PREPARATION OF ETHANOL WITH IMMOBILIZED MICROORGANISM

Abstract: Ethanol is prepared by fermenting a lactose-containing solution with a lactase-containing microorganism used in immobilised form.

Description: PREPARATIONOFETHANOLWITHIMMOBILIZEDMICROORGANISM Technical Field The present invention relates to a method of preparing ethanol by fermenting a lactose-containing solution with a lactase-containing microorganism. Background Art The preparation of alcoholic beverages by fermenting lactose, i.e. milk sugar, has been known from time im¬ memorial. Such a fermentation allows an immediate achievement of alcohol percentages of up to 3.0%, and through distillation the alcoholic strength may be in¬ creased. Up to the Second World War the preparation of alcohol based on whey could not be economically justified. Furthermore the alcohol based on whey could not com¬ pete with the alcohol based on potatoes and molasses as raw materials. During recent years the utilization of the lactose con¬ tent of the whey for preparing ethanol has, however, gained a renewed interest as a consequence -of the en¬ vironmental problems arising from draining of whey in waste water. The preparation of ethanol based on whey is in the present connection to be considered an alter¬ native to the conventional biological purification, since the sale of the ethanol processed may cover com¬ pletely or partially the expenses involved in the bio¬ logical purification. The development within the dairy industry, which concentrates the preparation of dairy products including cheese in still larger plants, has increased the problem concerning the removal of the whey. At the same time this development has made the preparation of ethanol from whey more attractive from an economical point of view. The article "Alkoholherstellung aus MoIke" in the periodical "Die Branntweinwirtschaft", from December 1977 discloses a method of preparing ethanol from whey. By this method a selected species of yeast, Saccharo yces fragilis, is propagated batchwise in two steps and subsequently transferred to the first of two fermenters connected in series, whey permeate from an ultra-filtration plant also being added to said fermenter. When the first fermenter has been filled to a predetermined level, an overflow is estab- lished to the second fermenter, and after filling this second fermenter a draining off to a separator is initiated. Part of the separated yeast float is returned to the first fermenter, whereas the wort freed from yeast is carried to a distilling plant through a buffer tank, the alcohol being continuously distilled in said plant. At the .fermentation the yeast species Saccharomyces fragilis is used in concentrations of 2 to 4 g of dry- ferment per liter of fermenting wort and a lactose concentration in the whey permeate of 5 to 9% of lac¬ tose anhydrate. The fermentation is carried out at a temperature of 25 to 30°C and a pH of 3.7 to 4.2. Typically, this fermenting process can only proceed continuously for up to 1 week since the lactose-con- taining medium is easily infected with microorganisms influencing the fermentation in an undesired manner. This feature especially applies to the use of Saccha- romyces fragilis, which compared to the majority of other Saccharomyces species is especially sensitive to infection. Saccharomyces fragilis is used by the known method for the known whey fermentation in spite of the parti¬ cularly high sensitivity thereof to infection because of its capability partly of cleaving lactose and part¬ ly of fermenting both the galactose and the glucose formed by the cleaving of lactose. The usual precautions against infection were to treat the yeast in question with diluted acid. This disin¬ fection cannot, however, be employed in connection with Saccharomyces fragilis because of its general sensitivity. This sensitivity thus also makes a cen- trifugation unsuitable for recirculating the yeast float, which on. the contrary may be performed in con¬ nection with for instance Saccharomyces cerviciae commonly used for fermenting molasses. The known method thus allows conversion velocities of lactose into ethanol of 1 to 5 σ of lactose per g of dry ferment per hour. The yield of the process corre¬ sponds to 75-80% of the yield theoretically obtain¬ able, i.e. that approximately 80% of the lactose is converted into ethanol, whereas the remaining lactose is lost as a consequence of growth of yeast, side re¬ actions and said infection. In accordance with the re¬ action scheme: C 12 H 22°11 + H 2° 4C 2 H 5 OH + 4C0 2 about 42 litersof whey with a lactose content of 4.4% are thus necessary for the preparation of 1 liter of 100% ethanol at a yield of 78%. Disclosure of Invention The present invention relates to a method of fermenting ethanol by fermenting a lactose-containing solution with a lactase-containing microorganism, said method being characterised by using the microorganism in im¬ mobilised form. The immobilisation of microorganisms is known per se and is for instance performed by encapsulating microor¬ ganisms in a polymer. Polyacrylamide is one of the most used polymers, but it suffers from the disadvantage that the polymerisation is difficult to perform whilst maintaining the vitality of the