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Utilisation of blackstrap molasses for the production of acetone and butanol by Clostridium acetobutylicum
AgricuhuralWastes4 (1982) 291-304
UTILISATION OF BLACKSTRAP PRODUCTION OF ACETONE
CLOSTRIDIUM
MOLASSES FOR THE AND BUTANOL BY
A CETOB UTYLIC UM
MAHMOUD FOUAD, ABOU-ZEID ALl & MOHAMED YASSEIN
Egyptian Sugar and Distillation Company, Organic Chemical Factories, Hawamdia, Giza, Egypt
ABSTRACT
The commonest carbohydrate raw material used in the aceton~butanol fermentation is blackstrap molasses. Blackstrap molasses contains relatively high levels qf sahs. especially divalent elements, which affect the microbial growth and the formation of acetone and butanol by Clostridium acetobutylicum. Therefore, treatments q[ blackstrap molasses by different techniques were tested in order to increase the titres of the microbial metabolites.
INTRODUCTION
Acetone and butanol are produced fermentatively, on a large scale, in the Organic Chemical Factories, Egyptian Sugar and Distillation Company. The raw materials used in the fermentative production of acetone and butanol are blackstrap molasses and rice- or corn-bran. The advantages provided by the use of molasses as a fermentation raw material have been summarised in an early patent by Beesch (1952) as follows: 1. 2. 3.
4. 5.
They are easy to handle as a liquid to be pumped. They possess the property of uniformity over fairly long periods. They give a yield of 30 ~o total solvents based on the amounts of fermentable sugars. The mixture of solvents consists of 60-65 % n-butanol and 35-40 oj~ acetone. Molasses mash is easy to sterilise at lower temperatures. The minerals contained in blackstrap molasses are of economic and nutritional importance. 291
Agrieultural Wastes 0141-4607/82/0004-0291/$02.75 ~L')Applied Science Publishers Ltd, England, 1982 Printed in Great Britain
292 6. 7. 8. 9. 10.
MAHMOUD FOUAD, ABOU-ZEID ALl, MOHAMED YASSEIN
Fermentation is run at 30 °C instead of 36 °C, providing a less favourable temperature for contaminating organisms. Tanks and equipment are much easier to clean and less blockage occurs. Molasses is normally the cheapest raw material. High concentrations of sugar can be fermented with correspondingly high yields of neutral organic solvents in a short period of time. 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 fodder.
The aim of this work was to increase the efficiencies and potentialities of the fermentation medium by improving the properties of blackstrap molasses. Egyptian blackstrap molasses is used in many fermentation processes, especially acetone and butanol, acetic acid, ethyl alcohol and fodder yeast productions. Blackstrap molasses contains a somewhat high level of salts, especially divalent elements, which partially inhibit the formation of acetone and butanol by Clostridium acetobutylicum. Therefore, treatments of Egyptian blackstrap molasses by different techniques were tested in order to remove the inhibitory effects of these divalent elements on the formation of acetone and butanol by CI.
acetobutylicum.
METHODS
Experimental organism Our experiments were concerned with an active strain of Cl. acetobutylicum, isolated from cultivated Egyptian bean roots.
Maintenance of microorganism The clostridium was maintained on soil medium. A mixture of soil (49 g), clean sand (49 g) and chalk (2 g) was distributed in small vials after steam sterilisation. One ml of a suspension of the clostridium in potato medium (of composition and growth conditions as inoculum medium) was added to each vial and mixed with the soil. The vials were then placed in a closed jar with active silica gel, where they dried out in 10-14 days. The vials were then held, dry, in a refrigerator at 5°C.
Inoculum medium The medium used as inoculum in the production of acetone and butanol contained the following ingredients (g litre- 1): wet potato mash, 250.0; glucose, 5.0; C a C O 3 , 2'0; (NH4)2SO 4, 1.5. The ingredients were thoroughly mixed and transferred into test tubes. The tubes were plugged and sterilised at 120 °C for 15 minutes. When the tubes attained room temperature, they were inoculated with CI. acetobutylicum. The inoculated tubes were incubated at 31 °C for three weeks to
USE OF BLACKSTRAP MOLASSES IN ACETONE BUTANOL FERMENTATION
293
obtain luxuriant growth and sporulation. The tubes were kept in a refrigerator at 5°C.
Fermentation media Clostridium acetobutylicum was grown anaerobically in different types of Egyptian blackstrap molasses: K o m o m b o molasses, Armant molasses, AbouKurkass molasses, Edfo molasses, clarified molasses and edible molasses. The composition of the medium (ingredients in glitre-1) was as follows: blackstrap molasses, 140.0; rice bran, 5-0; corn bran, 8.0; soybean meal, 3.0; corn steep liquor, 4.0; ammonium sulphate, 2.0; calcium carbonate, 3.6; phosphoric acid salt, 2.0; and 'slop' 100 ml at pH 6.0 6-3. The media were distributed in 1000 ml Erlenmeyer flasks, 750 ml in each. The flasks were plugged and sterilised at 120 °C for 15 min. When the flasks attained room temperature they were inoculated with a standard inoculum of Cl. acetobutylicum. The flasks were incubated at 31 °C for 96 h. At the end of the incubation period, the final pH value of the fermented media, the residual sugar, and the amounts of acetone, butanol and ethanol were estimated. 'Slop' is the residue left after the solvents have been distilled from the fermented culture medium. Determination of residual sugar. Somogyi's (1937) method was used. Determination of butanol and ethanol. Johnson's (1932) method was used. Determination of acetone. Goodwin's (1920) method was used.
