ENERGY RELATIONSHIPS IN CARBOHYDRATE ASSIMILATIONBY ESCHERICHIA COLI1

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

Medicine, Stanford, CaliforniaReceived for publication July 29, 1950

From studies employing the techniques of manometry, Clifton (1950) hasfurther developed the concept first enunciated by Kluyver (1930, 1931) that theenergy resident in a substrate molecule would not provide a propitious criterionfor predicting the extent of its assimilability by the bacterial cell. He noted, forexample, the same degree of assimilation with the monobasic organic compounds,lactate and pyruvate, despite the greater free energy of oxidation of the former;likewise with the dibasic compounds succinate and fumarate, oxidation was ac-companied by the same amount of assimilation, although the reaction in thefirst instance is attended by a greater free energy change (Clifton and Logan,1939).Thermodynamically it could be anticipated that, in a set of reactions involv-

ing the liberation of energy, the organism might profit more from those processesyielding greater portions of energy. Clifton, however, as did Kluyver, reasonedthat intermediates of these reactions, in serving as building blocks for the nu-merous syntheses carried on by the cell, would assume a significance as greatas, if not greater than the extent of energy evolved in oxidation. Along withKluyver's concept of the thermodynamically improbable state of the bacteriuim(1931), Clifton (1950) and others (van Niel, 1949) have further formulated theconcept of the cell as a transformer and converter of substrate substance ratherthan just as an engine transforming energy and subserving the themes of ther-modynamics.

In this paper is described a series of experiments designed to test the validityof the Kluyver concept as extended by Clifton.

EXPERIMENTAL PROCEDURES

Escherichia coli (K-12), an organism capable of utilizing any one of a numberof carbon substrates as the sole source of carbon and energy, was employedthroughout the experiments. Experiments with washed cells were first performedin order to obtain or to verify equations for the oxidative assimilation of thesugar substrates by the organism. In these experiments the bacterial cells weregrown on nutrient agar in Kolle flasks at 35 C for 20 hours. The cells were har-vested in M/15 phosphate buffer (pH 7.2) and filtered through a glass wool padto remove agar particles. Following centrifugation the washed cells were resus-pended in pH 7.2 phosphate buffer to give a concentration of approximately

I This paper is part of the thesis material submitted by the senior author in partialfulfillment of the requirements for the Ph.D. degree.

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1.5 X 1010 celLs per ml. Manometric experiments were carried out at 30 C aspreviously described (Siegel and Clifton, 1950).

In growth studies in synthetic media the role of possible adaptation was ruledout by prior cultivation of the organisms in the presence of the specific sugarto be studied. Solutions of D-glucose, D-lactose, or L-arabinose were made up indistilled water and later added to an equal volume of the basic salt solutionemployed in the previous study (Siegel and Clifton, 1950).

In preliminary growth asimilation studies 0.1 ml of a 20-hour culture in nu-trient broth incubated at 35 C was added to 30 ml of a M/20 sugar basic saltmedium in a 200-ml Erlenmeyer flask. The inoculated flasks were then shakenvigorously in a mechanical shaker for 24 hours at a constant air temperature of30 C. At 0, 6, and 24 hours, 1-ml samples of the suspension were removed andthe amounts of bacterial growth measured turbidimetrically with the Klett-Summerson photoelectric colorimeter; the increase in bacterial carbon was de-termined by the Van Slyke-Folch method (1940). Turbidity readings were con-verted into actual bacterial numbers with the aid of a standard curve previouslyprepared.

Clifton and Logan (1939) and Siegel and Clifton (1950) have shown thatoxidative synthesis occurs in similar measure in both suspensions and culturesof E. coli. Clifton (1937) has also obtained good carbon recoveries in aimila-tion studies using washed celLs with glucose and glycerol as substrates. Thisstudy, however, primarily deals with assimilation by young, growing cells, sincepreliminary trials indicated that more reproducible carbon balances were ob-tainable with them than with washed suspensions in buffer. It is possible thatwith large numbers of resting cells the element of diffusion of excess synthesizedmaterial out of the cell into the medium may account for the at times incon-sistent results.The cultures were incubated at 35 C in the single sugar basic salt medium,

the sugar being the one specifically under consideration. After 16 hours underthese conditions the cultures were diluted 1:10 in the synthetic medium. Man-ometric experiments and carbon determinations were then carried out as pre-viously described.

