THE INTERMEDIATE DISSIMILATION OF GLUCOSEBY AEROBACTER INDOLOGENES'

HOWARD REYNOLDS AND C. H. WERKMAN

Department of Bacteriology, Iowa State College, Ames, Iowa

Received for publication December 7, 1936

Harden and Walpole (1905) found that glucose fermented byAerobacter aerogenes yielded formic, lactic, acetic and succinicacids, carbon dioxide, hydrogen, ethyl alcohol, 2,3-butyleneglycol and acetylmethylcarbinol. Scheffer (1928) has confirmedthese as the the only products formed in appreciable quantities.On the basis of carbon balances obtained from analyses of com-pleted fermentations, the latter investigator proposed a break-down of glucose to methylglyoxal-hydrate which stabilized aslactic acid or split into formic acid and acetaldehyde-hydrate orinto pyruvic acid and hydrogen. The pyruvic acid yielded CO2and acetaldehyde, the latter with the acetaldehyde-hydrateformed acetylmethylcarbinol or was reduced to ethyl alcohol.2,3-Butylene glycol was formed by reduction of the carbinol,and formic acid was split into CO2 and H2.The formation of acetylmethylcarbinol and 2,3-butylene glycol

from glucose has been of interest as an example of the enzymaticcoupling of carbon compounds. Neuberg and Hirsch (1921)demonstrated that acetaldehyde is condensed to acetylmethyl-carbinol by yeast when added to fermentations of glucose andpresented evidence supporting the suggestion that the condensa-tion involves one molecule of added and one of biologically formedacetaldehyde (Neuberg and Simon, 1933).The reversion of acidity caused by Aerobacter is of interest in

connection with the work to be reported. Ayers and Rupp(1918), among others, found that this reversion can take place

1 Supported in part by Industrial Science Research funds of Iowa State College.603

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HOWARD REYNOLDS AND C. H. WERKMAN

under conditions not dependent upon an alkaline protein decom-position. These investigators were able to show that acetic acidfirst accumulated in the fermenting medium and subsequentlyunderwent a significant decrease during the fermentation ofglucose. Acetic acid, when added to a fermentation of glucoseby Aerobacter, was partially destroyed. The fate of the convertedacetic acid was not determined but they suggested that it waschanged to bicarbonate. In contrast with the findings of Ayersand Rupp, Scheffer (1928) found no evidence that Aerobacter wasable to bring about further conversion of acetic acid.

In the present investigation a study of the dissimilation ofglucose has been made by employing serial analysis, a method ofattack which Reynolds, McCleskey and Werkman (1934) pointedout to have certain advantages. We have found a destruction ofacetic acid with an increase in acetylmethylcarbinol and 2,3-bu-tylene glycol. A general discussion is given of the dissimilationof glucose by Aerobacter indologenes.

METHODS

Five-liter fermentations were carried out at 300C. in 6-literflasks provided with a delivery tube, permitting aseptic removalof samples, and an exit tube leading to a gas collector.The medium contained 2.5 per cent glucose, 0.5 per cent sodium

bicarbonate and 0.1 per cent dibasic ammonium phosphate. Theglucose was sterilized at 15 pounds for fifteen minutes in distilledwater. The remainder of the medium containing the buffer wasprepared as follows:The required quantities of bicarbonate and phosphate, with

phenolphthalein as indicator, were sterilized in distilled water.Heat sterilization converts the bicarbonate, in part, to carbonate,the conversion being indicated by an alkaline reaction to phenol-phthalein after autoclaving. After cooling, the carbonate wasreconverted to bicarbonate by forcing sterile carbon dioxidethrough the solution until the indicator was decolorized. Thetwo solutions were mixed aseptically at the time of inoculation.The inoculum consisted of a suspension in phosphate buffer

(pH = 7.0) of the 24-hour growth from a large agar plate. The

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GLUCOSE AND AEROBACTER INDOLOGENES

organism, Aerobacter indologenes, has been adequately describedby Burkey (1928). The culture was examined for purity, micro-scopically and by plating, before and after completion of fer-mentation.

