43

THE DEVELOPMENTAL EFFICIENCY OF THEAVIAN EMBRYO

BY JOSEPH NEEDHAM, M.A., PH.D.,

Fellow of Gonville and Caius College, Cambridge.

(From the Biochemical Department, University of Cambridge.)

(Received ist February 1927.)

(With Two Text-figures.)

CONTENTS.PAGE

Introduction . . . . . . . . . . . . . 4 3( A ) T h e C h a n g e i n t h e " C o e f f i c i e n t d ' U t i l i s a t i o n " o r " P l a s t i c E f f i c i e n c y C o e f f i c i e n t "

d u r i n g d e v e l o p m e n t . . . . . . . . . . . 4 3( B ) T h e C h a n g e i n t h e " R e n d e m e n t E n e r g ^ t i q u e " o r " E n e r g e t i c E f f i c i e n c y C o -

e f f i c i e n t " d u r i n g d e v e l o p m e n t . . . . . . . . . 4 6S u m m a r y . . . . . . . . . . . . - S 3

INTRODUCTION.

IN the consideration of embryonic metabolism it is natural to enquire what degreeof wastefulness in growth is shown by the developing embryo. Up to the presenttime this question has only been answered by treating the ontogenetic period asa whole. The efficiency of growth may vary, however, during that period and aknowledge of the variations in this factor with time might throw some light onthe chemical events of incubation. The calculations of this paper were made withthis end in view. They were possible because of the general balance-sheet ofchemical changes in the developing chick which Murray (16-20) and I myself (31-36)have built up.

(A) THE CHANGE IN THE "COEFFICIENT D'UTILISATION" OR"PLASTIC EFFICIENCY COEFFICIENT" DURING DEVELOPMENT.

The degree of efficiency with which the transference of yolk and albumen intoflesh and blood is effected may most conveniently be expressed by an efficiencycoefficient.

The efficiency coefficient as such corresponds to the " Coefficient d'Utilisation "of Terroine and Wurmser(33), the "Coefficient ficonomique" of Pfeffer(27), andthe "Plastic Equivalent" of Waterman(37). The best name for it would seem tobe "Plastic Efficiency Coefficient" (P.E.C. for short) for this shows that it hasnothing to do with energy content or expenditure and explains that it is a measureof efficiency of transfer of matter. It may be described as the ratio

dry weight of embryodry weight of absorbed solid '

44 JOSEPH NEEDHAM

and is designed to show the relationship between the substance combusted andthe substance stored, or in other words, the relative cost in gm. of solid of buildingthe embryo. The higher the efficiency coefficient, the smaller the amount of burntsubstance in relation to stored substance.

Table I. Efficiency Coefficient.

I

Day

oi23456

789

10II1213'4151617181920

2

CumulativeEfficiencyCoefficient

(Gray)

•67•64•62•61•60•63

•69

•73

•74

3 4 5

Increments

Storage ofdry weight

mg.

37

1219-430-844'368-2

10251607241-0409-457568673°797832

Combustionmg.

1

36

112032456080

i°5132164198236253259

Totalmg.

410183O5i76

" 3162241346541

739884966

10501091

6

Daily Plastic(incremental)

EfficiencyCoefficient

•75•70-67•69•60•58•60•63•67•69•75•77•77•76•76•76

7

PercentagePlastic

EfficiencyCoefficient

3343505765

66595044322828323131

Gray do) in his recent memoir on the chemical embryology of the trout findsthat its average Plastic Efficiency Coefficient (P.E.C.) is -63 which compares veryclosely with that of the frog, the chick, the silkworm, and Aspergillus. He workedit out for the chick from Murray's data(18) in a cumulative way, but a more in-stantaneous picture would be given if it were calculated on a daily basis. Howexpensive is it on each day of development to build what is built on that day?Table I, column 1 gives the day and column 2 the P.E.C. as given by Gray. Thiswould not be appreciably different if it were computed using Murray's figures foroxygen consumption (ao) instead of those for carbon dioxide production, as couldnow be done.

