• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    PURPOSE:To prepare high concentration ethanol, by bringing fixed yeast into contact and reacting with a medium comprising a nutrient necessary for the propagation of the yeast in a gel carrier and a fermentable sugar as a raw material of ethanol formation. CONSTITUTION:Fixed yeast, which has formed concentrated mycelium layer on the parts near the surface of a gel carrier, is brought into contact and reacted with a nutrient medium so that a fermentable sugar is assimilated to prepare ethanol. When the sugar concentration of the reaction solution is lowered, for example, to about not more than 20wt% of the initial sugar concentration, preferably about not more than 10mg/ml, the fermentable sugar is supplised. Thus, the repetition of the supply of the fermentable sugar at the stage of the decrease of sugar concentration in the reaction solution can produce ethanol having a high concentration of about 200mg/ml at the maximum. A method wherein a feed opening is formed on a part at the top of an inverted cone column and an outlet for the solution after reaction is set at its bottom can be applied.
    公开号:SU1181555A3
    申请号:SU802934908
    申请日:1980-06-12
    公开日:1985-09-23
    发明作者:Чибата Ичиро；Като Дзедзи；Вада Мицуру
    申请人:Танабе Сейяку Ко Лтд (Фирма)；
    IPC主号:
    专利说明:

    oo
    O1 O1
    O1 The invention relates to the alcohol industry, in particular to methods for producing ethanol.  The aim of the invention is to accelerate the process and increase the concentration of ethanol in the medium.  The method is carried out as follows.  Sugar to ethanol is microbiologically transformed by contacting ethanol-producing yeast or anaerobic microorganisms of the genus Saccharomyces or Zymomonas immobilized in a carrier gel with a nutrient medium containing fermentable sugar at a concentration of 50-100 mg / ml and other nutrients necessary for the growth of microorganisms. .  After reducing the sugar concentration in the nutrient medium to 2-10 mg / ml, additional feeding of the nutrient medium containing 100-400 mg / l of sugar is carried out until sugar concentration in the medium 50100 mg / ml, thereby supporting in the process of microbial logical rotation of sugar in ethanol is the growth of yeast or anaerobic microorganisms.  The process is carried out in a batch or continuous manner at 15-45 ° C to obtain ethanol in an environment with a concentration of at least 75-100 mg / l, then the medium containing ethanol is separated, followed by its separation.  Yeast or anaerobic microorganisms with activity aimed at ethanol synthesis, for example, Sacchrerevisiae i FO 2018 and Sacch viarum IFO 1167; yeast used to produce wine, Sacchcerevisia If6 0216, Sacch cerevisiae IFO 0233, Sacch cerevisiae ATCC 4111 and Sacch cerevisiae ATCC 4124, yeast to obtain Sacch sake ATCC 26422 sake; wine yeast Sacch cerevisiae IFO 1661, Sacch cerevisiae ATCC 4098 and Sacch cerevisiae; P.  Any of the above types of yeast or anaerobic microorganisms immobilized in the gel can be used, and the immobilization of the microorganisms is carried out as follows: A small amount of microbial cells is mixed with a solution of the gel base material and the resulting mixture is gelatinized into tablets or film pieces according to with known methods of gelatin-.  For example, the mixture is added dropwise to a solution of a gelling agent in order to obtain tablets or film pieces with a thickness or diameter of 2 mm-5 cm, containing 0.01-10 colonies of microbial cells per 100 g (wet weight) gels.  Further, the gels are incubated in a culture medium suitable for the growth of microorganisms at 15-45 ° C in order to obtain the desired immobilized microorganisms that form a dense layer of microbial cells within the surface layer of the gel carrier.  As the gel base material polysaccharides are used, for example, sulfated polysaccharide, sodium alginate, cellulose succinate.  Any of the sulfated polysaccharides, which contains at least 10%. at. , in the preferred embodiment - 12-62%. in the sulfate groups (-SO N) in its molecule, the sulfated polysaccharides include carrageenan, furcelllaran and cellulose sulfate.  Carrageenan contains approximately 20-30% c. at.  sulfate groups (SOjH) in the molecule, and furcellaran - 12-16% c. at.  sulfate groups in their molecule.  