CN103789396B - Method for rapidly screening high-yield ethanol saccharomycetes - Google Patents
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Abstract
The invention provides a method for rapidly screening high-yield ethanol saccharomycetes. The method comprises the following steps: drawing a growth curve of a bacterial strain at first, and determining the lg phase, the logarithmic phase and the stable phase of the bacterial strain based on the growth curve, preparing a liquid culture medium and subpackaging the liquid culture medium into a plurality of test tubes, and placing fermentation tubules full of the liquid culture medium upside down in one half of the test tubes, sterilizing the two kinds of test tubes filled with the culture medium by using high-pressure steam and then taking out and cooling the test tubes, inoculating the test tubes, which are filled with the liquid culture medium and without the fermentation tubules, with the bacterial strain, carrying out shaking culture to the last logarithmic phase and then taking out the test tubes for later use, during the culture period of the bacterial liquid, preparing an antibiotic mother liquor and carrying out filtration sterilization, taking out the fermentation tubules out of the test tubes filled with the liquid culture medium through sterile operation, placing the taken-out test tubes into the culture solution, which is cultivated to the last logarithmic phase previously, upside down, guaranteeing no bubble entering the tubules and adding a proper amount of antibiotic mother liquor to the tubules and transferring the tubules to static fermentation culture, after culture by one cycle, taking out the test tube liquid, and finally, observing the size of a gas column in each tubule, wherein the larger the gas column is, the higher the yield of ethanol is, and therefore, the target bacterial strain can be rapidly screened out.
Description
Technical field
The invention belongs to technical field of microbiology, relate to rapid preliminary screening microbe high-yield bacterial strain, particularly can utilize the rapid screening method of the microorganism of glucose high-yield ethanol under anaerobic.
Background technology
Bacterial screening is one of important research content of microbiological art always, is also one of difficult point needing emphasis to solve in related scientific research work.For the directed screening of microorganism as directed mutagenesis, the screening operation of its object mutant strain is complicated and important, and if there is no selection markers, the screening of object bacterial strain will more blindly, heavy.About the screening method of object bacterial strain, researcher both domestic and external has done a large amount of research and inquirement, it is also proposed many different thinkings.These thinkings alleviate some Tasks, and object and directivity are also clearer and more definite, but still workload is huge.Therefore be necessary to do further discussion to the screening method of object bacterial strain.
On the other hand, when energy dilemma forces human development to utilize new forms of energy, the yeast seeds of screening high-yield ethanol just becomes and transforms Mierocrystalline cellulose or hemicellulose generates one of key influence factor of ethanol.
For yeast, it utilizes the metabolic mechanism of glucose as follows:
Maybe can be converted in the substratum (as PDA substratum) of glucose at the substratum (as YEPD substratum) taking glucose as carbon source, yeast such as yeast saccharomyces cerevisiae can utilize glucose to carry out metabolism under aerobic and oxygen free condition.When in environment during aerobic, part glucose exhaustive oxidation can be obtained the energy required for growth and breeding by yeast, produce CO simultaneously
2; When in environment during anaerobic, yeast also can glucose fermentation, carries out inefficient production capacity metabolism, generates ethanol and CO simultaneously
2.
During aerobic: C
6h
12o
6+ 6O
2 6CO
2+ 6H
2o+ energy
During anaerobic: C
6h
12o
6 2CO
2+ 2C
2h
5oH+ energy (on a small quantity)
Due to this feature of metabolism of yeasts, someone proposes to utilize fermentation (Du Shi) tubule to carry out qualitative indication yeast and produces CO
2ability, thus the ability of indirect indications yeast producing and ethanol.But the shortcoming done like this is: yeast carries out aerobic metabolism period in cultivation early stage and growth and breeding, while glucose exhaustive oxidation is obtained energy, also produce CO
2, and produce CO
2efficiency higher, 1 molecule glucose exhaustive oxidation can generate 6 molecule CO
2; And in anaerobic metabolism, 1 molecule glucose only can transform generation 2 molecule CO
2.Like this, be not easy to distinguish the intratubular CO of fermentation
2or actually produced by aerobic metabolism anaerobic metabolism, or the ratio of the former with the latter has much, if these CO
2do not add differentiation and broadly all treat as that glucose fermentation produces, be easy to obtain false superior strain.
