barton · physiologicalcharacteristicsoflacticacid bacterianearthemaximumgrowth temperature ii....

PHYSIOLOGICAL CHARACTERISTICS OF LACTIC ACIDBACTERIA NEAR THE MAXIMUM GROWTH

TEMPERATURE

II. STUDIES ON RESPIRATION1'2

ROBERT M. STERN AND W. C. FRAZIERDepartment of Agricultural Bacteriology, University of Wisconsin

Received for publication February 25, 1941

Of the many physiological functions of the lactic acid bacterianone has received less attention than respiratory activity, in spiteof its obvious importance to the growth and activity of the cell.For this reason it seemed desirable to investigate respiration bothat optimum temperatures and at temperatures near the maximumfor growth and to attempt to correlate the results with otherphysiological activities.Hansen (1938) studied twelve strains of lactic acid bacteria and

reported Qo2 values for glucose varying from one with Thermo-bacterium cereale and T. helveticum to 124 for Microbacteriumflavtum. BarTon and Jacobs (1938) reported an uptake of asmuch as 1.5 molecules of oxygen per molecule of glucose per hourin studies on hemolytic streptococci. They also studied theactivators and inhibitors of the respiratory mechanism.

Wohlfeil (1930) and Wohlfeil and Ewig (1935), studying therelation of respiration to bacterial numbers during growth, founda correlation between oxygen uptake and numbers of bacteriaand observed a decrease in oxygen consumption per cell with time.They concluded that "bacteria respire during growth at thebeginning more intensively, not because they need more oxygen

1 This work was aided by a grant from the Wisconsin Alumni Research Founda-tion.

2 Published with the consent of the Director of the Agricultural ExperimentStation.

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ROBERT M. STERN AND W. C. FRAZIER

for building up their body substance, but only because there arefewer microbes present in the unit space and because there is moreoxygen available per organism." Martin (1931-2), studyingcurves of rates of growth and of oxygen consumption, found thatthe curve of the rate of oxygen uptake always became a straightline earlier than that of growth. The rate of oxygen consumptionper cell increased rapidly to a maximum near the end of the lagphase of the growth curve, and then declined.Casman and Rettger (1933), working with a variety of bacteria,

studied the relation of certain respiratory enzymes to the limita-tion of bacterial growth at high temperatures. They concludedthat "limitation of bacterial growth by temperatures higher thanthe maximum temperature of growth may be due to the inhibitionof the activities of certain catalytic oxidation-reduction mecha-nisms involved in cellular respiration." Among the enzymesstudied were oxidase, peroxidase, catalase, and succinodehydro-genasq.

EXPERIMENTAL METHODS

The oxygen uptake of "resting" and growing cells of Lacto-bacillus butlgaricus and of two strains of Streptococcus thermophiluswas studied.For the preparation of "resting" cells, cultures were grown in

carrot-liver-extract medium, Stern and Frazier (1941), and, afteran eight hour incubation, were centrifuged at 5,000' r.p.m. forten minutes; the supernatant was removed, the cells resuspendedin Allison's solution, (Allison and Hoover, 1934), and, afterseveral washings, again suspended in Allison's solution. Theturbidity of the suspension was brought to a constant Evelynreading. 'These suspensions were used immediately.The oxygen uptake was determined by the Warburg respirom-

eter in a manner similar to that suggested by Wilson (1938).Values for Qo2 were determined by drying three ml. of the sus-pension at 110"C. Values for the cubic millimeters of oxygenuptake per milligram of nitrogen per hour (Qo2) were determinedby the micro-kjeldahl method of Umbreit and Bond (1936).For studies of oxygen uptake of the growing cells, 100 ml. por-

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CHARACTERISTICS OF LACTIC ACID BACTERIA

tions of freshly prepared carrot-liver-extract medium were placedin Warburg flasks, previously rinsed with cleaning solution andthree or four times with sterile distilled water to remove bacteria.In the central cup of each Warburg flask 0.5 ml. of 20 per centsodium hydroxide and a strip of filter paper were placed toabsorb carbon dioxide. The flasks were inoculated with one ml.of the inoculating cultures, and were immediately connected tothe manometers, and placed in the water bath. After 10 minutes,oxygen uptake readings were made at designated intervals. Allmanometric readings are reported as cubic millimeters of oxygenat atmospheric pressure and at the desired temperature. Ratesof oxygen uptake per milligram of dry weight per unit time werecalculated by means of the Buchanan formula, as modified byStern and Frazier (1941).

