CN101200733B - Method for producing fuel ethanol by yeast hybrid fermentation whey - Google Patents
Method for producing fuel ethanol by yeast hybrid fermentation whey Download PDFInfo
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Abstract
A method of producing fuel ethanol by whey especially relates to the mixed fermentation of Kluyveromyces marxianus and saccharomyces cerevisiae and a method of producing the fuel ethanol by solidifying fermented whey thereof, which can shorten the fermentation period of the whey and improve the substrate utilization rate. Base on the prior yeast strain, the present invention uses the technical measures of fluid culture, cell solidification, proliferation culture, fermentations (mixed fermentation, mixed solidification fermentation and solidification serial fermentation), etc., to ensure that the period that the yeast uses the whey fermentation to produce the fuel ethanol is shorted to 48 hours; the alcohol degree is 5.0 percent (V.V); the substrate utilization rate is 98.2 percent; compared with the single Kluyveromyces marxianus fermentation, the fermentation period is shortened by 50 percent and the wine yield is improved by 31.6 percent.
Description
[technical field]: the present invention relates to a kind of method of utilizing whey to produce alcohol fuel, particularly relate to the mixed fermentation of two saccharomycete kinds and the method that the immobilization fermentation whey is produced alcohol fuel thereof.
[background technology]: whey is to produce the byproduct of cheese, casein food grade etc., is the liquid portion of separating from cow's milk when making the said products.Whey has higher nutritive value, and 55% (w/w) that it contains the milk nutritive ingredient mainly comprises lactose, protein, mineral substance and multivitamin etc.At present, still there is whey about half not to be utilized in the world and is discharged, because lactose-content is higher in the whey, the dissolved oxygen in the consume water, pollution power is bigger 100 times than the pollution power of the general sewage in city.Therefore reasonably development and use whey resource has become problem demanding prompt solution.
Lactose is a kind of disaccharides, need be decomposed into glucose under the effect of Sumylact L and semi-lactosi could be utilized by biology.General S. cervisiae can't directly utilize the lactose in the whey owing to lack Sumylact L in the cell.Known yeast that can ferment lactose mainly concentrates on genus kluyveromyces (Kluyveromyces) and mycocandida (Candida) etc.Problems such as the part yeast of these two genus can utilize lactose, but their are not so good as yeast saccharomyces cerevisiae in some characteristics aspect the production alcohol, exist fermentation period long, and sugared residual quantity height and fermented wine precision are not high.Present industrial whey mainly is processed to whey beverage, whey powder, whey cream, whey-protein, whey enriched material and lactalbumin powder etc., does not also utilize whey to produce the suitability for industrialized production of alcohol fuel on a large scale.
[summary of the invention]: the purpose of this invention is to provide a kind of novel method of utilizing whey to produce alcohol fuel, solve the problems such as not thorough, the sugared residual quantity height of whey fermentation alcohol and spirit yield are low of utilizing.A kind of method of efficiently utilizing whey to produce alcohol that the present invention relates to, to shorten the whey fermentation cycle, improve the utilization ratio and the fermented wine precision of whey, minimizing is to the pollution of environment, provide a potential resources for producing alcohol fuel, and provide a terms of settlement preferably for developing the whey resource.
For realizing purpose of the present invention, adopted following technical scheme, this method may further comprise the steps:
1). kluyveromyces marxianus and yeast saccharomyces cerevisiae are inoculated in respectively in the liquid nutrient medium (YEPD substratum), carry out seed culture.
2). direct mixed yeast fermentation: two primary yeast bacterium (kluyveromyces marxianus and the yeast saccharomyces cerevisiae) liquid seeds that will obtain inserts respectively (ratio is 3: 1 to 1: 3) and pitch time (time is 0 to 12 hour) by a certain percentage that (fermentative medium formula is: whey powder 12% in the fermention medium, lactose-content 100.2g/L wherein, pH6.0,110 ℃, 20min sterilization) carrying out mixed fermentation produces alcohol fuel.
