INDUSTRIAL AND ENGINEERING CHEMISTRY Edlund, K. R., unpublished work. Egloff, Schaad, and Lowry, J . Phys. Chem., 34, 1617-1740 (1930); J . Inst. Petroleum Tech., 16, 133-246 (1930). Frey and Hepp, IND. EKG.CHEM.,24, 282 (1932). Geniesse and Reuter, Ibid., 22, 1274 (1930); 24, 219 (1932). Haber, “Experimental-Untersuchungen uber Zersetaung und Verbrennung von Kohlen~vasserstoffen,”Habilitationsschrift, Munich, 1896. Hague, E. K,,and Wheeler, R. V.,J . Chem. SOC.,1929, 37893; Fuel, 8, 560-87 (19’29). Hall, U. S. Patent 1,194,289 (1916); British Patent 1594 (1915). Hofmann, K. A., “Lehrbuch der anorganischen Chemie,” 5th ed., P. 326, F. J&weg &z Sohn A-G., Brunschweig, 1924. Lenher, J. Am. Chem. Soc., 53, 3752-65 (1931).
Vol. 25, N o . 7
(15) Lomax, Dunstan, and Thole, J . Inst. Petroleum Tech., 3, 36-120 (1916). (16) Meffert, German Patent 99.254 (1897). (17) Meikle, British Patent 23,649 (1896). (18) Piotroivski and Winkler. Petroleum Z., 26, 763-80 (1930). (19) Rittman, British Patents 9162 and 9163 (1915). (20) Wheeler, R. V., and Wood, W. L., Fuel, 7, 535-9 (1928) ; J. Chem. Soc., 1930, 1819-28; F u d , 9, 567-74 (1930). (21) Wheeler, T. S., and Imp. Chem. Ind., Ltd., British Patent 332,998 (June 4, 1929). (22) Williams-Gardner, Fuel, 4, 430-40 (1925). RECEIVEDN w e m b e r 21, 1932. Presented before t h e Division of Petroleum Chemistry a t t h e 84th Meeting of the American Chemical Society, Denver, colo., August 22 t o 26, 1932.
Production of 2,3-Butylene Glycol by Fermentation Effect of Sucrose Concentration ELLISI. FULMER,L. h4. CHRISTENSEN, AND A. R. KENDALL, Iowa State College, Ames, Iowa
T
H E purpose of this study was to determine in a quantitative manner the influence of the concentration of sucrose, in a synthetic medium, upon the production of 2,3-butylene glycol by bacterial action, in order to find the optimum conditions for maximum yields of the glycol. The data show yields in the neighborhood of 50 grams of the glycol per 100 grams of sugar fermented under the optimum conditions described. These yields are of such a magnitude as to point t o industrial production. The research is being extended to increase these yields further, to speed up the rate of fermentation, to develop methods of recovery, and t o study the chemical properties of the glycol and its uses in the synthesis of other organic substances. There are many papers in the literature involving the determination of 2,3-butylene glycol and acetylmethyl carbinol; most of these determinations were qualitative and incidental. Hence, there will be briefly reviewed only those communications in which quantitative data were obtained under standardized conditions. One of the earliest references is that of PBr6 (28) who identified acetylmethyl carbinol as produced from mannitol by B. subtilis and B. naesentericics vdgatus, and from dextrose and glycerol by Tyrothrix tenuis. Grimbert ( 6 ) identified this chemical as produced from various sugars by B. tartiicus. Desmots (3)proved this material to be produced from various substrates by several bacteria including B. mesentericus vulyatus, B. fuscus, B. pauua, B. ruber, B. subtilis, and Tyrothrix tenuis. Harden and Walpole (9) were the first to prove the production of acetylmethyl carbinol and 2,3-butylene glycol by bacterial action on sugars. They found that about 27.2 per cent of the dextrose fermented by B. lactis aeroyenes, under anaerobic conditions, was converted into 2,3-butylene glycol. Walpole (26), using B. lactis aeroyenes in a nutrient medium containing 5 per cent sugar (dextrose or levulose), under anaerobic conditions, obtained yields of two optically active forms of the glycol, the diphenylurethan derivatives melting a t 199.5’ and 157’ C., respectively, with the former composing about 90 per cent of the mixture. Harden and Norris (8)found that 6 . coli communis converted 33 per cent of the dextrose into the glycol, calculated on the basis of sugar carbon.
