CN105838630A - Seed medium for culturing yeast cells and use thereof - Google Patents
Seed medium for culturing yeast cells and use thereof Download PDFInfo
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- CN105838630A CN105838630A CN201510020288.3A CN201510020288A CN105838630A CN 105838630 A CN105838630 A CN 105838630A CN 201510020288 A CN201510020288 A CN 201510020288A CN 105838630 A CN105838630 A CN 105838630A
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Abstract
The invention relates to a seed medium for culturing yeast cells and use thereof. The invention discloses a seed medium for culturing yeast cells able to produce ethanol by consuming glucose and xylose, the seed medium is mainly composed of molasses, corn steep liquor and water, wherein based on the total volume of the seed medium, the molasses has a concentration of 1.0-6.0% (v/v), the corn steep liquor has a concentration of 4.0-8.0% (v/v), and the concentration of the molasses is lower than that of the corn steep liquor. The seed medium can be used for preparation of a seed culture for yeast cells, and the seed culture can be used for preparation of ethanol.
Description
Technical field
The present invention relates to a kind of seed culture medium (seed medium) for cultivating the yeast cell that can be produced ethanol by consumption of glucose and xylose, particularly relate to a kind of seed culture medium being substantially made up of molasses (molasses), corn seep liquor (corn steep liquor) and water, can be used for culture yeasts bacterium cell, make this yeast cell under adverse circumstance environment, can effectively utilize biomass metabolism to generate ethanol.
Background technology
Cellulose biomass (cellulosic biomass) is a kind of rechargeable energy resource (renewable energy resources) that can be produced in large quantities via industry and agriculture and forestry running.Utilize chemical method or biological method that cellulose biomass is converted into biomass energy (biomass energy) [namely cellulose alcohol (cellulosic ethanol)] to be extensively studied and inquired into.Compared to chemical method, biological method is because coming into one's own especially for ecological environment is more friendly and energy demand is relatively low.
Utilizing cellulose biomass to during generating ethanol; need to first prepare cellulosic hydrolysate (cellulosic hydrolysate); this cellulosic hydrolysate is in addition to containing reducing sugar (reducing sugars); generally it is also accompanied by preparation process fermentation inhibitor [such as acetic acid, furfural (furfural), hydroxyl first furfural (hydroxymethyl furfural, HMF) and phenolic compound (phenolic compounds) etc.] produced by the degraded because of hemicellulose (hemicellulose) and reducing sugar.
The most existing many research discloses, and improves the technological means of ethanol production by saccharomyces cerevisiae is carried out genetic modification.But owing to fermentation inhibitor contained in cellulosic hydrolysate can suppress growth and the fermentation of saccharomyces cerevisiae, and make the utilization rate of reducing sugar cannot obtain effectively lifting, even can be lowered, and then affect the yield of ethanol.Such as, at E.Casey et al. (2010), FEMS Yeast Research, in 10:385-393, E.Casey et al. finds, in the YEP fermentation medium containing glucose and xylose (xylose), the existence of acetic acid can significantly reduce ethanol production rate, and can reduce the consumption rate of glucose and xylose, the most notable with inhibitory action that xylose is consumed the most again.For solving this problem, E.Casey et al. discloses the inhibitory action of acetic acid and can lower by adding ammonia constantly in the period of fermentation culture.Only this measure but can increase the pH value of fermentation medium, and then increases the risk of fouled by microzyme, and add alkali liquor constantly in the period of fermentation culture and more can improve the cost needed for fermentation processing procedure further.Therefore, the fermentation technique that development makes new advances is to lower the adverse effect that fermentation inhibitor (particularly acetic acid) is caused, and then improves the utilization rate of xylose and promote the yield of ethanol, it has also become an important research and development problem.
The by-product of numerous food product industry owing to being available for the nutrient source (nutrient source) needed for growth of microorganism rich in many, thus there is the value being available for recycling, existing many document illustrations can be used to cultivating microorganism and by the valuable product of production.Molasses (molasses) are a kind of by-products of sugar industry, mainly contain substantial amounts of sugar (including sucrose, glucose and fructose etc.) and water, can be used as the carbon source of culture medium, and food or the additive of feedstuff.Molasses can be divided into cane molasses (cane molasses) (total sugar content is of about 48-54%), beet molasses (beet molasses) (total sugar content is of about 48-52%), Citrus molasses (citrus molasses) (total sugar content is of about 40-48%) and Semen Maydis molasses (corn molasses) [the starch molasses that are otherwise known as (starch molasses), total sugar content is of about 50%] etc. according to source.It addition, corn seep liquor (corn steep liquor) is then a kind of by-product of corn wet milling (corn wet-milling) industry, mainly contains the protein of about 47%, the nitrogen source of culture medium can be used as.
