LactobaciUus acidophilus. The bacteria were cultivated at 30'C. juice was added). After twenty-four to thirty-six hours, the

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1 GROWTH AND FERMENTATION OF BACTERIA NEAR THEIR MINIMUM TEMPERATURE MILTON J. FOTER AND OTTO RAHN Laboratory of Bacteriology, New York State College of Agriculture, Cornell University, Ithaca, New York Received for publication May 25, 1936 The object of this paper was to study the minimum temperature of fermentation separately from that for growth, and to explain, if possible, the cause for a minimum temperature. Since both these life functions are chemical reactions, they should continue, though at a greatly reduced speed, until the medium freezes solid. This is certainly not always the case with the growth of bacteria; most of them completely cease to grow at temperatures 5 to 1 or more degrees above the freezing point. EXPERIMENTAL APPROACH A first set of experiments was carried out with the technique used by Rahn (1932 p. 134) using Streptococcus lactis, S. fecalis, S. glycerinaceus, S. cremoris, S. liquefaciens, S. mastitidis and LactobaciUus acidophilus. The bacteria were cultivated at 3'C. in a heavily buffered lactose broth (for the Lactobacillus, tomato juice was added). After twenty-four to thirty-six hours, the cultures were neutralized and centrifuged. The bacterial sediment, after being cooled to the desired temperature, was suspended in sterile milk of the same temperature., This series demonstrated the influence of low temperature upon fermentation, independent of growth. Some of the results are given in figure 1. I It was intended to make the inoculum so large that no multiplication would take place, but with Lactobacillus and S. glycerinaceus, this aim was not accomplished as the frequent plate counts revealed. The inoculum was, in million cells per cubic centimeter, with Lactobacillus 6, with S. glycerinaceus 18, with S. lactis 25, with S. fecalis 4 and with S. liquefaciens 5. The sigmoid curves of S. glycerinaceus show plainly that the number of cells slowly increased. 485

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3 GROWTH AND FERMENTATION OF BACTERIA 487 A second series was carried out by inoculating sterile milk with small amounts of the same bacteria, and studying their multiplication as well as their fermentation at different temperatures. Some of these results are shown in table 1. FERMENTATION The amount of acid formed by large numbers of cells of Lactobacillus at O to 2 was within the limits of error. Since the plate counts showed only 6 million cells per cubic centimeter, this does not disprove their ability to ferment. All streptococci produced distinct amounts of acid at 2 (figs. 1 and 2), but the fermentation ceased at a very low acidity. S. fecalis, S. cremoris and S. liquefaciens produced much more acid than the others. At 5, 1 and 15, the endpoints of fermentation became higher for each increase in temperature, but not proportionately, so that at 15, S. glycerinaceus and S. fecalis were lowest, while at, they were highest. From these data and from the plate counts, the fermenting capacities (milligram lactic acid per cell per hour) were computed (table 2). The cells of the large inocula appear to have a greater fermenting capacity. This is partly due to the impossibility of redistributing centrifuged streptococci uniformly in milk; the plate counts indicate the number of clumps rather than individual cells. This accounts also for some irregularities which do not occur in the experiments with small inocula. It must further be remembered that old bacteria (in large inocula) lose their viability before they lose their fermenting power; this would make the fermentation per viable cell appear larger (Rahn and Barnes, 1933). Still another factor might cause differences: the bacteria for the experiments with large inocula had been grown at 3, and it has never been proved that they really contain the same amount of fermenting zymases per cell as those grown at 5 or 1. Table 2 shows also the temperature coefficients of fermentation. By "temperature coefficient" is understood the increase 2 This range will be abbreviated here as which was the predominant temperature.

4 488 MILTON J. FOTER AND OTTO RAHN TABLE 1 Growth and acid formation of lactic bacteria at different temperatures (cells per cubic İOI; centimeter, acid in per cent of lactic acid) STREPTOCOCCUS FECALIS 8TREPTOCOCCIJ8 STREPTCOCCUSLACTISPHILUS LACTI8 LACTOBACILLUS ACIDO- TIME Per Per Plateteacid count Per cent Plate count cent Plate count cent acid acid.c. days 4, , , , , ,, 42 36,, ,, ,, ,, hours ,8 1,2 31,6 187, 1,138, 4,9, 11,, 46,, 81,, 143,, 27,, 35,, 61,, 345,, 4,8 3, 525, 1,4, 34,, 28,, 79,, 89,, 87,, I , 3,9 13, 28, 12, 22, 34, 51, 1,, 1,, 88 1, 795, 44,, 29,, 425,, 36,, , 7, 86, 265, 24, 192, 115, 15, 7, 31, 7,9, 124,, 191,, 144,,