RESULTS
Fermentation media Different types of Egyptian blackstrap molasses were used in the fermentation media. The amounts of acetone, butanol and ethanol produced depended on the type of blackstrap molasses used. The results obtained (Table 1) show that NagaHamadi molasses produced the maximum yield of total neutral solvents. The residual sugars in the fermentation medium were from 0.56 to 1.08 °/o of the initial amount. The purer molasses, centrifugally clarified and edible molasses, did not give as good production of solvents as the native molasses. The production of acetone and butanol was greatly affected by the amounts of Naga-Hamadi molasses added to the fermentation medium (Table 2). The total amount of acetone and butanol increased with the increase in Naga-Hamadi molasses concentration, reaching an optimum at 140 g litre- 1. The residual sugars in the fermentation medium ranged from 0.20 to 2.32 ~J0 of the initial amounts. Ethylenediaminetetraacetic acid, in the form of the disodium salt was used to remove excess of inorganic salts present in Naga-Hamadi molasses by complex formation. The results obtained (Table 3) show that EDTA affected the production
TABLE 1
6.0 6.1 6.0 6.0 6.1 5-9 6.0 5.8 5.8
Final pH valuer 0.73 0.81 0.76 0.77 0-68 0.84 0.96 1.00 1.08
Residual sugar (%)
t The initial pH value of the medium was adjusted to 6.0.
Komombo Armant Abou-Kurkass Edfo Naga-Hamadi Kous EI-Hawamdi Clarified Edible
Types of molasses (140 g litre-1)
645 588 559 571 611 538 487 411 387
Acetone (rag 100 ml-1 mash) 1253 1212 1306 1299 1381 1260 1199 1086 995
Butanol (rag 100 ml-1 mash)
43 50 57 40 11 15 63 52 32
1941 1850 1922 1910 2003 1813 1749 1541 1414
Total solvents (rag 100 ml- 1 mash)
BY Cl. acetobutylicum
Ethanol (mg 100 ml-1 mash)
EFFECT OF DIFFERENT TYPES OF EGYPTIAN BLACKSTRAP MOLASSES ON THE PRODUCTION OF A C E T O N E - B U T A N O L
0
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5.7 5.7 5.6 5"8 5.0 5.9 5-7 6.0 6.1 6.2 6.4 6.3 6.1 6.0 5.8
20 40 60 80 100 110 120 130 140 150 160 170 180 190 200
0"20 0"22 0.31 0-33 0.38 0.41 0.48 0.56 0.71 0'82 1-07 2.01 2.13 2-30 2.32
Residual sugar (%)
t The initial pH value of the medium was adjusted to 6-0.
Final pH value
Concentrations of Naga-Hamadi molasses (g litre- 1)
101 212 269 362 416 484 526 610 709 587 639 519 421 365 318
Acetone (mg 100 ml - 1 mash)
189 303 564 811 1084 1146 1224 1245 1378 1215 1125 1048 929 825 788
Butanol (rag 100 ml - 1 mash)
-2 10 24 8 12 49 26 11 96 5 81 77 100 106
Ethanol (rag 100 ml - 1 mash)
290 517 843 1197 1508 1642 1799 1881 2098 1898 1769 1648 1427 1290 1212
Total solvents (mg 100 ml - 1 mash)
TA B LE 2 EFFECT OF DIFFERENT CONCENTRATIONS OF NAGA-HAMADI MOLASSESON THE PRODUCTION OF ACETONE AND BUTANOL BY Cl. acetobutylicum
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TABLE 3
6.3 6.4 6.3 6.4 6.3 6-3 6.1 6.4 6.4 6.3 6-3 6.1 6.4 6.4
0.00 0.02 0.04 0.06 0-08 0-10 0-12 0.14 0.16 0.18 0.20 0.22 0-27 0-32
0.78 1.01 0.74 0.96 0-75 0.77 1.12 1-09 0.92 0-81 0.90 0.88 0.85 0.75
Residual sugar (~)
t The initial pH value of the medium was adjusted to 6.0.
Final pH valuet
ED TA (g litre-1)
638 556 648 590 658 625 590 585 638 611 595 638 614 640
Acetone (mg 100 ml-1 mash) 1315 1301 1442 1325 1315 1385 1200 1250 1235 1363 1280 1210 1315 1211
Butanol (rag 100 ml I mash) 70 70 59 50 125 73 80 80 100 36 100 135 70 80
Ethanol (mg 100 m l - l mash)
EFFECT OF R E D U C T I O N BY EDTA OF I N O R G A N I C SALTS IN N A G A - H A M A D I MOLASSES ON THE P R O D U C T I O N OF A C E T O N E A N D B U T A N O L BY
2023 1927 2149 1965 2098 2083 1870 1915 1973 2010 1975 1983 1999 1931
Total solvents (mg 100 ml - 1 mash)
Cl. acetobutylicum
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6"0 5"9 5'9 5"8 5"9 5"8 6"6 5"7 5"7 5"7 5'9 5"8 5"8 5"6 5"6 5'6
0'00 0"01 0"02 0"03 0"04 0'05 0"06 0"07 0"08 0"09 0"10 0"12 0"14 0"16 0"18 0"20
0"68 0"70 0"76 0"79 0"85 0"88 0"89 0"97 1'12 1"14 1'20 1"21 1"26 1'26 1'29 1'32
Residual sugar (%)
t The initial pH value of the medium was adjusted to 6.0.