Composition contained by molasses and corn seep liquor can be by yeast Absorption And Metabolism, and then can be used for supporting its growth and fermentation activity, the most existing many document illustrations, the culture medium containing molasses and/or corn seep liquor is used to carry out saccharomycetic cultivation, the growth of yeast cell number can be promoted, and then promote the yield of tunning.Such as, at Samuel Amartey and Thomas W.Jeffries (1994), Biotechnology letters., in 16:211-214, Samuel A. et al. discloses to be incubated at pichia stipitis bacterium (Pichia stipitis) CBS 6054 and obtains an inoculation source in the culture medium of the corn seep liquor comprising only 30g/L, and this inoculation source then is seeded in identical culture medium carry out fermentation reaction.Found that, compare down with the culture medium containing complicated ingredient, the culture medium using the corn seep liquor comprising only 30g/L can improve the growth rate of pichia stipitis bacterium CBS 6054 slightly to carry out fermentation reaction, and marginally improves xylose utilization rate and alcohol output.
At Kim Y.H.et al. (2007), J.Ind.Eng.Chem., in 13:153-158, Kim Y.H. et al. is in order to produce substantial amounts of beta glucan (β-glucan) (it is topmost polysaccharide in cell wall composition), saccharomyces cerevisiae JUL3 is incubated at the seed culture medium (seed medium) being made up of glucose sugar, yeast extract and peptone obtains an inoculation source, then this inoculation source is seeded to the molasses containing variable concentrations and cultivates in the culture medium of corn seep liquor.And the culture medium that experimental result only discloses the corn seep liquor using molasses and 17% (v/v) containing 6.4% (v/v) can increase saccharomyces cerevisiae JUL3 cell quality in culture.
nullAt VU V.H.and Kim K. (2009),J.Microbiol.Biotechnol.,In 19:1603-1611，VU V.H. et al. is in order to improve the cell density of the cultivation of saccharomyces cerevisiae KV-25，Saccharomyces cerevisiae KV-25 is incubated at YPD culture medium obtains an inoculation source，This inoculation source is then seeded to the molasses containing variable concentrations cultivate in the culture medium of corn seep liquor，Found that: molasses are respectively 10.25% (v/v) and 16.87% (v/v) with the optimization concentration of corn seep liquor，And use the culture medium being vaccinated the molasses containing this optimization concentration with this inoculation source and corn seep liquor can obtain the cell quality of 36.5g/L to carry out batch cultivation.
But, these previously researchs need to use substantial amounts of molasses and/or corn seep liquor to improve required cost during cultivating, do not apply the actual application in industry, and only announcement uses molasses and/or corn seep liquor to carry out cultivation as saccharomycetic nutrient source and can improve cell quality, do not disclose yeast impact in cellulosic hydrolysate, do not disclose the xylose utilization amount that can improve yeast in the cellulosic hydrolysate containing acetic acid, and then promote alcohol yied.
Summary of the invention
Then, at first aspect, the present invention provides a kind of seed culture medium for cultivating the yeast cell that can be produced ethanol by consumption of glucose and xylose, it is mainly made up of molasses, corn seep liquor and water, wherein the cumulative volume with this seed culture medium is basic for calculating, these molasses have scope and fall in the concentration of 1.0 to 6.0% (v/v), this corn seep liquor has scope and falls in the concentration of 4.0 to 8.0% (v/v), and the concentration of these molasses is less than the concentration of this corn seep liquor.
At second aspect, the present invention provides a kind of method of inoculum (seed culture) for preparing yeast cell, comprising: by yeast cell, the yeast cell of ethanol particularly can be produced by consumption of glucose and xylose, it is incubated in seed culture medium as above, thus in this seed culture medium, prepare the inoculum of this yeast cell, for after fermentation inoculation.
At the 3rd aspect, the present invention provides a kind of method preparing ethanol with biomass, comprising:
It is incubated at the yeast cell of ethanol can be produced by consumption of glucose and xylose in seed culture medium as above, thus in this seed culture medium, prepares the inoculum of this yeast cell；And
Inoculate this inoculum to ferment in these biomass；
Wherein, these biomass comprise glucose, xylose, and acetic acid.
Accompanying drawing explanation
Below in conjunction with the accompanying drawings and embodiment comes that the present invention is described in detail, so the present invention is in above-mentioned and other purposes and feature, can by referring to following description, examine attached claims and adjoint graphic and become apparent, in accompanying drawing with literary composition:
In the case of Fig. 1 shows and carries out fermentation reaction in the fermentation medium containing acetic acid, the different disposal condition gained saccharomyces cerevisiae inoculum comparison diagram on the ethanol production impact of fermentation processing procedure；And
In the case of Fig. 2 shows and carries out fermentation reaction in the fermentation medium containing acetic acid, the different disposal condition gained saccharomyces cerevisiae inoculum comparison diagram on the ethanol production impact of fermentation processing procedure.
Detailed description of the invention
Cellulose biomass contains the compositions such as substantial amounts of cellulose, hemicellulose and lignin, and these become the mutual voluble wrapping of branch to form complicated and tough and tensile network structure, and this can make to be restricted during utilizing cellulose biomass producing and ethanol in next life.Therefore, after this cellulose biomass would generally first pass through suitable pre-treatment (pretreatment), then carry out resolution process with enzyme it is hydrolyzed into reducing sugar (such as glucose with xylose etc.).But, cellulose biomass, after above-mentioned pre-treatment, can be supervened fermentation inhibitor (such as, acetic acid, furfural and hydroxyl first furfural etc.), and then affect the ability of yeast fermentation product ethanol processed.