It should be noted that, when growth conditions changes, yeast can be selected based on a certain metabolic way.As under aerobic conditions, yeast is based on aerobic metabolism, and at this moment consumed glucose major part by metabolism, but also has a few part to be consumed in ethanol fermentation mode with aerobic form; And under anaerobic, yeast is based on anaerobic metabolism, at this moment the consumed glucose overwhelming majority is fallen by metabolism with anaerobically fermenting form, but still has a seldom part to be consumed in aerobic metabolism mode.Therefore, in the whole culture cycle of yeast, aerobic metabolism and anaerobic fermentation can not strictly be distinguished.But we still can utilize yeast metabolic characteristic under anaerobic to screen object bacterial strain however.
Summary of the invention
The object of the invention is for above-mentioned Problems existing, a kind of method of screening the yeast strain of high-yield ethanol is quickly and accurately provided.The present invention utilizes yeast to be that this feature main carrys out design experiment scheme under anaerobic with ethanol fermentation.
Technical scheme of the present invention is:
A method for rapid screening high-yield ethanol yeast, the step of the method is:
1st step, first draw the growth curve of bacterial strain, judge its lag period, logarithmic phase, stationary phase accordingly;
2nd step, the preparation also some test tube liquid nutrient mediums of packing, wherein half is inverted into the fermentation tubule of filled with fluid substratum, by two kinds of test-tube culture medium high pressure steam sterilizations, takes out cooling;
3rd step, in the test tube liquid nutrient medium not putting fermentation tubule, access bacterial classification, shaking culture to logarithmic growth latter stage, is taken out stand-by just;
4th step, between bacterium liquid incubation period, preparation microbiotic mother liquor, filtration sterilization;
Fermentation tubule in 2nd step takes out by the 5th step, aseptic technique from test tube liquid nutrient medium, being inverted into above-mentioned 3rd step is cultured in the nutrient solution in logarithmic growth latter stage, ensure that in tubule, bubble-free enters, and add the antibiotic solution of appropriate 4th step preparation, it is carried out static fermentation culture;
6th step, after being cultured to one-period, take out the test tube liquid in above-mentioned 5th step, observe gas column size in fermentation tubule, gas column is larger, and ethanol production is higher, thus rapid screening can go out object bacterial strain.
The concrete operations of the inventive method are as follows:
(1) drafting of yeast strain growth curve
Under fixed in pre-stage test, to comprise medium component and content, culture temperature, pH, culture cycle, oscillation rate optimal culture condition, inoculation yeast bacterium is in liquid nutrient medium and cultivate, and take incubation time as X-coordinate, with the absorbance A of bacterium liquid
600for ordinate zou, draw the absorbance A of yeast nutrient solution
600with the growth curve of incubation time change, judge the lag period of strain growth, logarithmic phase, stationary phase accordingly;
(2) preparation of test tube liquid nutrient medium and sterilizing
Preparation is containing the substratum of glucose, the substratum that maybe can change into glucose as YEPD is as PDA substratum or bean sprout juice dextrose culture-medium or wort dextrose culture-medium 1000mL, be distributed into 100 test tube liquid, each 9.7mL, get 50 test tube liquid wherein, be inverted into the fermentation tubule being full of aforesaid liquid substratum 0.3mL wherein, careful operation is in order to avoid enter bubble in tubule, the test tube liquid nutrient medium of fermentation tubule will be housed, be designated as a, the test tube liquid nutrient medium of fermentation tubule is unkitted with other 50, be designated as b, simultaneously at 112 DEG C, sterilizing 20min under 0.5Mpa wet-hot steam, taking-up is cooled to room temperature,
(3) inoculation and cultivation
Inoculum size with 0.5% accesses yeast strain in the test tube liquid nutrient medium b being unkitted fermentation tubule, and at 30 DEG C, 150 r/min shaking culture are just to logarithmic phase latter stage, take out stand-by, are designated as c;
(4) microbiotic mother liquor
Prepare kantlex solution mother liquor and chloromycetin solution mother liquor respectively, concentration is 0.5g/100mL, two kinds of microbiotic mother liquor filtration sterilizations under aseptic technique respectively; The preparation of microbiotic mother liquor after inoculation bacterium liquid cultivation period is carried out;