Cellulose sulphate containing 1262% is suitable. at.  sulfate groups in the molecule.  The nutrient medium is prepared by mixing a nitrogen source in water, for example, a yeast extract that has been grown. corn liquor, peptone or t. P.  and one or more nutrients necessary for the growth of microorganisms.  These nutrients are selected from vitamins: thiamine, biotin, pantothenic acid, inositol, etc. P. minerals: phosphates, magnesium salts, calcium salts, sodium salts, potassium salts, etc. P. , ammonium salts: ammonium chloride, kgsh mixtures thereof.  The amount of these nutrients that you need to add is the following,% c. at. : nitrogen source 0.05-1.0, mineral substances 0.0001-0.1, ammonium 0.01-1.0, in addition, traces of vitamins can be added to the nutrient medium.  Then cooked. thus, the 3 nutrient medium is added with the fermentable sugar: glucose, fructose, sacharose, maltose, etc. P.  Molasses contains both fermented sugar and other nutrients necessary for the growth of a microorganism.  In connection with this, an aqueous solution of molasses can be used as a medium for growing a culture.  When implementing the proposed batch principle, the immobilized microorganisms are first contacted with the medium for growing the culture under agitation in order to convert the fermentable sugar in the medium into ethanol. When the sugar concentration drops to less than 20% of the initial concentration, preferably not exceeding 10 mg / ml, an additional amount of fresh medium for growing the sugar-containing culture is added to the conversion reaction system.  The newly added sugar is also converted to ethanol and its concentration in the medium decreases again.  The addition of fresh medium for growing a culture containing sugar can be repeated at certain intervals until a high concentration of ethanol is produced, for example, 100-200 mg / ml.  Additionally, the amount of fresh culture medium that is added to the conversion reaction system should contain fermentable sugar in relatively high concentrations of at least 250 mg / ml, preferably, along with nutrients other than fermentable sugar necessary for growth. immobilized microorganisms every time an additional amount of fresh nutrient broth is added, the sugar concentration in the fresh nutrient medium can be periodically increased. elichivat up to 400 mg / ml.  When implementing the proposed method on the principle of continuous operation, columnar reaction equipment packed with immobilized microorganisms is used.  A cylindrical column, a reversed conical column, a column equipped with a circulation pipe, or any series, or a combination on their base 554 can be used in pumps, columnar reaction equipment.  If a cylindrical column is used as the reaction equipment, the culture medium containing, in a preferred embodiment, 50-100 mg / ml of fermentable. the sugars along with nutrients other than fermentable sugars necessary for the growth of microorganisms are fed continuously through one of the ends of the column in order to convert the sugar into ethanol, and the medium containing the ethanol thus obtained flows out of the column through the other end.  The loading rate of the culture medium is adjusted so that the concentration of sugar in the stream leaving the column does not exceed 20% of its initial concentration, preferably not more than 10 mg / ml.  Then, an additional amount of fresh nutrient medium for growing a culture containing at least 200 mg / ml of sugar is continuously fed into the column, the loading rate of the medium for refining the culture is adjusted so that the concentration of CaIhara in the outflow is not more than 20% of its original concentration, t. e.  no more than 10 mg / mp.  When a conic-shaped reversed column is used as the reaction equipment, the conversion of the fermentable sugar to ethanol can be carried out more efficiently, because the carbon dioxide gas produced in the reaction can be removed more efficiently.  When using a cylindrical column equipped with a circulating tube, a portion of the medium passing through the column circulates through the circulation tube to the bottom of the column.  In this embodiment, the concentration of sugar in an additional amount of fresh nutrient medium for refining the culture, which must be continuously fed through the inlet tube to the bottom of the column, must be increased, for example, up to 250-400 mg / mp, in order to eliminate the decrease in sugar concentration caused by dilution with a circulating portion of broth.  