Column 6 gives the P.E.C. worked out for each day, the incremental P.E.C, andin Fig. 1 it is compared with Gray's. Both curves fall and then rise, and the lagin the cumulative one is not significant for each day's point bears, as it were, initself the effects of the previous days. The incremental P.E.C. shows the instan-taneous change.

There must be some significance in the deep trough through which the curvepasses between the seventh and twelfth days. Evidently at that period develop-ment is most expensive; the amount of burnt substance is greater relatively to the

The Developmental Efficiency of the Avian Embryo 45

amount of stored substance then than at any other time. This calls to mind thecorrelation suggested in a previous paper (23) between heat-production and mid-development, in which it appeared that both for the chick and the toad (Gaydaw)it is most expensive to double the weight of the animal when embryogenesis ishalf completed. In the chick this is between the seventh and twelfth days. Thismight be related to the fact that the growth-rate of dry solid is constant during thatperiod, but the fit is not exact for the constancy is hardly established by the seventhday and continues till the fifteenth. There is, moreover, no reason to suppose thatan increase of dry substance rather than water should necessarily lead to an increaseof catabolism. But the correlation of the intensity of protein combustion is muchmore exact, in fact, strikingly so, as may be seen from the vertical line in Fig. 1,

•to •

•ss

Days 5- 10 rr

O Gray: cumulative. © Needham: incremental.Fig. 1. Plastic efficiency coefficient. The vertical dotted line indicates the point of

maximum intensity of protein combustion.

and the inference that we have here to deal with an effect of Specific DynamicAction is difficult to resist. Another explanation is also available. It is just at thisperiod that the transference of fat into protein is probably going on, and, asTerroine, Trautman, and Bonnet (35) have shown, such a transference results in anextra energy-loss of 23 per cent. This might lead to an increased expenditure ofsubstance in a given amount of architectural enterprise. Probably more than onefactor is responsible.

The words "a given amount of architectural enterprise" suggest that an alter-native way of expressing the P.E.C. might be valuable. Instead of finding out howmuch material has to be burnt on a given day in order to construct a given amountof embryo, one could calculate how much material would have to be burnt on agiven day in order to construct 100 mg. of embryo. Such a value, which wouldobviously differ from the P.E.C. above described in being high when the develop-ment was wasteful and low when it was efficient, and which might be called the

46 JOSEPH NEEDHAM

percentage P.E.C, is given in column 7 of Table I. It gives a peak at 8-5 days ofdevelopment instead of a trough. There can be no doubt of the phenomenon ofmaximum inefficiency in the middle of the ontogenesis of the chick.

We have seen that the average P.E.C. for the whole of development is -68. It isinteresting to enquire which of the foodstuffs contributes principally to this degreeof efficiency. Knowing already that fat is the chief foodstuff combusted and thatprotein is the chief architectural material, it would be natural to predict that themost efficiently stored substance would be protein. The exact figures follow.

Carbohydrate storedburnt

Protein storedburnt

Fat storedburnt

Total solid storedDry weight of embryo at

195 days app.

mg.

107

298669

17002110

47935000

Reference

(26) Tab. I, col. 5(26) „ VIII, „ 325) „ III, „ 2

(23) „ V, „ 9(25) „ VIII, „ 3(25) „ IX, „ 3

P.E.C.

) *

( -98

I -

% of totalfoodstuff

combusted

5-6

302

91-4

Out of 100 gm. of protein in its diet, then, the embryo can store away 98, outof 100 gm. of carbohydrate 82, but out of 100 gm. of fat only 43. This could nothave been predicted from the combustion curves alone, but needed a considerationof the constitution of the embryo. The embryo has to thank protein absorptionfor its average P.E.C. level, and to a lesser degree that of carbohydrate. In the caseof animals such as the trout which burn large amounts of protein, the "foodstuffP.E.C." would be very different.

(B) THE CHANGE IN THE "RENDEMENT ENERGfiTIQUE" OR"ENERGETIC EFFICIENCY COEFFICIENT" DURING DEVELOPMENT.