Or
3). the mixed immobilization fermentation: two primary yeast bacterial classification that liquid culture is obtained mix the back immobilization by a certain percentage, and the fixed yeast gel particles carried out multiplication culture (proliferated culture medium is the YEPD liquid nutrient medium), then cultured immobilization mixed yeast gel particles is inserted producing fuel ethyl alcohol by ferment in the fermention medium.
A). two primary yeast bacterial classification are mixed back (ratio can be 3: 1 to 1: 3) by a certain percentage, mix (sodium alginate soln is prepared with the distilled water heating for dissolving) with 3.0% sodium alginate and form sodium alginate-yeast suspension.
B). it is 20 ℃ water-bath constant temperature that preparation 2mol/L calcium chloride solution places temperature, with syringe (being furnished with the 6# injection needles) sodium alginate-yeast suspension drop is splashed into granulation in the calcium chloride solution, and constant temperature was kept 1 hour, make the abundant immobilization of yeast, leave standstill after 10 hours and make gel particles.
C). the supernatant liquor that inclines, with the distilled water flushing fixed yeast once, put in the calcium chloride solution of 0.05mol/L balance then again after 24 hours, standby.
D). before inserting proliferated culture medium with 0.85% normal saline flushing fixed yeast once.The fixed yeast gel particles for preparing is linked in the proliferated culture medium, cultivated 24 hours for 30 ℃, carry out the propagation of fixed yeast.
E). the co-immobilization yeast gel particles behind the multiplication culture is filled into the glass column reactor with 10% inoculum size, inserts in the fermention medium producing fuel ethyl alcohol by ferment.
Or
4). the polyphone immobilization fermentation: the two primary yeast bacteria liquids that will obtain are cultivated seed and are carried out immobilization and immobilization seed multiplication culture respectively, two kinds of fixed yeast seeds behind the multiplication culture are inserted the immobilization fermentation of contacting in two glass column reactors respectively again and produce alcohol fuel.
A). be 3: 1 to 1: 3 respectively immobilization of ratio by volume with two primary yeast bacteria liquid seeds.
B). two kinds of fixed yeast gel particles that prepare are inserted in the proliferated culture medium 30 ℃ of multiplication culture 24 hours respectively.
C). two kinds of good fixed yeast gel particles of multiplication culture are respectively charged in two glass reaction posts, the whey fermentation substratum is introduced into immobilization kluyveromyces marxianus reaction column (figure one A post), stop charging after advancing to expire, elder generation's fermentation culture continues charging after 0 to 12 hour again, and following current to immobilized brewing yeast reaction column (figure one B post) carries out the alcohol fuel fermentation.
In the method for above-mentioned production alcohol fuel:
Used yeast be kluyveromyces marxianus (Kluyveromyces marxianus, TY-13) and yeast saccharomyces cerevisiae (Saccharomyces cerevisiae AY-5), is the laboratory and preserves bacterial strain.
Advantage of the present invention and positively effect:
The method of utilizing whey to produce alcohol fuel provided by the invention, based on kluyveromyces marxianus bacterial strain and Wine brewing yeast strain, by liquid culture, cell fixation, multiplication culture, in conjunction with mixed fermentation technology and immobilization technology, the cycle that can make yeast utilize whey fermentation to produce alcohol fuel shortens, and improves substrate utilization ratio and fermented wine precision.
[description of drawings]:
Fig. 1 is a polyphone immobilization fermentation device synoptic diagram.
Wherein, 1 triangular flask, 2 peristaltic pumps, 3 glass columns, 4 opening for feeds, 5 thief holes, 6 overflow ports, 7 discharge ports.Glass pillar reactor A and B volume are 2000mL, H/D=4.0.
[embodiment]:
Embodiment 1: kluyveromyces marxianus and yeast saccharomyces cerevisiae mixed fermentation whey are produced alcohol fuel
1, kluyveromyces marxianus and yeast saccharomyces cerevisiae are inoculated in the liquid nutrient medium, carry out the cultivation of liquid seeds.