Lemoigne (12, 13) found that the relative amounts of 2,3butylene glycol and acetylmethyl carbinol varied with the time of fermentation. The ratio of carbinol to glycol was 860 to 1718 at the end of 3 days, and a t the end of the seventh day was 5772 t o 3371. Harden and Xorris ( 7 ) found that Aerobacter avogenes converted 9.9 per cent of the glycerol used into 2,3-butylene glycol. Lemoigne (14-1 7 ) reported the action of three strains of the Bacillus proteus group upon dextrose. The amounts of the carbinol and glycol in milligrams produced after various time intervals were: 3 Days 16 60
Acetylmethyl carbinol 2,3-Butylene glycol
6 DAYS 60 140
18 DAYS 110 60
Breden and Fulmer (1) studied the fermentation of sucrose and xylose by Aerobacter faeni. The yields of glycol and carbinol may be summarized as follows in terms of grams of each chemical produced per 100 grams of sugar fermented: XYLOSF
2 3-Butylene glycol
&etylmethyl carbinol
Aerobic 10 5 2 6
Anaerobic 13 7 0 2
S~CROTE Aerobic Anaerotic 16 7 15 0 6 4 0 5
F’erhave (25) found the organisms Clostridium polyrnyxa and Aerobacter aerogenes to be especially active in the production of 2,3-butylene glycol from carbohydrates. The production of 2,3-butylene glycol and acetylmethyl carbinol by the action of yeast upon various substrates has been studied especially by Neuberg and Reinfurth (19), Neuberg and Rosenthal (ZO), Kluyver and Donker ( I O ) , Seuberg and Gorr (18), Seuberg and Simon ( a l ) , Kluyver, Donker, and visser’t Hooft (11), Elion (0, and others. hIE DIUM
AABD
BACTERIA
I n developing synthetic media for the growth of yeast, Fulmer, Kelson, and Shermood ( 5 ) and Sherwood and Fulmer (84) systematically varied the concentrations of the salts used in order t o determine optimum conditions for growth a t the given temperature. The same procedure was adopted here in developing the medium optimum for the maximum production of the 2,3-butylene glycol by Aerobacter pectinouorum a t 37.5” C . The best medium contained, per 100
Jull-, 1933
INDUSTRIAL AND ENGINEERISG
CHEMISTRY
799
the weighed residues from the extractions, and the solutions were analyzed by the refractometric method. The results agree reasonably well but are, in general, somevhat lower than those obtained by the weighing procedure. I n the discussion the average value is used to represent the yield of the glycol. SKALYSIS OF UKFERJIENTED SUCROSE. Samples of the medium containing 12 per cent of sucrose were hydrolyzed for various periods of time with varying concentrations of hydrochloric acid. The reducing sugars were determined by the Shaffer and Hartmann method (23). The most satisfactory procedure was as follows: A 5-cc. sample of the fermented medium, clarified as noted above, was diluted with METHODSOF AKALYSIS 45 cc. of mater and 5 cc. of concentrated hydrochloric acid. 2,3-BUTYLEXE GLYCOL. To each flask after fermentation The solution was heated a t 75" C. for 9 minutes, cooled was completed, as evidenced by the cewation of the formation quickly to 20" C., and immediately neutralized by the addiof acid, was added 1 cc. of 12 S sodium hydroxide. The tion of 5 cc. of 12 sodium hydroxide. The reducing sugar alkali caused a precipitation of suspended material, including was then determined. bacteria, leaving a clear solution for analysis. A 25-cc. DETERMINATIOK OF ACID PRODUCED. Every day each portion of the clear supernatant liquid was placed in a glass fermenting medium Tyas adjusted to a p H of 6.0 by the addiextraction tube which was so constructed that it, together tion of 1M sodium carbonate solution under sterile conditions. with a small funnel, could be suspended from an A. S.T. 11. The total acid produced is expressed in terms of the total extraction