In addition, the concentration of sugar in the culture enhancement medium, which is continuously loaded in the tank, can gradually increase to 250-400 mg / ml over time.  When implementing the proposed method, the immobilized microorganisms retain an extremely high productivity, aimed at ethanol synthesis, for a period of. long period of time, while ethanol is produced with a high concentration of, for example, 100-200 mg / ml.  In addition, a whole series of columns can be used in accordance with the continuous principle of operation.  For example, the continuous process is carried out using a series of cylindrical columns and supplying fresh nutrient medium for refining the culture in the first column at such a feed rate that the sugar concentration in the effluent from the first column decreases to a value not exceeding 20% of its initial concentration, in the preferred embodiment, to a value not exceeding 10 mg / ml, additional amount of fresh nutrient medium for growing a culture containing not more than 250 mg / ml of sugar is loaded into the second columns together with the fluid flowing from the first column, and so on. d. , repeat the same loading procedure for subsequent columns.  It is possible to use this column by adjusting the feed rate of the fresh culture medium so that the sugar concentration in some part of it is reduced to a value not exceeding 20% of its initial concentration by supplying an additional amount of fresh medium for the culture containing sugar. , to the part of the column where the sugar concentration decreases, then repeat this loading procedure in the subsequent parts of the column.  These embodiments of the proposed method can increase the ethanol yield per unit of time; in addition, ethanol can be formed continuously with high concentration and very efficiently.  The separation of the medium after the completion of the transformation reaction can be carried out by known methods, for example by distillation, centrifugation, decantation, etc. P.  When using a column, the broth can be easily separated as a stream flowing from it.  The activity of immobilized microorganisms aimed at the synthesis of ethanol is determined by determining the amount of ethanol produced, the concentration of fermentable sugar is determined in terms of glucose.  . Example 1  Yeast Saccharomyces sake ATCC 26422 is mixed with 20 ml of sterilized 4.5% c. about.  aqueous solution of carrageenan at 37 ° C.  The mixture is added dropwise from a 2% v drain tip. about.  an aqueous solution of potassium chloride (200 ml) to obtain spherical gels with a diameter of 4 mm.  The nutrient medium is prepared by mixing in water,% c. at. : Yeast extract 0.15, ammonium chloride 0.25, acid potassium biphosphate 0.55, magnesium sulfate heptahydrate 0.025, calcium chloride 0.001, citric acid 0.1 and sodium chloride 0.25 and maintain the pH at 5.0.  Concentration glucose is added to the resulting medium.  10% B. C.  and the resulting gels are incubated in containing gluco. 500 ml with gentle shaking for 60 hours to grow the yeast in gels.  The immobilized yeast thus obtained has an activity aimed at the synthesis of ethanol, which is equal to 50 mg of ethanol / ml of gel / h.  The immobilized yeast is in contact with a nutrient medium containing 10% B by 20 mi. C.  glucose (20 ml) for 1 hour  When the concentration of the remaining glucose in the medium drops to 2 mg / ml, an additional amount of fresh nutrient medium containing 40% c is added. about.  glucose (5 ml), and the conversion of glucose to ethanol continues for 1 hour under the same conditions.  Then the step of adding a medium containing 40% c. about.  glucose (5 ml), repeated three times with an interval of 1 h.  As a result, the immobilized yeast retains its activity aimed at ethanol synthesis at a level of 50 mg / ml gel / h during the reaction period, after carrying out the conversion reaction for 5 h, a medium containing ethanol at a concentration of 125 mg / ml (40 ml ).  Example 2  The method is carried out analogously to example 1, the activity directed to the synthesis of ethanol, the released immobilized yeast n decreases even after the extraction and the ethanol production stage is repeated ten times, resulting in a medium containing ethanol with a concentration of 125 mg / ml (40 ml) at the end of each conversion reaction that lasted for 5 hours.  