ENERGETICS AND ASSIMILATION IN THE COMBUSTION OFCARBON COMPOUNDS BY ESCHERICHIA COLI'

BENJAMIN V. SIEGEL AND C. E. CLIFTONDepartment of Bacteriology and Experimental Pathology, Stanford University

School of Medicine, Stanford, California

Received for publication July 29, 1950

In the preceding paper (Siegel and Clifton, 1950) on the oxidative assimilationof carbohydrates by Escherichia coli considerations of growth, assimilation, andenergetics made it evident that free energy relationships alone do not provideconclusive criteria concerning the extent of the assimilability of a carbohydrateutilized by the cell. This has been attributed to the more important role of pos-sible intermediates of the oxidative process that serve as building blocks for thenumerous syntheses carried on by the cell. Thus, lactose, with a molar AF forthe reaction of oxidative assimilation almost twice that of arabinose or glucose,is assimilated to a lesser extent during growth than the latter two sugars, ofwhich arabinose is more readily assimilated. In the studies to be described thisconcept has been more exhaustively investigated employing the methods andreasoning previously described (Siegel and Clifton, 1950) and with organic sub-strates other than sugars.

EXPERIMENTAL RESULTS AND DISCUSSION

Experiments performed with washed cells to obtain equations for the oxida-tive assimilation of the several organic substrates by the organism gave the re-sults shown in table 1. These values, in general agreement with those previouslyreported (Clifton, 1946), suggest the following equations for the oxidative as-similation reactions of succinate, fumarate, lactate, pyruvate, glycerol, andacetate, respectively:

C4H604 + 2.502 (CH20) + 3C02 + 2H20C4H404 + 202 - (CH20) + 3C02 + H20C3H603 + 202 (CH20) + 2C02 + 2H20C&H403 + 1.502 (CH20) + 2C02 + H20C3H803 + 2.502 (CH20) + 2C02 + 3H202C2H402 + 302 > (CH20) + 3C02 + 3H20

The carbon balances and manometric data attained in typical experiments ona basis of actual assimilation of substrate carbon by growing cells are presentedin table 2. Lactate was poorly utilized and acetate not at all during a 4.5-hourincubation period, although the inocula were from cultures in the synthetic me-dium containing the test substrate.The experimental data obtained with resting cells indicate that all the energy1 This paper is part of the thesis material submitted by the senior author in partial

fulfillment of the requirements for the Ph.D. degree.585

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available to the organism during assimilation is provided by the aerobic oxida-tion of the specific substrate, since the R.Q.'s rather closely correspond to thetheoretical values (table 1) based on the equations for oxidative assimilation.Greater discrepancies are noted, except with lactate, between the R.Q.'s ob-

TABLE 1Manometric observations on the oxidation of several substrates by washed suspensions of E.

coli at 30 C in m/15 phosphate buffer at pH 7.2

SUBSTRATE SACC PUMA- LACTATE PYRUVATE GLYCEROL ACETATZNATE RATE

mgC utilized....................... 0.883 0.680 0.672 0.726 0.803 0.702." 02 consumed.......... 1030 676 935 755 1465 1014A CO2 produced.......... 1152 974 960 950 1175 977% oxidized.......... 71.5 76.0 69.5 70.5 78.0 74.9R.Q. observed.......... 1.12 1.44 1.02 1.26 0.80 0.96R.Q. for complete combustion....... 1.14 1.33 1.00 1.20 0.86 1.00R.Q. for oxidative assimilation...... 1.20 1.50 1.00 1.33 0.80C assimilated/CO2-C* .......... 0.43 0.35 0.31 0.43 0.28 0.34

* Calculated by difference between mg C utilized and mg COr-C produced.