(5) ethanol fermentation
Under aseptic technique, fermentation tubule in a is taken out from test tube liquid, be inverted into rapidly in the nutrient solution c being cultured to logarithmic phase latter stage, careful operation is to make not enter bubble in fermentation tubule, then be rapidly in same nutrient solution and add the chloromycetin solution that kantlex solution that final concentration is 50 μ g/mL and final concentration are 25 μ g/mL respectively, after more than having operated, the Tube propagation liquid c that fermentation tubule is housed is continued quiescent culture to culture cycle at 30 DEG C;
Culture cycle comprises thalli growth stage of not producing and ethanol and thalline glucose fermentation producing and ethanol and CO
2stage;
(6) primary dcreening operation of ethanol high-yield bacterial strain
The above-mentioned nutrient solution c completing a culture cycle is taken out, observe in test tube liquid intratubular bubble production and the expel liquid situation of fermenting, according to fermentation intratubular gas column size can tentatively judge yeast producing and ethanol number, gas column is larger, side light ethanol production is higher, very promptly can filter out the object bacterial strain of high-yield ethanol like this; The number of producing and ethanol is determined by further detection by quantitative;
(7) primary dcreening operation result verification
The superior strain obtained by above-mentioned primary dcreening operation carries out producing and ethanol fermentation, then samples, suitably dilutes, and uses bio-sensing analyser method or liquid phase chromatography to carry out producing and ethanol analysis, to verify the accuracy of present method primary dcreening operation result.
advantage of the present invention and beneficial effect:
The present invention is different according to the metabolic characteristic of yeast under aerobic with oxygen free condition, reasonable arrangement place fermentation tubule time, ingenious eliminating its under aerobic conditions, produce CO
2to the interference that fermentation judges, ensure the intratubular CO of fermentation
2the gas overwhelming majority is by being produced with producing and ethanol fermentation, but not good oxygen metabolism produces.Make how much judge that yeast producing and ethanol ability has more accuracy according to bubble in fermentation tubule.In addition, present method is not only suitable for the saccharomycetic screening of general producing and ethanol, is particularly useful for the screening of yeast mutagenic mutant.This programme logicality is tight, simple to operate, easy, is one fast and exactly screening method.
Accompanying drawing explanation
Fig. 1 is the growth curve of yeast saccharomyces cerevisiae QD in YEPD.
Fig. 2 is the growth curve of schizosaccharomyces pombe 2.1621 in YEPD.
Fig. 3 is the growth curve of kluyveromyces marxianus 191l in YEPD.
Fig. 4 is yeast saccharomyces cerevisiae QD growth curve in a pda.
Fig. 5 is schizosaccharomyces pombe 2.1621 growth curve in a pda.
Fig. 6 is kluyveromyces marxianus 191l growth curve in a pda.
Fig. 7 is the growth curve of yeast saccharomyces cerevisiae QD in bean sprout juice dextrose culture-medium.
Fig. 8 is the growth curve of schizosaccharomyces pombe 2.1621 in bean sprout juice dextrose culture-medium.
Fig. 9 is that kluyveromyces marxianus 191l is at the growth curve in bean sprout juice dextrose culture-medium.
Embodiment
embodiment 1: the preliminary judgement of yeast glucose fermentation producing and ethanol ability in YEPD substratum
Can the yeast seeds of glucose fermentation producing and ethanol be respectively yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) QD, derive from Qingdao Beer Co., Ltd., schizosaccharomyces pombe bacterium (
schizosaccharomyces pombe) 2.1621, derive from DSMZ of the Chinese Academy of Sciences, kluyveromyces marxianus (
kluyveromyces marxianus) 191l, derive from Chinese industrial Culture Collection, three are openly bacterial classification.
Conveniently experimental technique, needs to test the producing and ethanol ability of every Yeasts in an experiment.
The viable bacteria concentration of inoculation bacterium liquid is 1.0 × 10
6~ 1.0 × 10
7cFU/mL; Yeast YEPD culture medium solution is the aqueous solution be made up of following component, and the content of each component is respectively yeast powder 10.0, peptone 20.0, glucose 20.0 by g/1000mL aqueous solution, and surplus is water, the pH value nature of substratum; The sterilization method of substratum is high pressure steam sterilization, and sterilising temp is 112 DEG C, and the time is 20min.
