THE BIOCHEMISTRY OF ACETONE FORMATION FROM SUGARS BY BACILLUS ACETOETHYLICUM. (From /he Depurtment of Z~moloyy, Cnirwsity oj Toronto! Toronto, Canada.) (Received for pllblication, Vel>ruary 27, 1025.) Acetone is found among the products of two distinct types of bacteriological fermentation. Anaerobic bacilli of the amylo- back type produce acetone and butyl alcohol from a large series of sugars, and the characteristic acid products are butyric and acetic acids. h second group of bacilli produce acetone and ethyl alcohol from sugars, and the volatile acids produced are acetic acid anti formic acid. ‘IIe first member of this group to be described was Btrcill~s nmerans by Schardinger (I), and the second was isolated, described, and named by Northrop, ;Ishe, and Senior (2) Bacillus acetoethylicunz. The two species differ in only one important characteristic; namely, Uacill~s nceto- ethylicunt alone is able to ferment galactose and lcvulose in a medium containing ammonium salts as the source of nitrogen. The investigation of this type of fermentation was continued by hrzberger, Peterson, and Fred (3, 4), and mention n-ill be made later of scx*eral important points cst’ablished by these xorkers. Neuberg and his associstcs have discussed the methotl by which acetone is produced by bacterial action, and they arc of the opin- ion that the following schemeholds for both the amylobacter and maccrans types of fermentation (5). .~cetald~h~dc~nldol-iacetoacetic acid-bacctone Northrop in his original paper reported that the neutral prod- ucts of the fermentation ol’ several sugars were 5 to 10 per cent acetone and 12 to 25 per cent ethyl alcohol, whereas, there was no acetone produced from glycerol, but the yield of ethyl alcohol rose to 40 per cent. An attempt has been made to study further these fermentations, to account for the difference in the products, 41 by guest on May 9, 2018 http://www.jbc.org/ Downloaded from
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THE BIOCHEMISTRY OF ACETONE FORMATION FROM SUGARS BY BACILLUS ACETOETHYLICUM.
(From /he Depurtment of Z~moloyy, Cnirwsity oj Toronto! Toronto, Canada.)
(Received for pllblication, Vel>ruary 27, 1025.)
Acetone is found among the products of two distinct types of bacteriological fermentation. Anaerobic bacilli of the amylo- back type produce acetone and butyl alcohol from a large series of sugars, and the characteristic acid products are butyric and acetic acids. h second group of bacilli produce acetone and ethyl alcohol from sugars, and the volatile acids produced are acetic acid anti formic acid. ‘IIe first member of this group to be described was Btrcill~s nmerans by Schardinger (I), and the second was isolated, described, and named by Northrop, ;Ishe, and Senior (2) Bacillus acetoethylicunz. The two species differ in only one important characteristic; namely, Uacill~s nceto- ethylicunt alone is able to ferment galactose and lcvulose in a medium containing ammonium salts as the source of nitrogen. The investigation of this type of fermentation was continued by hrzberger, Peterson, and Fred (3, 4), and mention n-ill be made later of scx*eral important points cst’ablished by these xorkers. Neuberg and his associstcs have discussed the methotl by which acetone is produced by bacterial action, and they arc of the opin- ion that the following scheme holds for both the amylobacter and maccrans types of fermentation (5).
.~cetald~h~dc~nldol-iacetoacetic acid-bacctone
Northrop in his original paper reported that the neutral prod- ucts of the fermentation ol’ several sugars were 5 to 10 per cent acetone and 12 to 25 per cent ethyl alcohol, whereas, there was no acetone produced from glycerol, but the yield of ethyl alcohol rose to 40 per cent. An attempt has been made to study further these fermentations, to account for the difference in the products,
and to utilize the experimental evidence obtained in formulating a biochemical scheme for this organism.
EXPERIMEnTTAL.
The parent culture used in this investigation was obtained by the courtesy of the American Museum of Natural History, New York. Stock cultures have been maintained for several years by periodic transfers in a medium containing maize meal and 1 per cent CaC03. For the purpose of these experiments active cul- tures were obtained in the following manner. Agar plate cul- tures grown aerobically were prepared from an active maize cul- ture. Individual colonies were transferred to two types of liquid media, one containing 3 per cent maltose and the other 3 per cent glycerol in a stock solution of mineral salts, calcium carbonate, and peptone. For several weeks the organism was kept active in these two media in order to rule out from our glycerol experi- ments any changes due to an initial transfer of the bacillus from a medium containing sugar. Both types of media support active fermentations.