Final pH valuer
K4(Fe(CN) 6) (g litre 1)
561 567 542 533 516 508 511 498 480 507 479 466 432 389 345 322
Acetone (mg 100 m l - 1 mash) 1470 1451 1401 1392 1378 1361 1387 1392 1316 1296 1212 1195 1171 1190 1163 1147
Butanol (mg 100 ml 1 mash) 45 39 44 30 22 18 27 66 84 33 11 18 21 47 29 17
Ethanol (mg 100 m l - 1 mash)
TA B LE 4 EFFECT OF TREATMENT OF NAGA-HAMADI MOLASSES WITH DIFFERENT CONCENTRATIONS OF POTASSIUM FERROCYANIDE
2076 2057 1987 1955 1916 1887 1925 1956 1880 1836 1702 1679 1624 1626 1537 1486
Total solvents (mg 100 ml mash)
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298
MAHMOUD FOUAD, ABOU-ZEID ALI, MOHAMED YASSEIN
of acetone and butanol by CI. acetobuty!icum. The yield increased with the increase of EDTA concentration reaching a maximum at 0.04g litre -1. At the end of the incubation period, the final pH value ranged from 6-1 to 6.4 depending on the amount of EDTA added to the fermentation medium. The residual sugars in the fermentation medium were ranged from 0.74 to 1.12 ~o of the initial amount. Naga-Hamadi molasses was treated with different concentrations of potassium ferrocyanide for the reduction in concentration of toxic metals such as Zn, Cu and Fe. Potassium ferrocyanide was added before sterilisation at different concentrations up to 0.2 g litre- 1. The other ingredients of the fermentation medium were as in the previous experiments. Table 4 shows that addition of potassium ferrocyanide to the fermentation medium did not improve the production of acetone and butanol. To remove the excess of toxic metals such as Zn, Cu, Mn and Fe present in NagaHamadi molasses, methylene blue was added before sterilisation in different concentrations up to 0.2g litre -1. The other ingredients of the fermentation medium were used. The results obtained (Table 5) show that methylene blue did not increase the production of acetone and butanol. Sulphuric acid was added in concentrations ranging from 0.0 to 0.3 ~o to try to reduce the toxic effect of metals such as Ca, Zn, Mn, and Fe by formation of insoluble salts. Table 6 shows that addition of sulphuric acid to the fermentation medium did not improve the Naga-Hamadi molasses. Naga-Hamadi molasses used in the medium contained different carbon sources; mainly sucrose, glucose and fructose. Therefore the effect of these carbon sources individually and in combination in the ratio of 3 : 2:1, respectively, as approximately present in molasses, was studied. Table 7 show that molasses itself played an important role in the production of acetone and butanol by CI. acetobutylicum. When molasses used in the fermentation medium was replaced by equivalent amounts of sucrose, glucose, and fructose separately, and in combination in the ratio of 3:2:1, the yield of acetone and butanol was high if the basal medium contained molasses (2010 mg100 ml - 1 mash), while in the case of sucrose, fructose and glucose it was 1783, 1701 and 1660 mg 100 ml - 1 mash respectively. If the three carbon sources were added in the ratio of 3:2:1 the total solvents were 1874mg 100m1-1 mash. The results obtained in the previous experiment indicated that the favourable effect of molasses on the production of acetone and butanol by CI. acetobutylicum was due to factors other than the sugars present in molasses. In this experiment molasses ash was prepared and added to the medium without molasses in different amounts ranging from 0.10 to 5-0g litre -~. When different amounts of molasses ash were added to the medium containing sucrose, glucose and fructose yield of solvents increased with the increase of ash concentration (Table 8), but the best production was hardly equal to that with molasses. At 0.30g litre -1 molasses ash, the amount of acetone and butanol
6-0 5.9 6-1 5-8 5-9 6-0 5-8 5.7 5.8 5.8 6.0 5.9 5.8
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.25 0.30
0.45 0.86 1.06 1.00 1-11 1-19 1-38 1.27 1.33 0.93 1.21 1.13 1.19
Residual sugar (%)
÷ The initial pH value of the medium was adjusted to 6.0.
Final pH valuer
Methylene blue (g litre- 1)
691 596 532 551 538 499 508 484 491 571 587 516 476
Acetone (mg 100 ml - ~ mash) 1264 1263 1260 1249 1265 1193 1137 1187 1205 1212 1181 1222 1165
Butanol (mg 100 m l - l mash) 140 81 46 52 117 65 19 30 0 72 42 70 79
Ethanol (rag 100 ml mash)
TA B LE 5 EFFECT OF TREATMENT OF NAGA-HAMADI MOLASSES WITH DIFFERENT CONCNETRATIONS OF METHYLENE BLUE
2095 1940 1838 1852 1820 1757 1664 1701 1696 1855 1710 1808 1720
Total solvents (mg lOOml 1 mash)
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TABLE 6
6-0 6.1 5.9 5.8 5.9 6.0 5.9 5.8 5.8 5.7 5.9 6.1 6.0
0.00 0.02 0-04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.25 0.30
0.45 0-91 0.61 0.53 1.21 0.95 1-13 1.10 1.13 1.27 1.01 0.89 0.56
Residual sugar (%)
t The initial pH value of the medium was adjusted to 6.0.
Final pH valuer
Concentrated H2SO 4 (g litre -1)
641 571 594 653 487 512 499 580 541 475 590 566 610
Acetone (mg 100 ml 1 mash) 1254 1243 1233 1194 1199 1193 1239 1220 1217 1135 1210 1246 1160
Butanol (mg 100 m1-1 mash)
110 70 98 115 77 58 101 46 83 76 61 74 190
2005 1884 1925 1962 1733 1763 1839 1846 1841 1686 1861 1886 1960
Total solvents (mg lOOm1-1 mash)
Cl. acetobutylicum
Ethanol (mg 100 ml - l mash)
EFFECT OF H 2 S O 4 IN NAGA-HAMADI MOLASSES MEDIUM ON THE PRODUCTION OF ACETONE AND BUTANOL BY
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TABLE 7
5.3
5.5 5.4 5-7 5.6
Sucrose + glucose + fructose~t (3:2:1 w/v)
Sucrose:~
Glucose S
Fructose~
1.30
1.23
1.16
1.01
.
0.48
Residual sugar (%)
t The initial pH value of the fermented medium was adjusted to 6.0. No growth of clostridia, no initial sugar. :~ In concentration equivalent to the sugars in the molasses.