apparatus, and hence was adapted for the con- amount of the sodium carbonate solution added during the tinuous extraction of liquids with an immiscible solvent. course of the fermentation. The temperature of the water bath was so regulated that about one drop of ether condensed each second, and the EFFECTO F VARYING CONCENTRATIOKS OF SUCROSE UPOX extraction was continued for 4 days a t this rate. This proPRODCCTIOK OF 2,S-BCTYLEKE GLYCOL longed extraction was found advisable for complete removal To each of the 200-cc. portions of the medium, with varying of the glycol. The ether was then evaporated and the residue heated for one hour a t 85" C., thus removing the neutral concentrations of sucrose, were added 2 cc. of inoculum of a volatile compounds such as ethyl alcohol and acetylmethyl 2-day old culture of Aerobacter pectinovorum grown on a carbinol, leaving the glycol behind. The flask was then similar medium containing 6 per cent sucrose. The pH placed in a desiccator for 2 days to remove moisture, and the of each flask was adjusted to 6.0 by the addition of 1 M amount of glycol determined by weighing. sodium carbonate solution. The cultures were incubated a t A considerable quantity of this material had been obtained 37.5" C. and the medium analyzed for 2,3-butylene glycol by this method in previous experiments and was subjected and sucrose when no further acidity developed. The results to purification. The material obtained by this extraction of these experiments are given in Table 11. method from the media was remarkably pur('. Using the purified material, data were obtained on the reading. of a COXCLUSIONS dipping refractometer in various concentrations of the glycol 1. Up to and including 8 per cent sucrose, all of the sugar in water (Table I). is fermented; a t higher concentrations (8 to 12 per cent) the TABLE I. DIPPINGREFRACTOMETER READIXGSFOR 2,3- percentage of sucrose fermented falls from 100 to 85 per cent. 2 . The rate of fermentation of the sucrose-that is, the AT 25" C. BUTYLESEGLYCOLAT VARIOIXCOSCESTRATIOXS average amount of sugar fermented per day-is a t a definite GLYCOL/~OO CC. REFRACTOMETER GLYCOL/~OO cc. HEFRACTOVETER WATER KEADIXG (R) WATER KE.ADIX( R ) maximum a t about 8 per cent. The average rate of ferExptl. Calcd." Exptl. Calcd.O, mentation a t 8 is nearly double that a t 1 per cent. cc. cc. CC. cc. 3. 2,3-Butylene glycol produced per 100 grams of sucrose 0 0.030 13.26 1.214 1.216 16.80 0.202 0.218 13.82 1,624 1.627 18.02 fermented is a t a maximum of 47 grams a t 8 per cent sucrose. 0.404 0.620 15.02 2.070 2.024 19.21 0.806 0,828 15.64 4.160 4.017 25.16 4. The acid produced per 100 grams of sucrose fermented 1.010 1.016 16.20 decreases a t first rapidly and then slowly with increase a Calculated by t h e following equation: glycol per 1000 cc. = 0.3351 R - 4.412 in concentration of sucrose. 5 . The ratio of acid to glycol is markedly affected by the It is evident that the refractometer reading is a linear concentration of the sucrose, dropping from a value of 1.88 function of the concentration of the glycol. I n order to for 1 per cent sucrose to a constant low level of 1.00 a t 8 per check the gravimetric method, 25 cc. of water were added to cent sucrose.
cc.: 0.250 gram ammonium chloride, 0.150 gram potassium monophosphate, 0.150 gram calcium chloride, 0.20 gram of magnesium sulfate, and sucrose. The optimum pH was found to be 6.0. I n preliminary studies three cultures mere used: Aerobacter faeni, Aerobacter motorium, and Aerobacter pectinovorum. The yield of the glycol increased slightly in the order named; hence, in the work here reported, Aerobacter pectinovorum was employed as the test organism. These bacteria were isolated and named by Burkey (21.