Scheffer (1928), on the basis of carbon and oxidation-reductionbalances, has suggested that small quantities of succiniic acidproduced by Aerobacter resulted from proteins (yeast and peptone)in the substrate. Under the conditions used in our experiments,no succinc acid could be found. Since the medium containedonly ammonium salts as a source of nitrogen, the results supportScheffer's suggestion.The gases evolved were collected in a graduated container over

water saturated with salt and carbon dioxide. Carbon dioxidewas determined as the decrease in volume of gas following itsabsorption in potassium hydroxide contained in a Hempel pipette.Hydrogen was determined by explosion after mixing with oxygen.Residual carbon dioxide in the liquor was determined by acidify-ing an aliquot portion to congo red with 25-per-cent sulphuricacid, heating and aerating under a small reflux condenser forone hour. The evolved carbon dioxide was collected in an excessof 0.3N barium hydroxide contained in a Brady (1914) absorptiontube. The excess base was titrated with 0.3N hydrochloric acidand phenolphthalein.

Glucose was determined by the method of Munson and Walker(1906) on samples previously deproteinated with phosphotungsticacid. Since acetylmethylcarbinol rapidly reduces Fehling's solu-tion it was necessary to make a correction. It has been found inthis laboratory that 1 mgm. of acetylmethylcarbinol when treatedby the method of Munson and Walker yields 3.14 mgm. ofcopper oxide.

Acetylmethylcarbinol was determined on separate portions ofthe fermented liquor according to Stahly and Werkman (1936).Ethyl alcohol was determined by slowly distilling an aliquot

portion of the liquor under a fractionating column until approxi-mately two-thirds had distilled over. The distillate was madealkaline to phenolphthalein and concentrated by one or two suc-cessive distillations to a volume of 100 ml. A 50 ml. portion

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HOWARD REYNOLDS AND C. H. WERKMAN

of the distillate was oxidized by the method of Stahly, Osburnand Werkman (1934) and the resulting acid solution titrated withstandard base. Under the conditions of the above oxidation,acetylmethylcarbinol is oxidized to acetic acid, each mole ofcarbinol giving two moles of acid. Since an appreciable quantityof the carbinol present distills over, under the conditions de-scribed, it is necessary to make the determination on a separateportion of the distillate and apply a correction. Recovery ofalcohol by the above oxidation method is 94 per cent. The total

TABLE 1Diasimilation of glucose by Aerobacter indologenes

Glucose fermented and products in millimoles per liter

GLUCOSE ETIYL ACETYL2,-BuC 3TIME FER- HYDNO- CARBON FORMIC AC ACETIC LACTIC METHYL CR1"E- R&EIOX

MUNTED GEN DIOXIDE ACID ROL ACI D CARC OLYCOLVERY INDEXBINOL

hours per cent

24 14.33 0 7.12 21.0 16.70 6.00 0.55 0.226 6.02 116.3 0.6828 32.70 0.725 24.15 21.30 - 1.00 0.452 13.87 -31 36.10 2.26 34.35 24.0 22.70 8.65 1.47 0.78 16.70 90.5 0.93535 41.00 3.44 46.50 30.0 30.65 6.80 3.29 0.452 22.30 102.5 0.93247 61.10 7.07 85.60 33.2 43.00 5.00 3.90 1.22 35.45 102.0 1.01364 82.50 17.32 110.50 31.75 55.20 2.90 4.13 0.452 49.80 95.3 0.90981 100.30 23.95 144.30 34.60 68.30 1.70 4.02 0.11 66.05 99.0 0.90110 123.80 34.20 189.0 34.60 82.50 1.10 3.68 0.89 79.40 97.0 0.943209 127.30 45.00 218.3 21.60 88.301 0.60 3.68 0 84.25 100.31 0.956

* Cf. Erb, Wood and Werkman. J. Bact. 31, 595 (1936). A perfect balance(Oxidized\OReduced gives an index = 1.0.

acid minus that due to acetylmethylcarbinol divided by 0.94gives the acid equivalent of the alcohol present in the distillate.Only ethyl alcohol was present.2,3-Butylene glycol was determined by the method of Brock-

mann and Werkman (1933). Unfermented sugar was removedby copper-lime precipitation (Hewitt, 1931) before carrying outthe alkaline distillation. Failure to do so results in carameliza-tion of the sugar, giving rise to compounds which interfere withthe determination of the glycol as well as with that of lactic acid.