Example 3  Immobilized yeast 20 ml prepared as in example 1.  Then they are placed in a cylindrical column (30 ml volume into which nutrient medium containing 10% c is loaded through one of the ends. about.  glucose, at a rate of 20 ml / h at 30 ° C, with the yeast emerging and leaving the column through the other end at the same speed.  .  After incubation for 60 hours at 30 ° C, the medium is continued to flow from. with the above rate, the broth feed rate is brought to 7 ml / hr, so that the effluent stream contains glucose with a concentration of not more than 10 mg / ml.  Under these conditions, the concentration of glucose in the nutrient medium, which is fed to the column, gradually increases in such a way that the concentration of glucose in it reaches 30%. about.  in terms of broth after 120 h.  During this period, the activity of immobilized yeast, aimed at ethanol synthesis, does not decrease, and as a result, the effluent from the column contains ethanol at a concentration of 150 kg / ml.  In addition, when the nutrient medium, containing 30%. about.  glucose, continuously enters the column at a rate of 7 ml / h, the immobilized yeast in the column retains its stable activity for more than 1 month, and the resulting stream contains ethanol with an average concentration of 146 mg / ml.  Example 4  The production of ethanol is carried out using cylindrical columnar reaction equipment containing three columns.  Each column (volume 30 mp) is prepared analogously to Example 3, t. e.  the columns are packed with immobilized yeast and supplied with a nutrient medium containing 10% B. C.  glucose, at a speed of 20 mi / hr for 60 hours.  The columns are then interconnected.  Nutrient medium containing 10%. l  glucose, served in the lower part of the first column through a pipe at a rate of 20 ml / h at.  The stream flowing from the column is fed to the second column along with a nutrient medium containing 40% B. C.  glucose through the loading tube at a rate of 5 ml / h.  The stream flowing from the second column is fed to the third column along with a nutrient medium containing 40% c. about.  glucose through the loading tube at a rate of 5 ml / h.  As a result, a stream containing ethanol at a concentration of 100 mg / ml at a rate of 30 ml / h flows out of the third column through the exit pipe.  Approximately 5.  The method is carried out analogously to example 4, only 20% of. about.  A 5% aqueous solution of molasses is used instead of 10% c. about.  glucose in the original nutrient medium (100 mg / ml in terms of glucose) and the concentration of an aqueous solution of molasses in the additional nutrient medium gradually increases to 60%. about.  for the purpose of the continuous production of a stream containing ethanol at a concentration of 142 mg / ml at a rate of 5 ml / h.  Example 6  The preparation of ethanol is carried out using a conical columnar reaction equipment.  In a conical column (volume 30 ml) placed immobilized yeast 20 ml, obtained according to example 1, serves a nutrient medium containing 10%. about.  glucose, in the lower part of the column through the loading tube at a speed of 20 ml / h with, the medium was removed from the column in its upper part through the pipeline at the same speed.  After incubation for 40 hours (the medium is fed at a superficial rate), the feed rate is changed to 6 ml / hr. the effluent contained glucose at a concentration not exceeding 10 mg / mp.  Under these conditions, concentration. glucose in the nutrient medium, which is fed into the column, gradually increases so that the concentration of glucose in it reaches 35%. about.  in terms of Wednesday after 72 hours  During this period, the activity of the immobilized yeast, aimed at ethanol synthesis, does not decrease and as a result, the ethanol concentration in the outflow is 175 mg / mp.  Example 7  The production of ethanol is carried out using a cylindrical columnar reaction equipment containing a pipe for a circulating flow, with which part of the medium flowing from the column is fed to the bottom of the column, t. e.  