5.9
Control (B) (without molasses)
Final pH valuer
Control (A) (molasses + other ingredients)
Carbon sources
.
388
435
498
585
641
Acetone (mg 100 m l - 1 mash)
.
1095
1187
1191
1212
1322 .
Butanol (rag 100 ml 1 mash)
177
79
94
77
55
1660
1701
1783
1874
2018
Total solvents (rag 100 m l - 1 mash)
acetobutylicum
Ethanol (rag 100 ml 1 mash)
EFFECT OF CARBON SOURCES (PRESENT IN MOLASSES) ON THE PRODUCTION OF ACETONE AND BUTANOL BY Cl.
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Final pH valuer 5.3 5.5 5.6 5.6 5.8 5.9 6.1 6.0 5-8 5.8 5.6 5.6 5.6 5-5 5.4 5.4
Ash concentration (g litre 1)
Control without molasses 0.10:~ 0.15 0.20 0.25 0.30 0-35 0-40 0.45 0.50 0.75
8
0.71 0.65 0-64 0.61 0.51 0.52 0.60 0-67 0.73 0.95 1.70 1-83 1-99 2.01 2.87
Residual sugar (%)
586 513 550 655 595 584 574 599 552 558 497 416 365 293 218
Acetone (rag 100 m l - l mash)
t The initial pH value of the fermented medium was adjusted to 6.0. - - No initial sugar, no growth of clostridia. :~ The medium contained sucrose, glucose, fructose in 3:2:1 ratio as in Table 7.
2.00 3.00 4.00 5.00
1.00
TABLE
1230 1370 1340 1310 1332 1400 1361 1253 1149 1082 1021 987 906 841 737
Butanol (mg 100 m l - 1 mash)
acetobutylicum
1 6 20 0 94 16 38 0 97 47 1 13 53 43 56
Ethanol (rag 100 m l - 1 mash)
EFFECT OF D I F F E R E N T A M O U N T S OF MOLASSES A S H ON T H E P R O D U C T I O N OF A C E T O N E AND B U T A N O L BY Cl.
1817 1889 1910 1965 2021 2000 1973 1852 1788 1687 1519 1416 1324 1177 1011
solvents (mg 100 ml-1 mash)
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USE OF BLACKSTRAPMOLASSESIN ACETONEBUTANOLFERMENTATION
303
produced was 2021 mg 100 ml-1 mash, and above this level of ash concentration it was decreased. At high concentration of molasses ash (5.0 g litre-1) the neutral solvents yield was very much depressed.
DISCUSSION
Blackstrap molasses is a local, cheap by-product obtained from sugar cane manufacture and it proved to be an effective carbon source in the production of acetone and butanol. On adding molasses to the fermentation medium, the yield of acetone and butanol was increased with the increase of molasses concentration reaching its optimum when the concentration was 140g litre-1 Methods, techniques and processes used in sugar cane manufacture are variable, therefore, blackstrap molasses from different sources differ in chemical composition. These variations in chemical composition affected the production of acetone and butanol by Cl. acetobutylicum. Naga-Hamadi molasses was more suitable for organic solvents production than other types of molasses. The other ingredients in the fermentation medium are all relatively cheap by-products of Egyptian manufacture. The molasses contains compounds essential for acetone-butanol production. Egyptian blackstrap molasses contains 50~0 total reducing sugars, nitrogen sources, organic acids and salts of Ca, Mg, Zn, Fe, Cu, Ni, Na, K and other ions. The presence of Zn, Mn, Cu, Fe, Na, K and Ca, in excess could retard the microbial growth and acetone butanol biosynthesis, therefore attempts were made to reduce the amount of these salts by centrifugation or addition of some chelating or precipitating compounds. E D T A improved solvent production to some extent, but the other additions tended to decrease production, possibly because these compounds, or excess EDTA, removed too much of the metal ions. Purification to produce clarified and edible molasses also reduced the solvent yield. A medium containing only the sugars of molasses did not give a good yield of solvents, but addition to the sugars of molasses ash gave results almost as good as molasses itself. All the results show that the value of molasses in production of solvents lies not only in its content of sugars as substrates for the fermentation, but in its content of metal ions and other substances essential for good microbial growth. Acetone butanol fermentation by micro-organisms is an economic procedure, especially in countries where the raw materials are available. Some authors claim that the microbiological production is more costly that the production of the solvents (acetone, butanol and ethanol) by petrochemical industries. Actually, the countries which have raw agricultural by-products can produce the solvents economically by fermentation. In the future, however, the crude petroleum will be exhausted and the microbiological production could then be flourishing.
304
MAHMOUD FOUAD, ABOU-ZEID ALI, MOHAMED YASSEIN REFERENCES AND BIBLIOGRAPHY
BEESCH, S. C. (1952). Acetone-butanol fermentation of sugars. Ind. Eng. Chem., 44, 1677-82. BROWN,R. W., WOOD, H. G. & WERKMAN,C. H. (1938). Growth factors for the butyl alcohol bacteria. J. Bact., 25, 206. DAVIES, R. & S~I'HENSON, M. (1941). Studies on the acetone-butyl alcohol fermentation. I. Nutritional and other factors involved in the preparation of active suspensions of CI. acetobutylicum (Weizmann), Biochem. J., 35, 1320-31. FOWLER, G. J. & SUBRAMANVAN,V. (1925). Studies relating to the acetone producing organisms. J. Indian Inst. Sci., 8A, 71-87. GABRIEL, C. L. (1928). Butanol fermentation process. Ind. Eng. Chem., 20, 1063-7. GOODWIN, L. F. (1920). The analysis of acetone by Messingers method. J. Am. Chem. Soc., 42, 39. JOHNSON, M. J. (1932). Determination of small amounts of ethyl and butyl alcohols. Ind. Eng. Chem. Anal. Ed., 4, 20. McCoY, E. (1946). Acetone-butanol fermentation, U.S. Patent 2,398,837. SoMo~vl, M. (1937). A reagent for the copper iodometric determination of very small amounts of sugar. J. Biol. Chem., 117, 771.
