fermentation time is short, and the industrialization production of ethyl alcohol is prepared suitable for agricultural stalk.
Description
Technical field
The present invention relates to a kind of S. cervisiaes, S. cervisiae and its breeding side more particularly, to a kind of resistance to mortifier
Method and application.
Background technique
Alcohol fuel be by living resources by a series of physics, biology, chemical change process and obtain, be a kind of clean
Only, renewable, the not clean emission of carbon of production process traffic fuel is the maximum biology transport fuel of current usage amount.Wherein, with
Agricultural crop straw etc. is that the lignocellulose-like biomass of representative has the characteristics that cheap, abundance, is fuel ethanol industrial
Large-scale development generally acknowledged raw material sources (Hossain Zabed, Jaya Narayan Sahu, Akter Suely, et
al.Bio-ethanol production from renewable sources:current perspectives and
technological progress.Renewable and Sustainable Energy Reviews,2017,71:475-
501.)。
Lignocellulosic material is monosaccharide, mainly glucose and xylose, microorganism hair by pretreatment and hydrolysis
Ferment monosaccharide can produce ethyl alcohol.Saccharomyces cerevisiae has many advantages, such as outstanding producing and ethanol ability and environment resistance, is widely used in ethyl alcohol
Production, but saccharomyces cerevisiae be used for cellulosic material fuel ethanol production, two major issues are faced, first is that the hair of xylose
Ferment, second is that the tolerance of various mortifiers.Lignocellulosic material generates various by-products in pretreatment and hydrolytic process
Object mainly includes weak acid class (formic acid, acetic acid and levulic acid), furans (furfural, 5~hydroxymethylfurfural) and phenols (cloves
Aldehyde, vanillic aldehyde and phenol etc.), their presence can seriously hinder cell growth and the alcohol fermentation of Wine brewing yeast strain
(Palmqvist E,Hahn B.Fermentation of Lignocellulosic hydrolysates.I:
Inhibition and detoxification.Bio-resource Technology, 2000,74 (1): 17-24.), therefore
The mortifier tolerance for improving saccharomyces cerevisiae is particularly important to the production of fuel ethanol industry metaplasia.
Therefore, it is necessary to consider that there are various mortifiers may inhibit utilization of the bacterial strain to xylose in cellulosic hydrolysate, need
It is further improved and is improved saccharomyces cerevisiae performance, improves its tolerance to mortifier, to construct a kind of resistance to mortifier
Saccharomyces cerevisiae.
Summary of the invention
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of wine of resistance to mortifier
Brewer yeast bacterium and its selection and application.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of S. cervisiae of resistance to mortifier, is named as ZR/SC-UV-2, classification naming are as follows: saccharomyces cerevisiae
(Saccharomyces cerevisiae) was preserved in China Committee for Culture Collection of Microorganisms on 06 13rd, 2019
Common micro-organisms center, deposit number are as follows: CGMCC No.17924, preservation address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number.
In an embodiment of the invention, the mortifier includes weak acid substance, furans or phenolic material
One or more of matter.
In an embodiment of the invention, the weak acid substance includes formic acid, acetic acid and levulic acid etc..
In an embodiment of the invention, the furans include furfural, 5 hydroxymethyl furfural etc..
In an embodiment of the invention, the phenolic substances includes syringaldehyde, vanillic aldehyde and phenol etc..
In an embodiment of the invention, the mortifier is mortifier present in ligno-cellulose hydrolysate.
The present invention also provides a kind of selections of the S. cervisiae of resistance to mortifier, for the wine brewing can utilize xylose
Saccharomycete is starting strain, obtains the S. cervisiae of resistance to mortifier by Uv-induced screening.
It in an embodiment of the invention, can be bacterial strain ZR-1, bacterial strain ZR-1 using the S. cervisiae of xylose
Classification naming are as follows: saccharomyces cerevisiae (Saccharomyces cerevisiae) was preserved in the micro- life of China on 07 08th, 2019
Object culture presevation administration committee common micro-organisms center, deposit number are as follows: CGMCC No.18090, preservation address are as follows: Beijing
The institute 3 of Chaoyang District North Star West Road 1.