Into the column packed with 25 ml of immobilized yeast prepared according to Example 1, then 20% V, o are fed.  an aqueous solution of molase (100 mg / mp in terms of glucose) in the lower part of the column through a tube at a rate of 25 ml / h at 30 ° C, as a result, the immobilized yeast floats and the medium is removed from the column through its upper part at the same rate.  After incubation for 40 h at (while the medium is being fed at the indicated rate), 50% c. Is fed into the column. about.  A solution of molasese (250 mg / mp in terms of glucose) at a rate of 10 ml / h, and a portion of the medium flowing from the column circulates to the lower part of the column through a tube at a rate of 100 ml / h.  The concentration of molase in the stream circulating through the pipe is reduced to a value not exceeding 10 mg / ml in terms of glucose.  Under these conditions, the activity of immobilized yeast, aimed at ethanol synthesis, does not decrease, and a liquid containing 125 mg / ml of ethanol continuously flows through the pipe, because the concentration of the aqueous solution of molase in the broth, which is loaded into the column, is diluted with medium circulating through a pipe.  .  Example 8  One colony of Zymomonas mobilis IFO 13756 is mixed with a sterilized solution (4.5% c. o,) carrageenan 20 ml at.  The mixture is added dropwise from a 2% v drain tip. about.  an aqueous solution of potassium chloride 200 ml in order to obtain spherical gels with a diameter of 4 mm.  Prepare the nutrient medium by stirring in water.  % at. about. : yeast extract 0.15, ammonium chloride 0.25, acid potassium biphosphate 0.55, magnesium sulfate heptahydrate 0.025, calcium chloride 0.001, citric acid 0.1 and sodium chloride 0.25, the pH of the mixture was adjusted to 6.8 .  Glucose with a concentration of 10% is added to the nutrient medium. about.  and the resulting gels are incubated in a 500 ml glucose-containing broth at 30 ° C for 90 hours in a nitrogen atmosphere in order to grow anaerobic microorganisms in them.  The activity of the thus-obtained immobilized anaerobic microorganisms, directed towards the synthesis of ethanol, is 77 mg of ethanol / ml of gel / h.  Immobilized anaerobic microorganisms 20 ml in contact with a nutrient medium containing 10%. l  glucose (20 ml), for 45 min.  When the concentration of the remaining glucose in the medium drops to 2 mg / ml, an additional amount of fresh nutrient medium containing 40% c is added. about.  glucose (5 ml), and the conversion of glucose to ethanol continues for 40 minutes under the same conditions.  The next step is the addition of a nutrient medium containing 40% of. about.  glucose (5 ml), repeated three times at 40-minute intervals.  B (As a result of immobilization, anaerobic microorganisms retain their activity aimed at ethanol synthesis at the level of 77 mg of ethanol / ml of gel / h and after reacting for 200 minutes, a broth is obtained containing ethanol with a concentration of 125 mg / ml (40 W).  Example 9  The immobilized anaerobic microorganisms obtained according to example 8 were decanted. The operations of example 8 were repeated using the immobilized microorganisms used to obtain ethanol.  The activity of the isolated immobilized anaerobic microorganisms, aimed at the synthesis of ethanol, does not decrease even after the extraction and preparation of ethanol for ten times, as a result, at the end of each conversion reaction lasting 200 minutes, a medium containing 125 mg / mp (40%) is obtained. ml)  Example 10  The method is carried out analogously to example 1, only immobilized anaerobic microorganisms according to example 9 in an amount of 20 ml are placed in the column, and then the nutrient medium containing 10% c is supplied. l  glucose at a rate of 30 ml / h at 30 ° C for 90 h.  The columns are then connected to the sequence n1.
    but. Nutrient medium containing 10% v.o. glucose, is fed into the lower part of the column through a pipe for feeding it at a rate of 30 ml / hr. The liquid flowing out of the column is sent to the second column along with a nutrient medium containing 40% v. glucose supplied through a tube at a rate of 7 ml / h. The liquid flowing out of the second column is supplied to the third column together with the nutrient medium containing 40% of v. O. glucose through a tube at a rate of 7 ml / h. Thus, the effluent liquid containing ethanol with a concentration of 100 mg / ml continuously flows from the third column through the outlet pipe at a rate of 44 ml / h.