RESULTS

Oxygen uptake by "resting" bacteria at optimum temperatures andat temperatures near the maximum

The oxygen uptake at 370C. and at 49.50C. with glucose as thesubstrate was determined. Figure 1 shows that during the first30 minutes oxygen uptake at 49.50C. was more rapid than at370C. QO, values for this period were 55 at 49.50C., and 33.9at 370C. When the respiratory activity was based on thenitrogen content of the cells, values for Qo2 at 370C. and at49.50C. were found to be 321 and 552, respectively. As respira-tion continued at 49.5'C., the rate of oxygen uptake decreasedrapidly, and at the end of 100 minutes the total uptake at thelower temperature was greater than that at the higher tempera-ture, being 398.6 cubic millimeters at 370C. and 356.5 cubicmillimeters at 49.50C. The results indicated a marked degree ofinactivation of respiration at the higher temperature.The effect of the temperature at which the cells had been

grown on the oxygen uptake at 370C. by "resting" cells wasstudied as follows: One culture was grown at 370C. throughseven transfers, another at 49.50C. through seven transfers, anda third through six transfers at 370C. and one transfer at 49.50C.The organisms grown at 49.50C. were incubated for five hours and

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those grown at 370C. for eight hours, at which times each culturehad just reached the peak of the logarithmic growth phase.

400

5300U

z

0/ -O OY10 IJE AT 3MC.I,-200i 0-*-* OXYGEN UPIWKEAT aSC.

0

10 20 30 40 50 60 70 80 90 100TIME IN MINUTES

FIG. 1. THE OXYGEN UPTAKE BY "RESTING" CELLS oF LACTOBACILLU 8 BULGARICUSAT 370C. AND AT 49.50C.

200

0

JI O--o mALL TRANSFERSGWNAT 37t.s0---O GRK/WN 49SCALL TRANSFERS CGAOWN AT 37t.EXCEPT THE LAST AT4O

%.

z

w

TIME IN MINUTES

FIG. 2. THE INFLUENCE OF THE TEMPERATURE AT WHICH LACTOBACILLUSBBULGARICUS WAS GROWN ON THE OXYGEN UPTAKE AT 370C. OF

"RESTING" CELLS OF THE ORGANISM

The cultures were prepared for oxygen uptake determinationsaccording to the methods previously described.

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CHARACTERISTICS OF LACTIC ACID BACTERIA 505

Figure 2 shows that the organisms grown exclusively at 370C.took up three times as much oxygen as those grown at 49.50C.and about twice as much oxygen as did the organisms with onlyone transfer at 49.5'C. Continued cultivation of the organismsat temperatures near the maximum for growth markedly de-creases the respiratory activity of the cells.

Therefore the influence on respiration at 370C. of the holdingof "resting" cells at the two temperatures for one, two, and four

LEGEND200 -O----OCELLS NOT HELD

W_-- CELLS HELD I HOUR AT 3rC.- CELLS HELD 2 HOURS AT 3tC.

~-,CELLS HELD4HOURSAT 17 C.

100 4 ,

0~~~~~~~~~~~~~~~~~~~~~0

0 10 20 30 40 50 60 70TIME IN MINUTES

FIG. 3. THE INFLUENCE OF HOLDING "RESTING" CELLS OF LACTOBACILLUSBULGARICUS AT 370C. FOR VARYING LENGTHiS OF TIME ON THEIR OXYGEN

UPTAKE AT 370C.

hours was determined. Figure 3 shows a small but gradualdecrease in the rate of respiration as the holding period at 37CC.was increased from zero to four hours. When the cells hadreceived no holding treatment, 202 cubic millimeters of 02 weretaken up in 70 minutes. For the cells held for one hour the up-take was 168.5 cubic millimeters, and for those held for two hoursand four hours the uptake was 154 and 126 cubic millimeters,respectively. The cells held for four hours took up 63 per cent,approximately, as much as those that were not held.A similar experiment at 49.50C. (fig. 4) shows that there was a

gradual decrease in the rate of respiration as the time of holding


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was increased from zero to two hours. The cells held for fourhours gave an entirely different type of curve. With them, therewas a very rapid rate of uptake for the first twenty minutes,greater even than for those organisms which had received noholding treatment. Later, the rate decreased rapidly, and in 70minutes the total oxygen uptake was less than 50 per cent of that