TABLE 2Comparative carbon balances in the oxidative assimilation of several substrates during the

growth of E. coli(Duration of experiment 4.5 hours)

SUBSTRATE, MG C SUCCI- PUMA- LACTATE PYRUVATE GLYCEROL ACETAT*NATE RATE

Initial substrate-C ......... ........ 5.51 3.14 3.31 4.45 5.40Cell-C after assimilation............ 0.55 0.44 0.37 0.51 0.72Cell-C before assimilation........... 0.35 0.21 0.29 0.20 0.51C stored.......................... 0.20 0.23 0.08 0.31 0.21Supernatant-C at end of experiment.. 5.04 2.57 3.19 3.76 5.15C02-C.......................... 0.29 0.30 0.12 0.39 0.10Total recovered..................... 5.53 3.10 3.40 4.46 5.46Total recovered,%.100.3 98.7 102.7 100.2 101.1

,l CO2 produced. 536 534 214 724 182Jsl 02 consumed.. 380 274 222 394 266R.Q. observed. 1.41 1.95 0.97 1.84 0.70

* Not utilized by organisms during the course of the experiment.

served with actively growing cells and the values postulated on the basis of ob-servations with washed cells. It has been the general observation that morereproducible results in regard to carbon balances are obtained with actively pro-liferating cells since the extent of assimilation is apparently not decreased bythe secretion of cellular material into the environment or by other secondarychanges. Furthermore the results obtained with cultures are based on experi-ments in which an excess of substrate was present rather than ones in which the

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19501 COMBUSTION OF CARBON COMPOUNDS 587

oxidation proceeded until the rate decreased to a level near that of the controlexperiment. A comparison of results obtained with washed cells and with cul-tures is presented in table 3. Such comparisons suggest that equations for oxida-tive assimilation are only approximations, the actual quantitative relationshipsthat exist being dependent to a great extent on the conditions under which as-

TABLE 3A comparison of the observed metabolic activities and extent of assimilation in suspensions and

cultures of E. coli

RATIO OF METABOLIC ACTIVITY ASSDIILATION (PER CENTPER MG C UTILIZEDCAS LTD

SU'BSTRATE (cultures/suspensions) CASMLTD

0s COt Washed cells Cultures

Succinate ........................... 0.70 0.87 30 41Fumarate.................. 0.50 0.65 25 43Lactate........................... 1.30 1.33 30 40Pyruvate ........................... 0.55 0.80 30 44Glycerol ........................... 0.58 0.49 20 67

TABLE 4Equations, heats of combustion, and free energy changes for the complete oxidation of several

substrates by E. coli

MEATS OF COMBUSTION FREE ENERGY CHANGEEQUATIONS -AH -AF*

CAL MOI.-l CAL MOLE71

C41104 + 3.502 4CO2 + 3H20 356,240t 383,235C4H404 + 302 4CO2 + 2H20 318,700t 349,302C3H60a + 302 3CO2 + 3H20 326,000t 339,710C3H403 + 2.502 - 3CO2 + 2H20 279,100§ 268,425C01.80 + 3.502 - 3CO2 + 4H20 395,63011 406,495C2H402 + 202 2CO2 + 2H20 208,000% 214,840

*All -AF values were calculated.t Huffman and Fox, 1938.t Kharasch, 1929.§ International Critical Tables, 1929.11 Parks, West, et al., 1946.% Parks and Huffman, 1932.

similation is studied. The results obtained can be evaluated, however, on thebasis of the thermodynamic reasoning employed in the preceding paper (Siegeland Clifton, 1950).

In table 4 are summarized the equations for the complete oxidation of thesubstrates under consideration as well as the pertinent data for the heats of com-bustion and free energy changes. As has already been pointed out, the maximumenergy available to the cell for useful work is measured most accurately by thefree energy change of the oxidative process. The AF values for oxidation were

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calculated from the data included in table 5 by subtracting the sum of the freeenergies of formation of the reactants from that of the products of the oxidation.With the knowledge (table 1) that the cell does not oxidize the substrate to

completion and on the assumption that the AF for the reaction of oxidativeasimilation may be attributable mainly to that part of the reaction that hasgone to carbon dioxide and water, it being further assumed that the hypothetical

TABLE 5Free energies and heat. offormation at £5 C

mUSTAN'Ic -AF -AHCALMSOUL1 CAL MOLrV

0(g, 0.2 atm.) 950CO,(g, 0.0003 atm.) 99,060 94,052*H20(1) 5,560 68, 317tC4H04(8) 179,360 22,5660tC0404(s) 157,230 194,880tCsHs0s(1) 124,300 161,700§C,H40,(s) 114,050 139,5001ICsHs,0(l) 113,060 159,800 SC,H402(1) 94,050 117,200**

g = gs, 1 - liquid, s = solid.* Prossen et al., 1944.t Wagman et al., 1945.$ Huffman and Fox, 1938.§ Calculated.JI Parks, Thomas, et al., 1936.% Parks, West, et al., 1946.* Parks and Huffman, 1932.