Determine that each bacterial strain optimal culture condition is culture temperature 30 DEG C, culture cycle 24h, oscillation rate 150r/min, pH value nature by pre-stage test.
(1) saccharomycetic growth curve is drawn
Under the optimal culture condition of fixed in pre-stage test, to comprise each composition of YEPD substratum content, culture temperature, culture cycle, pH, oscillation rate, inoculation yeast bacterium is in liquid nutrient medium and cultivate, and take incubation time as X-coordinate, with the absorbance A of bacterium liquid
600for ordinate zou, every 2h sampling once, survey the absorbance A of solution
600, draw the absorbance A of each yeast nutrient solution
600with the growth curve of incubation time change, judge the lag period of each bacterial strain, logarithmic phase, stationary phase accordingly, see accompanying drawing, wherein accompanying drawing 1 is yeast saccharomyces cerevisiae QD growth curve, accompanying drawing 2 is schizosaccharomyces pombe bacterium 2.1621 growth curve, and accompanying drawing 3 is kluyveromyces marxianus 191l growth curve.
(2) preparation of test tube liquid nutrient medium and sterilizing
Preparation, containing the YEPD substratum 1000mL of glucose, is distributed into 100 test tube liquid, each 9.7mL; Get 50 test tube liquid wherein, put into the inverted fermentation tubule being full of aforesaid liquid substratum 0.3mL wherein, careful operation is to make not produce bubble in tubule, the test tube liquid nutrient medium of fermentation tubule will be housed, be designated as a, and other 50 be unkitted fermentation tubule test tube liquid nutrient medium, be designated as b, simultaneously at 112 DEG C, sterilizing 20min under 0.5Mpa wet-hot steam, take out cooling.
(3) inoculation and cultivation
Inoculum size with 0.5% accesses yeast bacterium liquid, 150 r/min shaking culture 8h at 30 DEG C in the test tube liquid nutrient medium being unkitted fermentation tubule, and now bacterium liquid is in logarithmic phase latter stage, takes out stand-by, is designated as c.
(4) microbiotic mother liquor
Prepare kantlex solution mother liquor and chloromycetin solution mother liquor respectively, concentration is 0.5g/100mL, two kinds of microbiotic mother liquor filtration sterilizations under aseptic technique respectively; The preparation of microbiotic mother liquor after inoculation bacterium liquid cultivation period is carried out.
(5) ethanol fermentation
Under aseptic technique, fermentation tubule in a is taken out, be inverted into rapidly in the nutrient solution c being cultured to logarithmic phase latter stage, careful operation is to make not enter bubble in fermentation tubule, then be rapidly in nutrient solution and add the chloromycetin solution that kantlex solution that final concentration is 50 μ g/mL and final concentration are 25 μ g/mL respectively, after more than having operated, the tube culture c that fermentation tubule is housed is continued quiescent culture to culture cycle at 30 DEG C.
Culture cycle comprises thalli growth stage of not putting fermentation tubule and thalline glucose fermentation producing and ethanol and CO after putting into fermentation tubule
2stage.
(6) ethanol high-yield bacterial strain primary dcreening operation
The above-mentioned nutrient solution c completing a culture cycle is taken out, observe in test tube liquid intratubular bubble production and the expel liquid situation of fermenting, according to fermentation tubule in gas column size can tentatively judge yeast producing and ethanol number, gas column is larger, producing and ethanol is more, very promptly can filter out the bacterial strain of high-yield ethanol like this, the number of producing and ethanol by further detection by quantitative as liquid phase chromatography or bio-sensing analyser method are determined.
(7) primary dcreening operation result verification
The superior strain obtained by above-mentioned primary dcreening operation carries out producing and ethanol fermentation, and sampling, suitably dilutes, and uses SBA-40C type bio-sensing analyser to carry out producing and ethanol analysis, to verify the exactness of this programme.
