DE629694C - Process for the production of n-butyl alcohol and acetone by fermentation of protein-containing mashes - Google Patents

Description

Process for the production of n-butyl alcohol and acetone by fermentation proteinaceous mashes It has already been proposed to use proteinaceous sugar mashes fermented by bacteria that produce n-butyl alcohol and isopropyl alcohol. Even it has already been recommended that mashes containing corn starch be carried out by bacteria to ferment which readily, d. H. without the addition of other substances or organisms, Generate butyl alcohol and acetone. The invention differs from such Process in that no isopropyl alcohol is formed and that the used Bacteria cannot produce sterile cornmeal mashing without other assistance ferment significant amounts of n-butyl alcohol and acetone.

According to the invention, the production of n-butyl alcohol and acetone achieved by fermentation of a protein-containing sugar mash that the mash with a culture of an anaerobic bacterium (of the type of Clostridium Saccharobutyl-acetonicum) is fermented, which can be determined by the fact that it is a sterile molasses mash Vigorously fermented with the formation of the named products, gelatine not liquefied, Glycerine does not ferment and sterile grain flour mashing is not done without aids Formation of significant quantities of the named products fermented.

This bacterium is one of the anaerobic bacteria that can be found in the soil and also in plants that grow in or near the soil, for example in potatoes.

The following is a typical method by which such a Organism can be grown and isolated, but also other methods or modifications this method can be used.

Small pieces of potato, about 25 cc of freshly boiled water and a small amount of diastatic malt extract are placed in a flask closed with a sterile cotton ball and heated to about 60 to 65 ° C for 30 to 60 minutes. The temperature of the contents of the flask is then increased and held at 80 to 85 minutes for 50 minutes. The mass is then cooled and then kept in a thermostat at about 36 °, preferably reducing the oxygen tension. In the course of 24 hours, a violent fermentation will begin. A quantity is then taken from the flask and transferred to a sterile, fermentable cane sugar molasses. The latter is kept for a few days, for example 4 to 5 days, at 36 ° in the thermostat, heated to 80 ° for 10 minutes and suddenly cooled. The mass is then transferred to a sterile, fermentable sugar solution and kept at 36 ° in the thermostat, with preferably cfi oxygen tension being reduced. This heat deterrent and fractional breeding method is repeated several times, for example five to ten times. In this way the organism described can be preserved in the culture in a predominant amount.

The bacterium can be obtained in pure culture with the aid of known bacteriological methods for cultivating anaerobic bacteria, @ for example-a) by fractional culture in deep, sterile test tubes containing 5% potato dextrose agar. The vaccine for such fractionated cultures was removed from near the bottom of the test tubes after piercing under sterile conditions; b) by coating a potato dextrc: seagar anaerobic; c) by isolating a single cell with the help of a micromanipulator .. A large number of such single cells was removed. executed. Various media were used for culture, of which the one described below was found to be suitable. - 1 5 g of white potatoes were scraped and boiled in a flask with 100 ounces of distilled water for 1 to 1 1/2 hours and cooled to about 60 °. ; 5 g of powdered barley malt in a little water were added and the contents of the flask were kept at 55 to 65 ° for about 2 hours. Then 7 g of beet molasses were added. This resulted in a mixture containing about 1/2 "of sugar at a hydrogen ion concentration of pH 6 to 6.7. About 10 cc of this mixture and a small amount of dried beef heart were added to each tube and held for about 20 minutes at steam pressure of 0.14 atrn .. The pure cultures, which showed good growth and good fermentation with the formation of butyl alcohol and acetone, were controlled by known bacteriological methods, for example by fermenting a large number of substances containing carbohydrates.

The results of these controls indicated that a pure culture was obtained and that the pure culture had large yields of butyl alcohol and acetone fermentation of sterile molasses mash.