    Example 11. The method is carried out analogously to example 8, except that instead of glucose in a nutrient medium (100 mg / ml in terms of glucose), 20% of b.o. water solution of molasses, and the concentration of the latter is gradually increased to 60% B. o. with the aim of obtaining a medium containing ethanol at a concentration of 125 mg / ml (40 ml), as a result of the conversion of molasses to ethanol for 3 hours.
    Example 12. The method is carried out analogously to example 7. In column 5512
    Well placed immobilized anaerobic microorganisms 25 ml, which is prepared according to example 9, in the lower part of the column through the tube serves 20% v.o. an aqueous solution of molasses (100 mg / ml in terms of glucose) at a rate of 40 mg / h at 90 h. The flow from the upper part of the column through the tube has the same rate. Then in the column serves 50%. A molasses solution (250 mg / ml in terms of glucose) at a speed of 15 MP / h, at the same time a part of the medium flowing from the column circulates to the bottom of the column through a circulation pipe at a rate of 100 ml / h. The concentration of the aqueous solution of melas circulating through the tube is reduced to a value not exceeding 10 mg / ml in terms of glucose. Under these conditions, the activity of immobilized anaerobic microorganisms aimed at ethanol synthesis does not decrease, and a stream containing 125 mg / ml of ethanol continuously flows through the tube, because the concentration of the aqueous solution of molasses that is loaded into the column is reduced by the circulating medium.
    权利要求:
    Claims (2)
    [1]
    METHOD FOR PRODUCING ETHANOL, which provides for the microbiological conversion of sugar into ethanol by contacting ethanol-producing yeast or anaerobic microorganisms of the genus Saccharomyces or Zymomonas immobilized in a gel carrier with a nutrient medium containing enzymatic sugar and separating the medium containing ethanol, which is characterized by the aim of and increasing the concentration of ethanol in the medium, during the microbiological conversion of sugar to ethanol, they support the growth of yeast or anaerobic microorganisms in e carrier through an additional supply of nutrient medium containing 100-400 mg / ml of sugar, up to the concentration of sugar in the medium 50,100 mg / ml.
    [2]
    2. The method of pop. 1, characterized in that the additional supply of the nutrient medium is carried out after reducing the concentration of sugar in the nutrient medium subjected to contact with yeast or anaerobic microorganisms to 210 mg / ml.
    see is n ™ ns “>
    1 1181555
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    同族专利:
    公开号 | 公开日
    ZA803474B|1981-06-24|
    JPS5913193B2|1984-03-28|
    IN154144B|1984-09-22|
    JPS55165796A|1980-12-24|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    AU534911B2|1981-01-27|1984-02-23|Kyowa Hakko Kogyo K.K.|Preparation of alcohol by fermentation|
    JPS633596B2|1981-07-13|1988-01-25|Shinnenryoyu Kaihatsu Gijutsu Kenkyu Kumiai|
    JPS5823789A|1981-08-05|1983-02-12|Nippon Oil Co Ltd|Production of high-concentration ethanol|
    JPS5876095A|1981-10-30|1983-05-09|S Y Assoc:Kk|Preparation of alcohol|
    JPS5876096A|1981-10-31|1983-05-09|Mitsubishi Kakoki Kaisha Ltd|Continuous alcohol fermentation process|
    JPS5920664A|1982-07-27|1984-02-02|Asahi Chemical Ind|Deep-drawing molding sheet|
    JPH0369514B2|1982-09-22|1991-11-01|Shinnenryoyu Kaihatsu Gijutsu Kenkyu Kumiai|
    JPS59196095A|1983-04-22|1984-11-07|Hitachi Zosen Corp|Alcohol production by fermentation|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    JP54074972A|JPS5913193B2|1979-06-13|1979-06-13|
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