(CH20) assimilatory product is the same in each instance, we can write for theequations based on manometric data that

CJH604 + 2.502 -+ (CH20) + 3C02 + 2H20 ;AF = -274,013 ca(l)C11404 + 202 (CH20) + 3C02 + H20 ;AF = -265,470 (2)CsH608 + 202 (CH20) + 2C02 + 2H20 ;AF = -236,098 (3)C0H408 + 1.502-4 (CH20) + 2C02 + H20 ;AF = -189,240 (4)C&H80s + 02 (CH20) + C02 + H20 ;AF = -317,066 (5)C2H402 + 1.502 0.5(CH20) + 1.5C02 + 1.5H20;AF = -160,915 (6)

The AF values listed above were calculated on the basis of the percentage ofoxygen consumed (see table 1) rather than on the percentage postulated by theforegoing equations. Since the amounts of oxygen consumed are less and of car-bon assimilated greater with growing than with washed cells, the AF valuesgiven above probably axe fair approximations and would represent maximumamounts of free energy that could be available.Although it is becoming increasingly apparent that the major part of available

oxidation-reduction energy is converted by the cell into phosphate bond energy,evidence for such thinling has derived mainly from the degradation reactions of

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the longer carbon chain compounds, particularly hexoses. Thus, in the case ofthe substrates studied here, insuficient information is at hand to fully discussenergy utilization from the standpoint of the formation of energy-rich phosphatebonds. In this- connection it might be mentioned, however, that Lipmann andTuttle (1944) and Utter, Krampitz, and Werkman (1944), using Lactobacillusdelbrueckii, Micrococcus lysodeikticus, and other cells, have presented evidencethat indicates that acetyl phosphate is an intermediate in the oxidation of pyru-vate to acetate and carbon dioxide, and that the reaction is coupled with thephosphorylation of adenylic acid to form adenosine triphosphate. Lipmannpictures the process as follows:

CH3COCOOH + H3P04 2H CH3COOPO3H2 + C02 (7)Pyruvic acid Acetyl phosphate

2CH3COPOPH2 + Adenylic acid -* 2CHsCOOH + ATP (8)The fate of acetate in the oxidative scheme is discussed in the light of recentfindings (Ajl, 1950) and the energetics of the mechanism are fully analyzed else-where (Siegel, 1950).

Kalckar's demonstrations (1939) with kidney extracts that oxidation of di-carboxylic acids such as succinic and fumaric induces phosphorylation of varioussubstrates (such as adenylic acid and carbohydrates) is inapplicable here, sincein the experiments described here these compounds were employed singly.However, the implications for possible phosphorylation of intermediates maybe readily inferred. Kalckar (1939), for example, found phosphopyruvate as aproduct of fumarate oxidation. Hastings et al. (1940) have also reported support-ing evidence for the indirect formation of phosphopyruvate in the liver of rats.'They were able to demonstrate that liver glycogen formed from lactic acid con-tainling radioactive carboxyl carbon contained little or no radioactive carbon;and they postulated that the lactate molecule on the way back to carbohydrateloses its carboxylic group. This, however, is to be expected when pyruvic acidformation is only possible by way of dicarboxylic acid oxidation. In this reac-tion, according to a scheme described by Lipmann (1941), two moles of lactateare used for the formation of one mole of phosphorylated pyruvate that thenreturns eventually to carbohydrate. This might be a factor in accounting for thesmall amount of assimilation accompanying lactate oxidation (table 2), althoughgrowth was very limited during the experimental period.From the values of their respective efficiency coefficients (table 6) it can be

deduced that, of the dibasic organic compounds studied, fumarate is utilizedwith lesser loss of energy, and that in the case of the monobasic compoundspyruvate is utilized with the lesser loss of energy by the cells. Thus, despite thegreater free energy of oxidation of succinate over fumarate and of lactate overpyruvate (see equations 1 to 4), the amount of assimilation by the growing cellis about the same while the rate is greater with each of the second members ofthese pairs of compounds as substrates (table 2).

