FERMENTATION OF GLUCOSE BY SUSPENSIONS OFESCHERICHIA COLI

J. L. STOKESHopkins Marine Station, Pacific Grove, California

Received for publication October 29, 1948

In 1926 Rona and Nicolai (1926) reported that manometric measurements ofthe fermentation of glucose by Escherichia coli showed this decomposition to be atypical lactic acid fermentation with the formation of two moles of lactic acidper mole of glucose. This conclusion is in contrast with the one reached byHarden (1901), who, by direct chemical analyses, found that the fermentationcan be represented approximately by the equation:2C6H1206 + H20 = 2CH3CHOHCOOH +glucose lactic acid

CH3C00H + C2H5OH + 2CO2 + 2H2acetic acid ethanol

Harden's work received support from later investigations (Grey, 1914, 1918;Kay, 1926; Scheffer, 1928; Tasman, 1935; Tikka, 1935) also conducted on a scalelarge enough to permit chemical analyses. Although Harden's studies weremade with growing cultures of the bacteria, essentially similar results were laterobtained by the use of nonproliferating cell suspensions (Grey, 1918; Tasman,1935; Tikka, 1935). Even with cell-free extracts of E. coli a decompositionmore complicated than a lactic acid fermentation has been demonstrated (Kalnit-sky and Werkman, 1943).The discrepancy between the results of Rona and Nicolai and of Harden,

Grey, and others has never been explained or reinvestigated, although it hascaused much perplexity. The experimental support for Rona and Nicolai'sconclusion is rather meager; apart from a qualitative determination of lacticacid and the establishment that no gaseous products are formed directly from thesugar, their case rests upon manometric data that purportedly show the forma-tion of two moles of acid per mole of sugar.That no gaseous fermentation products were found by Rona and Nicolai is

understandable enough today; the stibsequent investigations of Stephenson,Stickland, and Yudkin (Stephenson, 1937) have clearly shown that cell sus-pensions of E. coli do not liberate gas from glucose under anaerobic conditionsif the organisms are grown in the presence of an abundant supply of oxygen.Thus, the method of preparing cell suspensions used by Rona and Nicolai-growing the organisms on the surface of agar media-would have precluded theappearance of gas in their experiments, other than through the decompositionof bicarbonate by acidic products. But it is also known that such cell suspen-sions yield, instead of the normally found mixture of C02 and H2, an equivalentamount of formic acid.

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From the evidence submitted in their publication, it is not clear how Ronaand Nicolai could clain the production of 2 moles of acid per mole of glucose;recalculation of their data pertaining to the two fermentations that were al-lowed to go to completion indicates that much less acid was formed:

GLUCOSE CONSUMED AC FORMED MOLES 01 ACD PEE MOLE

mg mm' no'0.15 18.7 11.5 0.610.30 37.4 44.5 1.19

One might, perhaps, consider the experiments of Cattaneo and Neuberg (1934)as providing support for Rona and Nicolai's contentions. By the use of driedcells of E. coli in the presence of toluene and glutathione, a quantitative con-version of hexose diphosphate to lactic acid was accomplished. Such a system,however, is far removed from that operating in a normal fermentation.

In view of the existing difficulty of accounting satisfactorily for the claimsput forward by Rona and Nicolai, it seemed advisable to repeat their manomet-ric experiments and to supplement and support the manometric data by chem-ical analyses of the fermentations.

EXPERIMENTS

Experiments were made with three strains of Ewcerichia coli, obtained fromdifferent sources to ensure that the results would have general significance.Strain PA4.1 was obtained from the culture collection of the Hopkins MarineStation; strain E was isolated from raw sewage by Dr. S. Elsden, to whom weare indebted for this culture; and strain S was isolated from a fecal suspensionstreaked on eosin methylene blue agar plates.