EFFECTOF, VARYING CONCENTRATIOSS OF SUCROSE UPOX P R O D U C T I O ~ ACTIOXOF Aerobacter pectinovorum
----
GLYCOL BY
--GLYVOL P E R 100 c c TIMETO FERMENDetd by GLucoL/100 T O T ~ L ACID/100 SUCROSE FERMENT GLUCOSE/IOO TiTION Detd. by refractive GRAMS ACID/200 GRAMS PER 1 GRAM.ICID/GRAM cc. SUCROSE FERMENTEDP E R I O D weighini index Av. SCCROSE cc SVCROSE D ~ Y SUCROSEGLYCOL Grams Grams Days Grams Grams Grams Grams Cc." $; cc. Cram Days cc. 98 4 1.7 87 0.245 4.04 99 7 0:90 0:so O:& 43.1 3.2 81 0.282 3.54 1:88 100 10 4.0 67 0.299 3.34 1.54 99 12 1:s2 1:65 1:74 4318 4.6 58 0.331 3.02 1.32 99 13 5.3 2.6% 53 0.382 1.19 99 14 2:96 2:i7 2:72 45:6 5.6 47 0.426 2.35 1.03 99 15 6.2 2.21 45 0.453 0.97 99 18 3:92 3:49 3:7l 46:9 7.4 2.28 46 0,439 0.98 93 23 7.8 46 0.366 2.75 0.98 88 23 4:42 3:SO 4,'ii 45:7 8.5 48 0.382 2.61 1.03 86 23 8.6 2.43 46 0.410 1.00 85 23 4'. 92 4'. 20 4: 56 44: 5 8.2 2.25 41 0.445 0.92 of M N a X O :I required t o maintain 200 cc. of medium a t pH = 6.0.
INDUSTRIAL AND ENGINEERING CHEMISTRY
800
k!KNOWLEDGhlENT
The bacteria used in the experiments were kindly furnished by C. H. Werkman of the Department of Bacteriology, Iowa State College.
LITERATURECITED Breden, C. R., with Fulmer, E. I., Iowa State COX J . Sci., 5, 133-53 (1931).
Burkey, L. A., Ibid., 3, 57-160 (1928). Desmots, H., Compt. rend. SOC. biol., 56,383-6 (1904j. Elion, L., Biochem. Z., 169,471-7 (1926). Fulmer, E. I., Nelson, V. E., and Sherwood, F. F., J. Am. C h m . SOC.,43,191-9 (1921). Grimbert, L., Compt. rend., 132,706-9 (1901). Harden, A., and Norris, O., Proc. Roy. SOC.(London), B85, 73-8 (1913). Harden, A., and Norris, S. G., Ibid., B84,492 (1912). Harden, A., and Walpole, G. S., Ibid., B77,399-405 (1906). Kluyver, A. J., and Donker, H. J. L., Veerslag. Akad. Wetensch. Amsterdam., 33,915-19 (1924).
Vol. 25, No. 5
(11) Kluyver, A. J., Donker, H. J. L., and Hooft, F. v’t, Biochem. Z., 161, 361-78 (1926). (12) Lemoigne, hi., Ann. inst. Pasteur, 27,856-85 (1913). (13) Lemoigne, M., Compt. rend.. 157,653-5 (1913). (14) Ibid., 170,131-2 (1920). (15) Ibid., 177,6 6 2 4 (1923). (16) Lemoigne, M., Compt. rend. SOC. b i d , 82,984-6 (1919). (17) Lemoigne, M., and Pettit, A.,Ibid., 88,467-8 (1923). (18) Neuberg, A., and Gorr, C., Biochem. Z., 154,495-502 (1924). (19) Neuberg, C., and Reinfurth, E., Ibid., 106, 281-91 (1920). (20) Neuberg, C., and Rosenthal, O., Ber., 57, 1436-41 (1924). (21) Neuberg, C., and Simon, E., Biochem. Z., 156,374-8 (1926). (22) PBr6, A., Ann. Inst. Pasteur, 10,417-48 (1896). (23) Shaffer, P. A , and Hartmann. A. F., J . Bid. Chem., 45, 371 (1920). (24) Sherwood, F. F., and Fulmer, E. I., J. Phys. Chem., 30,738-56 (1926). (25) Verhave, T. H., British Patents 335,280 and 337,025 (June 26, 1929). (26) Walpole, G. S., Proc. Roy. SOC.(London), B83,272-86 (1911). REC~IYED December 10, 1932.