The new bacterium was named »Clostridium Saccharobutyl-acetonicurn«. To facilitate the identification of this bacterium. it is described with the help of the table of the "Bociety of American Bacteriologists" Name: Clostridium Saccharobutyl-acetonicum,,.: Source: Potato: I. Morphology. Vegetative cells, mobile. Medium: potato and dextrose, pH of the medium: 7, o at the time of vaccination, Age: 18 hours, Temperature: 36 ° C, Shape: long and short rods; Chain formation; 2 to 12 A. Size: in length, un- 0.7 to z, 2, u colored, in width, Ends: rounded, Color: deep blue with methylene blue. Gram = positive - then negative (unstable), Iodine granules positive in old cells. _ .. spores. Medium: potato and dextrose, PH: 7, o at the time of vaccination, Age: - 48 hours, Shape: oval, Spurs: below the ends, 1.8 to 2.3, u in length, un- Orders of magnitude: 1.4 to 1.9, u colored, in width, Color: stains do not look easy with methylene blue. Me- ethylene blue enlarged apparently: the spurs. G.entian violet: Spo- ren dyed dark blue. II. Cultures. i. Nutrient agar sting cultures. Age: 5 days, Temperature: 36 ° C, Growth: not aerobic, Growth: anaerobic - low, predominantly preferably on the ground, Color: cream, Odor: not observed. Potato peel. - Age: 24 hours, Temperature: 36 ° C, Growth: not aerobic, Growth; anaerobic in 4 days, Shape: - sea urchin-shaped, Gloss: shimmering, optical character: opaque, Color: cream, Odor: like butyric acid, Surface: smooth, Consistency: slimy. III. Physiology. x. Optimal temperature for the cell wax tum: 3o to 40 "C, 2. Optimal hydrogen ion concentration a cane sugar molasses: 4, o to 6, o, 3. Gelatin puncture culture: no liquid becoming within 40 days, 4. Indole test: negative within 5 days, 5. Nitrate reduction: negative (absence of nitrite) 5 days (both aerobic as well as anaerobic), 6. Anaerobic organ mus, 7. Lead acetate agar: violent gas evolution ment within . 24 hours; no Blackening within of 5 days, S. Formed gases: CO .: and H_. Fermentation samples: a) are vigorously fermented: gas formation: Arabinose, dextrin; Galactose, lactose, Levulose, maltose, mannose, sucrose, Yylose, glucose; b) after a long time, the following are fermented: Gas formation: glycogen, mannitol, salazine, Inulin, sorbitol, melicitose, a-methyl- glucoside; c) turn very slightly fermented: soluble Strength (gas development and fermentation started after 37 days of storage in the thermostat), starchy substances, like corn, rye, wheat, rice, Barley; d) it does not ferment in the course of 85 days: cellulose, ouercit, glycerin, Erythritol, rhamnose, calcium lactate. The raw material used in these fermentation experiments was an artificial nutrient containing peptone and various inorganic salts. The inoculant used consisted of 8 drops of an 8 day old culture developed in a carbohydrate free medium. Of course, small fluctuations with different filtering or with the same filtering of an organism of this type can occur in the gas evolution or fermentation in such experiments, depending, among other things, on the age, the culture and the cultivation conditions to which the organism has been subjected. While it was mentioned above that a 4 day old culture could produce soluble marches e.g. B. fermented with a certain hesitation, an active, day-old culture eagerly attacked soluble starch. The: primary identifying features here highlighted as distinctive, however, do not vary.

The gelatin liquefaction sample was applied to a 10.8% solution a bacterial nutrient gelatine that contains 0.25 g of glucose per 100 cc of the solution contained. Bacterial nutrient gelatin is a nutrient that contains 3 parts of meat extract, Contains 5 parts peptone and 100 parts gelatin. The samples were with active, 20 Made hours of ancient cultures. There was good gas evolution in the pipes, however no liquid was observed, even after 40 days at 35 ° in the thermostat.

The bacterium does not ferment starchy substances such as corn or rye and wheat. Therefore, such materials cannot be used economically are not converted into fermentable substances by other means before the starch was, as in maltose, dextrose, and dextrins. These latter are easy to see ferment. This can be explained on the basis of the hypothesis that the organism does not secretes enough enzymes that convert the starch into substances which can be fermented by this organism. There were z. B. 3 1 of a mash from 5% corn flour for about 2 hours under a steam pressure of 1.2 atm. heated, vaccinated and kept at about 36 ° for 4 days. Butyl alcohol and acetone, if any originated, but to me in such small quantities that identification is not possible was. The isolated bacterium fermented vigorously cooked molasses mash, the high yields of butyl alcohol and acetone resulted in an effective use of the existing Zuckers made possible. At the time of inoculation, the mashes were. substantially free from dissolved oxygen.