Although glycerol is utilized during growth with the greatest efficiency (table6), the rate of assimilation is not so great as with succinate, fumarate, or pyru-

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vate (table 2). It should also be noted that the free energy change obtaining inthe oxidative assimilation of glycerol is greater than with any of the afore-men-tioned substrates.As was observed in the case of the carbohydrates, the values for the R.Q. and

for the ratio C assimilated to C02-C run lower generally for resting cells thanfor growing cells (cf. tables 1 and 6). Actually more carbon is assimilated and lessgiven off as carbon dioxide per unit of substrate carbon utilized by proliferatingcells as compared with washed suspensions. It might be of collative significance,here, to note that with resting cells this ratio is lowest for glycerol as substrate,but with growing cells the ratio is greatest.The significance of the somewhat higher than theoretical R.Q.'s observed in

table 2 has been discussed in the preceding paper. It was suggested there thatthe primary assimilation product or products may be more reduced than thoseimplied by the formula (CH20) representing a compound with the empiricalcomposition of carbohydrate.

TABLE 6

Efficiencies of synthesis by E. coli with respect to several substrates

INCREASE IN Stored cell-C Stored cell-CSUBSTRATE Cma-C, Mg mg-"-Mg cel-C Substrate-Cconsumed

Succinate ........................... 0.20 0.29 0.69 0.41Fumarate ............................ 0 . 23 0.30 0.77 0.43Lactate........................... 0.08 0.12 0.67 0.40Pyruvate ........................... 0.31 0.39 0.79 0.44Glycerol ........................... 0.21 0.10 2.10 0. 70

A closer perusal of table 2 reveals that, of all the substrates investigated withgrowing cells, glycerol is assimilated to the greatest extent, whereas acetate isnot utilized in the experimental period. Pyruvate, however, was utilized mostrapidly. The R.Q. for the oxidation of pyruvate is observed to be approximately2, which might suggest that the reaction that leads to the synthesis of acetylphosphate by the proliferating cells may be an oxidative decarboxylation coupledwith a phosphorylation as depicted in the equation:

CHCOCOOH + HsPO4 + 102 --+CHsCOOPO3H2 + CO2 + H20 (9)Banga, Ochoa, and Peters (1939), studying the relation of pyruvate oxidation

to phosphate concentration in brain dispersions, reported greater oxygen con-sumption with increasing concentration of phosphate. Ochoa (1941) furtherelucidated the problem of phosphate intervention by demonstrating the role ofadenylic acid in pyruvate oxidation. He found greatly increased total oxygenconsumption and --ph generation per mole of pyruvate upon the addition ofadenylic acid to dialyzed preparations. Experiments later performed by Long(1943) demonstrated that adenylic acid is chiefly or exclusively concerned with

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the postacetyl stage; it is not required by pyruvate either for aerobic or anaerobicbreakdown. But absence of adenylic acid must bring about a cessation of -phtransfer; and it seems likely then that adenylic acid enhances the completenessof oxidation by preventing a stagnation through removal of initially formedphosphorylated intermediates (Lipmann, 1946; Warburg and Christian, 1939).