In order to lower the adverse effect that the acetic acid as fermentation inhibitor is caused, to improve utilization rate and the yield of ethanol of xylose, applicant finds through result of study of joining hands, the be total to glucose fermentation of seed culture gained and the saccharomycetic inoculation inoculum of xylose is carried out with the seed culture medium containing molasses Yu corn seep liquor, can effectively promote the resistance of its Dichlorodiphenyl Acetate, this inoculum prepared by namely processing through the technology of the present invention, can be under the fermentation condition that there are acetic acid, it is effectively improved its xylose utilization amount, and then promotes alcohol yied.
Then, according to a kind of seed culture medium for cultivating the yeast cell that can be produced ethanol by consumption of glucose and xylose proposed by the invention, it is mainly made up of molasses, corn seep liquor and water, wherein the cumulative volume with this seed culture medium is basic for calculating, these molasses have scope and fall in the concentration of 1.0 to 6.0% (v/v), this corn seep liquor has scope and falls in the concentration of 4.0 to 8.0% (v/v), and the concentration of these molasses is less than the concentration of this corn seep liquor.
As used in this article, the remaining syrup (syrup) that the crystallization of sucrose (sucrose crystal) that term " molasses " means during the refined sugar coming from plant in removing massecuite (massecuite) is obtained afterwards, and be the thing being familiar with in the art.Molasses kind be applicable to the present invention is not particularly limited, it may include various commercial commercially available products.It is preferred that these molasses are selected from following constituted group: cane molasses, beet molasses, Citrus molasses, Semen Maydis molasses, and combinations thereof.In a preferred embodiment of the present invention, these molasses are cane molasses.
According to the present invention, basic for calculating with the cumulative volume of this seed culture medium, these molasses have scope and fall in the concentration of 3.0 to 4.0% (v/v).In a preferred embodiment of the present invention, with the cumulative volume of this seed culture medium for calculating basis, it is the concentration of 3.0% (v/v) that these molasses have.
As used in this article, term " corn seep liquor " means the concentrated solution in corn wet milling processing procedure by the impregnation liquid obtained by diluted acid soaking corn, and is the thing being familiar with in the art.The corn seep liquor kind being applicable to the present invention is not particularly limited, it may include various commercial commercially available products.
According to the present invention, basic for calculating with the cumulative volume of this seed culture medium, this corn seep liquor has scope and falls in the concentration of 5.0 to 7.0% (v/v).In a preferred embodiment of the present invention, with the cumulative volume of this seed culture medium for calculating basis, it is the concentration of 6.0% (v/v) that this corn seep liquor has.
As used in this article, term " water " includes, but are not limited to: deionized water, reverse osmosis water, distilled water and secondary water (ddH2O).In a preferred embodiment of the present invention, this water is deionized water.
The present invention also provides for a kind of method of inoculum for preparing yeast cell, comprising: be incubated at the yeast cell of ethanol can be produced by consumption of glucose and xylose in seed culture medium as above, thus in this seed culture medium, prepare the inoculum of this yeast cell.
As used herein, term " yeast cell " is intended to all yeast strain that can be produced ethanol by consumption of glucose and xylose, the yeast cell being applicable to the present invention includes, but it is not limited to, stems from following cell: Saccharomycodes species (Saccharomyces spp.), Pichia sp. ella species (Pichia spp.) and Candida species (Candida spp.).
According to the present invention, this yeast cell is recombinant type saccharomyces cerevisiae (recombinant Saccharomyces cerevisiae).Preferably, in the genomic DNA of this recombinant type saccharomyces cerevisiae, including coding Xylose reductase (xylose reductase, XR) gene, encoding xylulokinase (xylulose kinase, XK) gene, and the gene of encoding xylitol dehydrogenase (xylitol dehydrogenase, XDH).More preferably, fps1 gene [its encoding glycerol channel albumen (glycerin passage protein)] in the genomic DNA of this recombinant type saccharomyces cerevisiae and gpd2 gene [its encoding glycerol-3-phosphate dehydrogenase-2 (glycerol 3-phosphate dehydrogenase-2, GPD2)] it is deleted, destroys, or lost efficacy.
As used in this article, all or part of coding region meant by deleting gene " deleted (delete) " in term.
As used in this article, term " destroys (disrupt) " and means to carry out the deletion of nucleotide, insertion (insertion) or sudden change (mutation) in gene, and this gene is beyond expression and produces organized enzyme (active enzyme), or this gene expression generation is made to have the enzyme seriously being reduced activity (severely reduced activity).
As used in this article, term " makes inefficacy (disable) " and means that gene or its coded protein are deactivated (inactive), and then loses original activity or function.
In a preferred embodiment of the present invention, this yeast cell be by by strain with deposit numbering BCRC 920077 be deposited at Foodstuff Industrial and Development Inst. living resources preserve and research center (BCRC of FIRDI) saccharomyces cerevisiae [also with deposit numbering DSM 25508 be deposited at Germany microorganism fungus kind deposit center (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ)] genomic DNA in fps1 gene and gpd2 gene carry out deleting or destroying and prepare.
In another preferred embodiment of the present invention, this yeast cell is pichia stipitis bacterium (Pichia stipitis).