Two primary yeast bacterium (kluyveromyces marxianus and the yeast saccharomyces cerevisiae) liquid seeds that 2, will obtain is 3: 1 according to volume ratio, 2: 1,1: 1,1: 2,1: 3 and pitch time (two primary yeast joining days added kluyveromyces marxianus liquid seeds fermentation culture earlier and add the yeast saccharomyces cerevisiae liquid seeds after 0,1,2,3,6,12 hour at interval) are inserted respectively and carry out mixed fermentation production alcohol fuel in the fermention medium.
Table 1 mixed fungus fermentation and single yeast fermentation are relatively
| Subject | Alcoholic strength (v/v%) | Residual sugar (g/100mL) | Fermentation period (hour) | Substrate utilization ratio |
| The single yeast of mixed fungus fermentation | 4.6 3.8? | 0.23 0.58 | 88 96? | 97.70％ 94.20％? |
Annotate: single yeast refers to kluyveromyces, and inoculum size is 10%.
Table 2 mixed yeast fermentation bacterial strain ratio
| Bacterial strain ratio (kluyveromyces marxianus: yeast saccharomyces cerevisiae) | Alcoholic strength (v/v%) | Residual sugar (g/100mL) | Fermentation period (hour) |
| 3∶1 2∶1 1∶1 1∶2 1∶3 | 4.3 4.4 4.5 4.2 4.0 | 0.355 0.285 0.241 0.410 0.480 | 88 88 88 88 88 |
Table 3 mixes bacterium inoculation time (ratio 1: 1) at interval
| Incubation time (hour) | Alcoholic strength (v/v%) | Residual sugar (g/100mL) | Fermentation period (hour) |
| 0 1 2 3 6 10 12 | 4.4 4.4 4.5 4.6 4.4 4.3 4.2 | 0.271 0.285 0.259 0.230 0.295 0.385 0.420 | 88 88 88 88 88 88 88 |
Experimental result sees Table 1, table 2 and table 3, and the result shows: with two primary yeast ratios 1: 1, add Marx Shandong dimension yeast culture earlier and add yeast saccharomyces cerevisiae after 3 hours, when carrying out mixed fermentation production alcohol fuel, alcoholic strength is 4.6%v/v, fermentation period 88 hours, and the residual sugar value is 0.23g/100mL.Compare with single gram Marx Shandong dimension yeast fermentation, fermentation period shortens 8 hours, and alcohol output improves 21.05%.Embodiment 2: kluyveromyces marxianus and yeast saccharomyces cerevisiae co-immobilization fermented whey are produced alcohol fuel
1, cultured kluyveromyces and yeast saccharomyces cerevisiae seed liquor were mixed with volume ratio in 3: 1 to 1: 3 after, mix (sodium alginate soln with the preparation of distilled water heating for dissolving) again with 3.0% sodium alginate and form sodium alginate-yeast suspension.
2, to place temperature be 20 ℃ water-bath constant temperature to preparation 2.0mol/L calcium chloride solution 30mL, with syringe (being furnished with the 6# injection needles) sodium alginate-yeast suspension drop is splashed into granulation in the calcium chloride solution, and constant temperature kept 1 hour, made the abundant immobilization of yeast.The supernatant liquor that inclines with the distilled water flushing fixed yeast once, is put in the calcium chloride solution of 0.05mol/L balance after 24 hours then again, and is standby.Before inserting proliferated culture medium with 0.85% normal saline flushing fixed yeast once.
3, the co-immobilization yeast gel particles for preparing is inserted in the proliferated culture medium, 30 ℃ activate 24 hours.
4, will breed the kluyveromyces marxianus and the yeast saccharomyces cerevisiae of back co-immobilization, the inoculum size with 10% inserts carries out the alcohol fuel fermentation in the fermention medium, and leavening temperature is 30 ℃.
Experimental result sees Table 4, table 5 and table 6, the result shows: kluyveromyces marxianus and yeast saccharomyces cerevisiae ratio are when carrying out co-immobilization producing fuel ethyl alcohol by ferment at 1: 1, alcoholic strength reaches 4.8%v/v, fermentation period 60 hours, residual sugar 0.21g/100mL, fixed yeast can be reused 8 batches continuously.Co-immobilization mixed fungus fermentation and the fermentation of single immobilization kluyveromyces are compared, and fermentation period shortens 12 hours, and alcohol output improves 6.7%.