All three strains consisted of short gram-negative rods. They fermentedglucose and lactose in broth with the formation of acid and gas, formed indolein tryptone medium, did not grow with citrate as the sole source of energy,produced sufficient acid in glucose broth to change the color of methyl red indica-tor, and did not produce acetylmethylcarbinol. The strains were thereforetypical E. coli. Stock cultures were maintained on yeast extract, 2 per centglucose (YED) agar slants in the refrigerator and subcultured at bimonthlyintervals.Manometric experiments. The Barcroft-Warburg apparatus was used. Cell

suspensions were prepared from YED agar plate cultures incubated at 35 C forapproximately 18 hours. The cells were washed from the plates with 0.01M NaHCOs, centrifuged, and resuspended in sufficient 0.01 m NaHCOs to give aconcentration of 5 mm' of cells per ml. Two ml of suspension were used ineachWarburg vessel. Two-tenths ml of M/30 glucose were placed in one side cup;the other cup received 0.2 ml of 2 N HISO4 for the determination of initial orresidual bicarbonate. The atmosphere was either C02, or N2 containing 1 or 5per cent C02. The gases were freed of oxygen by passage over heated coppergauze. The bath temperature was 30.4 C, z1 0.1 C. Fermentations were

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allowed to continue until all the glucose had been utilized as indicated by thecessation of acid formation. This usually required 2 to 3 hours. The endog-enous fermentation was negligible. Acid formation was measured as C02 liber-ated from the interaction of the fermentation acids with the bicarbonate of thecell suspensions, and the total amount of acid formed was calculated from thedifference between initial and residual bicarbonate. The course of a typicalfermentation is shown in figure 1.The results of a series of experiments to determine the number of moles of

acid produced by E. coli per mole of glucose fermented are given in table 1.Without exception and regardless of the gas phase, the three strains produced anaverage of about 2.5 moles of acid per mole of glucose. All strains formed ap-proximately the same amount of acid. These values are greater than the 2moles of acid claimed by Rona and Nicolai and immediately rule out the possi-bility that each mole of sugar is converted into 2 moles of lactic acid. Theyindicate, on the contrary, a decomposition more in line with the results ofHarden, Grey, and others.That the C02 produced in the vessels is due entirely to the reaction of the

fermentation acids with the bicarbonate and does not arise, in part, metabolicallyfrom the glucose is shown by the close agreement between initial HCO3--C02and the sum of the residual HC037-C02 and the C02 output (table 2). If anymetabolic C02 had been formed, the sum of C02 liberated to the gas phase andresidual bicarbonate would have exceeded the initial HCOO--CO2 by an amountequal to that of the metabolic C02.

It was to be expected that the pressure changes would be due entirely to C02and not in part to hydrogen, since the cells were grown aerobically and wouldtherefore not contain hydrogenlyase, the enzyme that splits formic acid to C02and H2 (Stephenson, 1937). This hypothesis was further supported by theabsence of gas formation in fermentations in phosphate buffer. Finally, theabsence of hydrogen was firmly established by gas analyses at the terminationof several of the manometric experiments. This was necessary in view of thefollowing considerations:The close agreement between initial bicarbonate-CO2 and final bicarbonate

C02 pluS liberated gaseous C02 does not rigorously exclude the possibility thathydrogen might have been formed. Since E. coli, in common with other hetero-trophic bacteria, can fix C02 (Elsden, 1938; Wood et al., 1941), it is at leasttheoretically possible that H2 production might have occurred, counterbalancedby an exactly equivalent C02 assimilation. The gas analyses made by a pro-cedure similar to that of the "second method" of Dickens and Simer (Dixon,1943) showed convincingly that neither H2 nor any gas other than C02 and theinitially introduced nitrogen were present.The three strains of E. coli will form both acid and gas from glucose, providing

the cell suspensions are prepared from YED broth cultures (100 ml of mediumper 150-ml Florence flask) instead of from YED agar plates. Such cells, havingbeen cultivated under reduced oxygen tension, contain hydrogenlyase and thusproduce H2 and C02 in place of formic acid. Data for two of the strains grown inbroth are given in table 3.

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J. L. STOKES

400

300

200

100

20 60 100 140MINUTES

Figure 1. Fermentation of 6.66 pM of glucose by 10 mm3 of E. coli cells. A. CO, produc-tion with glucose. B. C02 production without glucose (endogenous fermentation).