In an embodiment of the invention, the condition of the ultraviolet mutagenesis are as follows: under the conditions of being protected from light, using 15~20W
Ultraviolet lamp irradiates 30~600s under the irradiation distance of 15~20cm, carries out ultraviolet mutagenesis.
In an embodiment of the invention, in irradiation process, the spore of starting strain is stirred, stirring rate
For 50~100r/min, during this, the Conidia persistence of bacterial strain is 5~10%.
In an embodiment of the invention, the selection of the S. cervisiae of the resistance to mortifier specifically include with
Lower step:
(1) starting strain primary dcreening operation: the S. cervisiae of xylose can be utilized as starting strain, starting strain is being coated with
It is activated on YPD plate, the activated starting strain of a ring is taken to be inoculated in YPD culture medium, stirred, carried out starting strain and train in advance
It supports；
(2) ultraviolet mutagenesis breeding: taking the cultured bacterial strain in YPD culture medium, and the training containing EDTA aqueous solution is added
It supports in ware, while stirring liquid, using ultraviolet light irradiation culture dish, then will be coated on suppression after liquid diluting in culture dish
On object plating medium processed；
(3) the mortifier plating medium having been coated with the separation screening of the S. cervisiae of resistance to mortifier: is protected from light culture
3-4d；On the mortifier plating medium, several single colonies that growth is fast and bacterium colony is big of preliminary picking are inoculated in inhibition
Fermented and cultured in object fermentation medium detects Xylose Content；Wood can efficiently be utilized by filtering out several according to xylose utilization situation
The bacterial strain of sugar；
(4) hydrolyzate fermentation secondary screening: the bacterial strain that step (3) screening obtains is inoculated into seed culture medium respectively, takes part
Bacterium solution, which is inoculated into hydrolysate fermentation culture medium, ferments, and detects Xylose Content, selects the highest bacterial strain of xylose utilization rate, obtain
To the S. cervisiae of the resistance to mortifier.
In an embodiment of the invention, the YPD culture medium is collective media, the composition of mass percentage
Are as follows: glucose 2%, peptone 2%, yeast extract 1%, surplus are water, and pH is 4.8~5.0；Solid plate need to be added additionally
2% agar.
In an embodiment of the invention, the molar concentration of the EDTA aqueous solution is 0.5~0.8mol/L.
In an embodiment of the invention, the mortifier plating medium includes the group of following mass percentage
Point: xylose 3.0~5.0%, peptone 1.0~3.0%, yeast extract 1.0~2.0%, mortifier 0.15~0.60%, agar
1.5~2.0%, surplus is water, and the culture medium is through 105~120 DEG C of high temperature, and sterilize 10~60min.
In an embodiment of the invention, the mortifier fermentation medium includes the group of following mass percentage
At: glucose 6.0~9.0%, xylose 3~5%, peptone 1.0~3.0%, yeast extract 1.0~2.0%, mortifier 0.15~
0.60%, surplus is water, and the culture medium is through 105~120 DEG C of high temperature, and sterilize 10~60min.
In an embodiment of the invention, the seed culture medium includes the composition of following mass percentage: Portugal
Grape sugar 3.0~5.0%, xylose 2.0~4.0%, peptone 1.0~3.0%, yeast extract 1.0~2.0%, surplus are water, pH
It is 4.0~5.5；
In an embodiment of the invention, the hydrolysate fermentation culture medium passes through in ligno-cellulose hydrolysate
Addition peptone and yeast extract are prepared, and the mass percentage of the peptone is 1.0~3.0%, the quality of yeast extract
Percentage composition is 1.0~2.0%, which sterilizes through 105~120 DEG C of high temperature, 10~60min.