UTILISATION OF BLACKSTRAP PRODUCTION OF ACETONE
CLOSTRIDIUM
MOLASSES FOR THE AND BUTANOL BY
A CETOB UTYLIC UM
MAHMOUD FOUAD, ABOU-ZEID ALl & MOHAMED YASSEIN
Egyptian Sugar and Distillation Company, Organic Chemical Factories, Hawamdia, Giza, Egypt
ABSTRACT
The commonest carbohydrate raw material used in the aceton~butanol fermentation is blackstrap molasses. Blackstrap molasses contains relatively high levels qf sahs. especially divalent elements, which affect the microbial growth and the formation of acetone and butanol by Clostridium acetobutylicum. Therefore, treatments q[ blackstrap molasses by different techniques were tested in order to increase the titres of the microbial metabolites.
INTRODUCTION
Acetone and butanol are produced fermentatively, on a large scale, in the Organic Chemical Factories, Egyptian Sugar and Distillation Company. The raw materials used in the fermentative production of acetone and butanol are blackstrap molasses and rice- or corn-bran. The advantages provided by the use of molasses as a fermentation raw material have been summarised in an early patent by Beesch (1952) as follows: 1. 2. 3.
4. 5.
They are easy to handle as a liquid to be pumped. They possess the property of uniformity over fairly long periods. They give a yield of 30 ~o total solvents based on the amounts of fermentable sugars. The mixture of solvents consists of 60-65 % n-butanol and 35-40 oj~ acetone. Molasses mash is easy to sterilise at lower temperatures. The minerals contained in blackstrap molasses are of economic and nutritional importance. 291
Agrieultural Wastes 0141-4607/82/0004-0291/$02.75 ~L')Applied Science Publishers Ltd, England, 1982 Printed in Great Britain
292 6. 7. 8. 9. 10.
MAHMOUD FOUAD, ABOU-ZEID ALl, MOHAMED YASSEIN
Fermentation is run at 30 °C instead of 36 °C, providing a less favourable temperature for contaminating organisms. Tanks and equipment are much easier to clean and less blockage occurs. Molasses is normally the cheapest raw material. High concentrations of sugar can be fermented with correspondingly high yields of neutral organic solvents in a short period of time. 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 fodder.
The aim of this work was to increase the efficiencies and potentialities of the fermentation medium by improving the properties of blackstrap molasses. Egyptian blackstrap molasses is used in many fermentation processes, especially acetone and butanol, acetic acid, ethyl alcohol and fodder yeast productions. Blackstrap molasses contains a somewhat high level of salts, especially divalent elements, which partially inhibit the formation of acetone and butanol by Clostridium acetobutylicum. Therefore, treatments of Egyptian blackstrap molasses by different techniques were tested in order to remove the inhibitory effects of these divalent elements on the formation of acetone and butanol by CI.
acetobutylicum.
METHODS
Experimental organism Our experiments were concerned with an active strain of Cl. acetobutylicum, isolated from cultivated Egyptian bean roots.
Maintenance of microorganism The clostridium was maintained on soil medium. A mixture of soil (49 g), clean sand (49 g) and chalk (2 g) was distributed in small vials after steam sterilisation. One ml of a suspension of the clostridium in potato medium (of composition and growth conditions as inoculum medium) was added to each vial and mixed with the soil. The vials were then placed in a closed jar with active silica gel, where they dried out in 10-14 days. The vials were then held, dry, in a refrigerator at 5°C.
Inoculum medium The medium used as inoculum in the production of acetone and butanol contained the following ingredients (g litre- 1): wet potato mash, 250.0; glucose, 5.0; C a C O 3 , 2'0; (NH4)2SO 4, 1.5. The ingredients were thoroughly mixed and transferred into test tubes. The tubes were plugged and sterilised at 120 °C for 15 minutes. When the tubes attained room temperature, they were inoculated with CI. acetobutylicum. The inoculated tubes were incubated at 31 °C for three weeks to
USE OF BLACKSTRAP MOLASSES IN ACETONE BUTANOL FERMENTATION
293
obtain luxuriant growth and sporulation. The tubes were kept in a refrigerator at 5°C.
Fermentation media Clostridium acetobutylicum was grown anaerobically in different types of Egyptian blackstrap molasses: K o m o m b o molasses, Armant molasses, AbouKurkass molasses, Edfo molasses, clarified molasses and edible molasses. The composition of the medium (ingredients in glitre-1) was as follows: blackstrap molasses, 140.0; rice bran, 5-0; corn bran, 8.0; soybean meal, 3.0; corn steep liquor, 4.0; ammonium sulphate, 2.0; calcium carbonate, 3.6; phosphoric acid salt, 2.0; and 'slop' 100 ml at pH 6.0 6-3. The media were distributed in 1000 ml Erlenmeyer flasks, 750 ml in each. The flasks were plugged and sterilised at 120 °C for 15 min. When the flasks attained room temperature they were inoculated with a standard inoculum of Cl. acetobutylicum. The flasks were incubated at 31 °C for 96 h. At the end of the incubation period, the final pH value of the fermented media, the residual sugar, and the amounts of acetone, butanol and ethanol were estimated. 'Slop' is the residue left after the solvents have been distilled from the fermented culture medium. Determination of residual sugar. Somogyi's (1937) method was used. Determination of butanol and ethanol. Johnson's (1932) method was used. Determination of acetone. Goodwin's (1920) method was used.