300

2501-

2

I-a.

z

ICJ

0

IL

LEGEND*a - CELLS NOT HELD0----0 CELLS HELD I HOUR AT 49.5rC.O-..-..O-CELLS HELD 2 HOURS AT 4.5"C-§ -- CELLS HELD4 HWRS AT 40.PC.

200[-

1501-

iool-

so[-

I I I I I I0 10 20 30 40 50 60 70

TIME IN MINUTES

FIG. 4. THE INFLUENCE OF HOLDING "RESTING" CELLS OF LACTOBACILLUSIuLGARICUS AT 49.50C. FOR VARYING LENGTHS OF TimE ON THEIR OXYGEN

UPTAKE AT 370C.

of the untreated cells. The complete lack of correlation betweenthe rate of respiration of the cells held for four hours and of thosenot held or held for lesser periods of time cannot be adequatelyexplained. A second experiment yielded similar results. Thetotal uptake for the cells that received no holding treatment was255.4 cubic millimeters, for those held one hour, 218.9 cubicmillimeters, for those held two hours, 177.8 cubic millimeters,and for the cells held four hours at 49.50C., 123 cubic millimeters.

Ift

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It is apparent that there is a somewhat greater inactivation ofrespiration for the cells held at 49.50C. than for those kept at370C., especially with those cells held for four hours.

Several other strains of lactic acid bacteria were studied tosupplement the work discussed. Determinations on oxygenuptake were made on suspensions of "resting" cells at tempera-tures suitable for the study of the organisms. Streptococcusthermophilus, strain C0 and strain Me were grown in the same

LEGEND50 Oxygen Uptake 0

0 -0 BY STRAIN MC AT 5OC. /_--4 BY STRAIN MC AT 37C. /

BY STRAIN C3 AT 0C. / /

40 if 9 BY STRAIN C3 AT 37 / / *

V //Z30 /

20Z /A

0 t

0 10 20 30 40 so

TIME IN MINUTES

FIG. B. THE OXYGEN UPTAKE OF "RESTING" CELLS OF STREPTOCOCCUSTHURMOPHILUS STRAIN C3 AND STRAIN MC AT 370C. AND AT 500C.

medium used for Lactobacillus bulgaricus, except that the finalpH was adjusted to 6.7. The "resting" cells were prepared asalready described.The oxygen uptake by Streptococcus thermophilus at 370C.

and at 500C. is shown in figure 5. At both temperatures strainMc took up oxygen at a more rapid rate than did strain C3.This was further emphasized by a comparison of the Qo2 valuesof the organisms at the two temperatures. For strain Mc theQo2 values- at 370C. and at 500C. were 8.5 and 10.3, respectively,

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and for strain C8 3.91 and 5.3, respectively. As with Lactobacillusbulgaricus, it was apparent that the rate of uptake was greaterat the higher than at the lower temperature.

TIME IN HOURSFIG. 6. THE GROWTH AND OXYGEN UPTAKE OF LACTOBACILLUS BULGARICUS IN

CARROT-LIVER EXTRACT MEDIUM AT 370C. AND AT 49.50C.

The difference between the rate of oxygen uptake of these twoorganisms raised the question as to whether both were strains ofStreptococcus thermophilus. Other workers have reported that

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CHARACTERISTICS OF LACTIC ACID BACTERIA 509

this organism takes up little or no oxygen. The observationsmade in this study, however, indicated considerable respiration,especially by the Mc strain. It is possible that strain Me maynot be Streptococcus thermophilus.

Because of the differences between activity at 37TC. and at49.50C. of resting cells, it was decided to study the respiratoryactivities of growing cells and the relationship between growthand respiration of Lactobacillus bulgaricus at 37TC. and at 49.50C.