TABLE 1Formation of acid from glucose by suspensions of E. coli

MKS ACIDOLS AC

STRIN MM2 GLUCOSE Percentage of COs in atmosphere E MOLE O1GLUCOSE

1 5 100

PA4.1 149.3 384 2.57149.3 372 2.49

S 149.3 355 2.38149.3 386 2.58149.3 389 2.60149.3 355 2.38

E 149.3 369 2.47149.3 380 2.54

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The exes of 300 mm' of gas, calculated as C02, over the total initial HCO37-C02 is that produced metabolicaly and not from the interaction of the fermenta-tion acids with bicarbonate. From the A HCO3-, acid productioniscomputed as1.3 moles per mole of glucose, i.e., 1.2 moles less than in fermentations withaerobically grown cells. This difference could readily be accounted for as dueto the decomposition of formic acid. As will be shown, this is exactly the amountof formic acid produced by cells grown under aerobic conditions. Also, such

TABLE 2Carbon dioxide balances in the fermentation of glucose by E. coli 8uspensioS

STRAIN

PA4.1 S E

mm3 mms mmInitial HCO3--CO- 451 420 428

C02-liberated.386 389 380Residual HCO3--CO 2.61 33 62

Total.447 422 442

TABLE 3Formation of acid and gas from 0.2 ml of m/30 glucose by E. coli suspensionsfrom broth cultures

E. COLI (S) E. COLI (Z)

mm' mm$

1. InitialHCO--CO..413 414

2. Gas liberated (CO2 + H).492 5033. Residual HCO-C02a.205 220

4. Total gas (2 + 3).697 723

5. Metabolic gas (4 minus 1).284 309

6. C02 due to acid formation (1 minus 3).208 194

7. Moles of acid per mole of glucose (6 *. 149.3) 1.37 1.30

decomposition should give rise to a 1:1 mixture of H2 and C02 and that is whatwas found.

Chemical analyses. Manometry shows that 2.5 moles of acid are producedper mole of glucose. This is in line with the amounts to be expected from theinvestigations of Harden, Grey, and others. One might, however, expect thatacidic products other than lactic acid had been produced, notably, formic,acetic, and succinic acids. It was therefore desirable to support this view furtherby chemical analyses.

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In preparing material for analysis, a pilot manometric experiment was alwaysmade in order to be certain that the particular batch of celLs used behavedtypically and also to indicate when the sugar was completely utilized. Along-side the Warburg vessels was placed a 500-ml Erlenmeyer flask, fitted with aninlet and outlet tube for aeration, which received exactly 100 times the quantityof cell suspension and glucose that went into each Warburg vesel. The flaskthus received 200 ml of cells suspended in 0.01 M NaHCO3 and 20 ml of M/30glucose (120 mg). The gas phase in the flask was either nitrogen with 5 percent C02 or C02 alone, the same as in the Warburg vessel. The flask culture wasshaken by hand at frequent intervals during incubation. At the conclusionof the manometric experiment, the flask culture was removed from the bathand acidified with 3 ml of 10 N H2SO4 to stop the fermentation and to preservethe fermented liquid from microbial contamination during storage. The cellswere removed by centrifugation, washed once with about 50 ml of water, and thetotal supernatant liquids were adjusted to exactly 300 ml. Aliquots were re-moved for chemical analysis. The remainder of the fermentation liquor wasstored in the refrigerator.Some difficulties were encountered in the analyses because of the small amounts

of metabolic products. About 150 ml of liquor, representing only 60 mg of theinitial glucose, was used for a complete analysis of ethanol, the various organicacids, and residual glucose. Semimicro methods, adequate for quantitativelymeasuring 0.5 mg to 1.0 mg of each of the expected end products, were required.The methods finally adopted were first tested on known solutions of each of thefermentation products and then on an artificial mixture of all of them, mixed inthe proportion normally found in the fermentations. Recoveries were of theorder of 90 per cent or better.

Residual glucose was tested for by the modified method of Luff-Schoorl(Browne and Zerban, 1941). Even with considerable amounts of fermented solu-tions, concentrated by evaporation, no trace of unfermented sugar was everdetected. Ethanol was determined by the dichromate oxidation method ofNorthrup et al. (1919). The total volatile acids were obtained by steam distilla-tion. Formic and acetic acids were determined by Duclaux distillation of analiquot of the volatile acid fraction. Because of the small quantities of acidsinvolved, it was necessary to use special precautions and very carefully tostandardize the distillation procedure in order to obtain accurate and reproducibleresults. The distillation fla was lagged with asbestos and covered with a tincan to reduce condensation. Also, it was essential to use a flame strong enoughto distill 25-ml fractions in 5j minutes with a reproducibility of i5 seconds;slower distillation gave erratic results. Care was taken to avoid any changein apparatus or procedure after establishment of the proper conditions for attain-ment of reproducible constants with known solutions of formic and acetic acids.Formic acid was also determined directly by the HgC12 method of Fincke (1913).This was done, usually, on an aliquot of the volatile acid fraction but occasionallyalso directly on the fermented liquor; the same results were obtained in both