The spores of this organism are very, heat-resistant. In one The spores resist medium with a hydrogen ion concentration of PH 4.3! Lyis 4.5 heating to about 85 ° for 15 minutes.

As a typical example of a fractional culture that applied In order to prepare the vaccine, the following procedure may be described; however, a different method can of course also be used.

About 0.5 cc of a pure culture containing free spores became transferred to a normal test tube 12 cm long and 1.3 cm in diameter. That The test tube contained about ten cc of sterile potato mash consisting of 7 to 100 per cent Potatoes and 1 to 4 per cent. Dextrose in water. the inoculated Tube was heated by immersing it in a beaker of water, so; that the Liquid level in the tube was below the water level in the beaker. That Water in the beaker was kept at a temperature of approximately 85 ° Tube remained in the beaker for 15 minutes, then was removed and immediately cooled by immersion in cold water. The tube was in the thermostat at about 35 held at 37 ° for 15 to 24 hours; during this time a violent continued Fermentation a.

These io cc of the fractionated culture were then used as the culture for uses a flask, the zoo uses up to 500 cc of a similar, sterile potato mash contained. After inoculation, the flask was left at 35 to 37 ° for about 6 to ao hours held in the thermostat. That way, a suitable amount of vaccine will be available for the mash to be fermented is produced. A vaccine that is 2 to 5 percent by volume of the Mash, gives good results, but also relatively larger or smaller quantities of the vaccine can be used.

The typical fermentation processes described below serve to illustrate the present invention; however, of course, other conditions, other proportions of vaccine, and other sugary materials can be used. Example 15o 1 tap water, 10000 g dark cane sugar molasses, 400 g corn flour glutin, 40 g ammonium sulfate, -45 ccm n / i-lactic acid, sterilization by boiling, duration: 60 minutes, inoculated with a 2 to 1/3% active Culture, fermentation time: 60 hours, yield: approx. 24% of the starting sugar (percent by weight), yield: 64.1 ° / o n-butyl alcohol, yield: approx. 359 ° / 0 acetone, yield: approx. approximately 6504, highest acidity value during fermentation: 2.2.

Example 2 15o 1 tap water, 7800 g dark cane sugar molasses, 1950 g beet molasses, 45 g glue, 25 g beet pulp,. 50 g ammonium sulphate, sterilization by boiling, duration: 60 minutes, inoculated with a 2 to 1/3% active culture (percent by volume), duration of fermentation: 7: 2 hours, yield: approximately 26% of the starting sugar ( Percent by weight), yield: approximately 64.5 n-butyl alcohol, yield: approximately 35.5% acetone, yield: approximately o ethyl alcohol, consumed sugar: approximately 75 '/ "highest acidity value during fermentation: 2.1.

Example 3 150 liters of tap water, 45oo g of beet molasses, 4500 g of corn (dextrose) syrup, 400 g of corn flour gluten, 25 ccm of conc. Sulfuric acid, sterilization by boiling, duration: 50 minutes, inoculation with a 5- to 1/2% active culture (percent by weight), fermentation time: 79 hours, yield: approximately: 280 /, the total amount of fermentable carbohydrates, yield: approximately 77 ° / o. n-butyl lalkohol, yield: approximately 23 ° / ° of acetone, yield: approximately o ethyl alcohol, Spent sugar: approximately 83 '/ "Highest acidity during fermentation: 47. Example 4 150 1 tap water, 9ooo g beet molasses; 25 cc kon For example sulfuric acid, 100 g corn flour glutin, sterilization by boiling, duration: 60 minutes, inoculated with a 20% active culture (percent by volume), fermentation time: 90 hours, yield: approximately: 20% of the starting sugar (percent by weight), yield : approx. 77 ° / ° n-butyl alcohol, yield: approx. 23 % acetone, yield: approx.