EXPERIMENTAL RESULTS

The experiments performed with washed cells to obtain equations for theoxidative assimilation of the sugar substrates by the organism gave the resultsshown in table 1. These values, in accordance with the reasoning of Barker(1936), Giesberger (1936), and Clifton (1937, 1946), lead to the following equa-tions for the oxidative assimilation of the three sugars:

C6H1oO5 + 302 - 2(CH20) + 3C02 + 3H20C6H1206 + 302 3(CH20) + 3C02 + 3H20C12H2011 + 602 6(CH20) + 6C02 + 5H20

The influence of the composition of the culture medium on the enzymaticactivity of microbial cultures has been analyzed by numerous investigators (Gale,

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1943), and it has become increasingly evident that the production of a givenenzyme may be greatly stimulated when the substrate that it attacks is a con-stituent of the culture medium. Before study of the quantitative aspects of as-similation, a comparison of the relative rate and extent of growth in the pres-ence of the various sugars was undertaken, with the results shown in table 2.The results recorded in table 2 indicate that growth is most rapid at first in

glucose and least rapid in arabinose. Growth increases in the latter medium aswell as in lactose until at the end of 24 hours these cultures show a greater totalincrement than in glucose, the bacteria in the arabinose medium in particularshowing a marked increase in number. The total increase in carbon content was

TABLE 1Manometric observations on the oxidation of arabinose, glucose, and lactose by washed

suspensions of E. coli at 30 C in m/15 phosphate buffer at pH 7.2

ARBINOSE CLUCOSE LACTOSE

mg carbon utilized....................... 0.553 0.660 0.718Jl 02 consumed.......................... 612 685 710p1 C02 produced......................... 636 739 756% oxidized.............................. 60 55 53R.Q.............................. 1.04 1.08 1.06C assimilated/WrC ............ ........ 0.63 0.67 0.77

TABLE 2Growth of E. coli in sugar basic salt media at 30 C

(Bacterial numbers are given in terms of 107 and bacterial carbon in milligrams per ml)

0 HOURS 6 HOURS 24 HoURsSUBSTRATE

Bact. Bact.-C Bact. Bact.-C Bact. Bact.-C

Arabinose ....................... 2.6 0.0126 8.2 0.039 346 0.379Glucose ........................ 2.6 0.0126 19.0 0.050 250 0.332Lactose ....................... 2.6 0.0126 14.0 0.046 296 0.365

also greatest in the arabinose medium. This observation of delayed growth is inline with previous experiments (Monod, 1942, 1947) that have demonstratedthat the enzymes involved in glucose utilization are constitutive whereas thoseconcerned with lactose and arabinose may be more dependent on the presenceof the specific sugar. It also suggested that, if further experiments were to beconducted in which time was a factor, the organisms would have to be grownin the presence of the specific substrate prior to the test runs.Although the incomplete oxidation of substrate and the inhibitory action of

poisons on assimilation (Clifton, 1937) as indicated in manometric studies lendcredence to the concept of oxidative assimilation, they do not wholly confirmit. The experimental demonstration of actual assimilation by the bacterial cellwould provide further support for this postulate. Winzler (1940) demonstratedan increase in the reducing sugar content of yeast during the course of the oxi-

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dative assimilation of acetate; van Niel and Anderson (1941) noted that theproduction of equimolar quantities of carbon dioxide and ethanol in the fermen-tation of glucose by yeast accounted for only 70Gper cent of the depleted sugar;Pickett and Clifton (1943) and Swanson and Clifton (1948) have succeeded inestablishing carbon balances for a pure strain of Saccharomyce8 cereviae in aglucose medium, and Siegel and Clifton (1950) have obtained good carbon re-coveries in a glucose milieu with suspensions or cultures of E. coli. The carbonbalances attained on the basis of actual assimilation of substrate carbon by