It may be surmised then that the coupling of the immediate process of pyruvicdecarboxylation with phosphorylation leads to the formation of an active C2compound and the generation of an abundance of -ph's. This compound mayeither degenerate to acetate or advance immediately to the next step, which isprobably a condensation with a C4 dicarboxylic acid (Barron, 1949). Ochoa's(1943) observations of 10 to 15 phosphorylations per mole of pyruvate furthersuggest a multiplicity of phosphorylated intermediates.An interesting paradox observed here is that though suspensions of resting

cells are seemingly able to assimilate acetate readily, cells in the cultures areunable to do so at an appreciable rate although acetate alone will support somegrowth on longer incubation. Free acetate, it is apparent, is a substrate whoseprimary breakdown products might not provide an abundance of appropriateintermediate building blocks for synthesis. The oxidation to completion of someof the acetate molecules might, however, provide energy for the assimilation ofother acetate molecules, assuming these to be appropriate building blocksadaptable to cell assimilation. The solution to this paradox may be in the con-jecture that in the initial small inocula employed in the growth experimentsinsufficient enzyme is present to catalyze the breakdown of acetate: fairly rapidgrowth does not take place, and no further enzyme is synthesized at an appre-ciable rate so that acetate remains unrespired. In the highly populated suspen-sions, on the other hand, there may be adequate enzyme available to induce res-piration and subsequent assimilation. Apropos of this manifestation is the factthat, generally in the case of the shorter chain carbon substrates, initial inoculaof growing cells three to five times as great as those used in the sugar substratesare required to bring about significant respiratory and assimilatory responses.Also, Long and Peters (1939) have noted that despite its appearance undervarious conditions as the primary product of pyruvate breakdown, free acetateseems to be what Lipmann (1946) has described as a degenerated intermediary.Apparently it cannot be respired by brain under conditions in which pyruvateeasily passes beyond the acetate stage.

Finally, comparisons of conversion efficiencies (cell-C/CO2-C) with those ofthe sugars, 1.36, 1.20, and 1.79 for glucose, lactose, and arabinose, respectively,reveal that the efficiency of utilization is less with the substrates of fewer carbonatoms. This is probably due in large measure to the circumstance that some ofthe molecules have to be oxidized completely to provide energy for the assimila-tion of others, or the intermediate building blocks obtaining from them. In thecase of longer carbon chains, particularly hexoses and at least one pentose(arabinose), the energy-evolving oxidative processes actually generate the inter-mediate blocks to be subsequently assimilated.

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SUMMARY

Further evidence has been adduced, employing a number of organic sub-strates, which corroborates the concept first enunciated by Kluyver (1930, 1931)that the molecular structure of the substrate and of the intermediate productsformed during its breakdown is of greater significance than the energy evolvedin predicting its potentialities as the starting point for the synthesis of cell mate-rials. This also bears out the conclusions formulated by Clifton from extensivemanometric studies that the energy resident in a substrate molecule would notprovide by itself a propitious criterion for predicting the extent of its assimila-bility by the bacterial cell. The efficiency of assimilation of different substratesby the growing cell is seen to be generally independent of the free energy ofoxidation of the substrate since a greater degree of assimilation is noted withfumarate than with succinate, with pyruvate than with lactate, or with thelatter compounds than the former on the basis of each compound.An interesting paradox has been observed in the utilization of acetate by grow-

ing and by resting cells. This discrepancy is described and a surmise suggestedby way of explanation.

REFERENCESAJL, S. J. 1950 Acetic acid oxidation by Escherichia coli and Aerobacter aerogenes. J.

Bact., 59, 449-507.BANGA, I., OCHOA, S., AND PETERS, R. A. 1939 Pyruvate oxidation in brain. VII. Some

dialysable components of the pyruvate oxidation system. Biochem. J., 33, 1980-1996.BARRON, E. S. G. 1949 Cellular metabolism. In The chemistry and physiology of growth.

Princeton Univ. Press, Princeton, N. J.CLIFTON, C. E. 1946 Microbial assimilations. Advances in Enzymol., 6, 269-08.HASTINGS, A. B., KISTIAKOWsKY, G. B., CRAMER, R. D., KLEMPERER, F. W., SoLOMoN,

A. K., AND VENNESLAND, B. 1940 Liver glycogen from lactic acid containing radio-active carboxyl carbon. Science, 91, 421.

HUFFMAN, H. M., AND Fox, S. W. 1938 Thermal data. X. The heats of combustionand free energies, at 250, of some organic compounds concerned in carbohydrate me-tabolism. J. Am. Chem. Soc., 60, 1400-1403.