5 GROWTH AND FERMENTATION OF BACTERIA 489 TABLE 1-Concluded.c. 15 TIME hours BTREPTOCOCCUS FECAgs Plate count 4,8 66,, 1,3,, 9,, 88,, 83,, Parcidnt STREPTOCOCCUS LACTIS Plate count 88 5, 25,5, 377,, 51,, 7,, 645,, 79,, 74,, Per cent acid IACTOBACILLUS ACIDO- PMLUS Plate count 7, 128,, 17,, 53,, 77,, 59,, Per cent acid k ,8 4,5, 12,, 58,, 86,, 1,2,, 1,12,, 1,4,, ,5, 289,, 69,, 925,, 1,117,, 99,, 89,, , 38, 6,6, 14,5, 33,, 193,, 267,, 45,, 865,, 53,, in the rate of fermentation (or eventually of growth) for a temperature increase of 1'C. It is designated as Qjo. The temperature coefficient for 1 increase is the square of the temperature coefficient for 5 increase, i.e., Qjo (Q)2. = The most puzzling experience is the low endpoint of acidity in the cultures at. Figure 2 shows the endpoints at different temperatures arranged in two groups with high and with low endpoint at. It is clear that fermentation must be very slow. But since the enzymes are present in the cells, it is difficult to understand why fermentation should not go on with time and

6 49 MILTON J. FOTER AND OTTO RAHN reach ultimately as high an endpoint as it does at higher temperatures. All curves plainly indicate that this is not the case. It seemed as if the enzymes in the cells were injured by prolonged cold. This was tested by holding milk cultures (inocu- TABLE 2 Rates and temperature coefficients of lactic fermentation S. glycerinaceus... S. liquefaciens... S. fecalis... S. tactics... L. acidophilus... S. fecalis S. lactis L. acidophilus... _ S. glycerinaceus... S. liquefaciens... S. fecalis... S. lactis... L. acidophilus... S. fecalis... S. lactis... L. acidophilus... I FUBMUTING CAPACITI (1-1 MIGH. ACID PEU CULL PER HOUR) - I 6 V Large inocula ?? Small inocula TU1MPURATU3U COUlTIUCIUNM FOR 1* INCRUASU *to 5 5 to 1 1*to 1 jto 2 to 3*to 37 to Large inocula Small inocula lated with large numbers of cells as described above) for different lengths of time at, and then raising the temperature suddenly to 3C. and measuring the rate of fermentation. The experiment with S. lactis is given in detail in table 3. The acid of the culture was neutralized just before raising the temperature.

7 GROWTH AND FERMENTATION OF BACTERIA 491 The table shows that after one week, the bacteria were injured: the plate count decreased, and so did the fermenting capacity. In four weeks, the enzyme content per cell had dropped to onefourth of the original. The six-week culture is for unknown reasons better preserved than any other. At 3, the injured cells replenish their enzyme content, but not completely as may be seen from the 2, 4 and 8 weeks old cultures compared with the first 6 hours of the and 3 days old cultures. In the oldest culture, the enzyme content per cell still increases after 12 hours; the injury caused by the cold lasted through 5 generations of cells. WetS~~~~~~W FIG. 2. MAXIMAL AMOUNTS OF ACID REACHED AT DIFFERENT TEMPERATURES BY INOCULATION WITH LARGE NUMBERS OF CELLS Left: species producing high acidity at. Right: species producing low acidity at. With S. fecalis, such injury could not be proved definitely. If it existed at all, it was slight. The difference between these two test organisms is that S. fecalis multiplies at, and is therefore capable of repairing or replacing inactivated enzymes while S. lactis does not multiply at (table 1). GROWTH As shown in table 1, only S. fecalis multiplies at while S. tactis dies slowly. At 5, S. tactis grew very slowly in 32 days to 1 million per cubic centimeter. The Lactobacillus multiplied