According to the present invention, this incubation step is to be carried out under aerobic condition (aerobic condition).
The present invention also provides for a kind of method preparing ethanol with biomass, comprising:
It is incubated at the yeast cell of ethanol can be produced by consumption of glucose and xylose in seed culture medium as above, thus in this seed culture medium, prepares the inoculum of this yeast cell；And
Inoculate this inoculum to ferment in these biomass；
Wherein, these biomass comprise glucose, xylose, and acetic acid.
According to the present invention, this yeast cell and incubation step thereof are as described above.
According to the present invention, these biomass are the mixed sugar liquid including glucose, xylose, and acetic acid.
According to the present invention, these biomass are to include the cellulosic hydrolysate of glucose, xylose, and acetic acid.
According to the present invention, this cellulosic hydrolysate is to be made by order cellulose biomass is carried out pre-treatment and hydrolysis process.
As used in this article, term " cellulosic hydrolysate " is to be used interchangeably with " ligno-cellulose hydrolysate " and " biomass hydrolysate (biomass hydrolysate) ", and means by product produced by the saccharifying (saccharification) of biomass.
According to the present invention, the acetic acid in these biomass has scope and falls in the concentration of 4 to 10g/L.Fall in the concentration of 5 to 8.5g/L it is preferred that the acetic acid in these biomass has scope.More preferably, the acetic acid in these biomass has scope and falls in the concentration of 6 to 8g/L.
According to the present invention, this fermentation step be substantially there is no molasses and corn seep liquor under conditions of carried out.
As used in this article, term " essentially without (substantially free of) " means that the composition being fully described and particularly pointed lacks significant quantity.It is preferred that this fermentation step is to be carried out under conditions of entirely without this composition, or the quantity of this composition does not have measurable impact (measurable effect) for this fermentation step.
According to the present invention, this fermentation step is to fall in scope to being carried out under conditions of the pH value of 5.0 to 6.5.It is preferred that this fermentation step is to fall in scope to being carried out under conditions of the pH value of 5.0 to 5.5.
According to the present invention, this fermentation step is to be carried out under anaerobic condition (anaerobic condition).
The present invention will be described further with regard to the following examples, however, it should be noted that such embodiment is for illustrating, and the restriction being not necessarily to be construed as in the enforcement of the present invention.
<embodiment>
General experiment material:
1. prepare the saccharomyces cerevisiae (Δ fps1 Δ gpd2 double mutant of Saccharomyces cerevisiae) of the double sudden change of Δ fps1 Δ gpd2:
Below the saccharomyces cerevisiae bacteria strain used in experiment is the saccharomyces cerevisiae of the double sudden change of Δ fps1 Δ gpd2, its be generally based on Zhang A et al. (2007) (with above-mentioned) and Hubmann G.et al. (2011) (with above-mentioned) when described in method and be produced.nullIn brief，First，According to Zhang A et al. (2007) (with above-mentioned) when described in method，By fermenting altogether, pentose [is obtained from applicant priority patent case TW I450963 (corresponding application case CN103695329A) with the saccharomyces cerevisiae BCRC 920077 of hexose，Also be deposited at DSMZ depositing numbering DSM 25508] fps1 gene knockout，Then by obtained Δ fps1 mutant strain according further to Hubmann G.et al. (2011) (with above-mentioned) when disclosed in method carry out knocking out of gpd2 gene，Obtain the saccharomyces cerevisiae bacteria strain (hereinafter referred to as " through the saccharomyces cerevisiae of double sudden changes ") of the double sudden change of Δ fps1 Δ gpd2 whereby.
2. in embodiment below, cane molasses and corn seep liquor for preparing seed culture medium are available from good year Fenghe enterprise stock company limited (Fonen And FonHer Enterprise Co. respectively, and Taiwan Sugar Industry Co., Ltd. (Taiwan Sugar Corporation) LTD), wherein cane molasses contain the sucrose of 435g/L, the glucose of 36.5g/L and the fructose of 86g/L, and corn seep liquor contains the water of 60.88% (w/w), the thick protein of 17.67% (w/w), the coarse ash of 6.49% (w/w) and the sulfur dioxide of 177.94ppm.
3. following experiments material is available from Jing Ming chemical inc (Echo Chemical Co., LTD): glucose and xylose.
4. following experiments material is available from SHOWA: carbamide.
5. following experiments material is available from Scharlau: acetic acid.
6. the YPD culture medium used in embodiment below has the formula being shown in Table 1 below.
The formula of table 1.YPD culture medium
7., in embodiment below, through the fermentation medium of the saccharomyces cerevisiae of double sudden changes, there is the formula being shown in Table 2 below for fermentation culture.
Table 2. is for the formula of the fermentation medium of the saccharomyces cerevisiae of the double sudden change of fermentation culture warp
8., in embodiment below, the fermentation medium for fermentation culture pichia stipitis bacterium BCRC 21775 (corresponding to ATCC 58376) has the formula being shown in Table 3 below.