In an embodiment of the invention, in step (1), the activation temperature of the starting strain is 28~32 DEG C,
Preferably 30 DEG C, activation time be 20~for 24 hours；Starting strain preculture temperature be 28~32 DEG C, pre-incubation time be 12~
18h, stirring rate are 120~180r/min.
In an embodiment of the invention, in step (2), cultured bacterial strain is taken, EDTA aqueous solution, bacterium is added
The volume ratio of strain and EDTA aqueous solution is 1:4~8, preferably 1:5.
In an embodiment of the invention, the irradiation distance of the ultraviolet lamp is 15~20cm, irradiation time 30
~600s, the power of ultraviolet lamp are 15~20W, and stirring rate is 50~100r/min in irradiation process, during this, bacterial strain
Cell survival rate is 5~10%.Present invention optimizes the intensity of ultraviolet light irradiation and times, because this will affect the prominent of cell
Become, under irradiation condition of the invention, desired bacterial strain can be obtained by screening.
In an embodiment of the invention, in step (3), on mortifier plating medium, the list of preliminary picking
The number of bacterium colony is 10~50, preferably 20；The dosage of the mortifier fermentation medium is 1~10ml, preferably
5ml, the fermented incubation time of bacterial strain are 48~72h.
In an embodiment of the invention, in step (4), the strain inoculated amount in the hydrolysate fermentation culture medium
It is 10~20%；Fermentation time is 48~72h.
The present invention also provides a kind of applications of the S. cervisiae of resistance to mortifier, in lignocellulose degradation hydrolyzate
Xylose；Wherein, under conditions of fermentation time is 48~72h, the degradation rate of xylose is 72.6%.
Compared with prior art, the invention has the following advantages that
(1) S. cervisiae of the resistance to mortifier in cellulosic hydrolysate weak acid class, furans and phenols it is resistance to
It is higher by property, it can directly apply in cellulosic hydrolysate, xylose is carried out using degradation, thus the lignocellulosic system of raising
The yield of ethyl alcohol in standby ethanol process；
(2) in cellulosic hydrolysate, it is more than 70% to xylose utilization rate therein, improves the benefit to hydrolyzate xylose
With rate, the industrialization production of ethyl alcohol is prepared suitable for agricultural stalk；
(3) fermentation time of bacterial strain of the invention is short, and using the xylose in cellulosic hydrolysate as substrate, ferment 48~72h
The utilization of 72.6% xylose can be thus achieved, shorter fermentation period can greatly improve the utilization rate of equipment.
Detailed description of the invention
Fig. 1 is consumption rate of 20 single colonies to xylose of preliminary picking after ultraviolet mutagenesis breeding；
Fig. 2 be hydrolyzate ferment picking during secondary screening bacterial strain to the consumption rate of xylose.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection scope.
Embodiment 1
A kind of S. cervisiae of resistance to mortifier, is named as ZR/SC-UV-2, classification naming are as follows: saccharomyces cerevisiae
(Saccharomyces cerevisiae) was preserved in China Committee for Culture Collection of Microorganisms on 06 13rd, 2019
Common micro-organisms center, deposit number are as follows: CGMCC No.17924, preservation address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number.
The mortifier includes one or more of weak acid substance, furans or phenolic substances.The weak acid
Substance includes formic acid, acetic acid and levulic acid etc..The furans include furfural, 5 hydroxymethyl furfural etc..The phenol
Substance includes syringaldehyde, vanillic aldehyde and phenol etc..
It is bacterium germination that the S. cervisiae of resistance to mortifier in the present embodiment, which is can utilize the S. cervisiae of xylose,
Strain, obtains the S. cervisiae of resistance to mortifier by Uv-induced screening.
In the present embodiment, starting strain is bacterial strain ZR-1, bacterial strain ZR-1 classification naming are as follows: saccharomyces cerevisiae
(Saccharomyces cerevisiae) was preserved in China Committee for Culture Collection of Microorganisms on 07 08th, 2019
Common micro-organisms center, deposit number are as follows: CGMCC No.18090, preservation address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number.
