THE USE OF MOLASSES FOR THE PRODUCTION OF ACETONE-BUTANOL

Transcription

1 THE USE OF MOLASSES FOR THE PRODUCTON OF ACETONE-BUTANOL Mohamed Yassein Mohamed Sugar and Distillation Company, Chemical Factories, Hawamdia - Giza, Egypt ABSTRACT By-Product This paper deals with conditions for the production of acetone and butanol by fermentation of molasses using Clostridium acetobutylicum. Conditions examined are age of inocu~u~./rate of fermentation, effect of temperature, origin and optimum concentration of molasses, contamination by iron and status of molasses as a source of carbohydrate. The process is solidly established in Egypt. NTRODUCTON The Egyptian Sugar and Distillation Company was incorporated in 1881 and currently owns seven sugar factories which, in 1984/1985, processed 7 million tonnes of cane and produced tonnes of sugar and tonnes of molasses. Two-thirds of the molasses was used by local companies and one third was allocated for export. The bulk of the locally processed molasses is used to produce acetone and butanol, ethyl alcohol, acetic acid, oxytetracycline and baker's yeast. Ethyl acetate, butyl acetate, thinners, adhesives, liquid C02 and fodder yeast are produced as by-products. Blackstrap molasses is a complex mixture, containing sucrose, reducing sugars, salts, and many other minor components originally present in the cane juice, or added or formed during manufacture. When molasses is used for fermentation, the media are generally deficient in available nitrogen and phosphate. The organisms can utilise ammonia nitrogen for nutrition and do not need complex nitrogenous sources, such as are required by certain strains. Ammonia may be supplied as a salt, such as ammonium sulphate, or as ammonium hydroxide. f ammonium sulphate is used, calcium carbonate, may be added to neutralise the sulfate ions remaining as the ammonium ion is metabolised. Ammonium hydroxide can be satisfactorily added at intervals to an active fermentation. Phosphate is supplied generally as superphosphate. The advantages of blackstrap molasses as a fermentation raw material have been summarised in an early patent by Beeschl as follows: 1. Easy to handle, as a liquid to be pumped. 2. Uniformity over fairly long periods. 3. The yield depends upon the amounts of fermentable sugars. 4. Blackstrap molasses mash is easy to sterilise at lower temperatures. Keywords: Molasses, fermentation, cane by-product, asetone, butanol

2 MOHAMED YASSEN MOHAMED 5. Molasses contains some minerals which contribute to nutrition. 6. The fermentation is conducted at 30 OC, instead of 36 OC, providing less favourable teniperature for contaminating organisms. 7. Tanks and equipment are much easier to clean, and less blockage occurs. 8. Blackstrap molasses is normally the cheapest raw material. 9. High concentrations of cane sugar can be fermented with correspondingly high yields of products in a short period of time. 10. Any residual sugar left in the mash can be utilised rapidly by yeasts with the production of ethanol, or the mash can be used as a source of fodder. This paper deals with experiments conducted to determine the optimum conditions for the production of acetone and butanol by fermentation of molasses. MATERALS AND METHODS CULTURE The experiments were carried out with active strains of Clostridium acetobutylicum, mainly isolated from Egyptian sources. The isolates were held in soil culture in a mixture of soil (49%), clean sand (49%) and chalk (2%). Portions were placed in small vials which were plugged and sterilised. One ml of potato culture suspension was added to each vial and mixed with the soil. The vials were then placed in a closed jar containing active silica gel, and dried out for days. Cultures for seeding experimental media were prepared by inoculating from the stock cultures to test tubes containing glucose-potato medium. The tubes were incubated for three days at 31 OC, to give luxuriant, sporulating growth. The tubes were held in the refrigerator at 5 OC ANALYTCAL METHODS The analytical methods adopted were Sucrose in cane molasses - Tate and Lyle's invertase method (Plews5). Butanol and ethanol - Johnson's method3. Acetone - Messinger's method (Goodwin2). Residual sugar - Somogyi's method6. Total nitrogen - modified Kjeldahl method (Miller and ~ ou~hton~). Fermentable materials EXPERMENTAL RESULTS The fermentation medium which produced the maximum total yield of neutral solvents contained the following ingredients (g/l): potato starch 250, glucose 5, calcium carbonate 2, and ammonium sulphate 1.5. For trial purposes the glucose was replaced by equivalent amounts of the following carbon sources; sucrose, fructose, lactose, starch, molasses, glycerol, arabinose, mannose, and galactose. The ph value was adjusted to 6.0. The media were sterilised, inoculated with a standard inoculum of C. acetobutylicum and incubated at 31 OC for 72 hours. The results in Table show that although the supply of carbon was the same in all cases the yields of acetone-butanol were variable depending on the source material. The arrangement of the different carbon sources in descending order of

3 TABLE. The rate of fermentation for some sugars, by C. acetobufylicum~ Final Total Potency Acid Acetone Butanol Ethanol solvents Sugar of Production p H (mg1100ml) (mg1looml) (mgllooml) (mg1100ml) growth values mash mash mash mash Glucose Sucrose Fructose Lactose Starch Molasses Glycerol Arabinose Mannose ++& Galactose Laboratory list No. of the strain is No. (10). The initial ph value of the fermented medium was adjusted to 6.0.