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cases. Since the quantities of formic and acetic acids determined by the twoprocedures were in good agreement, it is obvious that volatile fatty acids otherthan these were not present. Lactic acid was determined by the acid KMnO4method of Friedemann and Graeser (1933), either directly on the fermentedliquor or on an aqueous solution of an ether extract of the nonvolatile acid frac-tion. Succinic acid was measured in the Warburg apparatus with a succinicdehydrogenase preparation made from cormorant breast muscle. The procedureof Cohen (Umbreit et al., 1947) was modified to include disintegration of themuscle tissue in a Waring blender for about 20 seconds in between the first andsecond washing of the minced tissue. The final preparation had relativelysmall particles because of the blender treatment, was easy to pipette, and had ahigh activity, probably due to the finer dispersion of the tissue, so that determina-tions were completed in 20 minutes as compared to the 60 to 90 minutes normallyrequired. Difficulty was encountered in the ether extraction of the nonvolatileacids after mixing the acidified, concentrated alcohol- and volatile-acid-freefermentation liquor with anhydrous Na2S04. This was traced to the simul-taneous extraction of small amounts of the H2S04, initially used in excess for thepurpose of acidifying the solution. Although the amount of H2S04 extracted,about 1.0 ml of 0.01 N acid, would be insignificant in the determination of macroquantities of nonvolatile acids, it was equal to about 25 per cent of the total non-volatile acids being measured. This difficulty was eliminated by acidifying thenonvolatile acids with only sufficient H2S04 to give a pH of 2 prior to mixing withNa2SO4. This procedure eliminated excess H2SQO and therefore the extractionof the latter by ether.The results of the chemical analyses have been combined in table 4. Each

mole of glucose fermented gave rise to 0.8 moles of ethanol and of acetic acid,1.2 moles of formic acid, 0.1 to 0.2 moles of lactic acid, and 0.3 to 0.4 moles ofsuccinic acid. All the carbon, hydrogen, and oxygen of the fermented glucoseis accounted for by these end products. The recoveries, although somewhathigh, are reasonably good in view of the small quantities of metabolic productsinvolved. The chemically recovered equivalents of acid are substantially inagreement with the quantities determined manometrically. There were no sig-nificant differences between strains E and S.

Lactic acid, which is stated by Rona and Nicolai to be the only end productof the fermentation, actually is produced in only relatively small amounts, 0.1to 0.2 moles per mole of sugar fermented, which is less than that of any of theother organic acids. These quantities are also considerably less than the ap-proximately 1 mole of lactic acid found by Harden (1901), Scheffer (1928), andTasman (1935) in their experiments. At first, it was suspected that the lowyield of lactic acid might be due to the relatively high pH (7.1) at which thefermentations proceeded, since Tikka (1935) has shown such a correlation in hisexperiments. For example, he found 0.9 moles of lactic acid formed per mole ofglucose fermented at pH 6.4, but only 0.4 moles at pH 7.1, and 0.05 moles at pH7.6. In one experiment, conducted in bicarbonate buffer at pH 7.6, Tikka

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obtained 0.2 moles of lactic acid, a value which corresponds exactly with our ownresults. The profound effect of pH on the outcome of microbial fermentationshas frequently been observed (see, e.g., Mickelson andWerkman, 1938; Gunsalusand Niven, 1942).

In order to test this possibility, a fermentation was conducted with strain S,suspended in 0.01 M NaHCO3, and equilibrated with a gas phase of pure C02,resulting in a pH of 5.8. This experiment yielded essentially the same smallamount of lactic acid as fermentations at the higher pH; also the quantities ofthe other fermentation products were in agreement with those obtained at pH

TABLE 4Metabolic products formed in the dissimilation of glucose by suspensions of E. coli

E. COLI (E) E. COLI (s)

Gas phase.......................................... 5% COs in N2 5% CO0 in Nt CO0