RESULTS
Fermentation media Different types of Egyptian blackstrap molasses were used in the fermentation media. The amounts of acetone, butanol and ethanol produced depended on the type of blackstrap molasses used. The results obtained (Table 1) show that NagaHamadi molasses produced the maximum yield of total neutral solvents. The residual sugars in the fermentation medium were from 0.56 to 1.08 °/o of the initial amount. The purer molasses, centrifugally clarified and edible molasses, did not give as good production of solvents as the native molasses. The production of acetone and butanol was greatly affected by the amounts of Naga-Hamadi molasses added to the fermentation medium (Table 2). The total amount of acetone and butanol increased with the increase in Naga-Hamadi molasses concentration, reaching an optimum at 140 g litre- 1. The residual sugars in the fermentation medium ranged from 0.20 to 2.32 ~J0 of the initial amounts. Ethylenediaminetetraacetic acid, in the form of the disodium salt was used to remove excess of inorganic salts present in Naga-Hamadi molasses by complex formation. The results obtained (Table 3) show that EDTA affected the production
TABLE 1
6.0 6.1 6.0 6.0 6.1 5-9 6.0 5.8 5.8
Final pH valuer 0.73 0.81 0.76 0.77 0-68 0.84 0.96 1.00 1.08
Residual sugar (%)
t The initial pH value of the medium was adjusted to 6.0.
Komombo Armant Abou-Kurkass Edfo Naga-Hamadi Kous EI-Hawamdi Clarified Edible
Types of molasses (140 g litre-1)
645 588 559 571 611 538 487 411 387
Acetone (rag 100 ml-1 mash) 1253 1212 1306 1299 1381 1260 1199 1086 995
Butanol (rag 100 ml-1 mash)
43 50 57 40 11 15 63 52 32
1941 1850 1922 1910 2003 1813 1749 1541 1414
Total solvents (rag 100 ml- 1 mash)
BY Cl. acetobutylicum
Ethanol (mg 100 ml-1 mash)
EFFECT OF DIFFERENT TYPES OF EGYPTIAN BLACKSTRAP MOLASSES ON THE PRODUCTION OF A C E T O N E - B U T A N O L
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5.7 5.7 5.6 5"8 5.0 5.9 5-7 6.0 6.1 6.2 6.4 6.3 6.1 6.0 5.8
20 40 60 80 100 110 120 130 140 150 160 170 180 190 200
0"20 0"22 0.31 0-33 0.38 0.41 0.48 0.56 0.71 0'82 1-07 2.01 2.13 2-30 2.32
Residual sugar (%)
t The initial pH value of the medium was adjusted to 6-0.
Final pH value
Concentrations of Naga-Hamadi molasses (g litre- 1)
101 212 269 362 416 484 526 610 709 587 639 519 421 365 318
Acetone (mg 100 ml - 1 mash)
189 303 564 811 1084 1146 1224 1245 1378 1215 1125 1048 929 825 788
Butanol (rag 100 ml - 1 mash)
-2 10 24 8 12 49 26 11 96 5 81 77 100 106
Ethanol (rag 100 ml - 1 mash)
290 517 843 1197 1508 1642 1799 1881 2098 1898 1769 1648 1427 1290 1212
Total solvents (mg 100 ml - 1 mash)
TA B LE 2 EFFECT OF DIFFERENT CONCENTRATIONS OF NAGA-HAMADI MOLASSESON THE PRODUCTION OF ACETONE AND BUTANOL BY Cl. acetobutylicum
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6.3 6.4 6.3 6.4 6.3 6-3 6.1 6.4 6.4 6.3 6-3 6.1 6.4 6.4
0.00 0.02 0.04 0.06 0-08 0-10 0-12 0.14 0.16 0.18 0.20 0.22 0-27 0-32
0.78 1.01 0.74 0.96 0-75 0.77 1.12 1-09 0.92 0-81 0.90 0.88 0.85 0.75
Residual sugar (~)
t The initial pH value of the medium was adjusted to 6.0.
Final pH valuet
ED TA (g litre-1)
638 556 648 590 658 625 590 585 638 611 595 638 614 640
Acetone (mg 100 ml-1 mash) 1315 1301 1442 1325 1315 1385 1200 1250 1235 1363 1280 1210 1315 1211
Butanol (rag 100 ml I mash) 70 70 59 50 125 73 80 80 100 36 100 135 70 80
Ethanol (mg 100 m l - l mash)
EFFECT OF R E D U C T I O N BY EDTA OF I N O R G A N I C SALTS IN N A G A - H A M A D I MOLASSES ON THE P R O D U C T I O N OF A C E T O N E A N D B U T A N O L BY
2023 1927 2149 1965 2098 2083 1870 1915 1973 2010 1975 1983 1999 1931
Total solvents (mg 100 ml - 1 mash)
Cl. acetobutylicum
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0'00 0"01 0"02 0"03 0"04 0'05 0"06 0"07 0"08 0"09 0"10 0"12 0"14 0"16 0"18 0"20
0"68 0"70 0"76 0"79 0"85 0"88 0"89 0"97 1'12 1"14 1'20 1"21 1"26 1'26 1'29 1'32
Residual sugar (%)
t The initial pH value of the medium was adjusted to 6.0.
