the influence of the composition of the medium on

THE INFLUENCE OF THE COMPOSITION OF THEMEDIUM ON THE METABOLISM OF SOME SLOW-LACTOSE-FERMENTING BACTERIA OF INTESTINALORIGIN

A. D. HERSHEY AND J. BRONFENBRENNERDepartment of Bacteriology, Washington University Medical School,

Saint Louis, Missouri

Received for publication, June 20, 1936

In the course of earlier studies (Bronfenbrenner and Schlesinger,1918) of some slow-lactose-fermenting bacteria of intestinalorigin, it was observed that the appearance of acid and gas in thelactose fermentation test was remarkably influenced by thecomposition of the medium.The influence of buffer and of salts of organic acids upon sugar

fermentation in general have been investigated by Clark (1915),and Ayers and Rupp (1918), respectively. The question hasremained open, however, as to the specific manner in which thecomposition of the medium influences the development of thebacterial culture, and particularly, by what mechanism theconcentration of lactose determines the rate of utilization of thissugar.

It was the object of the present investigation to determinewhether the observed influence of concentration factors upon thefermentation of lactose by the slow fermenting strains was exertedmerely upon the rate of growth of the organism, or upon itsvariability, or whether special phases of metabolism were pri-marily involved. It was necessary, therefore, to submit to sepa-rate analysis the several manifestations of growth and metabo-lism, utilizing various media of known composition.The cultures studied consisted of a slow lactose-fermenting

strain of Escherichia coli, and a rapidly fermenting variantderived from it. The isolation and characteristics of these

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A. D. HERSHEY AND J. BRONFENBRENNER

strains have been described elsewhere (Hershey and Bronfen-brenner, 1935).The organisms were grown on a synthetic medium of the fol-

lowing composition: ammonium sulfate 0.5 gram, sodium chloride0.5 gram, calcium chloride 0.005 gram, magnesium sulfate 0.02gram, potassium acid phosphate 0.68 gram, dissolved in 100 cc.distilled water. Varying amounts of disodium succinate and oflactose were added to this basal medium as will be shown below.Sodium was used in preference to the ammonium salt of succuincacid so that the concentration of this acid could be varied inde-pendently of the nitrogen. For each experiment, 250-cc. amountsof the above-described medium were distributed in seven 1-literErlenmeyer flasks, and 10-cc. amounts in Dunham fermentationtubes of uniform size. These were sterilized under cotton plugs,and inoculated simultaneously from a twenty-four-hour agarculture. At this time the cotton plugs of two of the flasks werereplaced by rubber stoppers equipped for aeration of the cultures.All were incubated at 370C. The two sealed flasks were aerateddaily for a period of two hours for the purpose of determining theamount of carbon dioxide produced. At intervals of two daysone of the remaining cultures was centrifuged, after restoring thewater lost by evaporation, and the supernatant fluid was used foranalysis. Succinic acid was determined by titration of theprecipitated silver salt (Volhard). Lactose was determined bythe Schaffer-Hartmann micro method. Total organic acidswere titrated by the method of Van Slyke and Palmer. Thehydrogen ion concentration was determined colorimetrically.The amount of growth was measured gravimetrically after wash-ing the centrifuged cells and drying to a constant weight. Theamount of gas formed in the Dunham fermentation tubes wasmeasured in linear centimeters. The carbon dioxide produced inthe sealed flasks was absorbed into standard NaOH solution,precipitated as BaCO3, and titrated by difference. Each culturewas plated at the conclusion of its incubation period on a lactoseChina-Blue Rosolic-Acid medium (Bronfenbrenner 1918) tocontrol purity and variation. The results plotted in figures 1and 2 represent the means of two or more experiments.

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METABOLISM OF BACTERIA OF INTESTINAL ORIGIN 521

THE INFLUENCE OF THE CONCENTRATION OF LACTOSE IN MEDIACONTAINING LACTOSE AS SOLE SOURCE OF CARBON

In figure 1 are shown the changes brought about during thegrowth of a slow-lactose-fermenting strain in media containinglactose in concentrations of 0.5 and 3 per cent in the absence ofany additional source of carbon.

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FIG. 1. INFLUENCE OF THE CONCENTRATION OF LACTOSE ON METABOLIS3M

Subcultures on the indicator plates, and in lactose fermentationtubes, revealed no increase in fermenting power during the growthof the cultures before the twelfth day of incubation. Bacterialvariation,-therefore, did not occur in either of these media duringthe course of the experiments.