International critical tables. 1929 McGraw-Hill, New York. Refer to V, 166.KALcxAR, H. 1939 The nature of phosphoric esters formed in kidney extracts. Biochem.

J., 33, 631-641.KHARscH, M. S. 1929 Heats of combustion of organic compounds. J. Research Natl.

Bur. Standards, 2, 359-430.KLUYVER, A. J. 1930 Atmung, Garung und Synthese in ihre gegenseitigen Abhangigkeit.

Arch. Mikrobiol., 1, 181-196.KLUYVER, A. J. 1931 The chemical activities of microorganisms. Univ. London Press,

London.LEWIs, G. N., AND RANDALL, M. 1923 Thermodynamics and the free energy of chemical

substances. McGraw-Hill, New York and London.LIPMANN, F. 1941 Metabolic generation and utilization of phosphate bond energy. Ad-

vances in Enzymol., 1, 231-267.LIPM.ANN, F. 1946 Acetyl phosphate. Advances in Enzymol., 6, 231-267.LIPMANN, F., AND TuTTLE, L. C. 1944 Acetyl phosphate: chemistry, determination, and

synthesis. J. Biol. Chem., 153, 571-582.LONG, C. 1943 The in vitro oxidation of pyruvic and a-ketobutyric acids by ground prep.

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arations of pigeon brain. The effect of inorganic phosphate and adenine nucleotide.Biochem. J., 37, 215-225.

LONG, C., AND PETERS, R. A. 1939 Pyruvate oxidation in brain. V. Evidence derivedfrom the metabolism of a-ketobutyric acid. Biochem. J., 33, 759-773.

OCHOA, S. 1941 "Coupling" of phosphorylation with oxidation of pyruvic acid in brain.J. Biol. Chem., 138, 751-773.

OCHOA, S. 1943 Efficiency of aerobic phosphorylation in cell-free heart extracts. J.Biol. Chem., 151, 493-505.

PARKS, G. S., AND HUFFMAN, H. M. 1932 The free energies of some organic compounds.Am. Chem. Soc., Monograph No. 60.

PARKS, G. S., THOMAS, S. B., AND LIGHT, D. W. 1936 Studies on glass. XII. Some newheat capacity data for organic glasses. The entropy and free energy of dl-lactic acid.J. Chem. Phys., 4, 64-69.

PARKS, G. S., WEST, T. J., NAYLOR, B. F., FuJiI, P. S., AND MICCLAINB, L. A. 1946 Ther-mal data on organic compounds. XXIII. Modern combustion data for fourteen hy-drocarbons and five polyhydroxy alcohols. J. Am. Chem. Soc., 68, 2524-2527.

PROSSEN, E. J., JESSUP, R. S., AND RoSSINI, F. D. 1944 Heat of formation of carbondioxide and of the transition of graphite into diamond. J. Research Natl. Bur. Stand-ards, 33, 447-449.

SIEGEL, B. V. 1950 Thermodynamic aspects of a proposed mechanism for acetate oxida-tion in bacteria. Proc. Natl. Acad. Sci. U. S. In press.

SIEGEL, B. V., AND CLIFTON, C. E. 1950a Oxidative assimilation of glucose by Escherichiacoli. J. Bact., 60, 113-118.

SIEGEL, B. V., AND CLIFTON, C. E. 1950b Energy relationships in carbohydrate as-similation by Escherichia coli. J. Bact., 60, 573-583.

UTTER, M. F., KRAMPITZ, L. O., AND WERKMAN, C. H. 1944 Oxidation of acetyl phos-phate by Micrococcus lysodeikticus. J. Bact., 47, 412.

WAGMAN, D. D., KILPATRICK, J. E., AND TAYLOR, W. J. 1945 Heats, free energies, andequilibrium constants of some reactions involving 02, H20, C, CO, C02, and CH4.J. Research Natl. Bur. Standards, 34, 143-161.

WARBURG, O., AND CHRISTIAN, W. 1939 Isolierung und Kristallisation des Proteins desoxydierenden Garungsferments. Biochem. Z., 303, 40-68.

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