Table 3. is for the formula of the fermentation medium of fermentation culture pichia stipitis bacterium BCRC 21775
General experimental technique:
The most high-effect liquid chromatography (LC) (high performance liquid chromatography, HPLC) is analyzed:
nullIn the following embodiments，It is used for composition contained in the testing sample that HPLC analyzes and concentration (g/L) is with reference to American National Renewable Energy Laboratory (National Renewable Energy Laboratory,NREL) lab analysis program (the laboratory analytical procedures that the relevant standard biological matter promulgated is analyzed,LAPs)，And by using equipped with a refractive index detector (refractive index detector,RI detector) high-effect liquid chromatograph (DIONEX Ultimate 3000) be measured，Tubing string and operating condition used in it are as follows: analyzing tubing string is Aminex HPX-87H tubing string (BioRad)；Flowing phase: 5mM sulphuric acid (is assigned in water)；Flow velocity is controlled as 0.6mL/ minute；Sample injection volume is 20 μ L；Column oven (column oven) temperature controls at 65 DEG C；RI temperature controls at 45 DEG C.
In addition, for for comparison, the glucose (1.25-24g/L) of variable concentrations, xylose (1.25-24g/L), acetic acid (0.25-6g/L) and ethanol (0.938-20g/L) is used respectively as calibration standard product (controlstandard) and to carry out identical analysis.
Embodiment 1. is under the fermentation condition that there are acetic acid, the warp pair obtained by the seed culture medium of the corn seep liquor containing variable concentrations and the cane molasses of 3% inoculum of the saccharomyces cerevisiae suddenlyd change is for the impact of the alcohol yied of fermentation processing procedure
In the present embodiment, it is brought as test strain according to the saccharomyces cerevisiae through double sudden changes obtained by the 1st " the preparing the saccharomyces cerevisiae of the double sudden change of Δ fps1 Δ gpd2 " of " general experiment material " above.
Experimental technique:
First, the saccharomyces cerevisiae bacteria strain of double for this warp sudden changes is divided into 6 groups, including 5 experimental grouies (namely experimental group 1 to 5) and 1 matched group.Then, it is seeded to the bacterial strain of experimental group 1 to 5 respectively to have in the seed culture medium (10mL) of formula as shown in Table 4 below, and by the inoculation of matched group to YPD culture medium (10mL) as shown in Table 1.
The formula of the seed culture medium of each experimental group of table 4.
Afterwards, carry out cultivation in each group of bacterial strain is placed in constant-temperature shaking incubator (30 DEG C, 200rpm) and last 16 hours.Then, by the culture that formed in 15, it is centrifuged under 700g lasting 1 minute, then collecting cell precipitate and use fermentation medium as shown in Table 2 to fill part suspension thalline, thus obtained cell suspending liquid is brought as the inoculum of the saccharomyces cerevisiae through double sudden changes.
Afterwards, by each group of inoculum respectively with 2 × 106The inoculum concentration of cell/mL is inoculated in containing in the conical flask of the fermentation medium as shown in Table 2 of 100mL, then and carries out fermentation reaction in constant incubator (30 DEG C, 200rpm) under anaerobic condition and lasts 72 hours.After fermentation reaction starts the 6th hour and the 24th hour, to each group of NaOH adding 6N so that its pH value is maintained at 5.5.
Afterwards, by obtained each group fermentation culture medium in 12, be centrifuged under 000rpm and lasted 1 minute, and obtained tunning (fermentation product) be based on above the 1st " the high-effect liquid chromatography (LC) analysis " of " general experimental technique " when described in method to carry out the content analysis of ethanol.
Alcohol yied is by contained glucose content and Xylose Content in measured ethanol content and fermentation primary fermentation culture medium are substituted into following equation (I) and be calculated:
A=[B/ (C × 0.51+D × 0.48)] × 100 (I)
Wherein: A=alcohol yied (%)
Ethanol content (g/L) measured by B=
Glucose content (g/L) contained in C=fermentation primary fermentation culture medium
Xylose Content (g/L) contained in D=fermentation primary fermentation culture medium
Result:
Result measured by this experiment is shown in Fig. 1.It can be seen from figure 1 that compare down with matched group, the alcohol yied measured by the tunning of each experimental group all increases significantly, and can more they tend to along with the increase of the concentration of corn seep liquor substantially simultaneously.This experimental result shows, under the fermentation condition that there are acetic acid, by the inoculum of the saccharomyces cerevisiae through double sudden changes obtained by the seed culture medium of the corn seep liquor containing variable concentrations and the cane molasses of 3% (v/v), all can effectively promote the alcohol yied of fermentation processing procedure.
Embodiment 2. is under the fermentation condition that there are acetic acid, the warp pair obtained by the seed culture medium of the cane molasses containing variable concentrations and the corn seep liquor of 6% inoculum of the saccharomyces cerevisiae suddenlyd change is for the impact of the alcohol yied of fermentation processing procedure
In the present embodiment, use the test strain being same as 1 tool person of embodiment.
Experimental technique:
First, the saccharomyces cerevisiae bacteria strain of double for this warp sudden changes is divided into 7 groups, including 6 experimental grouies (namely experimental group 1 to 6) and 1 matched group.Then, it is seeded to the bacterial strain of experimental group 1 to 6 respectively to have in the seed culture medium (10mL) of formula as shown in Table 5 below, and by the inoculation of matched group to YPD culture medium (10mL) as shown in Table 1.