The present embodiment provides a kind of selections of the S. cervisiae of resistance to mortifier, comprising the following steps:
1, the preparation of culture medium or culture solution used in Breeding Process
The YPD culture medium is collective media, the composition of mass percentage are as follows: glucose 2%, peptone 2%, ferment
Female cream 1%, surplus are water, and pH is 4.8~5.0；Solid plate need to additionally add 2% agar.
The molar concentration of EDTA aqueous solution is 0.5mol/L；
Mortifier plating medium includes the composition of following mass percentage: xylose 3.0%, peptone 2.0%, yeast
Cream 1.0%, furfural 0.15%, hydroxymethylfurfural 0.15%, phenol 0.15%, agar 1.5%, surplus are water, culture medium warp
105 DEG C of high temperature, sterilize 60min；
Mortifier fermentation medium includes the composition of following mass percentage: glucose 8%, xylose 4%, peptone
2.0%, yeast extract 1.0%, furfural 0.2%, hydroxymethylfurfural 0.2%, phenol 0.2%, surplus is water, and the culture medium is through high temperature
105 DEG C of sterilizing 60min；
Seed culture medium includes the composition of following mass percentage: glucose 3.0%, xylose 2.0%, peptone
2.0%, yeast extract 1.0%, surplus is water, and pH is 4.8 or so；
Hydrolysate fermentation culture medium is prepared by adding peptone and yeast extract in ligno-cellulose hydrolysate, egg
The mass percentage of white peptone is 2%, and the mass percentage of yeast extract is 1%, which sterilizes through 105 DEG C of high temperature
60min。
2, the breeding of S. cervisiae
(1) starting strain starting strain primary dcreening operation: is taken one with oese in 30 DEG C of activation 22h on being coated on YPD plate
The activated starting strain of ring thallus is inoculated in YPD culture medium, stirring, under 30 DEG C, the stirring condition of 160r/min, to going out
Bacterium germination strain preculture 16h；
(2) ultraviolet mutagenesis breeding: taking the YPD culture medium containing bacterial strain, is that 1:5 is diluted with water according to thinner ratio,
Then 1mL is taken to be added in the culture dish of the EDTA aqueous solution containing 5mL (purple in order to guarantee subsequent domestication initial live thalline quantity
Prepare multiple Duplicate Samples when outer mutagenesis)；While stirring liquid with the speed conditions of 80r/min, shone using the ultraviolet lamp of 15W
Penetrate culture dish, irradiation distance 20cm, irradiation time 600s；During this, the cell survival rate of bacterial strain is 8%；
(3) separation screening of the S. cervisiae of resistance to mortifier: the bacterium solution after taking above-mentioned ultraviolet mutagenesis is according to thinner ratio
1:5 is diluted with water, and is coated on mortifier plating medium after dilution, and culture 72h is protected from light at 30 DEG C；In mortifier plate culture
On base, preliminary picking 20 single colonies that growth is fast and bacterium colony is big are inoculated in fermented and cultured in the mortifier fermentation medium of 5ml
60h detects Xylose Content after fermentation, and testing result is as shown in Figure 1, by fermentation results it is found that the wood of 1,2,5, No. 15 bacterium
Sugar utilization is apparently higher than control bacterium (control bacterium is starting strain), illustrates that this 4 plants of bacterium can be in the environment containing mortifier
Efficiently utilize xylose.This 4 bacterial strains that can efficiently utilize xylose are filtered out according to the consumption rate of Xylose Content；
(4) hydrolyzate fermentation secondary screening: 4 bacterial strains that step (3) screening obtains are inoculated into seed culture medium respectively, take portion
Point bacterium solution is 15% to be inoculated into hydrolysate fermentation culture medium and carry out fermentation 60h according to inoculum concentration, detects Xylose Content, detection knot
Fruit obtains the S. cervisiae of resistance to mortifier as shown in Fig. 2, select the highest bacterial strain of xylose utilization rate from Fig. 2.By Fig. 2 knot
It is found that different from the fermentation results in the pure sugar culture-medium containing mortifier, only No. 1 and No. 2 bacterium show higher fruit
Xylose utilization rate, wherein No. 1 bacterium is fermented in hydrolyzate under conditions of 72h, the degradation rate of xylose has reached 72.6%；And it is original
Starting strain, the degradation rate to xylose is only 20.6%, therefore, the S. cervisiae for the resistance to mortifier that the present embodiment obtains
It being capable of better xylose in degradation of fibers cellulose hydrolysate.