Initial pH.......................................... 7.1 5.8

moles per mole of glucose dissimilaed

End productsEthanol ............................... 0.84 0.77 0.82Formic acid .............................. 1.16 1.21 1.34Acetic acid............................... 0.81 0.78 0.70Lactic acid............................... 0.10 0.20 0.21Succinic acid............................. 0.34 0.39 0.27

per cent

RecoveriesCarbon................................. 102 108 102Hydrogen................................ 110 114 111Oxygen................................. 107 115 110

Redox index................................ 0.90 1.04 0.98

Equivalents of acidManometrically ........................... 2.54 2.60 2.38Chemically ............................... 2.75 2.97 2.79

7.1 (table 4). Obviously the hydrogen ion concentration is not the only factorwhich influences lactic acid formation.The high yields of lactic acid have invariably been associated with fermenta-

tions in media containing phosphate. Hence a number of experiments wereconducted in m/15 phosphate buffers at different levels of pH. The results (table5) leave no doubt that it is the combined effect of phosphate and low pH thatgives rise to yields of lactic acid of the order of magnitude of 1 mole per mole ofsugar fermented-yields which have so often been encountered ever since Har-den's investigations. The spectacular decrease in lactic acid formation at highpH levels is accompanied by an increase in ethanol and volatile acids; analysisof the latter has proved that it is chiefly formic acid production that is affected.This phenomenon is directly comparable with the findings of Gunsalus and Niven

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(1942), who showed that homofermentative lactic acid bacteria produce con-siderable amounts of ethanol and fatty acids at high pH. It would be importantto establish whether the behavior of the lactic acid bacteria, too, depends uponthe presence of phosphate.From the foregoing results it follows, therefore, that Harden's equation can

be considered to paraphrase the coli fermentation only under a restricted set ofenvironmental conditions. It is possible to view the coli fermentation, especiallythat with hydrogenlyase-deficient cells, as one in which the preliminary orglycolytic phase gives rise to the key intermediate, pyruvate. The subsequentfate of pyruvate will largely determine the nature of the fermentation. The

TABLE 5Effect of pH on the quantities of metabolic products formed in the fermentation* of glucose

by E. coli (S)

pH LACTIC ACID SUCCINIC ACID VOLATILE AaD ETHANOL

moles per mole ofglucsefermensed5.62 0.95 0.14 1.05 0.486.00 0.74 0.19 0.75 0.506.50 0.32 0.31 0.79 0.787.00 0.10 0.26 1.51 0.817.46 0.07 0.26 1.39 0.827.96 0.05 0.24 1.44 0.83

* Fermentations were conducted in M/15 phosphate buffer at the indicated pH's andunder an atmosphere of nitrogen.

pertinent reactions of pyruvate in coli probably are reduction, phosphoroclasticsplitting, and dismutation:(1) CH3COCOOH + 2H = CH3CHOHCOOH

pyruvic acid lactic acidHaPO4 ~~~~~(Utter and Werk-(2) CH3COC00H + H20 = CHICOOH + HCOOH m 1an,1943; Utter,

acetic acid formic acid Lipmann, andWerkman, 1945)

(3) 2CH3COCOOH + H20 = CH3CHOHCOOH + CHICOOH + C02(Krebs, 1937)

These are competitive reactions which are regulated by the conditions of thefermentation and which, in turn, determine the quantities of end productsformed. Below pH 7 reduction of pyruvate to lactic acid occurs to a consider-able extent, whereas above pH 7 this reduction is limited in favor of a phos-phoroclastic split of pyruvate to acetic and formic acids. This view is strength-ened by the observation that in the absence of phosphate the amount of lacticacid formed is not influenced by pH-and even more so by the lack of C02production in the experiments with aerobically grown cells, which rules out theoccurrence of the Krebs dismutation reaction.