Lactic acid was determined by the method of Friedemann and

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GLUCOSE AND AEROBACTER INDOLOGENES6

Kendall (1929) on the residue remaining from the 2,3-butyleneglycol distillation.The combined residues remaining after the previously described

distillation for ethyl alcohol were acidified to congo red and steam-distilled according to the method of Olmstead, Whitaker andDuden (1929). Volatile acids in the distillate were determinedas described by Osburn, Wood and Werkman (1933). It wasfound that acetylmethylcarbinol does not interfere with theirmercuric oxide oxidation method for the determination of formicacid. Only formic and acetic acids were present.

EXPERIMENTAL

Periodic analysis of the fermentation of glucose by Aerobacterindologenes (table 1) reveals that three products, i.e., formic acid,acetic acid and acetylmethylcarbinol undergo such decreases fol-lowing previous accumulation as to be assigned significant rolesas intermediates.

It is evident that if the biological conversion of a sugar suchas glucose into final products occurs in such manner that thesugar and its decomposition products can be equated chemicallythroughout the fermentation, the following relationships hold:each mole of glucose disappearing must be accompanied by theappearance of a definite number of millimoles of a given initialproduct, so that, graphically, the relationship between productand converted substrate is linear, assuming no change in the char-acter of the fermentation. This linearity should hold exceptwhere a product is subject to further conversion, such as thedecomposition of formic acid to carbon dioxide and hydrogenwhen the sum of carbon dioxide (or hydrogen) and formic acidshould give the linear relationship. With these considerationsin mind it may be possible to gain some conception as to the fateof those products showing characteristics of intermediates.

In figure 1, ethyl alcohol, carbon dioxide, formic acid and thesum of formic acid and carbon dioxide have been plotted againstglucose fermented. The curve for ethyl alcohol is linear through-out. The sum of carbon dioxide and formic acid likewise showsthe linear relationship, showing an intimate connection between

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608 HOWARD REYNOLDS AND C. H. WERKMAN

carbon dioxide and formic acid and indicating that carbon dioxideis formed by decomposition of the intermediately formed acid.

Carbon Dioodda +

ICONkazoarbo Dixd +m&ui

Formic Acidps to Formic Acid

0 1

440 - 0 M lwoczooGLUCOSE FE2MENTED (Millimoles per Lit-r)

FIG. 1

If the decrease in acetic acid (table 1) be attributed to its con-version to one of the above products, we should expect the linearrelationship to be destroyed, unless the change, for example, toethyl alcohol be accompanied by a simultaneous and equivalent

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GLUCOSE AND AEROBACTER INDOLOGENES6

decrease in the formation of the alcohol from glucose. Occur-rence of the latter phenomenon is highly improbable.

I_

* 5Sunm of Acety methwtcorbInol,Z,-Butyjlem Gljycol and

0 a°f d i E5-5tqloma CT

g 4> C~~Z,5~8utyj-ne GIycoI

400~~~~~~~~'

300Acetic Acid

~Zoo

o I" I UIhIb H

oa 40 00 - 1Z lco ZOGLCO5E FERMENTED (Millimoles per Liter)

FIG. 2

With the small quantities of lactic acid, there remain onlyacetylmethylcarbinol and 2,3-butylene glycol to account for theconverted acetic acid. In figure 2, these products have beenplotted as millimoles per mole of glucose fermented against milli-