Final pH valuer
K4(Fe(CN) 6) (g litre 1)
561 567 542 533 516 508 511 498 480 507 479 466 432 389 345 322
Acetone (mg 100 m l - 1 mash) 1470 1451 1401 1392 1378 1361 1387 1392 1316 1296 1212 1195 1171 1190 1163 1147
Butanol (mg 100 ml 1 mash) 45 39 44 30 22 18 27 66 84 33 11 18 21 47 29 17
Ethanol (mg 100 m l - 1 mash)
TA B LE 4 EFFECT OF TREATMENT OF NAGA-HAMADI MOLASSES WITH DIFFERENT CONCENTRATIONS OF POTASSIUM FERROCYANIDE
2076 2057 1987 1955 1916 1887 1925 1956 1880 1836 1702 1679 1624 1626 1537 1486
Total solvents (mg 100 ml mash)
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MAHMOUD FOUAD, ABOU-ZEID ALI, MOHAMED YASSEIN
of acetone and butanol by CI. acetobuty!icum. The yield increased with the increase of EDTA concentration reaching a maximum at 0.04g litre -1. At the end of the incubation period, the final pH value ranged from 6-1 to 6.4 depending on the amount of EDTA added to the fermentation medium. The residual sugars in the fermentation medium were ranged from 0.74 to 1.12 ~o of the initial amount. Naga-Hamadi molasses was treated with different concentrations of potassium ferrocyanide for the reduction in concentration of toxic metals such as Zn, Cu and Fe. Potassium ferrocyanide was added before sterilisation at different concentrations up to 0.2 g litre- 1. The other ingredients of the fermentation medium were as in the previous experiments. Table 4 shows that addition of potassium ferrocyanide to the fermentation medium did not improve the production of acetone and butanol. To remove the excess of toxic metals such as Zn, Cu, Mn and Fe present in NagaHamadi molasses, methylene blue was added before sterilisation in different concentrations up to 0.2g litre -1. The other ingredients of the fermentation medium were used. The results obtained (Table 5) show that methylene blue did not increase the production of acetone and butanol. Sulphuric acid was added in concentrations ranging from 0.0 to 0.3 ~o to try to reduce the toxic effect of metals such as Ca, Zn, Mn, and Fe by formation of insoluble salts. Table 6 shows that addition of sulphuric acid to the fermentation medium did not improve the Naga-Hamadi molasses. Naga-Hamadi molasses used in the medium contained different carbon sources; mainly sucrose, glucose and fructose. Therefore the effect of these carbon sources individually and in combination in the ratio of 3 : 2:1, respectively, as approximately present in molasses, was studied. Table 7 show that molasses itself played an important role in the production of acetone and butanol by CI. acetobutylicum. When molasses used in the fermentation medium was replaced by equivalent amounts of sucrose, glucose, and fructose separately, and in combination in the ratio of 3:2:1, the yield of acetone and butanol was high if the basal medium contained molasses (2010 mg100 ml - 1 mash), while in the case of sucrose, fructose and glucose it was 1783, 1701 and 1660 mg 100 ml - 1 mash respectively. If the three carbon sources were added in the ratio of 3:2:1 the total solvents were 1874mg 100m1-1 mash. The results obtained in the previous experiment indicated that the favourable effect of molasses on the production of acetone and butanol by CI. acetobutylicum was due to factors other than the sugars present in molasses. In this experiment molasses ash was prepared and added to the medium without molasses in different amounts ranging from 0.10 to 5-0g litre -~. When different amounts of molasses ash were added to the medium containing sucrose, glucose and fructose yield of solvents increased with the increase of ash concentration (Table 8), but the best production was hardly equal to that with molasses. At 0.30g litre -1 molasses ash, the amount of acetone and butanol
6-0 5.9 6-1 5-8 5-9 6-0 5-8 5.7 5.8 5.8 6.0 5.9 5.8
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.25 0.30
0.45 0.86 1.06 1.00 1-11 1-19 1-38 1.27 1.33 0.93 1.21 1.13 1.19
Residual sugar (%)
÷ The initial pH value of the medium was adjusted to 6.0.
Final pH valuer
Methylene blue (g litre- 1)
691 596 532 551 538 499 508 484 491 571 587 516 476
Acetone (mg 100 ml - ~ mash) 1264 1263 1260 1249 1265 1193 1137 1187 1205 1212 1181 1222 1165
Butanol (mg 100 m l - l mash) 140 81 46 52 117 65 19 30 0 72 42 70 79
Ethanol (rag 100 ml mash)
TA B LE 5 EFFECT OF TREATMENT OF NAGA-HAMADI MOLASSES WITH DIFFERENT CONCNETRATIONS OF METHYLENE BLUE
2095 1940 1838 1852 1820 1757 1664 1701 1696 1855 1710 1808 1720
Total solvents (mg lOOml 1 mash)
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6-0 6.1 5.9 5.8 5.9 6.0 5.9 5.8 5.8 5.7 5.9 6.1 6.0
0.00 0.02 0-04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.25 0.30
0.45 0-91 0.61 0.53 1.21 0.95 1-13 1.10 1.13 1.27 1.01 0.89 0.56
Residual sugar (%)
t The initial pH value of the medium was adjusted to 6.0.
Final pH valuer
Concentrated H2SO 4 (g litre -1)
641 571 594 653 487 512 499 580 541 475 590 566 610
Acetone (mg 100 ml 1 mash) 1254 1243 1233 1194 1199 1193 1239 1220 1217 1135 1210 1246 1160
Butanol (mg 100 m1-1 mash)
110 70 98 115 77 58 101 46 83 76 61 74 190
2005 1884 1925 1962 1733 1763 1839 1846 1841 1686 1861 1886 1960
Total solvents (mg lOOm1-1 mash)
Cl. acetobutylicum
Ethanol (mg 100 ml - l mash)
EFFECT OF H 2 S O 4 IN NAGA-HAMADI MOLASSES MEDIUM ON THE PRODUCTION OF ACETONE AND BUTANOL BY
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5.3
5.5 5.4 5-7 5.6
Sucrose + glucose + fructose~t (3:2:1 w/v)
Sucrose:~
Glucose S
Fructose~
1.30
1.23
1.16
1.01
.
0.48
Residual sugar (%)
t The initial pH value of the fermented medium was adjusted to 6.0. No growth of clostridia, no initial sugar. :~ In concentration equivalent to the sugars in the molasses.
5.9
Control (B) (without molasses)
Final pH valuer
Control (A) (molasses + other ingredients)
Carbon sources
.
388
435
498
585
641
Acetone (mg 100 m l - 1 mash)
.
1095
1187
1191
1212
1322 .