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The three reaction equations for the decomposition of pyruvic acid do notaccount for the formation of ethanol, nor of succinic acid. In connection withthe latter, the present investigation poses a new problem. Since 1938 succinicacid production in sugar fermentations has been generally considered as resultingfrom the addition of C02 to pyruvic acid and the subsequent reduction of thecondensation product, oxaloacetic acid (Wood and Werkman, 1938; Krebs andEggleston, 1940; Wood, 1946). The experiments here reported render thisinterpretation doubtful for the fermentation by formic hydrogenlyase-deficientE. coli. The C02 analyses at the termination of the manometric experiments,as well as the close agreement between initial bicarbonate-CO2 and the sum ofthe residual HC0ij-C02 and C02 production resulting from acid formation,show conclusively that C02 was not assimilated during the fermentation. Never-theless, appreciable amounts of succinic acid were formed. It would appear,therefore, that this substance must have arisen by a mechanism other than the"Wood-Werkman reaction," and it seems possible that a condensation of a 3-carbon compound with formic acid rather than with C02 may have occurred.The possibility is, of course, not excluded that other mechanisms may be in-volved, such as the postulated condensation of acetate with a C2 or C3 compound(Slade and Werkman, 1943; Kalnitsky, Wood, and Werkman, 1943). However,some implications of the present data seem to favor the former supposition.According to the three equations representing the modes of decomposition of

pyruvic acid, the formation of acetic acid should be accompanied by the libera-tion of an equimolar quantity of a 1-carbon compound. It is reasonable toassume that the other 2-carbon product, ethanol, is formed by a similar mecha-ni8m. But in that event the molecular ratio of ethanol and acetic acid, on the onehand, and of the 1-carbon compound, here restricted to formic acid, on the other,should be unity. And this is evidently not the case; in the three fermentationslisted in table 4 the quantities of the 2-carbon products are far in excess of theformic acid. By postulating a condensation of a 3-carbon intermediate substancewith formic acid as one of the steps leading to succinic acid production, the totalextent of formic acid formation can be computed by adding the presumable as-similated quantity, 1 mole for each mole of succinic acid, to that finally observedas remaining in the fermented liquid. In doing so, the following figures result:

QUANTITY IN MOLES O0

"Corrected"Cs-products Formic acid Succinic acid formic acid (formic

+ succinic acids)

Fermentation 1 ...... 1.65 1.16 0.34 1.50Fermentation 2 ...... 1.55 1.21 0.39 1.60Fermentation 3 ...... 1.52 1.34 0.27 1.61

Average.... 1.57 1.57

This reveals that the "corrected" values for formic acid production closelyapproximate those of the C2 compounds, so that the expected 1:1 ratio of C2and Ci products is actually realized.

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Alternatively, a mechanism of succinic acid formation involving condensationsof 2-carbon compounds would fit the data only if the latter substances couldoriginate from the breakdown of sugar without being accompanied by 1-carbonproducts. No evidence exists at present for such a degradation.As for ethanol, the most probable mode of its formation now appears to con-

sist in a reduction of acetic acid. These problems are being investigated further.

ACKNOWLEDGMENTS

These investigations would not have been possible without the constantadvice and help of Professor C. B. van Niel. I am also indebted to him forassistance in the preparation of the manuscript. The capable assistance of Mr.William Byrne with the experiments on the effect of pH on the coli fermen-tations in phosphate buffer is gratefully acknowledged.

SUMMARY

The claim of Rona and Nicolai, based on manometric experiments, that aero-bically grown cells of Escherichia coli ferment glucose with the production of twomoles of lactic acid per mole of sugar could not be confirmed. Instead, our mano-metric experiments show that 2.5 moles of acid are formed. Chemical analysesdemonstrate that 0.8 moles each of ethanol and of acetic acid, 1.2 moles of formicacid, 0.2 moles of lactic acid, and 0.4 moles of succinic acid are produced in thefermentations. These end products, in the amounts formed, account com-pletely for all the glucose dissimilated.The pH greatly influences the yields of metabolic products in fermentations

conducted in phosphate, but not in those in bicarbonate buffer.The quantitative data strongly suggest that succinic acid originated by a

condensation of a 3-carbon compound with formic acid.

REFERENCESBROWNE, C. A., AND ZERBAN, F. W. 1941 Sugar analysis. 3d ed. John Wiley and Sons,

New York.CATTANEO, C., AND NEUBERG, C. 1934 Umstellung der Coli-Garung auf reine Milchsaure-

Garung. Biochem. Z., 272, 441-444.DIXON, M. 1943 Manometric methods. 2d ed. Cambridge University Press, Cambridge,

Eng.ELSDEN, S. R. 1938 The effect of CO2 on the production of succinic acid by Bact. coli

commune. Biochem. J., 32, 187-193.FINCKE, H. 1913 Nachweis und Bestimmung der Ameisensaure. Biochem. Z., 51, 253-

287.FRIEDEMANN, T. E., AND GRAESER, J. B. 1933 The determination of lactic acid. J. Biol.