Experiment I.-A preliminary qualitative examination was made of the products obtained by the fermentation of gIucose, maltose, and glycerol. Two points of theoretical significance were established. Previous reports contain very little information regarding the nature of the gas which is evolved. Northrop does not mention the subject, and in the experiments of Arzberger, Peterson, and Fred the gas was estimated quantitatively as COz. That H, is evolved in sugar and glycerol fermentations can be shown by collecting samples of gas, removing the CO2 by means of NaOH, and exploding the residue with air. This is in accordance with the facts relat- ing to similar bacteriological fermentations.
The second point of importance is that traces of pyruvic acid are present at various times in fermenting media containing glycerol or glucose. This was first indicated satisfactorily during an examination of old test-tube cultures, using the method recommended by Simon and Piaux (6) and Quastel (7). To a small volume of culture add 2 cc. of Hz0 and supersatu- rate with solid (NH4)2SOa in the cold. Add a crystalof sodiumnitroprusside and 2 cc. of NH40H. Shake and allow to stand at room temperature. A blue colour gradually develops, and in dilute solutions of pyruvic acid its intensity is proportional to the concentration of the acid. The test is essentially Rothera’s test for acetone, and when this substance is present in the medium it is impossible to establish the presence of traces of pyruvic acid in the mixture owing to the purple colour which develops. This diffi- culty can be overcome in the following manner. A sample of the culture
to be tested is heated for 5 minutes at 50°C. and 20 mm. of pressure. A little capryl alcohol is added to prevent foaming. Acetone distills over without decomposition of any pyruvic acid which may be present. The test outlined above can be then applied to t.he residue. The blue colour obtained varies from a greenish blue to a pure indigo, according to the amount of pyruvic acid in solution, and for this reason difficulty was en- countered when attempts were made to estimate pyruvic acid by theuse of a standard solution and the calorimeter. A method has been devised, however, which has given satisfactory results in experiments during which the utilization of pyruvic acid by the bacillus was followed. The following standardized conditions are necessary. To 4 cc. of distilled Hz0 add 1 cc. of the culture and an excess of (NHr)$SO4 crystals. Run in from a pipette 2 cc. of a 1 per cent solution of sodium nitroprusside, freshlyprepared,and 1 cc. of MLOII. Allow to stand for 10 minutes, and shake at intervals. Compare the colour which develops with a series of test-tubes containing from 1 to 20 per cent of CuSO4 in II20 by means of a block comparat.or. These tubes must be previously standardized in terms of pyruvic acid. In this way concentrations of pyruvic acid from zero to 0.2 per cent can be measured.
The remaining products found in our preliminary experiments were those which have b,een identified by previous workers.
Experiment Z.-In order to study more completely the occurrcncc of pyruvic acid in the fermentation, and its relation to acetone formation, the following experiment was performed. Three series of test-tubes con- taining maltose, glucose, and glycerol respectively in 3 per cent conccn- tration were prepared, and inoculated. The basis of these media was a mineral salt solution containing also0.5 per cent of peptone and 1 per cent of CaC03. The glycerol tubes were inoculated from stock glycerol cultures in an active condition, and the sugar-containing tubes from maltose cul- tures. They were incubated at 38”C., and at intervals a tube of each type was examined qualitatively for acetone and pyruvic acid. Observations regarding gas evolution were also made. The results are summarized in Table I.
In the tubes containing sugar pyruvic acid was present during the first half of the fermentation period. Later the free acid disappeared, and acetone accumulated. The maltose tubes were more active than the glucose ones, but the amount of pyruvic acid which could be detected was by far the greater in the glucose medium. In the glycerol fermentation a trace of pyruvic acid was present on the 5th day and more definite amounts on the 21st day of incubation. The loss of acetone in all the tubes was due to evaporation. There appeared to be two possible explanations for the almost complete absence of pyruvic acid and acetone in the products of the glycerol fermentation: (n) the organism only
produces the acid from glycerol with difficulty, and pyruvic acid being the intermediate in acetone formation, little of the latter takes place; and (b) pyruvic acid is formed readily from glycerol, but it is converted with equal rapidity into some other characteristic product, most probably ethyl alcohol.