TABLE 1Growth and oxygen uptake by Lactobacillus bulgaricus in carrot-liver-extract medium

at 37'C. and at 49.50C.

ORGANISMS GROWN AT 37 C. ORGANISMS GROWN AT 49.50C.

TIMEC Oxygen OxygenDry weight Oxygen uptake per Dry weight Oxygen uptake perper ml.upae

mgm. of dry per ml. Oxygen m o rof culture uptake weight per of culture uptake weight per

unit time unit time

hours mgm. mm' mm' mgm. mmt mm$

1 0.0040 155.8 0.0060 155.02 0.0415 326.0 529.0 0.0480 378.0 552.03 0.1385 567.0 149.7 0.1385 732.0 207.531 0.2465 771.0 108.7 0.2030 845.0 67.14 0.3700 1,175.0 132.2 0.2525 924.5 35.14i 0.5320 1,704.0 118.6 0.3030 995.5 25.65 0.7590 2,385.0 106.6 0.3470 1,050.0 16.15j 0.8880 2,926.0 65.8 0.3580 1,097.0 13.46 1.0250 3,622.0 71.6 0.3635 1,140.0 11.8

26 1.1900 4,105.0 43.57 1.3180 4,575.0 40.1 0.3900 1,220.0 10.6

Figure 6 shows that at 37TC. the curve of oxygen uptake issimilar to that of growth, indicating a close relationship betweengrowth and oxygen uptake. At seven and one-half hours,growth had practically ceased while respiration continued at arapid rate to the twelfth hour when the determinations werediscontinued.No such correlation was evident at 49.50C. In this case the

rate of oxygen uptake by the growing cells was very rapid in thefirst three hours, being proportionately greater than the rate ofgrowth. Later the rate of respiration decreased anid by the fourth


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hour the rate of growth was greater than that of respiration. Thisrelationship existed until growth had ceased, after which oxygenuptake continued at a slow rate throughout the duration of theexperiment.

Table 1 lists values for the cubic millimeters of oxygen uptakeper milligram of dry weight per unit time. These data areparticularly worth noting since they effectively emphasize thedifferences in respiratory activity between the organisms grownat 49.500. and those grown at 3700. The uptake per milligramof dry weight in the second hour at 49.5TC. was 552 cubic milli-meters, and at 3700. it was 529 cubic millimeters. During thistime the organisms grown at the higher temperature were in thelogarithmic growth phase, while those grown at the lower tempera-ture were still in the lag phase. At the third hour the dry weightsper ml. of culture were the same at both temperatures, but therewas considerably more uptake per milligram of dry weight at49.50C. than at 37CC. After this time respiration by the organismsgrown at the higher temperature dropped off rapidly, and by thesixth hour when growth just about had ceased, the oxygen uptakeper milligram of dry weight during the preceding half hour wasonly 11.8 cubic millimeters. By the third hour the culture,grown at 370C., was in the logarithmic growth phase. Duringthe next three hours the oxygen uptake decreased at a relativelyslow rate, and at the end of seven hours, at which time growthpractically had ceased, the uptake per milligram of dry weightwas 40.1 cubic millimeters. Thus, at the same stage of thegrowth cycle more than three times as much oxygen was takenup per milligram of dry weight at 3700. as at 49.50C.As a whole the results of the studies on oxygen uptake reveal

a more rapid respiration at 49.500. than at 37CC., and that con-tinued incubation of either "resting" cells or growing cells resultsin a considerable decrease in the rate of activity at the highertemperature.

DISCUSSION

It is shown that the rate of respiration is stimulated and thatthe respiratory enzymes are soon inactivated at temperaturesnear the maximum for growth, and that shortly thereafter growth