Embodiment 2
In the present embodiment, starting strain is bacterial strain ZR-1, bacterial strain ZR-1 classification naming are as follows: saccharomyces cerevisiae
(Saccharomyces cerevisiae) was preserved in China Committee for Culture Collection of Microorganisms on 07 08th, 2019
Common micro-organisms center, deposit number are as follows: CGMCC No.18090, preservation address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number.
The present embodiment provides a kind of selections of the S. cervisiae of resistance to mortifier, comprising the following steps:
1, the preparation of culture medium or culture solution used in Breeding Process
The YPD culture medium is collective media, the composition of mass percentage are as follows: glucose 2%, peptone 2%, ferment
Female cream 1%, surplus are water, and pH is 4.8~5.0；Solid plate need to additionally add 2% agar.
The molar concentration of EDTA aqueous solution is 0.5mol/L；
Mortifier plating medium includes the composition of following mass percentage: xylose 3.0%, peptone 2.0%, yeast
Cream 1.0%, furfural 0.15%, hydroxymethylfurfural 0.15%, phenol 0.15%, agar 1.5%, surplus are water, culture medium warp
105 DEG C of high temperature, sterilize 60min；
Mortifier fermentation medium includes the composition of following mass percentage: glucose 6.0%, xylose 3%, peptone
2.0%, yeast extract 1.0%, furfural 0.15%, hydroxymethylfurfural 0.15%, phenol 0.15%, surplus is water, culture medium warp
105 DEG C of high temperature, sterilize 60min；
Seed culture medium includes the composition of following mass percentage: glucose 3.0%, xylose 2.0%, peptone
2.0%, yeast extract 1.0%, surplus is water, pH 4.8；
Hydrolysate fermentation culture medium is prepared by adding peptone and yeast extract in ligno-cellulose hydrolysate, egg
The mass percentage of white peptone is 2%, and the mass percentage of yeast extract is 1%, which sterilizes through 105 DEG C of high temperature
60min。
2, the breeding of S. cervisiae
(1) starting strain primary dcreening operation: starting strain is activated for 24 hours on being coated on YPD plate in 28 DEG C, takes one with oese
The activated starting strain of ring thallus is inoculated in YPD culture medium, stirring, under 28 DEG C, the stirring condition of 120r/min, to going out
Bacterium germination strain preculture 18h；
(2) ultraviolet mutagenesis breeding: taking the YPD culture medium containing bacterial strain, is that 1:4 is diluted with water according to thinner ratio,
Then 1mL is taken to be added in the culture dish of the EDTA aqueous solution containing 5mL (purple in order to guarantee subsequent domestication initial live thalline quantity
Prepare multiple Duplicate Samples when outer mutagenesis)；While stirring liquid with the speed conditions of 50r/min, shone using the ultraviolet lamp of 15W
Penetrate culture dish, irradiation distance 20cm, irradiation time 600s；During this, the cell survival rate of bacterial strain is 10%；
(3) separation screening of the S. cervisiae of resistance to mortifier: the bacterium solution after taking above-mentioned ultraviolet mutagenesis is according to thinner ratio
1:4 is diluted with water, and is coated on mortifier plating medium after dilution, and culture 48h is protected from light at 28 DEG C；In the mortifier plate
On culture medium, preliminary picking 10 single colonies that growth is fast and bacterium colony is big are inoculated in the mortifier fermentation medium of 1ml and ferment
72h is cultivated, detects Xylose Content after fermented and cultured；Xylose can efficiently be utilized by filtering out 4 according to the consumption rate of Xylose Content
Bacterial strain；
(4) hydrolyzate fermentation secondary screening: 4 bacterial strains that step (3) screening obtains are inoculated into seed culture medium respectively, take portion
Point bacterium solution is 10% to be inoculated into hydrolysate fermentation culture medium and carry out fermentation 72h according to inoculum concentration, and detection Xylose Content is therefrom chosen
The highest bacterial strain of xylose utilization rate is selected, the S. cervisiae of resistance to mortifier is obtained.