Expwintcnt J.-During our preliminary experiment.s in which B. ncetoe- thylicum was grown for about twenty generations in a glycerol medium it was always possible to detect. acetone in the products by qualitative methods. To obt.ain more definite information regarding the time and extent of this formation the following experiment was performed. Two flasks containing 1,500 cc. of 3 per cent maltose and 3 per cent glycerol
medium respectively were prepared, and sterilized in the usual way. Each contained an equal amount, of filter paper, cut into thin strips. The flasks were inoculated with 200 cc. of an active culture in the same type of medium. Samples of 100 cc. lvere withdrawn at intervals under sterile conditions, and distilled. Quantitative measurements of acetone and ethyl alcohol were made, acetone by the Mcssinger method and ethyl alcohol by osida- tion with potassium dichromatte and sulfuric acid, followed hy a titration of the excess dichromate wit,11 potassium thiosulfatr. Tt is unnecessary to describe t.hc mcthotls of procedure in detail. These observat,ions wcrc con- tinued until both fermentations had finished. and the results are given in Table II. This esperiment was performed three times.
This expcrimcnt indicates that during the glycerol fermentation there is a small amount of acetone formed during the second half of the fermont.ation period. Samples of culture after the 8th day gave a definite purple colour when Rothera’s test for acetone was made, showing that the quantitative measurements obtained by the iodoform method were not due ‘to the ethyl alcohol. At no time during this experiment was it possible to detect pyruvic acid in either flask. These fermentations wcrc more active than test-tube cultures, and these observations suggest that the occur- rence of free pyruvic acid is determined not only by the nature of the substrate but also by the activit,y of individual fermentations.
I?.rlj&r/nc?~( d.--Xcting on thcx It\-pothesis that in the last experiment no acetone was formed in thr early stages of lhc glycerol fermc~nlation owing to the al)senct of pyruvic: arid in the products, several cspctrimcnts of tlbe following general type w(‘re performed. ‘I’\vo flasks containing 1,300 cc. of glycerol medium were prepared and sterilized. ‘1’0 one flask was added just previous to its inoculation 100 cc. of II,0 containing 4 gm. of pyruvic acid (IJnstman Kotlak Companyj, neutralized with SaOFI. The solution of pyruvate had t)cxc>n sterilized by passage through a ISerke- fcld filter. Uoth flasks were inoculated with 200 CC. of an active cult,urc in glycerol medium. .It intervals during the pcbriotl of incubation quantita- t.ive measurements of pyruvic acid. acetone, and ethyl alcohol w(xrc made. On the 2nd clay we observed that almost the whole of the pgruvic arid had been utilized by the organism, and that already acetone had hrrn pro- duced. Ily 111~ 4th c!ay all tllc. pyrliric acid had been dest.royetl, and the acetone content of the medium showed an incrrasc of 200 per cent. Ethyl alcohol formation and gas production were also more rapid in the flask which containcti pyruvic acid. On the 8th day of incubation a second quantity of pyruvic acicl, 3 gm.. x-as neutraliz4 and sterilized. The sblu- tion contained 3 gm. of glycrrol. and was made up to 100 cc. This was added to the flask which had received the first batch. The etrect on the general appearance of the fermentation was very pronounced. Gas produc-
tion was greatly stimulated, and the surface of the medium was covered with filter paper and calcium carbonate. The pyruvic acid was again rapidly utilized, and the effects on the rates of formation of ethyl alcohol and acetone are indicated in Table III and Fig. 1.
900
800
300 / I /I .!/ I I I I -cd -1
Days0 2 4 6 8 10 12 14 16
FIG. 1. Curves relating to two glycerol fermentations. To one flask, continuous curves, pyruvic acid was added at the commencement of and during the fermentation.