The P.E.C. or Plastic Efficiency Coefficient is based on analyses of actual material.Terroine and Wurmser(33) in their classical paper on Growth Energy have argued,following Tangl(32), that a better idea of the fundamental nature of growth andespecially embryonic growth can be got by dealing in energy rather than matter.They therefore define the "Rendement Energ6tique" analogously to the PlasticEfficiency Coefficient as

Energy laid up in the organismEnergy in the raw materials _ Energy in the raw materials at

at zero hour the end of development

U'U-UB

which is only another way of writing

Energy storedor

Energy storedEnergy absorbed Energy stored + Energy in solid burnt

The Developmental Efficiency of the Avian Embryo 47

This can be calculated from the results obtained by the following investigatorsand works out thus:

Tangl(32) chick ... ... ... 66 per cent.Farkas(s) silkworm ... 87 ,,Glaser(s) minnow ... ... ... 78 ,,Faure-Fremiet and Vivier du Streel(6) frog ... 82 ,,Barthelemy and Bonnetd) frog ... ... ... 75 „

They proceed to point out, however, that this "Rendement Energ^tique brut"contains a fallacy and that to get the "Rendement Energ^tique r^el" the basalmetabolism must be taken into account. Tangl's " Entwicklungsarbeit" (Ea) failsto allow for the fact that all the time the embryo is growing it is also eating andevery cell as soon as formed begins a normal metabolic life; it is thus only a measureof the total embryonic metabolism. In just the same way the "Rendement Ener-getique brut" fails to allow for the fact that some of the energy absorbed by theembryo is expended in basal metabolism, maintenance energy, "energie d'entre-tien," etc. to which the embryo is committed by the mere circumstance of beingalive at all. Thus of the energy in the material combusted only a certain fractionought really to be included in the calculation of the efficiency, for the rest is ear-marked for the upkeep of that part of the building already constructed and cannotbe termed in any sense a waste. The " Rendement Energetique brut" does not takeinto account the fact that every cell embarks upon a basal metabolism as soon asit is completed. A calculation of the true growth energy must therefore allow for-this according to the following formula:

Energy laid up in the organism U'

Energy in the raw Energy in the raw Enerev of u ~ (UR+ UF)materials at zero - materials at the end + M a i n t ~ a n c e

hour or development

The denominator is now the energy absorbed for growth and non-basal metabolismonly. The strict correspondence between observed and calculated heat-productionfound by Bohr and Hasselbalch(i) suggests that the energy not allotted to one orother of the above headings will be very small. There is, however, some doubtwhether the usual notions of basal metabolism can be applied to so rapidly changinga system as the embryo. Basal metabolism is that amount of energy used in main-taining a steady state, but can the embryo be considered to be in a steady stateeven momentarily? Perhaps the conceptions of Terroine and Wurmser are notapplicable to the cells of a developing metazoon though they may be quite satis-factory for moulds and bacteria.

Terroine and Wurmser (33) not wishing to place confidence in the law of surfaces,especially as applied to Aspergillns niger, determined their UB or basal metabolismfrom experiments in which growth was hastened or retarded by adjustments ofthe pH of the culture medium. Such a procedure is not possible in the case ofthe chick where the limits within which normal development will proceed aresomewhat narrow. In the exploratory calculations of this paper it will therefore

48 JOSEPH NEEDHAM

unfortunately be necessary to have recourse to the law of surfaces using Meeh'sformula(15) and Rubner's constant(J8). The calculation can evidently not be exactbecause we do not know how these quantities vary during the embryogeny of thechick, but it is worth while to probe the matter and see what happens. The relevantfigures are shown in Tables II and III.

Table II.

I

Day

01234

6

89

10111213141516

\l1920

Total

2 3

Surface of the embryo

Totalsq. mm.