TABLE 3

Comparative carbon balances for the oxidative assimilation of three sugars during the growthof E. coli in synthetic media at 30 C

(Carbon recoveries are expressed as percentage of carbon added as sugar; duration ofexperiment 4.5 to 5.0 hours)

GLUCO(4. IffOM r &cTosz (4.5 ARABINOSE (5.0GLUCOSE (4.5 ous) HOUS) HOU(S)OSUBSTRATE _____ts

mg % mg % mg %

Initial substrate carbon.5.29 2.36 3.54Cell carbon after assimilation of sugar... 0.43 0.20 0.39Cell carbon before assimilation ofsugar.0.09 0.08 0.05

Carbon stored.0.34 6.4 0.12 5.1 0.34 9.6Supernatant carbon at end of experi-ment.4.72 89.0 2.14 90.7 3.01 85.0

Carbon dioxide carbon ................. 0.25 4.7 0.10 4.2 0.19 5.4Total recovered.5.31 100.1 2.36 100.0 3.54 100.0

R.Q.1.10 1.10 1.18Cell-C/COrC.1.36 1.20 1.79

growing cells with glucose, latose, or arabinose as the substrate are presentedin table 3.

DISCUSSION

Under the experimental conditions attending the assimilation and growth ofE. coli in this investigation, all the energy available to the organism must bemade available by the aerobic oxidation of the specific substrate, since the R.Q.'sare unity (table 1) and the carbon recovery near 100 per cent (table 3). Theorganic foodstuff not only serves as oxidizable material for provision of the en-ergy required for the endergonic synthetic reactions but is also an ultimate sourceof structural material for the synthetic activities of the cell. The thermodynamicnotation of Lewis and Randall (1923) is used throughout the discussion to fol-low of the relative roles of each of these functions in cellular activity.Although the energy released in the complete oxidation of the substrates un-

der consideration may be expressed by the equations:

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C&H1006 + 502 5C02 + 5H20;AH -559,000 cal (1)CeH1206 + 602 6CO2 + 6H20;AH -673,000 cal (2)CnH22011 + 1202 12CO2 + 11H20;AH -1,344,000 cal (3)

where AH is the heat of combustion, the maximum energy available to the cellfor useful work can be described accurately only in terms of the free energy ofthe oxidative process. At constant temperature and pressure the two quantitiesAF and AH are related to one another by the fundamental thermodynamic equa-tion

AF = AH- TAS (4)

where T is the absolute temperature and AS is the entropy of the system underconsideration. AH and AF are negative when heat and free energy, respectively,are liberated.The standard free energies of formation of CO2 and H20 are -99,060 and

-56,560, the respective heats being -94,052 and -68,317 cal. Here the C02is considered to be at 0.0003 atmosphere and 02 at 0.2 atmosphere pressure.These values and those of the other compounds concerned are listed in table 4.The entropy of formation from the elements of each compound was calculatedby the use of the values 1.36 (Jacobs and Parks, 1934), 31.23 (Giauque, 1930),and 49.03 (Giauque and Johnson, 1929) entropy units (cal deg.-') per mole ofcarbon, hydrogen, and oxygen, respectively. The free energy of formation ofarabinose was calculated from the heat of combustion (International CriticalTables, 1929) and the entropy value from the difference between its S298 and thecorresponding values for the entropies of the elements contained therein (Parksand Huffman, 1932). The free energy of formation of lactose was derived fromthe latest experimental findings of Anderson and Stegeman (1941). Until fairlyrecently the free energy of lactose was determined from the heat of combustion(International Critical Tables, 1929) and the entropy of glucose (Parks et al.,1929), the entropy of the disaccharide hexose unit and of glucose being assumedto be almost identical (Burk, 1929). The entropy of formation derived on thisassumption is -528 e.u., and with a listed value of -535,000 cal for AH givesa AF of formation of -377,926. This value was rejected in favor of the higherexperimentally determined value (see table 4).