The information obtained by these experiments justifies several important conclusions. In the first place it is clear that Bacillus acetoethylicum utilizes very rapidly any free pyruvic acid in a
glycerol medium. This results in the formation of acetone earlier and larger in amount than in the control flask. The two processes do not coincide in time, but about half of the acetone formation takes place after pyruvic acid has disappeared from the medium. These facts suggest that in the formation of acetone from pyruvic acid there are intermediates formed which accumulate. The
clear that from the pyruvic acid some other derivative is obtained, most probably ethyl alcohol. This conclusion is supported by the form of the ethyl alcohol curve in Fig. 1.
Eqxri~~cnt 6.-The purpose of this experiment eras to determine the products of the fermentation of pgruvic acid, glycerol, and a mixture of the two. The following flasks were prepared, and sterilized.
Flask A. 200 cc. of mineral salt, solution + ‘2 gm. glycerol + 1 gm. peptone + 2 grn. CaCOa.
Flask 13. 200 cc. of mineral salt solution + 2 gm. pyruvic acid $ 1 gm. peptone + 3 grn. CaC03.
Flask C. 200 cc. of mineral salt solution + I gm. glycerol + 1 gm. acid + 1 gm. peptone f 2 gm. CaC&.
The pyruvic acid was in each ease neutralized with ?;aOII, and sterilized by the filtration mrthod. Each flask was inoculated~~itl~lOcc. of an active culture in t,he glycerol medium. \Vhen fermentation had ceased in all the flasks certain quantitakive and qualitative (1(:tc~rrrlinatiorls ncre madeafter tlistiltiug IO0 cc. portions. The results are given in Tal)le II’.
The whole of the pyruvic acid in the mixture was utilized, but not in the pure pyruvic acid fcrmcntation. The weight of vola- tile acid in each cast was determined on the basis of equal weights of acet’ic and formic acids, a ratio which approximates to that found by Arzhergcr, Peterson, and Fred (3) for the products from glucose. ($ualit.ativc tests left no doubt that acctonc is formed in large amounts from pure pyruvic acid.
The following is an attempt to summarize those observations of previous workers and those contained in this report which throw light on the biochemical nature of the ferment,ation of different media by Uacill~~s acetoethybicun~.
1. Northrop,: Ashe, and Senior point out that, during the fer- mentation of glucose the ratio between the weights of acetone and ethyl alcohol in the products fell from 2.9 on the 3rd day to 2.2 on the 5th day, and continued at this figure until the end of the
fermentation (2). This observation can be verified and ampli- fied by considering the curve in Fig. 2 which is based on the results contained in Table II relating to the maltose fermentation. The form of the curve is an indication that at the commencement of the fermentation, and during several hours subsequently, the sole
neutral product is ethyl alcohol. Acetone formation commences later, and owing to its gradually increasing rate the ratio between the products falls to 2.2. These facts alone would suggest that acetone and ethyl aIcoho1 are formed by independent biochemical processes, but we have shown in Experments 3 and 4 that both products are formed from pyruvic acid. Also the recurrence of the ratio 2.2 and its persistence for several days is significant.
Days0 2 4 6 8 i0 12 14 16 FIG. 2. Curve showing the varying ratio between the weights of ethyl
alcohol and acetone in the products during the course of a maltose fermen- tation (Experiment 21.
The more justifiable conclusion would seem to be that ethyl alco- hol has a 2-fold origin: (A) by a process in which acetone is not involved, and (B) a series of chemical changes by which definite quantities of both substances are produced. The curve in Fig. 2 according to this interpretation represents a gradual change from (A) to (A + B), at which point the ratio 2.2 is established.
2. The yields of acetone and ethyl alcohol from glucose are reduced, and the yield of volatile acid is increased when the acid
products are neutralized at regular intervals with NaOH.’ This being the case it is not possible that ethyl alcohol is formed with acetic acid from acetaldehyde by the Cannizzaro reaction:
2 CH,.CHO + Hz0 = CHJ.COOH + CH,.CHzOH
3. The composition of the volatile acid fraction obtained by the fermentation of sucrose and starch has been determined by Arz- berger, Peterson, and Fred.2 The figures in Table V are taken from their report, and below we include the percentage figures relating to equimolecular portions of the two acids, formic and acetic.