The formula of the seed culture medium of each experimental group of table 5.
Afterwards, carry out cultivation in each group of bacterial strain is placed in constant-temperature shaking incubator (30 DEG C, 200rpm) and last 16 hours.Then, by the culture that formed in 15, it is centrifuged under 700g lasting 1 minute, then collecting cell precipitate and use fermentation medium as shown in Table 2 to fill part suspension thalline, thus obtained cell suspending liquid is brought as the inoculum of the saccharomyces cerevisiae through double sudden changes.
Afterwards, by each group of inoculum respectively with 2 × 106The inoculum concentration of cell/mL is inoculated in containing in the conical flask of the fermentation medium as shown in Table 2 of 100mL, then and carries out fermentation reaction in constant incubator (30 DEG C, 200rpm) under anaerobic condition and lasts 72 hours.After fermentation reaction starts the 6th hour and the 24th hour, to each group of NaOH adding 6N so that its pH value is maintained at 5.5.
Afterwards, by obtained each group fermentation culture medium in 12, it is centrifuged under 000rpm and is lasted 1 minute, and obtained tunning be based on above the 1st " the high-effect liquid chromatography (LC) analysis " of " general experimental technique " when described in method to carry out the content analysis of ethanol, and according to example 1 above when described in calculation calculate the alcohol yied of each group.
Result:
Result measured by this experiment is shown in Fig. 2.As it is clear from fig. 2 that compare down with matched group, the situation that the alcohol yied measured by the tunning of each experimental group is all improved is the highest with the alcohol yied measured by the tunning of experimental group 3 the most again.This experimental result shows, under the fermentation condition that there are acetic acid, the alcohol yied of fermentation processing procedure all can be effectively promoted, wherein with containing 3% (v/v) cane molasses and the best results of the seed culture medium of 6% (v/v) corn seep liquor by the inoculum through the saccharomyces cerevisiae of double sudden changes obtained by the seed culture medium of the cane molasses containing variable concentrations and the corn seep liquor of 6% (v/v).
Impact under the fermentation condition of the pH value that embodiment 3. is different
In the present embodiment, use the test strain being same as 1 tool person of embodiment, and use and simulate the biomass containing acetic acid (such as containing acetic acid and fermentation medium that pH value is 5.0,5.5 or 6.0, cellulosic hydrolysate), and in the case of inquiring into and carrying out fermentation reaction in this fermentation medium, by the sugared and utilization of xylose and the impact of alcohol yied for the glucose of fermentation processing procedure of the inoculum of the saccharomyces cerevisiae of the double sudden change of this warp obtained by the seed culture medium of the cane molasses containing 3% (v/v) and the corn seep liquor of 6% (v/v).
Experimental technique:
First, the saccharomyces cerevisiae bacteria strain of double for this warp sudden changes is divided into 6 groups, including 3 experimental grouies (namely experimental group 1 to 3) and 3 matched groups (namely matched group 1 to 3).Then, the bacterial strain of experimental group 1 to 3 is seeded to respectively in the seed culture medium (10mL) containing 3% (v/v) cane molasses and 6% (v/v) corn seep liquor, and the bacterial strain of matched group 1 to 3 is seeded to respectively in YPD culture medium (10mL) as shown in Table 1.
Afterwards, carry out cultivation in each group of bacterial strain is placed in constant-temperature shaking incubator (30 DEG C, 200rpm) and last 16 hours.Then, by the culture that formed in 15, it is centrifuged under 700g lasting 1 minute, then collecting cell precipitate and use fermentation medium as shown in Table 2 to fill part suspension thalline, thus obtained cell suspending liquid is brought as the inoculum of the saccharomyces cerevisiae through double sudden changes.
Afterwards, by each group of inoculum respectively with 2 × 106The inoculum concentration of cell/mL is inoculated in containing in the conical flask of the fermentation medium as shown in Table 2 of 100mL, then and carries out fermentation reaction in constant incubator (30 DEG C, 200rpm) under anaerobic condition and lasts 72 hours.After fermentation reaction starts the 6th hour and the 24th hour, each group is added the NaOH of 6N so that the pH value of experimental group 1 and matched group 1 is maintained at 5.0, experimental group 2 is maintained at 5.5 with the pH value of matched group 2, and the pH value of experimental group 3 and matched group 3 is maintained at 6.0.
Then, by obtained each group fermentation culture medium in 12, it is centrifuged under 000rpm and is lasted 1 minute, and obtained tunning be based on above " the high-effect liquid chromatography (LC) analysis " of " general experimental technique " when described in method to carry out the content analysis of ethanol, xylose and glucose, and according to example 1 above when described in calculation calculate the alcohol yied of each group.
Result:
Result measured by this experiment is shown in table 6 below.
Alcohol yied, glucose content and the Xylose Content measured by tunning respectively organized by table 6.