The S. cervisiae of the resistance to mortifier of the present embodiment is for the cellulose during degraded cellulose production ethyl alcohol
Xylose in hydrolyzate；Wherein, under conditions of fermentation time is 72h, the degradation rate of xylose has reached 55.2%；Compared to original
Starting strain, the degradation rate to xylose are only 20.6%, therefore, the S. cervisiae energy for the resistance to mortifier that the present embodiment obtains
Xylose in enough better degradation of fibers cellulose hydrolysates.
Embodiment 3
In the present embodiment, starting strain is bacterial strain ZR-1, bacterial strain ZR-1 classification naming are as follows: saccharomyces cerevisiae
(Saccharomyces cerevisiae) was preserved in China Committee for Culture Collection of Microorganisms on 07 08th, 2019
Common micro-organisms center, deposit number are as follows: CGMCC No.18090, preservation address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number.
The present embodiment provides a kind of selections of the S. cervisiae of resistance to mortifier, comprising the following steps:
1, the preparation of culture medium or culture solution used in Breeding Process
The YPD culture medium is collective media, the composition of mass percentage are as follows: glucose 2%, peptone 2%, ferment
Female cream 1%, surplus are water, and pH is 4.8~5.0；Solid plate need to additionally add 2% agar.
The molar concentration of EDTA aqueous solution is 0.5mol/L；
Mortifier plating medium includes the composition of following mass percentage: xylose 5.0%, peptone 1.0%, yeast
Cream 2%, furfural 0.20%, hydroxymethylfurfural 0.20%, phenol 0.20%, agar 2.0%, surplus are water, and the culture medium is through height
120 DEG C of temperature, sterilize 20min；
Mortifier fermentation medium includes the composition of following mass percentage: glucose 9.0%, xylose 5%, peptone
1.0%, yeast extract 2%, furfural 0.20%, hydroxymethylfurfural 0.20%, phenol 0.20%, surplus is water, and the culture medium is through height
120 DEG C of temperature, sterilize 20min；
Seed culture medium includes the composition of following mass percentage: glucose 5.0%, xylose 4.0%, peptone
1.0%, yeast extract 2.0%, surplus is water, pH 4.8；
Hydrolysate fermentation culture medium is prepared by adding peptone and yeast extract in ligno-cellulose hydrolysate, egg
The mass percentage of white peptone is 1%, and the mass percentage of yeast extract is 2%, which sterilizes through high temperature of 120 DEG C
20min。
2, the breeding of S. cervisiae
(1) starting strain starting strain primary dcreening operation: is taken one with oese in 32 DEG C of activation 20h on being coated on YPD plate
The activated starting strain of ring thallus is inoculated in YPD culture medium, stirring, under 32 DEG C, the stirring condition of 180r/min, to going out
Bacterium germination strain preculture 12h；
(2) ultraviolet mutagenesis breeding: taking the YPD culture medium containing bacterial strain, is that 1:8 is diluted with water according to thinner ratio,
Then 1mL is taken to be added in the culture dish of the EDTA aqueous solution containing 5mL (purple in order to guarantee subsequent domestication initial live thalline quantity
Prepare multiple Duplicate Samples when outer mutagenesis)；While stirring liquid with the speed conditions of 100r/min, using the ultraviolet lamp of 20W
Irradiate culture dish, irradiation distance 15cm, irradiation time 30s；During this, the cell survival rate of bacterial strain is 5%；
(3) separation screening of the S. cervisiae of resistance to mortifier: the bacterium solution after taking above-mentioned ultraviolet mutagenesis is according to thinner ratio