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2 A. D. HERBSHEY AND J3. BRONFENBRENNER

The growth curves (fig. 1 A) show a more rapid development ofthe culture in the medium containing the higher lactose concen-tration, with an early interruption and subsequent autolysis ofthe non-aerated cultures. The flask examined at eight days wassterile, and almost water-clear.Early lactose fermentation (fig. 1 B) was more rapid in the

medium containing lactose in the higher concentration, but ceasedwith the development of high acidity. The apparent increase inlactose content of the medium between the fourth and sixth dayscan be due only to relative suppression of monosaccharide fer-mentation by the high acidity reached during this period, whileactivity continues unchecked. Accumulated monosaccharidesliberated on cleavage of lactose by this enzyme would give nearlytwice the copper reduction value of the original lactose. Thisincrease was invariably observed when a pH of 4.8 was reached.During this period organic acid formation (fig. 1 D) was nearlystationary. Inhibition of glucolysis does not appear in the carbondioxide curve (E) presumably because daily aeration of thisculture prevented development of high acidity.The hydrogen-ion concentration (fig. 1 C) and organic acids

(fig. 1 D) also reached an earlier maximum value in the mediumcontaining 3 per cent lactose. This effect should be attributedto the increased oxygen demand in this medium, resulting inrelatively anaerobic conditions of growth unfavorable for theoxidation of organic acids, while acid formation by glucolysis isat its height. In the aerated cultures (not shown in the figures),although lactose was fermented even more rapidly, high aciditywas not reached, and the development of the culture was unin-terrupted. The accumulation of acid thus depends on the balancemaintained between simultaneous fermentation of sugar and ofthe resulting organic acids, as first clearly stated by Ayers andRupp (1918).While the carbon dioxide curves were obtained from cultures

not strictly comparable with those not subjected to aeration, theygive, nevertheless, a direct measure of the relative rates of respira-tion in the two media. The effect of lactose concentration oncarbon dioxide production (E) is slight compared with its effect

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on removal of lactose (B). This is another expression of thelimiting effect of oxygen supply on the rate of oxidation of theproducts of sugar cleavage, in contrast to the rate of cleavageitself.Gas formation in fermentation tubes is primarily a measure of

the ability of the organisms to develop anaerobically in a mediumcontaining fermentable substances. It has no direct relation,therefore, to carbon dioxide production measured in aerobiccultures. The curves (F) showing gas formed in the Dunhamfermentation tubes again illustrate the more rapid utilization oflactose when it is present in the higher concentration. Carbondioxide: hydrogen ratios were roughly 1: 2 in lactose media.

THE INFLUENCE OF THE CONCENTRATION OF LACTOSE AND OF

SUCCINATE RESPECTIVELY IN MEDIA CONTAINING

BOTH SOURCES OF CARBON

A second series of experiments was carried out, identical withthose described above except that both lactose and sodium succi-nate were supplied in the basal medium. Two media were used,one containing 0.5 per cent lactose and 2.5 per cent succinate; theother 2.5 per cent lactose and 0.5 per cent succinate. Theseproportions were chosen to give extreme values for the ratio oflactose to succinate. Changes taking place during the growth ofthe slow lactose-fermenting strain in these media are shown infigure 2.The first of the above represents an almost perfectly balanced

medium. The curves obtained by analysis of the cultures aresmooth throughout. The pH (fig. 2 C) remained practicallyunchanged during the course of the experiment, while the totalamount of organic acids (D) exclusive of carbon dioxide wasconstantly decreasing. The rate of removal of succinate (G) wasgreater than the rate at which acids formed from the fermentationof lactose accumulated.

In the medium containing 2.5 per cent lactose and 0.5 per centsuccinate, lactose fermentation, the formation of organic acids,and total gas production proceeded more rapidly than in themedium containing 0.5 per cent lactose and 2.5 per cent succinate.


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524 A. D. HERSHEY AND J. BRONFENBRENNER

This increase in the metabolic activity was similar in general tothat observed in the absence of succinate (fig. 1). However, inthe presence of the latter, growth (A) and fermentation (B) were

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FIG. 2. INFLUENCE OF THE CONCENTRATIONS OF LACTOSE AND OF SUCCINATEON METABOLISM

only temporarily suppressed by the accumulation of acid (D).The cultures survived, there was a reversion of pH (C) and fer-mentation of lactose was resumed. It can be seen that reversion

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METABOLISM OF BACTERIA OF INTESTINAL ORIGIN

in this case was not due to exhaustion of the sugar, but apparentlyonly to the decreasing rate of fermentation of lactose as its concen-tration diminished. The presence of succinate did not appearto affect lactose fermentation other than through its buffer-actionfavorable to survival of the culture. Differences in the respectivecurves showing lactose removal (B) in figures 1 and 2 are whollyreferable to differences in the corresponding growth curves (A).