4 MOHAMED YASSEN MOHAMED 959 production of acetone and butanol was as follows: blackstrap molasses, soluble starch, sucrose, glucose, fructose, galactose, lactose, mannose, arabinose, glycerol. The final ph value of the fermentation medium was between 5.3 and 6.0 (Yassein7). Age of inoculum The effect of the age of the inoculum culture on the production of acetone and butanol by C. acetobutylicum was studied on the medium which was selected as best. The flasks were sterilised and inoculated under aseptic conditions with a 1 standard inoculum of C. acetobutylicum at 13 different ages from 12, to 100 hours. The flasks were incubated at 31 "C for 96 to 144 hours. TABLE 11. Effect of inoculum age on the production of acetonebutanol* No. of The age of incubation Final Residual Total sugar ** solvents inoculum(h) period values (yo) (dl) (days) * Strain No. (10) was used ** nitial sugar concentration was adjusted at 7.0%. The results in Table 1 show that the yield increased with increase in the age of the inoculum culture, reaching an optimum at 24 hours, beyond which the total yield declined; however the yield reached a minimum at about 36 hours and after about 72 hours was back to optimum level. This is not of practical interest, as it is not advantageous to grow inocula so long, but it does suggest that a new cycle of young growth occurs. Rate of fermentation The rate of fermentation was studied by analysing samples taken at intervals to determine total solvents and residual sugars. The rate of production of solvents,

5 was rapid and almost constant at first and after 60 hours about 94% of the original sugar had been converted and 92% of ultimate solvent yield produced. After 60 hours the rate dropped sharply and the reaction was virtually complete at 96 hours. (Table 111). TABLE ll. Effect of incubation period on the production of acetone and butanol. Final Residual Total ncubation PH sugar* solvents period (h) values (911) * nitial sugar concentration was adjusted at 7.0% Effect of temperature The effect of temperature on the production of acetone and butanol shown in Table V indicate that the fermentation is very sensitive to temperature. There is a clearly defined optimum in the range 32 "C to 33 "C. Concentration and origin of molasses The effect of different concentrations of molasses was investigated using the best basal fermentation medium in each case. Samples of Egyptian molasses from various sources and a sample of Pakistani molasses were treated out at sugar concentrations ranging from 5 to 15% without altering the other ingredients. The results in Table V show that the amounts of acetone-butanol produced differed from one type to another and the higher concentrations of sugar were toxic; the effect first appeared at 9.0% sugar. The toxicity may be due to the presence of metals in molasses (specially sodium and potassium) or to unidentified organic substances. The yields from the different types of molasses increased with increase in the initial concentration reaching optimum levels at concentration 8.0%. The sources of the different types of molasses arranged in descending order of yields of solvents were as follows: Pakistanian; Naga-Hamadi; Komombo; Abou- Kurkas; Edfo; Armant; Kous; Refinery. Contamination by iron A major difference between laboratory and plant conditions is that some plant fermenters are made of iron. Pieces of fermenter metal weighing 5 to 10 g were added to fermentation flasks containing the best basal fermentation medium. This

6 MOHAMED YASSEN MOHAMED 96 1 ratio of metal surface to medium volume was much higher than in factory plant. n tests with 18 different strains of the bacterium the effect of iron was to reduce the mean yield of solvents from to g/l. Commercial fermenters should be of stainless steel, with smooth finish to facilitate cleaning. TABLE V. Effect of temperature on the production of acetone and butanol. Final Residual Total Temperature PH sugar * Acetone Butanol solvents ' OC values (mg1100 ml mash) * lntial sugar was adjusted at 7.0% Conditioning of bacteria Since the toxicity of Egyptian molasses appeared at 9.0% sugar, it was possibly of advantage to acclimatise the bacteria to this concentration of sugar. The results showed that acclimatisation seems to have had varied effects according to strain and the proportion of inoculum. Effect of brown sugar Tests were conducted with partial replacement of the sugar in molasses by sugar as brown granulated. When molasses alone was used in the fermentation medium at 6% sugar, the yield of acetone and butanol was 2120 mg/100 ml mash, while

7 TABLE V. Effect of concentrations of sugar with Egyptian and Pakistanian molasses. Concent- Armant ration of sugar Abou-Kurkas Final Edfo T.S Komombo Final T.S. Kous T.S. - Total solvents. gll Naga-Hamadi Refinery Pakistanian ph ,

8 MOHAMED YASSEN MOHAMED 963 in the cases of 4.0% sugar as molasses + 2.0% brown sugar and 5.0% sugar as molasses ;t 1.0% brown sugar, the yields were 2012 and 1891 respectively. The effectiveness of molasses is not related simply to its carbohydrate content and studies are being continued to investigate the effect of other factors present in molasses. t is possible that changes should have been made in the medium to compensate for the nitrogen not supplied in the brown sugar. CONCLUSON The production of sucro-based chemicals by fermentation of blackstrap molasses by microorganisms is practical and economical, especially in countries where the raw materials are readily available. Some authors claim that microbiological production is more costly than the production of sucro-based chemicals by petrochemical industries. Some day, however, the crude petroleum will be exhausted, and then microbiological production will flourish. REFERENCES 1. Beesch, S.C. (1952). Acetone-butanol fermentation of sugars. nd. Eng. Chem., 44, Goodwin, L.F. (1920). The analysis of acetone by Messinger's method. J. Am. Chem., SOC., 42, Johnson, M.J. (1932). Determination of small amounts of ethyl and butyl alcohols. nd. Eng. Chem. Anal. Ed., 4, Miller, L. and Houghton, J.A. (1945). Modifications of Kjeldahl. J. Biol. Chem., 159, Plews, R.W. (1970). Analytical methods used in sugar refining. p Somogyi, M. (1937). A reagent for the copper iodometric determination of very small amounts of sugar. J. Biol. Chem., 117, Yassein, M. (1975). Methods and trials for the production of acetone and butanol from blackstrap molasses by G. acetobutylicum. Technical report prepared for the United Nations ndustrial Development Organization. 8. Yassein, M. (1977). Chemical and biochemical studies on by-products of acetone-butanol fermentation. M.Sc. thesis; Egyptian Sugar and Distillation Company; Chemical Factories; Hawamdia, Giza, Egypt.