Chem., 100, 291-308.GREY, E. C. 1914 The enzymes which are concerned in the decomposition of glucose and

mannitol by Bacillus coli communis. Proc. Roy. Soc. (London), B, 87, 472-484.Giwy, E. C. 1918 The enzymes concerned in the decomposition of glucose and mannitol

by Bacillus coli communis. Part II. Experiments of short duration with an emulsionof the organisms. Proc. Roy. Soc. (London), B, 90, 75-92.

GUNSALUS, I. C., AND NIVEN, C. F., JR. 1942 The effect of pH on the lactic acid fermenta-tion. J. Biol. Chem., 145, 131-136.

HARDEN, A. 1901 The chemical action of B. coli communis and similar organisms on carbo-hydrates and allied compounds. J. Chem. Soc., 79, 610-628.

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KALNITSKY, G., AND WERKMAN, C. H. 1943 The anaerobic dissimilation of pyruvate by. cell-free extract of E8scherichia coli. Arch. Biochem., 2, 113-124.

IKALNTSKY, G., WOOD, H. G., AND WERKMAN, C. H. 1943 C02-fixation and succinic acidformation by a cell-free enzyme preparation of E8cherichia coli. Arch. Biochem., 2,269-281.

KAY, H. D. 1926 Note on the variation in the end-products of bacterial fermentationfrom increased combined oxygen in the substrate. Biochem. J., 20, 321-329.

KREBS, H. A. 1937 Dismutation of pyruvic acid. Biochem. J., 31, 661-671.KREBS, H. A., AND EGGLESTON, L. V. 1940 Biological synthesis of oxalacetic acid from

pyruvic acid and C02. Biochem. J., 34, 1383-1395.MICKELSON, M., AND WEREMAN, C. H. 1938 Influence of pH on the dissimilation of glu-

cose by Aerobacter indologenes. J. Bact., 36, 67-76.NORTHRUP, J. H., ASHE, L. H., AND SENIOR, J. K. 1919 Biochemistry of Bacillus aceto-

ethylicum with reference to the formation of acetone. J. Biol. Chem., 39, 1-21.RONA, P., AND NicoLAI, H. W. 1926 tJber den Fermentsstoffwechsel der Bacterien. I.

Mitteilung. Atmung und Glykolyse bei Bakterium coli. Biochem. Z., 172, 82-104.SCHEFFER, M. A. 1928 De Suikervergistung door Bakterien der Coli-Groep. Disserta-

tion, Delft.SLADE, H. D., AND WERKMAN, C. H. 1943 Assimilation of acetic and succinic acids con-

taining heavy carbon by Aerobacter indologenes. Arch. Biochem., 2, 97-111.STEPHENSON, M. 1937 Formic hydrogenylase. Ergeb. Enzymforsch., 6, 139-156.TASMAN, A. 1935 The formation of hydrogen from glucose and formic acid by the so-

called "resting" B. coli. II. Biochem. J., 29, 2446-2457.TIRKA, J. 1935 tiber den Mechanismus der Glucosevergarung durch B. coli. Biochem.

Z., 279, 264-288.UMBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. B. 1947 Manometric techniques and

related methods for the study of tissue metabolism. Burgess Publ. Co., Minneapolis.UTTER, M. F., LIPMANN, F., AND WERK-MAN, C. H. 1945 Reversibility of the phosphoro-

clastic split of pyruvate. J. Biol. Chem., 158, 521-531.UTTER, M. F., AND WERKMAN, C. H. 1943 Role of phosphate in the anaerobic dissimila-

tion of pyruvic acid. Arch. Biochem., 2, 491-492.WOOD, H. G. 1946 The fixation of carbon dioxide and the interrelationships of the tri-

carboxylic acid cycle. Physiol. Revs., 26, 198-246.WOOD, H. G., AND WERKMAN, C. H. 1938 The utilization of C02 by the propionic acid

bacteria. Biochem. J., 32, 1262-1271.WOOD, H. G., WERNMAN, C. H., HEMMINGWAY, A., AND NIER, A. 0. 1941 Heavy carbon

as a tracer in heterotrophic carbon dioxide assimilation. J. Biol. Chem., 139, 365-376.

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