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ceases. Furthermore it has been pointed out, Stern and Frazier(1941), that the maximum amount of growth of Lactobacillusbulgaricus obtained at 49.50C., depends largely on the number ofcells in the inoculum. At 370C. differences due to the size of theinoculum are apparent only during the early stages of growth,and in all cases no apparent differences in the maximum amountof growth are observed. At 49.50C., however, increase in num-bers is considerably more rapid when the larger inocula areemployed. Wide variations of a similar nature were obtainedin investigations on the influence of age of inoculating cultureon the growth and activity of the organisms at the two tem-peratures.On this basis it was postulated that since more of the respiratory

enzymes are present in younger and larger inocula, there will bemore respiratory activity, and thus more growth will take placethan when smaller or older inocula are employed. Further it wasshown that growth of cultures at 49.50C. ceased at about thesame time regardless of the size of the inoculum employed; hencethe cessation of growth was due to the inactivation of certainenzymes necessary for growth and the inability of the organismsto produce at 49.50C. these enzymes, probably the respiratoryenzymes. Observations also revealed that when organisms wereplaced at optimum temperatures after reproduction had ceasedat the higher temperature, growth soon was initiated and theturbidity was similar to that which normally occurred at optimumtemperatures. The inference is that at 49.50C. the enzymesresponsible for growth, perhaps the respiratory enzymes, weresoon inactivated, while at 370C. no such inactivation was evident,and growth continued until limited by the acid produced.

SUMMARY

1. The oxygen uptake of "resting" cells of Lactobacillus bul-garicus was more rapid during the first 30 minutes at 49.50C.than at 370C., but after this time the rate dropped rapidly at thehigher but not at the lower temperature.

2. There was somewhat greater inactivation of the oxygenuptake by the ''resting" cells of Lactobacillus bulgaricus at 49.50C.

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512 ROBERT M. STERN AND W. C. ZIER

than at 3700., especially with the suspensions of cells held for thelonger periods of time.

3. There was a marked decrease at 37TC. in the rate of oxygenuptake of "resting" cells of Lactobacillus bulgaricus when theorganisms were cultivated at 49.50. instead of at 3700.

4. Marked differences were found in the rate of oxygen uptakeof "resting" cells of two strains of Streptococcus thermophilus.With strain Mc considerable oxygen was taken up while withstrain C3 only a small amount of respiratory activity was observed.

5. The growth curve and the curve of oxygen uptake of L.bulgaricus were very nearly the same during the period of activegrowth at 3700. After reproduction had ceased, respirationcontinued at a rapid rate. At 49.500. the rate of oxygen uptakewas very rapid during the early stages of growth, but after severalhours of incubation there was a marked inactivation of the respir-atory mechanisms, which was soon followed by a decrease in therate of growth.

REFERENCESALLsON, F. E., AND HoOVER, S. R. 1934 An accessory factor for legume nodule

bacteria. J. Bact., 27, 561-581.BARRON, E. S. G., AND JACOBS, H. R. 1938 Oxidations produced by hemolytic

streptococci. J. Bact., 36, 433-449.CASMAN, E. P., AND RETTGER, L. F. 1933 Limitation of bacterial growth at

higher temperatures. J. Bact., 26, 77-123.HANSEN, P. A. 1938 The respiration of rod-shaped lactic acid bacteria. Zentr.

Bakt. Parasitenk., II., 98, 289-297.MARTIN, D. S. 1931-2 The oxygen consumption of Escherichia coli during the

lag and logarithmic phases of growth. J. Gen. Physiol., 15, 691-708.STERN, R. M., AND FRAZIER, W. C. 1941 Physiological characteristics of lactic

acid bacteria near the maximum growth temperature. I. Growth andacid production. J. Bact., 42, 479-499.

UMBREIT, W. W., AND BOND, V. S. 1936 Analysis of plant tissue. Applicationof a semi-micro-kjeldahl method. Ind. Eng. Chem., Anal. Ed., 8,276-278.

WILsoN, P. W. 1938 Respiratory enzyme systems in symbiotic nitrogen fixation.I. The "resting cell" technique as a method for study of bacterialmetabolism. J. Bact., 85, 601-623.

WOELEIL, T. 1930 Ueber die Abhangigkeit der Atmungsintensitit atmenderBakterienaufschwemmungen von der Bakterienzahl. Zentr. Bakt.Parasitenk., I., Orig., 117, 202-212.

WOHLFEIL, T., AND EWIG, W. 1935 Ueber Atmung und G§rung und ihre Ab-hangigkeit von der Bakterienzahl. Zentr. Bakt. Parasitenk., I.,-Orig., 138, 419-424.


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barton · physiologicalcharacteristicsoflacticacid bacterianearthemaximumgrowth temperature ii....

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