It is not always necessary to add proteinaceous nutrients. Beet molasses and certain cane molasses contain sufficient amounts of assimilable proteins. The mash does not have enough assimilable material Protein so will uses a mixture of higher and lower proteins in sugary materials, however, non-sugary protein can also be added. A plentiful one Amount of assimilable nitrogenous substance is desirable.

In the fermentations described above, the mashes were fermented at 36 ° C. The optimal temperature for fermentation is around 36 ° C, i.e. in the interval between 30 and 40 ° C.

If the mash has been sterilized or prepared for vaccination, it is it is not absolutely necessary to keep the air away from the mash. However, it is it is desirable that dissolved oxygen not be present in such amounts that local anaerobic conditions can easily develop.

The titratable acidity of the mash is considerably lower during the entire fermentation process than when using other butyl alcohol acetone-producing organisms that are known, for example when using Bacillus granulobacter pectinovorum, Bacillus acetobutylicum and Clostridium acetobutylicum. For example, usually grows the titratable acidity of a slurry containing about 3 to 3.5% sugar, in the first 1 5 to 24 hours of values about o, 5 to o, 6 cc of a n / r o-alkali solution to ro cc of the mash, to about 2.2 cc. Fermentation is usually complete in 2 to 31112 days from the time of inoculation. The organism does not give good yields if the titratable acid value of the mash rises above approximately 2.7 during fermentation.

For all titrations to determine acidity or alkalinity litmus paper was used as an indicator. One of the main functions of the organic Nutrient is the supply of proteins for the growth of bacteria. Different Organic nutrients of animal or vegetable origin can be used for example potatoes, corn gluten, rye, barley, wheat, rice, beet pulp, Soybean meal, cottonseed meal, copra meal, flaxseed meal, malt sprouts, slaughterhouse fluids, Casein, glue, sewage sludge, bone meal, contaminated ethyl alcohol and others proteinaceous substances and ureas.

Substances that contain inorganic nitrogen, ie substances that contain an inorganic nitrogen radical, such as sulfates, carbonates, bicarbonates, chlorides, phosphates, acetates, butyrates, hydroxides and lactates of ammonium, have a clear effect on fermentation. The fermentation is completed in a shorter time by the fact that the consumption of carbohydrates is increased, the ratio of butyl alcohol to acetone in the fermentation product obtained being changed. If inorganic, nitrogenous substances are present, more acetone and less butyl alcohol are obtained. Water-soluble, inorganic, nitrogen-containing substances are used in such small amounts that their ammonia content is, for example, about 0.02 to 0.2 percent by weight, based on the mash; however, larger amounts can also be used. But even mashes to which no inorganic nitrogen has been added have produced fermentation products that contain 7 5 to 85% butyl alcohol. A similar mash containing water-soluble, inorganic nitrogen was fermented under similar conditions. About 65 to 73 '/ "of butyl alcohol and a correspondingly larger proportion of acetone were obtained. The fermentation time can be shortened in this way to 6 to 24 hours.

The acidity that is favorable for the mash before inoculation is approximately 0.2 to 2.5 cc of titratable acidity on zo cc of mash against litmus. This matches with a hydrogen ion concentration of about pH 4.2 to 6.7, but at some Types of sugar, the pH of the mash can be 7.5 or even higher. Mashing with a higher or lower acidity can preferably, but not necessarily, can be adjusted by adding an acid. You can add sulfuric acid, hydrochloric acid, Use lactic acid, butyric acid or phosphoric acid. You can do that to dull you Hydroxide or the carbonate of ammonium or of an alkali or an alkaline earth metal use. The best yields are obtained at lower sugar concentrations with a titratable acid content of approximately 2.7 ccm of a n / ro sodium hydroxide solution on ro ccm mash during fermentation. The titratable acidity in mashes from dark cane sugar molasses rarely exceeded 2.5 cc. In other mashes, like In beet mashes and dextrose syrups, the maximum acidity can be much lower be.