Since we are dealing here with several different reactions that involve essen-tially the same components, it was-considered expedient to employ an alternatemethod to those generally employed for computing free energy changes. The freeenergies of formation of the several components were evaluated as functions oftemperature with the aid of equation (4), AF and AH now representing the freeenergy and heat of formation, respectively, and AS the entropy of formation.For very small ranges of temperature the difference in heat capacities betweenproducts and reactants can be considered equal to zero, and AH and AS thenbecome independent of temperature. As a consequence the equation for the freeenergy change reduces to AF = AH - TAS. Thus, although the heat of reactionand entropy change are constant, the free energy change varies with the tem-

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perature. The free energy change of the reaction could then be calculated bycombining the standard free energies of formation of the several components.The free energy changes for the oxidation of the several substrates can be cal-

culated from the data included in table 4 by subtracting the sum of the freeenergies of formation (A&Ff) of the reactants from that of the products of theoxidation. A sample calculation, dealing with the oxidation of glucose, may serveto make this clear.

CsHisO6(s) + 602(0.2 atm) -- 6CO2(0.0003 atm) + 6H20(l) (1)AFf = -218,720 -5700 -594,360 -339,360

AFf products = -933,720 calAFf reactants = -224,420

therefore, AF oxidation = -709,300

TABLE 4Free energies and heats offormation at 25 C

SUBSTANCE-LH

CAL MOLE-1 CA&L XOLV-1

02(g, 0.2 atm.) 950 0C02(g, 0.0003 atm.) 99,060 94,052*H20(l) 56,560 68,317tC,H1o00a(s) 180,533 252,000tC0.H12°6(8) 218,720 305,730§CisHnsOn1(s) 418,200 592,900

g = gas, 1 = liquid, s = solid.* Prossen et al., 1944.t Wagman et al., 1945.t Calculated.§ Huffman and Fox, 1938.|| Anderson and Stegeman, 1941.

The values for AF of oxidation as calculated according to the above schemeare -592,817 for arabinose, -709,300 for glucose, and -1,381,280 cal forlactose.The free energy of combustion of a sugar by the bacterial cell (at 30 C) will

not be exactly as given above, although of a quite similar order of magnitude,since the values generally employed in the computations for both reactants andproducts are referrable to the standard state at 25 C. However, tlke free energycorrections for concentration and for a temperature range of 5 C are not muchgreater than the uncertainty of some of the experimentally determined valuesand, moreover, amount to but a small fraction of the total free energy change.Hence, for purposes of this discussion, corrections for concentration and tem-perature will not be considered.

In the manometric observations on the cellular oxidation of the sugars, it wasnoted (see table 1) that the oxidations did not go to completion. The free energyof any one reaction would then be some fraction of that obtaining in the total

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oxidation. Exactly what this fraction would be cannot be determined since theamounts and natures of the intermediates formed in the reaction are not known.As a first approximation, the AF for the reaction of oxidative assimilation maybe ascribed only to that part of the reaction which has gone to carbon dioxideand water, it being assumed that the AF of the hypothetical (CH20) assimilatoryproduct is the same in all cases. Although this approximation is admittedlyarbitrary, its use for comparative purposes may not prove too inept. On thisassumption we can write for the equations based on manometric data that

C5H1005 + 302 2(CH20) + 3C02 + 3H20;AF = -355,690 (5)C6H1206 + 302 > 3(CH20) + 3C02 + 3H20;AF = -354,650 (6)C12H22011 + 602 ) 6(CH20) + 6C02 + 5H20;AF = -690,640 (7)