Yeast Growth curve is see accompanying drawing 1, accompanying drawing 2, accompanying drawing 3, due to YEPD substratum Middle nutrition material abundance and be convenient to utilize, yeast enters logarithmic phase very soon after of short duration lag period, according to growth curve: the lag period that yeast saccharomyces cerevisiae QD grows is 0-2h, logarithmic phase is enter stationary phase after 2-10h, 10h; The delayed growth phase of schizosaccharomyces pombe bacterium 2.1621 is 0-2h, logarithmic phase is enter stationary phase after 2-10h, 10h; The delayed growth phase of kluyveromyces marxianus 191l is 0-2h, logarithmic phase is 2-10h, cultivate 10h enters stationary phase later;
Relatively gas column size in each strain fermentation tubule, obtains result see subordinate list 1
Note: how much be divided into 5 grades according to aerogenesis in fermentation tubule:
A " ++++" represent aerification in fermentation tubule,
3/4 gas is full of in b " +++ " expression fermentation tubule,
L/2 gas is full of in c " ++ " expression fermentation tubule,
1/4 gas is full of in d "+" expression fermentation tubule,
E "-" represents in fermentation tubule do not have gas
the checking of primary dcreening operation result
Producing and ethanol Analysis offermehtations is carried out, to verify the exactness of primary dcreening operation result to the aerogenesis body bacterial strain listed in upper table.The concentration of preparation ethanol standardized solution is 40mg/100mL, after fermented liquid stoste dilutes 40 times, records the ethanol content of each bacterial strain as following table 2
Note: in upper table, ethanol production value is measured value after fermenation raw liquid dilutes 40 times
As can be seen from data in table 2, the ethanol production of yeast saccharomyces cerevisiae QD is relatively high, demonstrates the primary dcreening operation result listed in table 1, illustrates that this experimental program is correct.
embodiment 2: the preliminary judgement of saccharomycetes to make fermentation potato dextrose medium producing and ethanol ability
Saccharomycetes to make fermentation culture medium solution changes potato glucose (PDA) substratum into, the aqueous solution be made up of following component, the content of each component is respectively potato 200.0, glucose 20.0 by g/1000mL aqueous solution, and surplus is water, pH value nature.
Its compound method takes 200g potato, clean peeling is cut into small pieces, add water well-done (boil 20 ~ 30 minutes, can be poked by glass stick), by four layers of filtered through gauze, add glucose, continue heated and stirred mixing, slightly supply moisture to 1000 milliliter again, packing test tube after cooling, jump a queue, wrap up, after 112 DEG C of sterilizing 20min, take out cooling.
Except substratum and preparation method thereof is different, culture cycle is 48h, sample interval is except 4h, other as culture temperature, oscillation rate, inoculation viable bacteria concentration, growth curve draftings, test tube liquid subpackage, inoculate all identical with embodiment 1 with the primary dcreening operation, primary dcreening operation result verification etc. of cultivation, microbiotic mother liquor, ethanol fermentation and high-yield ethanol bacterial strain.
Figure 4 shows that the growth curve of yeast saccharomyces cerevisiae QD in PDA nutrient solution, the lag period of known strain growth is 0-4h, logarithmic phase is enter stationary phase after 4-24h, 24h; Fig. 5 is the growth curve of schizosaccharomyces pombe bacterium 2.1621 in PDA nutrient solution, and the lag period of known strain growth is 0-4h, logarithmic phase is enter stationary phase after 4-24h, 24h; Fig. 6 is the growth curve of kluyveromyces marxianus 191l in PDA nutrient solution, and the lag period of known strain growth is 0-4h, logarithmic phase is enter stationary phase after 4-24h, 24h.
With following table 3 be each bacterial strain aerogenesis bubble situation
Note: how much be divided into 5 grades according to aerogenesis in fermentation tubule:
A " ++++" represent aerification in fermentation tubule,
3/4 gas is full of in b " +++ " expression fermentation tubule,
L/2 gas is full of in c " ++ " expression fermentation tubule,
1/4 gas is full of in d "+" expression fermentation tubule,
E "-" represents in fermentation tubule do not have gas
the checking of primary dcreening operation result
In PDA substratum, the aerogenesis of each bacterial strain steeps the basic simlarity in situation and YEPD substratum, remains yeast saccharomyces cerevisiae QD aerogenesis and steeps more, carry out producing and ethanol Analysis offermehtations, to verify the exactness of primary dcreening operation result, obtain as following table 4 data above-mentioned each bacterial strain
Note: in upper table, ethanol production value is measured value after fermenation raw liquid dilutes 40 times
As can be seen from data in table 4, in PDA substratum, the ethanol production remaining yeast saccharomyces cerevisiae QD is higher, demonstrates the primary dcreening operation result listed in table 3, illustrates that this experimental program is correct.