1005198380586§47

11701550200025103080375°449052906150713081709280

10700

Dailyincrements

98-518220626132338045°510570670740800860980

104011101420

i°599

4

gm. cals.producedin basal

metabolismdaily

increments

9217219424630535§424481

537632698754811924981

10501350

5 6

Calories evolved(Bohr and Hasselbalch)

gm. calsoutput

per day

Heatabsorbed

1

i!" 5151200276396552780

IOOI124014601710i9602160

Dailyincrements

243631364976

120156228221239220250250200

In Table II, column 1 gives the day of development, and column 2 the calcu-lated surface of the embryo, obtained according to the formula

where S is the surface, K Rubner's constant for the chicken, 10-4, and W the weighttaken from Murray's figures. Column 3 gives the increments of surface each day,the amounts by which the surface is increased in each interdiurnal period. Column 4shows the calories produced in basal metabolism, assuming with Voit(36) that inthe chicken this amounts to 0-943 g111- ca^s- Pe r SCI- n"11- surface. This representsthe quantity of inevitable loss n the weight of embryo formed each day. In thenext column, No. 5, are placed the number of gm. cals. evolved as measured byBohr and Hasselbalch 00 in each period of 24 hours and if this column is comparedwith column 9 of Table III where the heat-production calculated from the oxygenconsumption is given, it will be seen that the agreement is fair, though the experi-mental is always rather lower than the calculated value. The fact that the calcu-

50 JOSEPH NEEDHAM

lated value assumes fat only to be burnt would not entirely account for this.Returning to Table II, however, it can at once be seen that the basal metabolismin column 4 invariably exceeds the total amount of heat put out as given in column 6which is the incrementation of column 5. Thus there is not enough heat put outto account for the amount that ought to be produced in maintenance energy alone.However, there cannot be much doubt that the basal metabolism as here calculatedis absurdly high, for if all the increments are added up the result is 61,000 calories,in other words, about four times as much as the total energy known to be lost bycombustion. We must therefore suppose either that the surface formula does nothold in embryonic life or that the high temperature (370) in which developmentproceeds leads to a lower basal metabolism than would be expected. Lusk(i4,.p. 141)says that the minimum requirement for energy is seen to be present when thefasting organism is surrounded by an atmosphere having a temperature of 30°to 350. Most important of all, however, is the probability that Rubner's constantfor the hen does not hold for the embryonic chick. It is quiescent, its muscleshave no tonus or very little, its respiratory muscles are inactive, and its heart aloneis requiring constantly a supply of energy. Since the metabolism is proportionalto the superficial area of the animal, it may well be asked what is happening in anembryo at the minute stage when its percentage growth-rate is 1400 (Schmalhausen(39)). The large surface in proportion to its weight which the very young embryomust have explains the fall in metabolic rate and rate of heat-production whichhas been brought to light by so many investigators, e.g. Le Breton and Schaeffer(ia),and Shearer (30). Columns 2 and 3 do not begin with nearly such small figures asdo columns in which weight is expressed.

Evidently it is not possible at present to calculate the " Rendement Energetiquereel" or "Real Energetic Efficiency" (R.E.E.); all that can be done is to calculatethe "Rendement Energetique brut" or "Apparent Energetic Efficiency (A.E.E.).

This is done in Table III. Column 1 shows the time of development, column 2the energy stored in the embryo taken from Murray's table and expressed asgm. cals. per gm. dry weight of embryo, column 3 the same expressed as actualcalories present in the embryo each day (cumulative). Column 4 shows the incre-ments of calories, in other words the amounts of potential energy stored in theembryonic body each day. In order to check this and to show that the data balanceproperly column 5 shows the energy present in the extra-embryonic part of theegg as determined with the bomb calorimeter by Tangle). It will be seen thatthe rest of the egg loses, in addition to combustion losses, 250 gm. cals. betweenthe eighth and the ninth days, while the embryo gains 232; a sufficient agreement.The figure of 34,193 gm. cals. seen at the bottom of column 4 representing thenumber of cals. contained in the finished embryo agrees sufficiently well with thevalue given by Tangl of 32,000; the latter was measured directly, the former wasobtained by the addition of all the increments.