In the medium containing the higher concentration of lactoseand the smaller amount of succinate, succinic acid removal waswholly suppressed (fig. 2 G). This may be attributed in part tothe production of additional small amounts of this acid resultingfrom fermentation of lactose (fig. 1 G). Whether there is a directeffect of the concentration of succinate on its utilization is notclear from these experiments. Unquestionably considerationsof oxygen demand are again pertinent here. In the more vigor-ously respiring culture, in which conditions of oxygen-starvationprevail, fermentation of succinate as well as of other aerobicallyfermented acids is inhibited. This mechanism, which has notbeen considered heretofore in this connection, perhaps defines theextent of the so-called protein-sparing action of fermentable sugarin bacterial cultures.

METABOLISM OF THE RAPIDLY LACTOSE-FERMENTING VARIANT

The following experiment, in which the behavior in a lactosemedium of a rapidly fermenting variant is compared with that ofthe parent culture, illustrates several points in connection withthe previous experiments. The synthetic medium alreadydescribed, containing 0.5 per cent lactose and 2.5 per cent sodiumsuccinate, was used. The curves are given in figure 3.The rates of growth for the two strains as indicated in the curves

(fig. 3 A) were nearly identical. For this reason the findings areparticularly instructive. In the culture of the variant (V) strain,lactose (B) was completely removed by the fourth day, whereastwelve days were required in the case of the parent culture (P).In the more actively fermenting (V) culture, the removal oforganic acids proceeded more slowly than their formation, re-sulting in the acid and alkaline phases (C) typical of bacterial

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fermentation of sugars. Carbon-dioxide production (D) wassomewhat more abundant in this culture, but the difference inrespiration rate was much less than the difference in the rate offermentation (B) and of total gas production (E). The essentialdifference between the two bacterial cultures is their content ofthe enzyme lactase, which is the limiting factor in the fermenta-tion of lactose by these organisms (Hershey and Bronfenbrenner,1935). Since, in the removal of lactose and to a large extent inthe formation of gas in closed fermentation tubes, only theanaerobic processes of fermentation are concerned, it is in thesetwo curves (B and E) that the difference between the metabolismof the two cultures is reflected. But only a small part of thecarbon dioxide output of the aerated culture is derived fromanaerobic processes, e.g., that represented by the equation:HCOOH -- H2 + CO2. The remainder is furnished by aerobicoxidations. Thus, the total carbon dioxide formed has twolimiting factors: the rate of sugar fermentation, and the rate ofrespiration as determined by the oxygen supply. Accordinglythe amount of carbon dioxide produced is somewhat greater inthe rapidly fermenting culture than in the parent culture receivingthe same amount of oxygen, but a much greater increase isobtained when the latter is continuously aerated (fig. 3 D). Asimilar discrepancy between the rates of fermentation (B) andrespiration (E) appears in figures 1 and 2, as an effect of varyingthe concentration of lactose. There it is seen to be largely inde-pendent of the succinate supplied in the medium.

OXYGEN UPTAKE OF CELL SUSPENSIONS IN SOLUTIONS OF PURELACTOSE

From the results of the foregoing experiments, it was not clearwhether the effect of changes in the concentration of lactose inthe medium was merely one of growth stimulation, with resultingaccumulation of lactase, or whether there was a direct relationbetween the concentration of lactose and the rate of lactaseactivity. The following experiments were designed to answerthis question by excluding the effect of growth stimulation. Forthis purpose the respiration of suspensions of washed cells of the


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slow lactose-fermenting strain in lactose solutions of differentconcentration was measured during short periods of time underconditions in which multiplication of cells would be a negligiblefactor. To exclude the possibility that impurities in the lactose,or products of its decomposition during sterilization by heat, andnot the concentration of lactose itself, were responsible for theeffects observed (fig. 1), the oxygen uptake in unheated lactosesolutions prepared from a twice recrystallized preparation of thesugar was controlled by a parallel experiment using autoclavedsolutions of commercial lactose. All substrates were dissolved inM/20 phosphate buffer of pH 7.0. The measurements were madein the Barcroft apparatus at 37.50C., in vessels designed to permit

TABLE 1Effect of concentration on oxidation of lactose

SUBSTRATE 02 UPTAKE PER CENTINCREASE

cm. per 10 minutes

Purified lactose:Initial concentration, 075 per cent..... 0.30; 0.35; 0.35 150Final concentration, 2.5 per cent............. 0 77; 0.84; 0.78 J