As seen from Table 6, no matter by being inoculated in the case of pH value is maintained at and carries out fermentation culture in the fermentation medium of 5.0,5.5 or 6.0 by the inoculum obtained by different seed culture mediums, respectively organize the remaining glucose content measured by tunning and be all 0g/L.Additionally, for alcohol yied and Xylose Content, alcohol yied measured by the tunning of each experimental group is all higher than corresponding matched group institute tool person, and the remaining Xylose Content measured by the tunning of each experimental group is all less than corresponding matched group institute tool person.Especially, the alcohol yied measured by the tunning of experimental group 1 is to be similar to 2 tool persons of matched group, and the alcohol yied measured by the tunning of experimental group 2 is to be similar to 3 tool persons of matched group.This experimental result shows, under the fermentation condition that there are acetic acid, no matter pH value is maintained at 5.0,5.5 or 6.0, all can effectively be promoted the xylose utilization amount of fermentation processing procedure by the inoculum through the saccharomyces cerevisiae of double sudden changes obtained by the seed culture medium of the cane molasses containing 3% (v/v) and the corn seep liquor of 6% (v/v), and then promote alcohol yied.Therefore, when pH value reduces over time in fermentation reaction, the NaOH usage amount being used to adjust pH value can be saved.
Embodiment 4. under the fermentation condition that there are acetic acid, by containing 3% cane molasses and 6% corn seep liquor seed culture medium obtained by the inoculum of pichia stipitis bacterium (Pichia stipitis) BCRC 21775 (corresponding to ATCC 58376) for the impact of alcohol yied of fermentation processing procedure
In the present embodiment, pichia stipitis bacterium BCRC 21775 (corresponding to ATCC 58376) (preserving and research center purchased from the living resources of the Foodstuff Industrial and Development Inst. in Taiwan) is brought as test strain, and use and simulate the biomass (such as, cellulosic hydrolysate) containing acetic acid containing acetic acid and fermentation medium that pH value is 6.0 or 6.5.
Experimental technique:
First, pichia stipitis bacterium BCRC 21775 is divided into 4 groups, including 2 experimental grouies (namely experimental group 1 and 2) and 2 matched groups (namely matched group 1 and 2).Then, the bacterial strain of experimental group 1 and 2 is seeded to respectively in the seed culture medium (10mL) containing 3% (v/v) cane molasses and 6% (v/v) corn seep liquor, and the bacterial strain of matched group 1 and 2 is seeded in YPD culture medium (10mL) as shown in Table 1 respectively.
Afterwards, carry out cultivation in each group of bacterial strain is placed in constant-temperature shaking incubator (30 DEG C, 200rpm) and last 16 hours.Then, by the culture that formed in 15, it is centrifuged under 700g lasting 1 minute, then collecting cell precipitate and use fermentation medium as shown in Table 3 to fill part suspension thalline, thus obtained cell suspending liquid is brought as the inoculum of pichia stipitis bacterium BCRC 21775.
Afterwards, by each group of inoculum respectively with 2 × 106The inoculum concentration of cell/mL is inoculated in containing in the conical flask of the fermentation medium as shown in Table 3 of 100mL, then and carries out fermentation reaction in constant incubator (30 DEG C, 200rpm) under anaerobic condition and lasts 72 hours.After fermentation reaction starts the 6th hour and the 24th hour, add the NaOH of 6N so that the pH value of experimental group 1 and matched group 1 is maintained at 6.0 to each group, and the pH value of experimental group 2 and matched group 2 is maintained at 6.5.
Then, by obtained each group fermentation culture medium in 12, be centrifuged under 000rpm and lasted 1 minute, and obtained tunning be based on above " the high-effect liquid chromatography (LC) analysis " of " general experimental technique " when described in method to carry out the content analysis of ethanol.
Alcohol yied is to be calculated by contained Xylose Content in measured ethanol content and fermentation primary fermentation culture medium is substituted into following equation (II):
E=[F/ (G × 0.48)] × 100 (II)
Wherein: E=alcohol yied (%)
Ethanol content (g/L) measured by F=
Xylose Content (g/L) contained in G=fermentation primary fermentation culture medium
Result:
Result measured by this experiment is shown in table 7 below.
The alcohol yied measured by tunning respectively organized by table 7.
As seen from Table 7, the alcohol yied measured by the tunning of each experimental group is all higher than corresponding matched group institute tool person.Especially, the alcohol yied measured by the tunning of experimental group 1 is higher than 2 tool persons of matched group.This experimental result shows, under the fermentation condition that there are acetic acid, no matter pH value is maintained at 6.0 or 6.5, the inoculum of the pichia stipitis bacterium BCRC 21775 (corresponding to ATCC 58376) obtained by the seed culture medium of the cane molasses containing 3% (v/v) and the corn seep liquor of 6% (v/v) all can effectively promote the alcohol yied of fermentation processing procedure.Therefore, when pH value reduces over time in fermentation reaction, the NaOH usage amount being used to adjust pH value can be saved.
Comprehensive above experimental result, can demonstrate,prove: under the fermentation condition that there are acetic acid, can effectively be promoted the xylose utilization amount of fermentation processing procedure by the saccharomycetic inoculum containing molasses with the be total to glucose fermentation obtained by the seed culture medium of corn seep liquor with xylose, and then promote alcohol yied.
The all patents and the document that are quoted in this specification are merged in this case as reference data using its entirety.If conflicted, this case detailed description (comprising in being defined in) will be got the upper hand.