Columns 6 to 9 give the figures relating to the energy lost in combustion.Column 6 shows Murray's figures for the mg. of dry solid burnt per gm. dryweight of embryo per day (a measure of the metabolic rate) and column 7 expresses.

The Developmental Efficiency of the Avian Embryo 51

this as actual number of mg. burnt each day. Column 8 translates this into inter-diurnal averages, so that the amount of substance combusted in producing thecorresponding value of column 4 is there shown for the interdiurnal periods. Incolumn 9 this is seen converted into gm. cals., assuming that 100 percent, insteadof the true 92 per cent, of the total solid burnt is fat, and that 1 gm. of fat produceson its combustion 9300 gm. cals. The total of this column amounts to 1700 gm.cals., not very far from the 1650 gm. cals., the Ea of Tangl. In column 10 Tangl'svalues for column 9 are given, and it may be noticed that they are very close tothe newer ones. An error exists here owing to the fact that no account has beentaken of the energy left behind in incompletely combusted materials, but as thechief of these is uric acid, and—using the data of Stohmann and Langbein(3O forthe calorific value of uric acid, 2750 gm. cals. per gm.—the cals. locked up in this

A.E.E.

6s--

60 •

I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I I I I '

S" (o IT 2 o

Fig. 2. Apparent energetic efficiency (Rendement Energe'tique brut).

way only amount to 16 on the nineteenth day or much less than i per cent, of thetotal combusted, this error is negligible. It may also be noticed, by comparingcolumn 7 with Table I, column 4, that the solid combusted calculated from thecarbon dioxide output differs very little from that calculated from the oxygenintake. The variations would perhaps be significant for some purposes but notfor the present one. Finally column 11 shows the A.E.E. (" Rendement Energe'tiquebrut").

It is diagrammatically represented in Fig. 2. Starting at a low level it slowlyrises, gaining in speed till at the fourteenth day it is rising rapidly but soon after-wards it falls off. At the initial stages of development, the efficiency is very lowand rises rapidly in the middle of development to attain a constant level by thetime of hatching. As can be seen by the value at the base of column 11 the valuefor the whole of development works out at 66-5, which is exactly what Tangl found.Since the basal metabolism is included in this estimate and since that would

52 JOSEPH NEEDHAM

naturally be expected to be very high in the early stages when the embryo is veryminute and has a large surface in proportion to its size, one would naturally predictthat the efficiency, the A.E.E., would be very low then. Its subsequent rise to aconstant level might be associated with the decrease in importance of basal meta-bolism. It is interesting to note that it finishes up very close to the values obtainedfor the R.E.E., the "Rendement Energdtique r6el," of mammalian post-embryonicgrowth by Kellner and Kohler(n) and Fingerling, Kohler, and Reinhardt(7). Butat the present time there is no means of telling what this latter coefficient wouldshow in the ontogenesis of the chick; it would certainly be much higher than theA.E.E. in the earlier stages but afterwards it might either fall or remain constant.It is difficult to see how the basal metabolism of the embryo could be measured.

Another way of interpreting Fig. 2 would be by the biogenetic law, for the lowefficiency of the early stages may not be due to a high basal metabolism then. Asa general rule the "lower" the animal the more wasteful it is: Horace Brown, forexample (3), showed that a yeast cell would ferment its own weight of maltose at300 C. in 2-2 hours and at 400 C. in 1-3 hours, during which time it was not repro-ducing and as far as could be seen was not doing any work at all. This metaboliclevel would be about 100 times as high as that of an adult man. The rise in effi-ciency (A.E.E.) during the development of the chick may perhaps be thought of asa recapitulatory phenomenon.