1:8 is diluted with water, and is coated on mortifier plating medium after dilution, and culture is protected from light at 32 DEG C for 24 hours；It is flat in the mortifier
On plate culture medium, preliminary picking 50 single colonies that growth is fast and bacterium colony is big are inoculated in the mortifier fermentation medium of 10ml
Fermented and cultured 72h detects Xylose Content after fermented and cultured；Wood can efficiently be utilized by filtering out 4 according to the consumption rate of Xylose Content
The bacterial strain of sugar；
(4) hydrolyzate fermentation secondary screening: 4 bacterial strains that step (3) screening obtains are inoculated into seed culture medium respectively, take portion
Point bacterium solution is 20% to be inoculated into hydrolysate fermentation culture medium and carry out fermentation 48h according to inoculum concentration, and detection Xylose Content is therefrom chosen
The highest bacterial strain of xylose utilization rate is selected, the S. cervisiae of resistance to mortifier is obtained.
The S. cervisiae of the resistance to mortifier of the present embodiment is for the cellulose during degraded cellulose production ethyl alcohol
Xylose in hydrolyzate；Wherein, under conditions of fermentation time is 72h, the degradation rate of xylose has reached 33.6%；Compared to original
Starting strain, the degradation rate to xylose are only 20.6%, therefore, the S. cervisiae energy for the resistance to mortifier that the present embodiment obtains
Xylose in enough better degradation of fibers cellulose hydrolysates.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring substantive content of the invention.
Claims (10)
1. a kind of S. cervisiae of resistance to mortifier, which is characterized in that the saccharomyces cerevisiae of the resistance to mortifier is named as ZR/SC-
UV-2, classification naming are as follows: saccharomyces cerevisiae (Saccharomyces cerevisiae) was preserved in China on 06 13rd, 2019
Microbiological Culture Collection administration committee common micro-organisms center, deposit number are as follows: CGMCC No.17924, preservation address are as follows: north
The institute 3 of the Chaoyang District Jing Shi North Star West Road 1.
2. the S. cervisiae of one plant of resistance to mortifier according to claim 1, which is characterized in that the mortifier includes weak
One or more of acid, furans or phenolic substances,
The weak acid substance includes formic acid, acetic acid and levulic acid etc.；
The furans include furfural, 5 hydroxymethyl furfural etc.；
The phenolic substances includes syringaldehyde, vanillic aldehyde and phenol etc.；
The mortifier is mortifier present in ligno-cellulose hydrolysate.
3. a kind of selection of the S. cervisiae of resistance to mortifier as described in claim 1, which is characterized in that with being capable of benefit
It is starting strain with the S. cervisiae of xylose, obtains the S. cervisiae of the resistance to mortifier by Uv-induced screening.
4. a kind of selection of the S. cervisiae of resistance to mortifier according to claim 3, which is characterized in that described ultraviolet
The condition of mutagenesis are as follows: under the conditions of being protected from light, 30~600s is irradiated under the irradiation distance of 15~20cm using 15~20W ultraviolet lamp,
Carry out ultraviolet mutagenesis.
5. a kind of selection of the S. cervisiae of resistance to mortifier according to claim 4, which is characterized in that irradiation process
In, the spore of starting strain is stirred, stirring rate is 50~100r/min, during this, the Conidia persistence of bacterial strain
It is 5~10%.