Although the present invention is described with reference to above-mentioned specific concrete example, it will be apparent that a lot of modifications and variations can be made under without departing substantially from scope and spirit of the present invention.Therefore it is intended that the present invention is only limited by such as examining attached claims those shown with literary composition.In the application, saccharomyces cerevisiae (saccharomyces cerevisiae) DSM 25508 is preserved in address on the 20th in December in 2011 and is positioned at the German Culture Collection GmbH of this thorough 7B D-38124 Brunswick of silver-colored great sweet smell.
Claims (19)
1. one kind for cultivating, can to produce the yeast of ethanol by consumption of glucose and xylose thin
The seed culture medium of born of the same parents, it is characterised in that this seed culture medium is mainly by molasses, corn seep liquor and water
Being constituted, wherein the cumulative volume with this seed culture medium is basic for calculating, and these molasses have scope and fall 1.0
To the concentration of 6.0% (v/v), this corn seep liquor has scope and falls in the concentration of 4.0 to 8.0% (v/v), and should
The concentration of molasses is less than the concentration of this corn seep liquor.
Seed culture medium the most according to claim 1, it is characterised in that with this seed culture medium
Cumulative volume is for calculating basis, and these molasses have scope and fall in the concentration of 3.0 to 4.0% (v/v).
Seed culture medium the most according to claim 1, it is characterised in that with this seed culture medium
Cumulative volume is for calculating basis, and this corn seep liquor has scope and falls in the concentration of 5.0 to 7.0% (v/v).
Seed culture medium the most according to claim 1, it is characterised in that these molasses are selected from following
The group constituted: cane molasses, beet molasses, Citrus molasses, Semen Maydis molasses, and combinations thereof.
5. the method being used for preparing the inoculum of yeast cell, it is characterised in that the method
Including: it is incubated at basis by the yeast cell of ethanol can be produced by consumption of glucose and xylose
In seed culture medium according to any one of Claims 1-4, thus preparation should in this seed culture medium
The inoculum of yeast cell.
Method the most according to claim 5, it is characterised in that this yeast cell selected from by under
The group that row are constituted: recombinant type saccharomyces cerevisiae and pichia stipitis bacterium.
Method the most according to claim 6, it is characterised in that the base of this recombinant type saccharomyces cerevisiae
Because group DNA includes encoding the gene of Xylose reductase, the gene of encoding xylulokinase and encoding xylitol
The gene of dehydrogenase.
Method the most according to claim 6, it is characterised in that this recombinant type saccharomyces cerevisiae
Fps1 gene and gpd2 gene in genomic DNA are deleted, destroy, or lose efficacy.
Method the most according to claim 5, it is characterised in that this incubation step is at aerobic condition
Lower carried out.
10. the method preparing ethanol with biomass, it is characterised in that the method includes:
It is incubated at basis by the yeast cell of ethanol can be produced by consumption of glucose and xylose
In seed culture medium according to any one of Claims 1-4, thus preparation should in this seed culture medium
The inoculum of yeast cell；And
The inoculum inoculating this yeast cell ferments in these biomass；
Wherein, these biomass comprise glucose, xylose and acetic acid.
11. methods according to claim 10, it is characterised in that this yeast cell selected from by
Following constituted group: recombinant type saccharomyces cerevisiae and pichia stipitis bacterium.
12. methods according to claim 11, it is characterised in that this recombinant type saccharomyces cerevisiae
Genomic DNA includes encoding the gene of Xylose reductase, the gene of encoding xylulokinase and coding xylose
The gene of alcoholdehydrogenase.
13. method according to claim 11, it is characterised in that this recombinant type saccharomyces cerevisiae
Genomic DNA in fps1 gene and gpd2 gene be deleted, destroy, or lost efficacy.
14. methods according to claim 10, it is characterised in that this incubation step is at aerobic bar
Carried out under part.
15. methods according to claim 10, it is characterised in that this fermentation step is substantially
Carried out under conditions of there is no molasses and corn seep liquor.
16. methods according to claim 10, it is characterised in that this fermentation step is to fall in scope
Carried out under conditions of the pH value of 5.0 to 6.5.
17. methods according to claim 10, it is characterised in that this fermentation step is at anaerobism bar
Carried out under part.
18. methods according to claim 10, it is characterised in that the acetic acid in these biomass is dense
Degree is 4 to 10g/L.
19. methods according to claim 10, it is characterised in that these biomass are cellulose hydrolysis
Liquid.
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| CN102586340A (en) * | 2012-02-11 | 2012-07-18 | 江门市杰士植物营养有限公司 | Preparation method of molasses for recycling |
| CN103181468A (en) * | 2011-12-29 | 2013-07-03 | 徐州观音猪业有限公司 | Pig feed formula |
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| CN103181468A (en) * | 2011-12-29 | 2013-07-03 | 徐州观音猪业有限公司 | Pig feed formula |
| CN102586340A (en) * | 2012-02-11 | 2012-07-18 | 江门市杰士植物营养有限公司 | Preparation method of molasses for recycling |
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| CN110734937A (en) * | 2018-07-20 | 2020-01-31 | 远东新世纪股份有限公司 | Process for producing lactic acid |
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