It seems most likely that the energy set free during the oxidation of the sub-strate is probably released in parcels of a definite size, a step at a time, with thebulk of the energy being released by the interaction of the oxidative systems(Ball, 1944). Whether the energy released at each one of these steps is capableof being harnessed for work by the cell or whether the cell can further subdividethese parcels of energy for its own use is unknown. During the course of some ofthese steps energy may appear in a low-grade form (heat) incapable of accom-plishing much work, or may possibly be wasted in its entirety. Most of theinformation we possess concerning energy utilization revolves around the highenergy phosphate bond (--ph) formed in glycolytic or oxidative foodstuff dis-integration reactions. And it is becoming increasingly evident that in all cellsthere exists a propensity to convert the major part of available oxidation-reduc-tion energy into phosphate bond energy (Lipmann, 1941).Ochoa (1943) found that during the oxidation of carbohydrate by muscle as

many as 6 --,ph could be formed for each molecule of oxygen consumed. Accept-ing 12,000 calories as the energy necessary to form such a bond (Lipmann, 1941)and the possibility, on the basis of the number of moles of oxygen actually con-sumed, that during oxidative assimilation there would be formed 18 --ph forarabinose or glucose and 36 for lactose, this total phosphate bond energy in theoxidation of the substrates would then amount to 216,000 cal for arabinose orglucose and 432,000 for lactose.Thus the energy stored in high energy phosphate bonds as a result of the lac-

tose oxidation would presumably be twice as great as that of either glucose orarabinose, the oxidation of the latter two resulting in -ph bond formation ofequal extent per mole of substrate oxidatively assimilated. From considerationsof the possible contribution of phosphate bond energy to the endergonic reac-tions of the cell it could be expected that the extent of synthesis would be greatestin cells active in a lactose medium and equal in either a glucose or arabinosemilieu. The same conclusion would be reached from a consideration of the freeenergy changes in the reactions of oxidative synthesis (equations 5 to 7), theAF's being almost equal for glucose and arabinose and greatest for lactose. Yetas shown in table 3, the proportional increase in stored carbon with growth isgreatest with arabinose as substrate, less with glucose, and least with lactose; re-

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sults that would certainly not be anticipated from the standpoint of the ener-getics of the systems concerned.The process of assimilation is one in which certain calculations may be made

concerning efficiencies of energy utilization. Baas-Becking and Parks (1927) con-sidered a process in terms of its free energy efficiency based on the ratio of thefree energy increase of the products to the free energy decrease in the reaction.Although this quotient is representative of the over-all efficiency of a process, itis not amenable to application with the experimental data at hand. An efficiency

increase in cellular carboncoefficient modified from Foster (1949) and defined as substrate carbon consumedgives the values presented in table 5. Arabinose, on the basis of the economiccoefficient of synthesis, is most efficiently utilized by E. coli and lactose the least.As for energy involved in maintenance, there is little evidence as to its magni-

tude or the reactions involved (Goddard, 1948). Since only young growing cul-tures are dealt with (expt. C), little concern need be placed on energy of mainte-

TABLE 5Efficency of synthesis during growth of E. coli in relation to various carbohydrate substrates

(Data from table 3)

ECONOIC CORmICaNT,lSU87RATZ SUBSTRATE CARBON INCREASE IN CELL OR

CONSUMD CARBON Incree iln cellular-CSubstrate-C consumed

mg mgArabinose...................... 0.53 0.34 0.64Glucose...................... 0.57 0.34 0.59Lactose...................... 0.22 0.12 0.55

nance. As Tamiya and Yamagutchi (1933) have shown, most of the energy inyoung growing cultures is devoted to synthesis. Also, according to Terroine(1922) and Terroine and Wurmser (1922), the true efficiency coefficient in sucha situation is independent of maintenance energy.In the situation where the energy derived from the oxidative process accumu-

lates in the energy-rich phosphate bond, the organisms would be able to utilizeall three substrates about equally effectively. This is borne out in table 6. Thecoefficients here may not be of too great significance, because they are predi-cated upon approximations and upon manometric data obtained with restingcells. However, they are included for what light they may throw on the possibleefficiency of energy transfer by high energy phosphate bond formation (Lip-mann, 1941; Kalckar, 1943) and to indicate that calculated --,ph efficiencycoefficients alone are not reliable guides for the prediction of the extent of as-similation.Tamiya (1932) formulated a synthesis quotient as a measure of conversion

substrate carbon stored as cell materialsubstrate carbon oxidized to carbon dioxide