6. a kind of selection of the S. cervisiae of resistance to mortifier according to claim 3, which is characterized in that including with
Lower step:
(1) the S. cervisiae of xylose can be utilized as starting strain, starting strain starting strain primary dcreening operation: is coated on YPD
It is activated on plate, activated starting strain is taken to be inoculated in YPD culture medium, stirred, carry out starting strain preculture；
(2) ultraviolet mutagenesis breeding: taking the cultured bacterial strain in YPD culture medium, and the culture dish containing EDTA aqueous solution is added
It is interior, while stirring liquid, using ultraviolet light irradiation culture dish, then mortifier will be coated on after liquid diluting in culture dish
On plating medium；
(3) the mortifier plating medium having been coated with the separation screening of the S. cervisiae of resistance to mortifier: is protected from light culture 3-4d；
On the mortifier plating medium, several single colonies that growth is fast and bacterium colony is big of preliminary picking are inoculated in mortifier hair
Fermented and cultured in ferment culture medium detects Xylose Content；Xylose can efficiently be utilized by filtering out several according to xylose utilization situation
Bacterial strain；
(4) hydrolyzate fermentation secondary screening: the bacterial strain that step (3) screening obtains is inoculated into seed culture medium respectively, takes part bacterium solution
It is inoculated into hydrolysate fermentation culture medium and ferments, detect Xylose Content, select the highest bacterial strain of xylose utilization rate, obtain institute
State the S. cervisiae of resistance to mortifier.
7. a kind of selection of the S. cervisiae of resistance to mortifier according to claim 6, which is characterized in that
The molar concentration of the EDTA aqueous solution is 0.5~0.8mol/L；
The mortifier plating medium includes the composition of following mass percentage: xylose 3.0~5.0%, and peptone 2.0~
3.0%, yeast extract 1.0~2.0%, mortifier 0.15~0.60%, agar 1.5~2.0%, surplus is water, culture medium warp
105~120 DEG C of high temperature, sterilize 10~60min；
The mortifier fermentation medium includes the composition of following mass percentage: glucose 6.0~9.0%, and xylose 3~
5%, mortifier 0.15~0.60%, agar 1.5~2.0%, surplus is water, and the culture medium is through 105~120 DEG C of high temperature, sterilizing
10~60min；
The seed culture medium includes the composition of following mass percentage: glucose 3.0~5.0%, xylose 2.0~4.0%,
Peptone 2.0~3.0%, yeast extract 1.0~2.0%, surplus are water, and pH is 4.0~5.5；
The hydrolysate fermentation culture medium is prepared by adding peptone and yeast extract in ligno-cellulose hydrolysate, institute
The mass percentage for stating peptone is 2.0~3.0%, and the mass percentage of yeast extract is 1.0~2.0%, the culture medium
It sterilizes through 105~120 DEG C of high temperature, 10~60min.
8. a kind of selection of the S. cervisiae of resistance to mortifier according to claim 6, which is characterized in that step
(1) in, the activation temperature of the starting strain is 28~32 DEG C, preferably 30 DEG C, activation time is 20~for 24 hours；Starting strain
Preculture temperature is 28~32 DEG C, and pre-incubation time is 12~18h, and stirring rate is 120~180r/min.
9. a kind of selection of the S. cervisiae of resistance to mortifier according to claim 6, which is characterized in that
In step (2), take cultured bacterial strain, be added EDTA aqueous solution, the volume ratio of bacterial strain and EDTA aqueous solution be 1:4~
8, preferably 1:5；
Described on mortifier plating medium in step (3), the number of the single colonie of preliminary picking is 10~50, preferably
It is 20；The dosage of the mortifier fermentation medium is 1~10ml, preferably 5ml, the fermented incubation time of bacterial strain is 48~
72h；
In step (4), the strain inoculated amount in the hydrolysate fermentation culture medium is 10~20%；Fermentation time is 48~72h.
10. a kind of application of the S. cervisiae of resistance to mortifier as described in claim 1, which is characterized in that the resistance to inhibition
The S. cervisiae of object is for xylose in lignocellulose degradation hydrolyzate；Wherein, under conditions of fermentation time is 72h, xylose
Degradation rate be 72.6%.
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