embodiment 3: the preliminary judgement of saccharomycetes to make fermentation bean sprout juice dextrose culture-medium producing and ethanol ability
Saccharomycetes to make fermentation culture medium solution changes bean sprout juice dextrose culture-medium into, is the aqueous solution be made up of following component, and the content of each component is respectively soybean sprout 100.0, glucose 50.0 by g/1000mL aqueous solution, and surplus is water, pH value nature.
Compound method claims fresh soybean sprout 100g, is placed in beaker, then adds 1000 mL water, and little fire boils 30 min, by filtered through gauze, supplies dehydration, namely makes 10% bean sprout juice.5g glucose is added by every 100 mL10% bean sprout juices.Then packing, jump a queue, wrap up.Take out after high pressure steam sterilization 112 DEG C of sterilizing 20min.
Except substratum and preparation method thereof difference, other is as all identical with embodiment 2 in culture condition, sample interval, inoculation viable bacteria concentration, growth curve drafting, test tube liquid subpackage, inoculation and the primary dcreening operation of cultivation, microbiotic mother liquor, ethanol fermentation and high-yield ethanol bacterial strain etc.
Accompanying drawing 7 is the growth curve of yeast saccharomyces cerevisiae QD in bean sprout juice glucose culture solution, and the lag period of known strain growth is 0-4h, logarithmic phase is enter stationary phase after 4-24h, 24h; Accompanying drawing 8 is the growth curve of schizosaccharomyces pombe bacterium 2.1621 in PDA nutrient solution, and the lag period of known strain growth is 0-4h, logarithmic phase is enter stationary phase after 4-24h, 24h; Accompanying drawing 9 is the growth curve of kluyveromyces marxianus 191l in PDA nutrient solution, and the lag period of known strain growth is 0-4h, logarithmic phase is enter stationary phase after 4-24h, 24h.
With following table 5 be each bacterial strain aerogenesis bubble situation
Note: how much be divided into 5 grades according to aerogenesis in fermentation tubule:
A " ++++" represent aerification in fermentation tubule,
3/4 gas is full of in b " +++ " expression fermentation tubule,
L/2 gas is full of in c " ++ " expression fermentation tubule,
1/4 gas is full of in d "+" expression fermentation tubule,
E "-" represents in fermentation tubule do not have gas
the checking of primary dcreening operation result
In bean sprout juice dextrose culture-medium, aerogenesis bubble situation and the basic simlarity in YEPD substratum of each bacterial strain, remain yeast saccharomyces cerevisiae QD aerogenesis and steep more, carry out producing and ethanol Analysis offermehtations to above-mentioned each bacterial strain, to verify the exactness of primary dcreening operation result, obtain as following table 6 data
Note: in upper table, ethanol production value is measured value after fermenation raw liquid dilutes 40 times
As can be seen from data in table 6, in bean sprout juice dextrose culture-medium, the ethanol production remaining yeast saccharomyces cerevisiae QD is higher, and the primary dcreening operation result in the result verification of quantitative analysis table 5, illustrates that this experimental program is correct.
Claims (3)
1. a method for rapid screening high-yield ethanol yeast, is characterized in that: the step of the method is:
1st step, first draw the growth curve of bacterial strain, judge its lag period, logarithmic phase, stationary phase accordingly;
2nd step, the preparation also some test tube liquid nutrient mediums of packing, wherein half is inverted into the fermentation tubule of filled with fluid substratum, by two kinds of test-tube culture medium high pressure steam sterilizations, takes out cooling;
3rd step, in the test tube liquid nutrient medium not putting fermentation tubule, access bacterial classification, shaking culture to logarithmic growth latter stage, is taken out stand-by just;
4th step, between bacterium liquid incubation period, preparation microbiotic mother liquor, filtration sterilization;
Fermentation tubule in 2nd step takes out by the 5th step, aseptic technique from test tube liquid nutrient medium, being inverted into above-mentioned 3rd step is cultured in the nutrient solution in logarithmic growth latter stage, ensure that in tubule, bubble-free enters, and add the antibiotic solution of appropriate 4th step preparation, it is carried out static fermentation culture;
6th step, after being cultured to one-period, take out the test tube liquid in above-mentioned 5th step, observe gas column size in fermentation tubule, gas column is larger, and ethanol production is higher, thus rapid screening can go out object bacterial strain.