Table 4 co-immobilization mixed fungus fermentation and the fermentation of single fixed yeast are relatively
| Subject | Alcoholic strength (v/v%) | Residual sugar (g/100mL) | Fermentation period | Substrate utilization ratio |
| The single fixed yeast of co-immobilization mixed fungus fermentation | 4.8 4.3? | 0.21 0.44 | 60 72? | 97.90％ 95.60％? |
Annotate: single fixed yeast is the immobilization kluyveromyces, and inoculum size is 10%.
Table 5 co-immobilization yeast fermentation bacterial strain ratio
| Bacterial strain ratio (kluyveromyces marxianus: yeast saccharomyces cerevisiae) | Alcoholic strength (v/v%) | Residual sugar (g/100mL) | Fermentation period (hour) |
| 3∶1 2∶1 1∶1 1∶2 1∶3 | 4.4 4.5 4.8 4.2 4.0 | 0.265 0.260 0.210 0.425 0.510 | 60 60 60 60 60 |
Table 6 co-immobilization mixed fungus fermentation stability test (bacterial strain ratio 1: 1)
| Algebraically (60 hours per generations) | Alcoholic strength (v/v%) | Residual sugar (g/100mL) | The particle situation |
| 1 2 4 8 9 10 | 4.8 4.8 4.7 4.5 3.8 3.2 | 0.210 0.224 0.255 0.365 0.580 0.980 | Particle is intact, and the good springiness particle is intact, and the good springiness particle is intact, and the good springiness particle is better, and the better particle of elasticity has breakage, and the relatively poor more particle breakage of elasticity is nonelastic |
Embodiment 3: immobilization kluyveromyces marxianus and immobilized brewing yeast polyphone fermented whey are produced alcohol fuel
1, with two primary yeast bacteria liquid seeds is 3: 1 to 1: 3 respectively immobilization of ratio by volume.
2, two kinds of fixed yeast gel particles that prepare are inserted in the proliferated culture medium 30 ℃ of multiplication culture 24 hours respectively.
3, two kinds of good fixed yeast gel particles of multiplication culture are respectively charged in two glass reaction posts, the whey fermentation substratum is introduced into immobilization kluyveromyces marxianus reaction column (figure one A post), stop charging after advancing to expire, elder generation's fermentation culture continues charging after 0 to 12 hour again, and following current to immobilized brewing yeast reaction column (figure one B post) carries out the alcohol fuel fermentation.
The experimental result of post A and post B fixed yeast different ratios sees Table 7, the experimental result of the independent fermentation culture different time of post A sees Table 8, the result shows: post A (kluyveromyces marxianus) is 1: 1 with post B (yeast saccharomyces cerevisiae) fixed yeast optimal proportions, between the independent fermentation culture of post A the most in good time is 3 hours, with this understanding, mean residence time 48 hours, alcoholic strength reach 5.0% (V/V), and the residual sugar value is 0.18g/100mL.
Table 7 fixed yeast polyphone fermentation strain ratio
| Bacterial strain ratio (kluyveromyces marxianus: yeast saccharomyces cerevisiae) | Alcoholic strength (v/v%) | Residual sugar (g/100mL) | Mean residence time (hour) |
| 3∶1 2∶1 1∶1 1∶2 1∶3 | 4.5 4.7 4.8 4.5 4.3 | 0.300 0.290 0.220 0.315 0.425 | 48 48 48 48 48 |
The independent fermented incubation time of table 8 post A (the bacterial strain ratio is 1: 1)
| Incubation time (hour) | Alcoholic strength (v/v%) | Residual sugar (g/100mL) | Mean residence time (hour) |
| 0 1 2 3 6 10 12 | 4.7 4.7 4.8 5.0 4.2 3.8 3.8 | 0.290 0.280 0.255 0.180 0.405 0.680 0.680 | 48 48 48 48 60 72 72 |
Claims (3)
1. method of using kluyveromyces marxianus and yeast saccharomyces cerevisiae mixed fermentation whey to produce alcohol fuel, it is characterized in that the direct mixed fermentation of two primary yeasts, with two primary yeast bacteria liquid seeds according to 3: 1 to 1: 3 ratio of volume ratio, insert the kluyveromyces marxianus fermentation culture earlier, insert yeast saccharomyces cerevisiae after 0 to 12 hour again and carry out the alcohol fuel fermentation, fermentation period 88 hours, alcoholic strength reaches 4.6V/V%.