This value is an expression of the conversion of the carbon source into cell sub-

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stance and carbon dioxide exclusively. Conversion coefficients, as calculatedfrom data from growth experiments recorded in table 3, are 1.79 for arabinose,1.36 for glucose, and 1.20 for lactose, as compared with 0.63, 0.67, and 0.77with resting cells (table 1). Comparison of these values suggests that duringactual growth of the cells some sugar may be assimilated as such or may beassimilated after undergoing chemical change without loss of carbon as carbondioxide. They also suggest that the intermediates obtaining in arabinose oxida-tion are assimilable with the least expenditure of energy, whereas those fromlactose are assimilated with the greatest loss of energy on the part of the cell.When AF's for complete oxidation are compared on a CH20 basis rather than

on molar quantities, the values are 118,000, 118,000, and 115,000 cal for arabi-nose, glucose, and lactose, respectively. Since three-fifths of the former sugarand one-half of the latter sugars are combusted to carbon dioxide and water byresting cells, 70,800, 59,000, and 57,500 cal would be the AF values for the por-tions oxidized to completion. In the growth experiments, ratios of C assimilated

TABLE 6Efficiency in oxidative assimilation as a function of high energy phosphate bond formation

SUBSTRTE ENERGY AS AF OXIDATIVE -,ph EFFICIENyASSIMILATION COEFFICIIENT*

cal cal

Arabinose ...................... 216,000 355,000 0.61Glucose........... ...... 216,000 354,000 0.61Lactose ....................... 432,000 690,000 0.63

* Energy going into ph bond formationFree energy of oxidative assimilation by resting cells'

to C02-C were 1.79, 1.36, and 1.20 for arabinose, glucose, and lactose, respec-tively. On the basis of these ratios it can be calculated that the AF's duringgrowth were 42,300 cal for arabinose, 50,000 for glucose, and 52,300 for lactose,with maximum AF values by difference of 75,700, 68,000, and 62,700 cal for theasimilation products from the three sugars. It is quite apparent that of the threecarbohydrates under consideration the organism is generally able to utilizearabinose most efficiently, glucose somewhat less, and lactose least for assimila-tory purposes during growth, and that consideration of energetics alone doesnot provide adequate criteria for theoretical considerations of assimilation.

ACKNOWLEDGMENT

The authors are indebted to Professor George S. Parks, who has called atten-tion to several of the important references listed in this paper.

SUMMARY

Evidence has been presented which indicates that energy production and sub-strate assimilation are not concomitant criteria in predicting the developmentof the cell; lactose with a molar AF for the reaction of oxidative assimilation

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almost twice those of arabinose and glucose, or with a AF per CH20 unit almostequal to those for the other sugars, being assimilated to a lesser extent duringgrowth than the latter two sugars, of which arabinose is more readily assimilated.This is attributed to the production of intermediates during the degradation ofarabinose, which are more readily fitted into the patterns and arrangements ofprotoplasmic structure than are those produced from glucose or lactose.

Thus, though a smaller yield of energy is derived from respiration duringgrowth in arabinose, it is either more effective for synthesis than that stemmingfrom lactose from which the energy produced by the respiring cells is twice asgreat per mole of sugar utilized, or arabinose or its dissimilation products areasimilated more readily.The Kluyver concept as extended by Clifton and others thus seems to be ex-

perimentally established for at least the three carbohydrates covered in thisinvestigation. From other studies in progress it appears to be applicable toassimilation of organic substrates generally.

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Sci., 45, 363-375.BARKER, H. A. 1936 The oxidative metabolism of the colorless alga, Prototheca zopfii.

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