2. method according to claim 1, is characterized in that the method concrete operations are as follows:
(1) drafting of Yeast Growth curve
Under fixed in pre-stage test, to comprise medium component and content, culture temperature, pH, culture cycle, oscillation rate optimal culture condition, inoculation yeast bacterium is in liquid nutrient medium and cultivate, and take incubation time as X-coordinate, with the absorbance A of bacterium liquid
600for ordinate zou, draw the absorbance A of yeast nutrient solution
600with the growth curve of incubation time change, judge the lag period of strain growth, logarithmic phase, stationary phase accordingly;
(2) preparation of test tube liquid nutrient medium and sterilizing
Preparation maybe can change into the substratum PDA of glucose or bean sprout juice dextrose culture-medium or wort dextrose culture-medium 1000mL containing the substratum YEPD of glucose, be distributed into 100 test tube liquid, each 9.5m ~ 9.8mL, get 50 test tube liquid wherein, be inverted into the fermentation tubule being full of aforesaid liquid substratum 0.2mL ~ 0.5mL wherein, careful operation is in order to avoid enter bubble in tubule, the test tube liquid nutrient medium of fermentation tubule will be housed, be designated as a, the test tube liquid nutrient medium of fermentation tubule is unkitted with other 50, be designated as b, simultaneously at 112 DEG C, sterilizing 20min under 0.5Mpa wet-hot steam, taking-up is cooled to room temperature,
(3) inoculation and cultivation
Inoculum size with 0.5% accesses yeast strain in the test tube liquid nutrient medium b being unkitted fermentation tubule, and at 30 DEG C, 150 r/min shaking culture are just to logarithmic phase latter stage, take out stand-by, are designated as c;
(4) microbiotic mother liquor
Prepare kantlex solution mother liquor and chloromycetin solution mother liquor respectively, concentration is 0.5g/100mL, two kinds of microbiotic mother liquor filtration sterilizations under aseptic technique respectively; The preparation of microbiotic mother liquor after inoculation bacterium liquid cultivation period is carried out;
(5) ethanol fermentation
Under aseptic technique, fermentation tubule in a is taken out from test tube liquid, be inverted into rapidly in the nutrient solution c being cultured to logarithmic phase latter stage, careful operation is to make not enter bubble in fermentation tubule, then be rapidly in same nutrient solution and add the chloromycetin solution that kantlex solution that final concentration is 50 μ g/mL and final concentration are 25 μ g/mL respectively, after more than having operated, the Tube propagation liquid c that fermentation tubule is housed is continued quiescent culture to culture cycle at 30 DEG C;
Culture cycle comprises thalli growth stage of not producing and ethanol and thalline glucose fermentation producing and ethanol and CO
2stage;
(6) primary dcreening operation of ethanol high-yield bacterial strain
The above-mentioned nutrient solution c completing a culture cycle is taken out, observe in test tube liquid intratubular bubble production and the expel liquid situation of fermenting, according to fermentation tubule in gas column size can tentatively judge yeast producing and ethanol number, gas column is larger, side light ethanol production is higher, very promptly can filter out the object bacterial strain of high-yield ethanol like this; The number of producing and ethanol is determined by further detection by quantitative.
3. method according to claim 2, is characterized in that the method also comprises:
(7) primary dcreening operation result verification
The superior strain obtained by above-mentioned primary dcreening operation carries out producing and ethanol fermentation, then samples, suitably dilutes, and uses bio-sensing analyser method or liquid phase chromatography to carry out producing and ethanol analysis, to verify the accuracy of present method primary dcreening operation result.
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| CN101215529A (en) * | 2007-12-26 | 2008-07-09 | 江南大学 | Alpha-ketoglutaric acid high yield bacterium, screening method thereof and production of alpha-ketoglutaric acid from the same by fermentation method |
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