8 492 MILTON J. FOTER AND OTTO RAHN at 5 within 2 days from 7, to 265, cells per cubic centimeter, but no further, and after 36 days, the number gradually decreased. This probably corresponds to Orla-Jensen's statement (1919) that he observed growth of streptococci in glucose TABLE 3 Effect of the time of holding at C., on Streptococcus lactis AFTER INCUBATION AT 3 C. FOR TIMUOF AJ'IUR HOLDING HOLDING 2 hours 4 hours 6 hours 8 hours 12 hours 24 hours days 3 days 1 week 2 weeks 4 weeks 6 weeks 8 weeks days 3 days 1 week 2 weeks 4 weeks 6 weeks 8 weeks days 3 days 1 week 2 weeks 4 weeks 6 weeks 8 weeks , (BEFORE NEUTRAL- IZING) MILLONS OF BACTssIA PER CUBIC CENTIMETER TOTAL, IN PER CENT OF LACTIC ACID, PRODUCED AFTER NEUTRALIZATION I- - _ FERMENTING CAPACITY IN 1Ot mgm O to 6 hours 6 to 12 hours 12 to 24 hours < broth without acid formation. It can be easily calculated from the fermenting capacity,.65 X 1-1 mgm., that the 265, cells per cubic centimeter observed would require 27 days to produce.1 per cent lactic acid.

9 GROWTH AND FERMENTATION OF BACTERIA From the plate counts can be computed the maximal growth rates for each temperature, and from these the temperature coefficients of growth (table 4). The most rapid rate obtained is that by S. lactis at 3; S. fecalis grows a little more slowly while the Lactobacillus requires about three times as many hours. Even at 1, S. fecalis grows more slowly than S. lactis while at 5, it grows twice as fast, and at, it still grows while the other does not. TABLE 4 Shortest generation times in hours ORGANISM S. fecalis S. lactis...o L. acidophilus.co TEMPERATURE COEFFICIENTS (Qlo) OF GROWTH to 5 5 to 1 1to 15 to 2 to 3 to 37 to 15., 2 3, 37 4 S. fecalis S. lactis..., L.. acidophilus.lusco TABLE 5 Endpoints of growth, in millions of cells per cubic centimeter S. fecalis ,3 1,12 1,95 1,13 S. lactis ,17 68 L. acidophilus ,23 1, The temperature coefficients show the usual picture of biological experiments; as the temperature approaches the minimum, Qio increases distinctly above the value known for ordinary chemical reactions. Interesting also is the effect of temperature on the "crop," i.e., the maximal number of cells (table 5). Near the minimum temperature, growth ceases early, and the crop is small. The largest crops were obtained over a range of about 1 from the temperature of fastest growth downwards.

10 494- MILTON J. FOTER AND OTTO RAHN DISCUSSION OF THE MINIMAL TEMPERATURE A clear line must be drawn between the temperature responses of the growth processes and those of the processes furnishing energy. This is not always done. Belehr~dek (1935) gives an extensive compilation of minimum temperatures (biological zero) of biological processes arranged in groups where he distinguishes between "growth" and "metabolic processes," but places increase of chlorophyl and repair of injury under "metabolic processes." If these are placed under "growth," the minimum temperature of metabolic processes is almost always the freezing point, while that for growth is almost always higher. This should be expected. Enzymatic processes such as respiration or fermentation, though they may take place within the cell, should hardly be handicapped by hardships of the cell. Growth processes are so interlinked that the retardation of any single reaction might prevent completely the functioning of others. However, the rate of fermentation in our experiments is progressively slower at low temperatures while the temperature coefficient of chemical reactions changes so little in the narrow range of biological events that it is constant for all practical purposes. It has been shown in table 3 that within a week at, the enzyme content of the cell is decreased. The rates of fermentation have been computed, however, from the amount of acid formed during the first days, where injury can have been only slight. The temperature coefficients between and 5 (table 2) are lowest with the two streptococci which grow at, and which consequently can repair and replenish the enzyme while the two species which cannot do this show a high temperature coefficient. Still, this is not quite sufficient to prove that the rate of fermentation has really a constant temperature coefficient, and that only such events as the gradual deterioration interfere. As to the lower final acidity at lower temperatures, the injury of the enzyme through cold is not quite sufficient for an explanation. It suffices only for those temperatures where cells do not multiply while even with S. fecalis (fig. 1), the final acidity increases from to 15. The common assumption regarding the endpoint of fermentation is that the concentration of products