immobilised microor¬ ganism. Furthermore the monomer used for the immobili¬ sation is toxic. For some time a number of polymer carbohydrates have been known to be applicable for immobilising micro¬ organisms. Examples of carbohydrates described in this connection are i-carrageenan, χ-carrageenan, fur= cellaran, cellulose sulfate, ' alginates, and pectinates. These substances are remarkable for their extensive use within the foodstuff industry, e.g. as a thicken¬ ing agent in jam, ice, and as foaming stabilizers in beer. The immobilisation of microorganisms by means of these substances may be carried out by initially mixing an aqueous solution of the polymer carbohydrate and the microorganisms and subsequently causing a formation of gel from the resulting mixture, e.g. through cooling or through contacting with cations such as potassium ions, ammonium ions, calcium ions, magnesium ions, ' — QMPr WIPO ■V.V stanno ions, ferri ions or aluminium ions. The immobilisation may for instance be carried out by transferring the mixture of carbohydrate and micro¬ organisms dropwise to an aqueous solution containing one of the ions causing formation of gel. As a result, guttiform solid particles containing microorganisms are obtained. Previously polymeric carbohydrates were technically ap¬ plied to immobilisation of Escherischia coli having aspartase activity, cf. the article "Immobilization of Escherischia coli cells having aspartase activity with carrageenan and locust bean gum", Enzyme Microb. Technol. , 1979, Vol. 1, April. The technical use of polymeric carbohydrates has, how— ever, not previously been applied to immobilisation of lactase-containing microorganisms. According to the invention it turned out to be possible to immobilise lactase-containing microorganisms in such a manner that the immobilised cells may be used for fermenting lactose-containing solutions. Thus it turned out that the lactase-containing microorganisms _ through the immobilisation achieve a quite unusual re¬ sistance to infection. According to the invention one or more microorganisms selected among Torula cremoris, Torula sphaerica, To- rula lactosa, Saccharomyces fragilis, Saccharomyces lactis, and Candida pseudotrooicalis may be used. According to a preferred embodiment of the invention the microorganism Saccharomyces fragilis may be used for the method. It turned out surprisingly that the usually highly sensitive Saccharomyces fragilis may be immobilised by using carbohydrate-containing poly¬ mers whilst maintaining completely the vitality and capability of lactose—fermentation of the organism. An immobilised lactase-containing microorganism may therefore be used for a substantially longer period by the technical method than the corresponding non- immobilised miσroorganismn. This feature is of par¬ ticular importance to the fermentation of lactose- containing media, since the microorganisms used there¬ for ' are typically propagated to highest degree on a glucose-containing medium and therefore have an indu¬ cing period at the beginning of the fermentation. Thus typically 6 to 8 hours lapse from the time the icro- organism is brought into contact with the lactose- • containing medium, to the time the lactose fermenta¬ tion occurs. This factor implies that the fermenting plant is unproductive in this period. An extension of the time of application of the microorganism therefore implies especially at lactose fermentations an increa¬ se of the productivity of the fermenting plant. By the usual industrial fermenting processes on a lar¬ ger scale it is therefore usually difficult to purify the used substrate sufficiently, whereby the polymer particles are often inactivated upon some time as a consequence of blocking caused by impurities. According to a preferred embodiment of the invention, the lactose-containing solution is ultrafiltrated whey. As a result it turned out to be possible to avoid to a great extent an inactivation of the immobilising material used, and whey permeate thus proved to be particularly suited for fermenting with an immobilised microorganism, especially an immobilised species of yeast. According to an advantageous embodiment of the inven¬ tion the fermenting is carried out by means of a single fermenter. By continuous fermentation of whey permeate it has for reaction-kinetic reasons been necessary to ferment in at least two fermenters, a lactose concentration of about 1% being maintained in the overflow between the two fenrenters. Diis is due to the fact that the quite par- ticular factor applies to the lactose fermentation, that the galactose formed by the influence of the lac- tase on the lactose, inhibits the lactase. This par¬ ticular enzyme-kinetic factor implies that in practise it has been necessary to use several fermenters con- nected in series in order to achieve an optimal pro¬ ductivity. However, according to the invention it turned out sur¬ prisingly that fermentation of a lactose solution with immobilised lactase-containing microorganisms may be carried out in a single fermenter while maintaining the high productivity. Beyond the reduction of the fermenting volume with a factor of about 5 to 10 ren¬ dered possible by the high concentration of microor¬ ganisms in the polymer