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HOWARD REYNOLDS AND C. H. WERKMAN

moles of fermented glucose. It will be seen that a constant rateof formation is indicated by a straight line parallel with theabscissa. The curves for 2,3-butylene glycol and acetic acidshow a reciprocal relationship while a graph of the sum of acetyl-methylcarbinol, 2,3-butylene glycol and one-half the acetic acidis approximately linear. The data indicate that the acetic acidintermediately formed, is condensed and reduced to 2,3-butyleneglycol.The suggested conversion of acetic acid to 2,3-butylene glycol

probably takes place through preliminary reduction of the acidto acetaldehyde, followed by condensation of the latter to acetyl-methylcarbinol and subsequent reduction of the latter to 2,3-butylene glycol. The catalytic reduction of acetic acid to acetal-dehyde is well known in organic chemistry. That acids can bereduced biologically by bacteria has been adequately demon-strated for the butyl forms by Reilly et al. (1920). The biologicalcondensation of aldehydes to carbinols has already been men-tioned. Condensation of acetaldehyde to acetylmethylcarbinolby ultraviolet radiation without intervention of biological systemshas been demonstrated by Dirscherl (1931) and by Barak andStyle (1935). The reduction of acetylmethylcarbinol to 2,3-butylene glycol by Aerobacillus and butter cultures has beenshown by Hammer, Stahly, Werkman and Michaelian (1935).The suggested conversion is consistent with established chemicaland biological principles.

Neuberg, Nord and Wolff (1920) have reported the fixation ofacetaldehyde in the fermentation of glucose by Aerobacter andthe conversion of acetaldehyde to equal molar mixtures of aceticacid and ethyl alcohol by the same organism. (Neuberg andWindisch, 1925.) We have identified both acetaldehyde andpyruvic acid as fixation products in the fermentation. In 1914,experimental results led Neuberg to suggest the bacterial decom-position of pyruvic acid according to the equation:

CH2 CO COOH + HOH = CHsCOOH + HCOOH

Recently we have reported the isolation of phosphoglycericacid formed by colon-aerogenes organisms from glucose (Werk-

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GLUCOSE AND AEROBACTER INDOLOGENES

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HOWARD REYNOLDS AND C. H. WER N

man et al. 1936). These findings, in addition to recent investiga-tions pointing to the phosphorylated trioses as primary inter-mediates in the biological decomposition of hexoses (cf. Lohmann,1930; Harden, 1933; Embden, Deuticke and Kraft, 1933; Meyer-hof, 1933; Meyerhof and Kiessling, 1933; 1935, Tikka, 1935)suggest that the mechanism of muscle glycolysis of Embden andMeyerhof has its counterpart among bacteria.

In figure 3 is given a series of reactions assumed to occur in thedissimilation of glucose by bacteria of the genus Aerobacter asdetermined with a typical species, Aerobacter indologenes.

Acetic and pyruvic acids, acetaldehyde and acetylmethyl-carbinol will serve as hydrogen acceptors for the oxidation of theintermediate triose (dioxyacetone-phosphate, glycerolphosphoricacid?). Since formic acid is decomposed and the ratio of hydro-gen to carbon dioxide becomes progressively less than one, someor all of the reductions of equations under 5 can take place withutilization of hydrogen liberated by reaction 7.The previously mentioned linear relationship between glucose

fermented and the sum of formic acid and carbon dioxide suggeststhat intermediately formed formic acid is changed in accordancewith reaction 7. Since, however, the same linearity will occurin the presence of 4b, if the latter proceeds at a uniform rate, thepossibility that pyruvic acid is converted in both ways cannotbe excluded. Unpublished results show that no hydrogen isevolved when glucose is fermented by A. indologenes in the pres-ence of an excess of acetic which is reduced according to 5b.Evidence failed to indicate further conversion of acetic acid asin the Thunberg-Wieland (Neuberg and Simon, 1933b) series,while the quantitative relationship between the acid and 2,3-butylene glycol support the previously suggested reduction andcondensation of the acid to the glycol.

SUMMARY

A series of reactions is proposed to account for the dissimilationof glucose by organisms of the genus Aerobacter.

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GLUCOSE AND AEROBACTER INDOLOGENES 613

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614 HOWARD REYNOLDS AND C. H. WEREMAN

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