Excellent butyl alcohol yields and rapid fermentations were achieved with mashes whose pH values were at least 4.0 and less than 5.0 . As far as is known, no organism that produces butyl alcohol provides good yields if the pH of the mash when the culture is added to the mash is less than 5.0. It was z. B. a molasses mash of about 3 to '/ 2' / 0 sugar concentration, the pH of which was about 4.2, inoculated with a culture of about 2 to 1 / g%. There was a total yield of fermentation products within a fermentation time of 60 hours, which accounted for more than 25 percent by weight of the sugar originally present in the unfermented mash.

The mash can be sterilized by boiling, it is cooled and fermented in metal vessels such as steel containers. sterilization under pressure is unnecessary, although pressure sterilization as well as temperatures below 100 ° C can be used. Usually it is sufficient to boil the mash for 3o to 6o Minutes. Excellent results have been obtained by boiling the mash 3o to 6o In steel vessels for minutes and leave to ferment in the same containers.

It has been found that when there are no solid particles in the mash are present; before sterilization a small amount of solid, water-insoluble Fabrics; which have no effect on the mash can be added. the Presence of such solid particles assists in a certain way a fast and perfect fermentation: Such substances are, for example, finely powdered coal, Sawdust, straw, starch, cellulose; Beet pulp, etc.

The fermentation products contain: n-butyl alcohol: 65 to 85 percent by weight, acetone: 1 5 to 3 5 percent by weight.

While using different, types of fermentable material the relationship between the individual fermentation products can shift, is this in molasses mashes that contain no substances other than water, carbohydrates and organic Proteins were added in such a way that about 1 part acetone and about 2 to, 6 or more parts of butyl alcohol are produced. The organism described. causes a more different ratio than any known butyl alcohol acetone producing organism. Namely, it causes a larger build-up of butyl alcohol in a proportionally high yield of acetone.

The gaseous fermentation products consist of about 6o to 65 0 / a carbon dioxide and about 35 to 40% hydrogen (volume percent).

The sugary solutions can be used economically, and good yields can be obtained - in mashes that have a sugar content of about 2 to 517o or. more; based on the weight of the mash. About 75 to 85% or more of the sugary material in the mash can persist be consumed if the concentration does not exceed 50/0. Higher concentrations sugary substances can be used, but only lesser ones are used Amounts of the total amount of viable. Substances in butyl alcohol and acetone. split. For example, there is a sugar consumption. of 8o% in mashes that contain 60J0 sugar included, typical.

Ethyl alcohol is only rarely produced by the organism described. In the few cases where ethyl alcohol was found it was only in traces or in amounts up to about: 21 /, present. In the fermentation of cane sugar reams Ethyl alcohol is not usually formed, but certain types of cane molasses give sometimes the mentioned small amounts of ethyl alcohol. When processing and Fermentation of numerous cane sugar molasses mashes, it has been shown that the organism described does not normally produce ethyl alcohol.

The expression »zucl (#: received«, - as used in the description and in The claims used refer to sugars such as sucrose. Dextrose, Levulose and maltose and on hydrolyzed carbohydrates, which are similar to sugars are and can be viewed as such; for example dextrins, inulin and Sugar juices that are left over from processing corn sugar. These Sugar juices mainly contain dextrose and / or dextrins.

Claims (4)

PATEN TA N'S P RÜG H9 i. Process for 'obtaining n-butyl alcohol and acetone by fermentation of protein-containing mashes, characterized in that the mashes are fermented with a culture of an anaerobic bacterium (of the type of Clostridium Saccharobutyl-acetoenicum), which is determined thereby. it can ferment a sterile molasses niche with the formation of the products mentioned, gelatine not liquefied; Glycerine does not ferment, nitrates are not reduced and sterile maize meal mashes are not fermented with the formation of significant quantities of the named products without auxiliary agents. 2. The method according to claim i, characterized in that that the pH of the mash to a value between 4.0 and 7.5, preferably between 4, o and 5, o, is set. 3 .. procedure according to. Claim i, characterized in that. because: ß the acidity of the mash approximately 0.2 to 2.7 ccm of a normal titration acid in io ccm corresponds to the mash i. 4. The method according to claim i to 3,