2. method of using kluyveromyces marxianus and yeast saccharomyces cerevisiae mixed fermentation whey to produce alcohol fuel, it is characterized in that the fermentation of two primary yeast bacterium mixed immobilizations, be to carry out co-immobilization after 3: 1 to 1: 3 the mixed with two primary yeast bacteria liquid seeds according to volume ratio, then co-immobilization yeast gel grain is inserted producing fuel ethyl alcohol by ferment in the whey fermentation substratum, fermentation period 60 hours, alcoholic strength reaches 4.8V/V%.
3. method of utilizing whey to produce alcohol fuel, it is characterized in that the fermentation of two kinds of immobilized yeast polyphones, be 3: 1 to 1: 3 ratio immobilization respectively by volume with kluyveromyces marxianus and yeast saccharomyces cerevisiae two primary yeast bacteria liquid seeds, be respectively charged into again in two glass reaction posts, the whey fermentation substratum is introduced into immobilization kluyveromyces marxianus reaction column, stop charging after advancing to expire, elder generation's fermentation culture continues charging after 0 to 12 hour again, and following current to immobilized brewing yeast reaction column carries out the alcohol fuel fermentation, mean residence time 48 hours, alcoholic strength reaches 5.0V/V%.
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| CN103333811B (en) * | 2013-06-21 | 2014-10-01 | 武汉轻工大学 | Kluyveromyces marxianus as well as composition and application thereof |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1170597A (en) * | 1980-10-20 | 1984-07-10 | Bernard Malige | Method for cultivating microorganisms, particularly yeasts, on lactoserum with an association of judiciously selected strains and of specific mutants of a strain |
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| CA1170597A (en) * | 1980-10-20 | 1984-07-10 | Bernard Malige | Method for cultivating microorganisms, particularly yeasts, on lactoserum with an association of judiciously selected strains and of specific mutants of a strain |
Non-Patent Citations (7)
| Title |
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| M. I. Gonzfilez Siso.THE BIOTECHNOLOGICAL UTILIZATION OFCHEESEWHEY: A REVIEW.Bioresource Technology57.1996,57第4页乙醇发酵部分. * |
| T. Roukas et al.E[hanol15production from deproteinized whey by/ -galactosidasecoimmobilized cells of Saccharomyces cerevisiae.Journal of Industrial Microbiology7.1991,第15页-第18页. |
| T. Roukas et al.E[hanol15production from deproteinized whey by/-galactosidasecoimmobilized cells of Saccharomyces cerevisiae.Journal of Industrial Microbiology7.1991,第15页-第18页. * |
| UOSENBERG, M. et al.Improved Ethanol Production from Whey withSaccharomyces cerevisiae Using Permeabilized Cells ofKluyveromyces marxianus.Acta Biotechnol.15 4.1995,第388页-390页材料和方法及结果和讨论部分. |
| UOSENBERG, M. et al.Improved Ethanol Production from Whey withSaccharomyces cerevisiae Using Permeabilized Cells ofKluyveromyces marxianus.Acta Biotechnol.15 4.1995,第388页-390页材料和方法及结果和讨论部分. * |
| 谢继志等.乳清酒发酵条件及发酵动态的研究.中国乳品工业27 1.1999,27(1),第3-6页. |
| 谢继志等.乳清酒发酵条件及发酵动态的研究.中国乳品工业27 1.1999,27(1),第3-6页. * |
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