It may be noted also that if the embryo continued to behave as wastefully allthrough incubation as it does in the beginning there would not be enough energyin the egg to provide for it: unless the egg were increased to about one and a halftimes its present size. Even then there would be no reserve yolk at hatching. Isthe increase in efficiency due to change of substrate or increasing complexity ofembryonic machinery? This is a problem which much future work in chemicalembryology will be required to solve. Apparently no conclusions about the sub-stance combusted can be drawn from the A.E.E. For the R.E.E., on the other hand,Terroine, Trautman, Bonnet, and Jacquot(34) have obtained a value of 38 whenprotein was the principal foodstuff and 58 in the case of sugar. Terroine, Trautmanand Bonnet (35) give further a value of 44 for fat. It is true that these figures wereall derived from experiments with moulds, Sterigmatocytis nigra and Aspergillusorhizae, so that it is doubtful whether they can be directly compared with such asmay be found to hold for homoiothermic organisms. If, however, they do form avalid series there, one might predict, bearing in mind the fact that protein, thoughcombusted in greatest amount at the mid-point of incubation, never preponderatesabsolutely in the solid burnt, that the R.E.E., if it ever becomes possible to plot it,will fall markedly as development proceeds.

Since the A.E.E. rises with age it resembles the percentage of total solids, thepercentage of fat, the latent period of growth in tissue cultured fragments, thetotal metabolism and the rate of the heart-beat; a miscellaneous collection of factors.But, having in mind the valuable generalisation of Murray (17) that embryonicdevelopment is symmetrically diphasic in character, we may enquire whether itmoves rapidly at first, then slowly, like the growth rate, or slowly at first, then

The Developmental Efficiency of the Avian Embryo 53

rapidly, like the metabolic rate. This question can only be answered by the state-ment that the rise in A.E.E. resembles the fall in metabolic rate. It is slow at firstand later more rapid. Thus it would seem as if the furious intensity of combustionwith which the embryo begins its life was associated with great wastefulness, whilelater on greater economy would accompany greater frugality. It may be notedthat there is no trough on the A.E.E. curve and that it attains an adult value shortlybefore the end of development. The calorific value of the embryonic tissue alsorises during development, and Murray's graph (17, P. 42O shows that it goes up ina curve shaped rather like that for the A.E.E., as in Fig. 2. The two may be related,for the richer in potential energy the embryonic body becomes per unit weight,the more efficient the transfer of energy from the yolk and white might be expectedto be. The increasing calorific value of the substance transferred would tend tonullify this tendency but might not abolish it altogether.

Though the curves for P.E.C. and A.E.E. are different, it is interesting to findthat the average P.E.C. for all development is -68 while the average A.E.E. is 66 percent. Out of 100 gm. of solid presented to it, the embryo can store 68; out of 100 gm.calories presented to it, the embryo can store 66.

Finally, the embryo can be compared with other engines. Its business is tostore as much energy as is given it with as little loss as possible. The object of thesteam-engine is to produce as much mechanical work from the energy given itwith as little loss as possible. The efficiency of this process is not great: in thelocomotive engine, which is notoriously wasteful, it may not exceed 15 per cent,and Wimperis(38) gives a value of 22 per cent, for the internal combustion engineworking on producer-gas. However, a much better comparison is between theembryo and the boiler or the electric battery for these machines do not alter theform of the energy passing through them. According to Low(13), a Lancashireboiler presented with 100 calories in the form of coal only wastes 28: an efficiencyof 72 per cent., and, according to Cooper (4), an average electric battery will giveback 74 per cent, of the electrical energy put into it. The average A.E.E. of the chick,the silkworm, the minnow, and the frog embryo is 77 per cent, but the R.E.E. wouldbe somewhat higher. It is interesting that the efficiency of the embryo should beof the same order as that of other machines.

SUMMARY.1. The "Coefficient d'Utilisation" or Plastic Efficiency Coefficient (P.E.C.) has

been calculated for each day during development. It has a trough which is deepestbetween the eighth and ninth days; development is therefore most expensive atthis point. The correlation between this and the point of greatest intensity ofprotein combustion is exact.