Butanol (rag 100 ml 1 mash)
177
79
94
77
55
1660
1701
1783
1874
2018
Total solvents (rag 100 m l - 1 mash)
acetobutylicum
Ethanol (rag 100 ml 1 mash)
EFFECT OF CARBON SOURCES (PRESENT IN MOLASSES) ON THE PRODUCTION OF ACETONE AND BUTANOL BY Cl.
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Final pH valuer 5.3 5.5 5.6 5.6 5.8 5.9 6.1 6.0 5-8 5.8 5.6 5.6 5.6 5-5 5.4 5.4
Ash concentration (g litre 1)
Control without molasses 0.10:~ 0.15 0.20 0.25 0.30 0-35 0-40 0.45 0.50 0.75
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0.71 0.65 0-64 0.61 0.51 0.52 0.60 0-67 0.73 0.95 1.70 1-83 1-99 2.01 2.87
Residual sugar (%)
586 513 550 655 595 584 574 599 552 558 497 416 365 293 218
Acetone (rag 100 m l - l mash)
t The initial pH value of the fermented medium was adjusted to 6.0. - - No initial sugar, no growth of clostridia. :~ The medium contained sucrose, glucose, fructose in 3:2:1 ratio as in Table 7.
2.00 3.00 4.00 5.00
1.00
TABLE
1230 1370 1340 1310 1332 1400 1361 1253 1149 1082 1021 987 906 841 737
Butanol (mg 100 m l - 1 mash)
acetobutylicum
1 6 20 0 94 16 38 0 97 47 1 13 53 43 56
Ethanol (rag 100 m l - 1 mash)
EFFECT OF D I F F E R E N T A M O U N T S OF MOLASSES A S H ON T H E P R O D U C T I O N OF A C E T O N E AND B U T A N O L BY Cl.
1817 1889 1910 1965 2021 2000 1973 1852 1788 1687 1519 1416 1324 1177 1011
solvents (mg 100 ml-1 mash)
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USE OF BLACKSTRAPMOLASSESIN ACETONEBUTANOLFERMENTATION
303
produced was 2021 mg 100 ml-1 mash, and above this level of ash concentration it was decreased. At high concentration of molasses ash (5.0 g litre-1) the neutral solvents yield was very much depressed.
DISCUSSION
Blackstrap molasses is a local, cheap by-product obtained from sugar cane manufacture and it proved to be an effective carbon source in the production of acetone and butanol. On adding molasses to the fermentation medium, the yield of acetone and butanol was increased with the increase of molasses concentration reaching its optimum when the concentration was 140g litre-1 Methods, techniques and processes used in sugar cane manufacture are variable, therefore, blackstrap molasses from different sources differ in chemical composition. These variations in chemical composition affected the production of acetone and butanol by Cl. acetobutylicum. Naga-Hamadi molasses was more suitable for organic solvents production than other types of molasses. The other ingredients in the fermentation medium are all relatively cheap by-products of Egyptian manufacture. The molasses contains compounds essential for acetone-butanol production. Egyptian blackstrap molasses contains 50~0 total reducing sugars, nitrogen sources, organic acids and salts of Ca, Mg, Zn, Fe, Cu, Ni, Na, K and other ions. The presence of Zn, Mn, Cu, Fe, Na, K and Ca, in excess could retard the microbial growth and acetone butanol biosynthesis, therefore attempts were made to reduce the amount of these salts by centrifugation or addition of some chelating or precipitating compounds. E D T A improved solvent production to some extent, but the other additions tended to decrease production, possibly because these compounds, or excess EDTA, removed too much of the metal ions. Purification to produce clarified and edible molasses also reduced the solvent yield. A medium containing only the sugars of molasses did not give a good yield of solvents, but addition to the sugars of molasses ash gave results almost as good as molasses itself. All the results show that the value of molasses in production of solvents lies not only in its content of sugars as substrates for the fermentation, but in its content of metal ions and other substances essential for good microbial growth. Acetone butanol fermentation by micro-organisms is an economic procedure, especially in countries where the raw materials are available. Some authors claim that the microbiological production is more costly that the production of the solvents (acetone, butanol and ethanol) by petrochemical industries. Actually, the countries which have raw agricultural by-products can produce the solvents economically by fermentation. In the future, however, the crude petroleum will be exhausted and the microbiological production could then be flourishing.
304
MAHMOUD FOUAD, ABOU-ZEID ALI, MOHAMED YASSEIN REFERENCES AND BIBLIOGRAPHY
BEESCH, S. C. (1952). Acetone-butanol fermentation of sugars. Ind. Eng. Chem., 44, 1677-82. BROWN,R. W., WOOD, H. G. & WERKMAN,C. H. (1938). Growth factors for the butyl alcohol bacteria. J. Bact., 25, 206. DAVIES, R. & S~I'HENSON, M. (1941). Studies on the acetone-butyl alcohol fermentation. I. Nutritional and other factors involved in the preparation of active suspensions of CI. acetobutylicum (Weizmann), Biochem. J., 35, 1320-31. FOWLER, G. J. & SUBRAMANVAN,V. (1925). Studies relating to the acetone producing organisms. J. Indian Inst. Sci., 8A, 71-87. GABRIEL, C. L. (1928). Butanol fermentation process. Ind. Eng. Chem., 20, 1063-7. GOODWIN, L. F. (1920). The analysis of acetone by Messingers method. J. Am. Chem. Soc., 42, 39. JOHNSON, M. J. (1932). Determination of small amounts of ethyl and butyl alcohols. Ind. Eng. Chem. Anal. Ed., 4, 20. McCoY, E. (1946). Acetone-butanol fermentation, U.S. Patent 2,398,837. SoMo~vl, M. (1937). A reagent for the copper iodometric determination of very small amounts of sugar. J. Biol. Chem., 117, 771.