matrix used, a considerable simplification of the apparatuses necessary is thus obtained at the lactose fermentation. By the method according to the invention it is prefer¬ red to add small amounts of air during the fermenta¬ tion, preferably about 0.01 to 0.05 liter of air per liter of fermentation volume per minute. The oxygen content in the draff from the whey fermen- OMPI * j/ WIPO- tations as conditioned by the biochemical oxygen con¬ sumption however becomes less than usual because less residues, dead cells, are present in the fermented product. Moreover a centrifuga ion of high liquid a- mounts is avoided by using immobilised yeast. This advantage is due to the above increase in productivity, which implies that the same productivity may be ob¬ tained by the method according to the invention com¬ pared to the use of the non-immobilised yeast. This is rendered possible by using a fermenting volume of a- bout 1/5 to 1/10 of the volume used by the known tech¬ nique. Mode for Carrying out the Invention Exam le Preparation of ethanol through fermentation of whey permeate with immobilised Saccharomyces ' fragilis. As ferment a sample of the strain Saccharomyces fragi= lis stored in test tubes on bias agar of the composi¬ tion: 3 g of yeast extract 5 g of meat peptone 3 g of malt extract 10 g of glucose 20 g of agar up to 1000 ml of water, is used. Molasses filled in bottles is inoculated with this culture by means of a platinum needle. After two days the content of the molasses bottles is transferred into two-liter Erlenmeyer flasks containing a liter of sterile nutrient solution of the following composition: O: 130 g of molasses 3 g of diamonium hydrogen phosphate up to 1000 ml of water, whereby the pH-value is ad¬ justed at 4.0 by means of sulphuric acid. The nutrient solution also used in the molasses bot¬ tles is sterilised before inoculation through auto- clavering at 121 C for 30 minutes. The yeast is cultured at 30°C in said molasses bot¬ tles and Erlenmeyer flasks in a shaking thermostat. After 48 hours the molasses used is decanted off, whereafter the flasks are filled with fresh molasses (nutrient solution) . After 24 hours the operation is repeated, and the following day the produced yeast is harvested. The yeast is suspended in a volume of 3% of sodium al- ginate, and the mixture is transferred dropwise into a cold 0.1 molar solution of calcium chloride. The immo¬ bilised yeast is stored therein at 3 C for 24 hours. As lactose-containing substrate, a 9% by weight solu- tiόn of lactose in water is used, and the fermentation is carried out in a thermostatically controlled fer¬ menter provided with an over¬ flow functioning at a volume of more than 1 liter. The total volume of the fermenter is 1 1/2 liter, and 0.5 liter of immobilised yeast corresponding to about 40 g of dry ferment is filled therein. Subsequently, 0.5 liter of 9% by weight of lactose solution is added, and the fermenting temperature is kept at 32 C. After an adapting period of 6 to 8 hours, the fermen- tation starts, which is registered by means of a de¬ velopment of carbondioxide and a drop in the specific gravity of the fermenting solution. Subsequently, 2.5 liter of 9% by weight of lactose solution is added per hour by means of a hose pump. The fermenting is carried out for 2 to 4 weeks whilst producing an amount of ethanol corresponding to 4.5% by volume determined through gas chromatography in the outlet of the fermenting unit. _____ -

Claims: Claims : 1. A method of preparing ethanol by fermenting a lactose-containing solution with a lactase-containing microorganism, c h a r a c t e r i s e d by the micro- organism being used in immobilised form. 2. A method as .claimed in claim 1, c h a r a c¬ t e r i s e d by using as microorganism one or more selected among Torula cremoris, Torula sphaerica, To¬ rula lactosa, Saccharomyces fragilis, Saccharomyces lactis and Candida pseudotropicalis. 3. A method as claimed in claim 2, c h a r a c- t e r i s e d by the microorganism being Saccharo= yces fragilis. 4. A method as claimed in claim 1, 2 or 3, c h a- r a c t e r i s e d by the microorganism being immo¬ bilised by encapsulation in a polymer. 5. A method as claimed in claim 4, c h a r a c¬ e r i s e d by the polymer being a carbohydrate. 6. A method as claimed in claim 5, c h a r a c- t e r i s e d by the carbohydrate being selected among alginates, pectinates, carrageenans and furcellaran. 7. A method as claimed in claim 5, c h a r a c¬ t e r i s e d by the polymer being calcium al inate. 8. A method as claimed in any one of the preceding claims, c h a r a c t e r i s e d by the lactose-con¬ taining solution being ultra-filtrated whey. 9. A method as claimed in any one of the preceding claims, c h a r a c t e r i s e d by the fermenta¬ tion being performed by use of a single fermenter. 10. A method as claimed in any of the preceding claims , c h a r a c t e r i s e d by adding small ampunts of air during the fermentation, preferably about 0.01-0.05 liter of air per liter of fermenta¬ tion volume per minute.

Inventors: BAYER N, GODTFREDSEN S
Applicants: BAYER N, DANSKE SPRITFABRIKKER, FORENEDE BRYGGERIER AS, GODTFREDSEN S
Application year: 1981
Main classification: C12P7
IPC codes: C12N11, C12P7