Autoclaved commercial lactose:Initial concentration, 0.5 per cent............ 0.31; 0.33; 0.34 150Final concentration, 2.5 per cent.............0.83; 0.80; 0.75 1

the addition of substrate during the course of the experiment.The cell suspension was placed in the vessel with an initial sub-strate, and three consecutive readings were made at ten-minuteintervals. * Then the new substrate was admitted, and additionalreadings taken in the same manner. Table 1 shows the relationbetween concentration of the two samples of lactose, and oxygenuptake. Manometer values are expressed in linear centimeters.It may be seen that there is a direct relation between the concen-tration of lactose and the oxygen uptake, and that the values forpurified unheated lactose solutions are identical with those for theautoclaved commercial preparation.As a further control on the preceding experiment, the effect on

the rate of respiration in the presence of lactose of the addition

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of a small quantity of glucose (comparable to the amount ofmonosaccharides which might be liberated by hydrolysis duringsterilization) was measured. This is shown in table 2.Here it is seen that the stimulation by small amounts of mono-

saccharide is of greater magnitude, and of shorter duration, thanthat provided by increased concentration of lactose alone. It isimprobable, therefore, that the influence of the concentration oflactose in the preceding experiments can be attributed to productsof its decomposition by heat.

TABLE 2Effect of added glucose on the oxygen uptake in lactose

SUBSTRATE 02 UPTAKE PER CENT INCREASE

cm. per 10 minutes

Purified lactose, 0.5 per cent............... 0.57; 0.62; 0.59 700, rapidlyAdded glucose, 0.02 percent.6.9 ; 4.8 ; 2.8 J decreasing

TABLE 3Growth and fermentation in purified lactose media

ODAY 1DAY 2DAYS 3DAYS 4DAYS

MEDIUM

0.5 per cent lactose, unheated ....... 6.8 6.8 1 0 7.0 2 0 6.8 2 0 6.8 2 03.0 per cent lactose, unheated....... 6.86.8 2 05.6 3 55.0 4 174.8 4 203.0 per cent lactose, autoclaved... ... 6.6 6.6 2 0 5.6 3 12 5.0 4 17 4.8 4 200.5 per cent lactose, unheated, plusGlucose 0.01 percent.6.86.8 2 06.8 2 07.0 3 07.0 3 0Glucose 0.10 per cent............. 6.86.82 06.8 3 06.6 3 06.5 4 0Glucose 0.20 per cent.............. 6.86.6 2 56.6 3 66.5 4 66.4 4 6

FERMENTATION AND GROWTH IN PURE LACTOSE MEDIA

Experiments with the bacterial suspensions showed clearlythat the fermentative activity of the cells is dependent on theconcentration of lactose itself. It was thought desirable tore-examine also the effect of concentration on growth, usingpurified, unheated lactose. Fermentation tubes containing


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different amounts of lactose, with or without added glucose, and1 per cent peptone, M/20 phosphate buffer, and China-BlueRosolic-Acid indicator (Bronfenbrenner, 1918) were prepared.One series of tubes were autoclaved, in another, filtered solutionsof purified lactose were added to the sterile medium. Both setswere inoculated with the slow fermenting strain, and observeddaily for comparison of turbidity, pH, and total gas. Resultsare shown in table 3. It is again evident that the early growthand fermentation of the slow fermenting strain in lactose mediais a function of lactose concentration, and that this effect is nottraceable to impurities or to products of the decomposition oflactose incidental to sterilization of the medium by heat.

SUMMARY

1. The metabolism of a slow-lactose-fermenting strain of theEscherichia coli type, and of its rapidly fermenting variant, inlactose-containing synthetic media, have been studied. Figuresare given showing the development of the cultures and theaccompanying metabolic changes as they occur in these media inrelation to the lactose concentration, and to the presence of anadditional source of carbon.

2. Variation within the culture does not play any part in theearly course of fermentation by these bacteria.

3. The multiplication rate, and the velocity of lactose fer-mentation, are markedly influenced by the concentration of lac-tose in the medium. The effect on the rate of fermentation isdirect, as well as dependent on changes in the rate of growth.

4. The rate of fermentation varies with the bacterial popula-tion of the culture, while the rate of respiration is largely inde-pendent of it. This divergence between the rates of fermentationand respiration is discussed without reference to "phases ofgrowth."

5. The presence of sodium succinate as an accessory source ofcarbon does not appear to influence early lactose fermentation,except through its buffer-effect on the pH of the culture.

6. Succinic acid is removed from the medium more rapidly as

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the ratio of the concentration of succinic acid to that of lactose isincreased.

REFERENCESAYERS, S. H., AND Rupp, P. 1918 Jour. Inf. Dis., 23, 188.BRONFENERENNER, J. 1918 Jour. Med. Res., 39, 25.BRONFENBRENNER, J., AND SCHLESINGER, M. J. 1918 Proc. Soc. Exp. Biol.

and Med., 16, 44.CLARK, W. M. 1915 Jour. Biol. Chem., 22, 87.HERSHEY, A. D., AND BRONFENBRENNER, J. 1935 Jour. Bact., 31, 451.


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the influence of the composition of the medium on

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