2. The "Rendement Energetique brut" or Apparent Energetic Efficiency hasbeen calculated for each day during development. It rises, changing more rapidlytowards the end than at the beginning; thus it resembles the metabolic rate ratherthan the growth rate. The "Rendement Energetique reel" or Real Energetic

54 JOSEPH NEEDHAM

Efficiency cannot at present be calculated for the basal metabolism of the embryois unknown and it is not certain whether the usual conceptions of basal metabolismcan be applied to a rapidly growing and changing organism.

My thanks are due to Professor Sir Frederick G. Hopkins, F.R.S. for his en-couragement and to Miss M. Stephenson, Mr J. T. Mason, Mr H. W. Phear, andMr J. P. Moyle for various interesting suggestions. I am also indebted to DrDorothy Needham for valuable help and to the Government Grant Committee ofthe Royal Society for a grant towards the cost of these researches.

REFERENCES.

(1) BARTHELEMY and BONNET (1926). Bull. Soc. Chim. Biol. 8, 1071.(2) BOHR and HASSELBALCH (1903). Skand. Arch. f. Physiol. 14, 398.(3) BROWN (1914). Annals ofBotany, 28, 197.(4) COOPER (1901). Primary Batteries, p. 288. London.(5) FARKAS (1908). Archiv f. d. ges. Physiol. 98, 490.(6) FAURE-FREMIET and DU STREET, (1921). Bull. Soc. Chim. Biol. 3, 480.(7) FINGERLING, KOHLER, and REINHARDT (1914). handwirtsch. Versuchst. 84, 149.(8) GAYDA (1921). Arch, di Fisiol. 19, 211.(9) GLASER (1912). Biochetnische Zeitschr. 44, 180.

(10) GRAY (1926). Brit. Journ. Exp. Biol. 4, 215.(11) KELLNER and KOHLER (1900). Landtoirtsch. Versuchst. 53, 474.(12) LE BRETON and SCHAEFFER (1923). Travaux de Vlnstitut de Physiol., Faculttde Mid. Strasbourg.(13) Low (1920). Heat Engines, p. 219. London.(14) LUSK (1919). The Science of Nutrition, 3rd ed. Saunders, Philadelphia, p. 141.(15) MEEH (1879). Zeitschr. f. Biol. 15, 425.(16) MURRAY (1925). Journ. Gen. Physiol. 9, 1.(17) (1925). Journ. Gen. Physiol. 9, 603.(18) (1926). Journ. Gen. Physiol. 9, 405.(19) (1926). Journ. Gen. Physiol. 9, 789.(20) (1926). Journ. Gen. Physiol. 10, 337.(21) NEEDHAM (1925). Physiol. Reviews, 5, 1.(22) (1926). Brit. Journ. Exp. Biol. 3, 189.(23) (1926). Brit. Journ. Exp. Biol. 4, 114.(24) (1926). Brit. Journ. Exp. Biol. 4, 145.(25) (1927). Brit. Journ. Exp. Biol. 4, 258.(26) (1927). Brit. Journ. Exp. Biol. 5, 6.(27) PFEFFER (1895). Pringsheim's Jahrbuch, 27, 205.(28) RUBNER. Cited in Lusk, as above, p. 119.(29) SCHMALHAUSEN (1926). Arch. f. Entwicklungsmcch. 108, 322.(30) SHEARER (1922). Proc. Roy. Soc. B, 92, 410.(31) STOHMANN and LANGBEIN (1891). Journ. f.prikt. Cheni. 44, 380.(32) TANGL (1903). Arch.f. d. ges. Physiol. 93, 327.(33) TERROIME and WURMSER (1922). Bull. Soc. Chim. Biol. 4, 519.(34) TERROINE, TRAUTMAN, BONNET, and JACQUOT (1925). Bull. Soc. Clum. Biol. 7, 351.(35) TERROINE, TRAUTMAN, and BONNET (1926). Ann. Phys. et Phys.-chem. Biol. 2, 172.(36) VOIT (1901). Zeitschr. f. Biol. 41, 120.(37) WATERMAN (1912). Folia Microbiologica, 4 , 1.(38) WIMPERIS (1922). The Internal Combustion Engine, London, p. 6.