Both acids were produced from pure pyruvic acid by this organism, and probably during those fermentations in which pyruvic acid is an intermediate they are formed according to the equation:
4. Lactic acid accumulates rapidly during the first stages of a glucose fermentation. The rate of production then falls to a con- stant at which it continues until the end of the fermentation. There is no evidence that lactic acid is an intermediate in the formation of other products.3
On the basis of the above experimental evidence the following scheme for the fermentation of glucose has been formulated.
CHs - CHOH . COOH
T -i- Hz
CsH,zOe + CHI . CO * COOH f Ha --j CH, * CHO + CO2
1 + Hz0 ’ -i- Hz
L CHs - COOH + H . COOH CHa . CHaOH
1 Arzberger, Peterson, and Fred (3), p. 473. 2 Arzberger, Peterson, and Fred (3), p. 470. 3 Arzberger, Peterson, and Fred (3), p. 476.
This scheme is that designated (A) in Section 1 of this discussion. When acetaldehyde is produced more rapidly than it is being reduced, and therefore present within the cell in the free state, the following series of changes takes place.
2 CHI . CHO = CHI - CIIOH - CH2 . CIIO
Alclol.
CHI . CHOH . CIIa - CHO 0 CHa - CHOH - CH, - COOH +I= +
CHs. CHO Hz CH, - CHzOII
CHI . CIIOH . CIIz . COOH +T=
CIIs . CO - CI-IZ . COOH + .
CII, . CIIO 112 CHa . CH,OH
CIII . CO . CH, . COOH = CIIs - CO . CHa CO2
Acetoacetic acid.
The question at once arises as to whether the above scheme for the glucose fermentation enables one to give an adequate explanation for the almost complete absence of acetone in the products of a glycerol fermentation, or in other words why does Section B of the scheme not come into operation? Let us con- sider the difference between molecules of glucose and glycerol, and assume that equal weights of pyruvic acid and acetaldehydc are produced from them.
By the change from a glucose to a glycerol substrate you change the mass relationship between acetaldehyde and Hz, and the process of reduction to ethyl alcohol is thereby facilitated. Acetalde- hydc rarely occurs free within the cell during the glycerol fer- mentation, and consequently aldol condensation and acetone formation do not take place. When pyruvic acid is added to the glycerol medium it is fermented more rapidly than the glycerol, and a new relationship CH3.CHO : Hz is established which approximates more to that obtaining during the latter half of a glucose fermentation, and consequently acetone formation occurs.
We have shown that the bacillus is abIe to form acetone from pure pyruvic acid. If this process involves aldol condensation
followed by oxidation it is necessary to postulate some acceptor for H,. The evidence points to acetaldehyde as this substance.
The general significance of this type of work in the larger field of carbohydrate oxidation has been adequately treated in recent reviews by Dakin (8) and Shaffer (9).
SUMMARY.
1. The fermentation of glucose, maltose, and glycerol by Bacil- lus acetoethylicum has been studied from the point of view of acetone and ethyl alcohol production.
2. Pyruvic acid has been shown to be an intermediate com- pound in these fermentations.
3. Pure pyruvic acid is fermented, and from it acetone, ethyl alcohol: and volatile acids are formed.
4. A biochemical scheme for these fermentations has been postulated.
BIBLIOGRAPHY.
1. Scharclinger, F., Cents. Bnkt., 1. Abt., 1905, xiv, 7i2. 2. Northrop, J. II., Ashe, L. II., and Senior, .J. I<., J. Bid. Chem., 1919,
xssis, 1. 3. Arzherger, C. F., Peterson, IV. H., and Fred, E. IS., J. b’iof. Chem.
1020 sliv s , , ‘ 165. 4. Peterson, \V. H., and Fred, E. B., J. Biol. Chml., 1920, sliv, 29. 5. Seubrrg, Cr., and Arinstein, B., Biociwn~. Z., 1921, csvii, 269. 6. Simon, I,.-J., and Piaux, L., Bzrll. Sot. claim. bid., 1924, vi, 4i7. 7. Quastel, J. H., Biochon. J., 1924, xviii, 365. 8. Dakin, R. D., Oxidations and reductions in the animal body, London
and Sew York, 1922. 9. Shaffer, P. A., Physiol. Reu., 1923, iii, 394.