Foaming and Priming of Boiler Water Peculiar Behavior in an Experimental Boiler C. W. FOULK AND KERMITGROVES,Department of Chemistry, The Ohio State University, Columbus, Ohio
I
N B E G I S S I K G this in-
follows: Inside diameter The experimental steel boiler described is of v e s t i g a t i o n , i t w a s inof drum, 10.2 cm. (4 inches), such design that pure water and salt solutions l e n g t h of d r u m , 168 c m . (5 tended to use t h e w a t e r when boiled in it exhibit in every respect a foamf e e t , 6 inches), and inside ditube boiler described below for ing and priming behavior that is the direct opameter of water tube, 2.5 cm. repeating the work of Joseph and posite of that usually found in steam boilers. (1 inch). The boiler was f i e d Hancock (6) and of Hancock (4). by gas. These investigators had found These results are due entirely to the design of that, in t h e i r e x p e r i m e n t a l the boiler and are in no sense to be ascribed to PROCEDURE.The s a l t s o l u boiler [a simple steel drum 48 any abnormal behavior of the water or salt solutions used were prepared from em. (18 inches) long and 31 cm. distilled w a t e r and salt of the tions. Water was thrown into the steam line be(12 inches) in diameter], finely grade employed i n a n a l y t i c a l cause of waves moving through the steam drum. chemistry. Before the beginning divided s o l i d m a t t e r h a d n o of the work the boiler had been W h e n a wave crest reached the steam outlet, a effect on the priming,’ that is, opened and thoroughly cleaned. t h e p a s s i n g of l i q u i d JTater slug of water went over. The height of these Since the capacity of the pump into the steam line. These ret h a t c a m e w i t h the boiler was waves was determined by the size of the steam not sufficient to maintain a consults were so contrary to popular bubbles emerging f r o m the water tube. Because stant water level during a run, belief and t o published laborathe practice of b e g i n n i n g each steam bubbles are smaller in salt solutions than in tory experiments in glass vessels experiment at the same water level water, the waves were at their highest when the (2) that i t s e e m e d d e s i r a b l e v-as adopted. The w a t e r i n t h e s t e a m was to repeat the work, particularly boiler was charged with pure water, and consemeasured by a chemical method. in a different t y p e of b o i l e r . quently there was more priming with pure water The total ejectate-that is, the I n order to have an appropriate mixture of steam and water-was than with strong salt solutions. b a c k g r o u n d for t h e proposed passed through a condenser and c o l l e c t e d in p o r t i o n s , usually experiments with solid matter, a series of runs was made with salt solutions alone. The of 250 cc. each. These portions were then titrated for chlorides, the amount of chloride found being obviously proportional to the results of this preliminary work were more perplexing than liquid boiler water that had gone over with the steam. When a those of Joseph and Hancock. The experiments indicated salt other than a chloride was used, about 100 p. p. m. of sodium t h a t t h e throwover of water became less as the concentration chloride were added. I n making the calculations, account of the dissolved salt increased. This was so unexpected and was taken of the increasing concentration resulting from the so out of accord with existing knowledge on foaming and evaporation. priming t h a t the question of solid matter was dropped for Since close agreement among quantitative results in the time being, and the study of this peculiar behavior of the foaming and priming experiments is not t o be expected, five salt solutions was taken up. runs were made as a rule for each condition investigated, and the results averaged. Each curve, therefore, is the average EXPERIhIEKTAL BOILER of a number of experiments. Figures 1 and 2 describe the general construction and Because the results were so contrary t o what was expected, appearance of the boiler. Its essential dimensions were as it was desirable to have a mass of evidence in their support, and accordingly four different compounds, each a t various 1 The word “priming” is used in this paper merely to indicate the water concentrations, were used. Runs were also made a t other in the steam. No special meaning other than this is intended.