11 GROWTH. AND FERMENTATION OF BACTERIA prohibits further action of the enzyme. If decreased temperature had any effect at all, we should expect it to increase the endpoint. We are coping here with an unknown factor which may be due to a change of equilibrium in the enzyme reaction, or to a change in the water itself though water polymers are probably not the cause. The reason for the cessation of growth at 5 by S. lactis and L. acidophilus while S. fecalis grows even at is still more difficult to find. Since the fermentation below 5 is greatly decreased, it seemed possible that not enough calories were produced to supply the growth mechanism with the minimum energy required for synthesis. This led to the calculation of the amount of energy needed for the synthesis of one cell. Streptococci grow so very poorly without carbohydrates that it seems safe to con- TABLE 6 Lactose required for the doubling of one cell recorded in 1-1 mgm. 495 oo S. fecalis S. lactis L. acidophilus sider the lactic fermentation as practically the only source of energy. If we multiply the generation time (in hours, as given in table 4) by the milligrams of lactic acid per cell per hour, we have the amount of acid formed, or of sugar consumed, for the formation of one cell. The results of this computation are given in table 6. It reveals the surprising fact that the bacteria do not require more food at low temperatures. At low or medium temperatures, the energy consumption is constant, but at temperatures near the optimum, the food consumption per generation increases; the bacteria utilize their food less economically. If this constant sugar requirement were extrapolated below 5 for S. lactis, we should still have growth at, very slow to be sure, but not impossibly slow. The product of fermenting capacity X generation time is a constant, namely, 2 X 1-1 mgm. From 5 to, the fermenting capacity drops to one-

12 496 MILTON J. FOTER AND OTTO RAHN third, consequently the generation should increase 3 times, which would make it 112 hours. Such generation times at low temperatures are not impossible. Therefore, since this event does not take place, it is not the fault of too slow fermentation. S. fecalis utilizes the lactose most economically. S. lactis needs almost twice as much lactose, and the large Lactobacillus 1 times as much in order to make two cells out of one. The most common explanation of growth cessation due to low temperatures is probably the assumption that the various interlinking reactions are influenced differently by a change of temperature, and that they may get "out of step" and upset the growth mechanism. Aside from this BelehrAdek lists four other explanations: (1) Accumulation of toxic products in the cell (this might be the result of the disturbed growth mechanism just mentioned). (2) Change in permeability or ion absorption. (3) Solidification of important lipids. (4) Too great viscosity of protoplasm. This last point, he states as follows: "Such an increase (in viscosity) would considerably hinder free movements of reacting molecules with the result that the biochemical reactions in the cell would be brought to a standstill." This statement is far too general. "Free movement" will usually be interpreted as meaning the rate of diffusion. As far as crystalloids are concerned, this is not hindered appreciably by an increase in viscosity, not even by a change from sol to gel (Gortner, 1929, p. 21). If the term "free movement" is to include selective permeability of the membranes, we are facing a different problem which is probably linked with point (3), solidification of lipids. Point (1) could hardly be considered with organisms having such an enormous surface as bacteria; any product of normal or abnormal cell metabolism could easily diffuse out. Points (2) and (3) can probably not be determined with such small organisms as bacteria.

13 GROWTH AND FERMENTATION OF BACTERIA 497 SUMALRY When large numbers of cells of streptococci are placed in milk at low temperature, lactic acid fermentation takes place even at with all species tested. The rate of fermentation is greatly decreased at, more so with the species which cannot grow at than with those that do. With Streptococcus lactis, incapable of growing at, the enzyme content is distinctly decreased by one week's holding at, and after 4 to 8 weeks, the cells require a number of generations before they come back to their original fermenting capacity. With Streptococcus fecalis, capable of multiplying at, such an effect was not noticed. The total acid produced by each species is lowest at, and increases with temperature. The difference is smaller with those species which grow at, but it is noticeable in all cases (fig. 2) and cannot be explained merely by a deterioration of the enzyme. No explanation can be given for the fact that Streptococcus fecalis and Streptococcus glycerinaceus grow at while Streptococcus lactis and Lactobacillus acidophilus die at this temperature, and can hardly multiply even at 5. The amount of lactose consumed during the doubling of one cell is constant at low and medium temperatures, but increases towards the optimal temperature. Streptococcus lactis requires twice as much lactose as Streptococcus fecalis, and Lactobacillus acidophilus needs 1 times as much to double its cell. The largest bacterial crops were obtained at temperatures between the optimum and 1 below the optimum. Near the minimum, the crops decreased decidedly. REFERENCES BibLEHRADEK, J Temperature and Living Matter. Berlin, Borntraeger. GORTNER, R. A Outlines of Biochemistry. New York, Wiley and Sons. ORLA-JENSEN, S Lactic Acid Bacteria. Copenhagen, Host and Son. RAEN, OTTO 1932 Physiology of Bacteria. Philadelphia, Blakiston. RAEN, OTTO, AND BARNES, M. N Jour. General Physiol., 16, 579.