organisms. All cultures conformed with the usual cultural and serological characteristics of the species or groups designated.

Transcription

1 THE FERMENTATION OF GLYCEROL BY STREPTOCOCCI I. C. GUNSALUS Ain J. M. SHERMAN Laboratory of Bacteriology, College of Agriculture, Cornell Univer8ity, Ithaca, New York Received for publication July 1, 1942 Although glycerol has been used as a test substrate in studies of the fermentation reactions of lactic acid bacteria the results obtained have not been completely satisfactory. Orla-Jensen (1919), using the amount of titratable acidity produced as a criterion of fermentation, has reported only one-fourth to one-half as much acid formed from glycerol as from glucose under analogous conditions. In addition, cultures reported as non-glycerol-fermenters frequently gave slightly higher-titrations than controls in the basic medium without glycerol. Less acid production from glycerol than from other substrates has also been reported by Fred, Peterson and Davenport (1920). Among the workers who detected fermentation by decrease in ph, several have reported variable results and slight or delayed fermentation (Edwards, 1932; Rudert, 1940; Simmons and Keogh, 1940). During studies on the dehydrogenases of Streptococcus mastitidis, Gunsalus and Wood (1942) observed several strains of this species, which is not generally reported to ferment glycerol, to dehydrogenate glycerol using methylene blue as hydrogen acceptor. Gunsalus and Sherman (1942) extended this observation to include acid production. Since these observations suggested additional need for a study of the metabolism of glycerol the present work was undertaken. This report deals with the production of acid from glycerol by growing cultures. METHODS The streptococci used were from the departmental culture collection; some had previously been received from other laboratories interested in this group of organisms. All cultures conformed with the usual cultural and serological characteristics of the species or groups designated. A strain of Staphylococcus aureus, a pneumococcus, and several cultures of lactobacilli were used in some experiments for comparative purposes. A base medium consisting of 1 per cent tryptone and 1 per cent yeast extract, to which the substrates were added before sterilization, was used throughout the experiments. The amount of growth was measured turbidimetrically using a nephelometer similar to that described by Stier, Arnold and Stannard (1934). The readings, as recorded, are an approximately linear function of the quantity of growth over the range used. Each scale unit is equal to mgm. of bacterial nitrogen per 10 ml. of medium. Fermentation was detected by determining final ph after growth by means of a Beckman ph meter using a glass electrode. 155

2 156 I. C. GUNSALUS AND J. M. SHERMAN RESUILTS During early experiments the time of incubation was observed to be an important factor in determining the final ph reached during glycerol fermentation. This effect is much more marked than with other substrates. Since the final ph was reached in 10 days at this time and temperature were selected as satisfactory. One-half per cent substrate is sufficient for the cultures to reach their limiting ph in the base medium used. Glycerol as a substrate for Streptococcus mastitidis Using 5 strains of Lancefield group B streptococci the value of glycerol, as source of energy for growth, was studied by deternining the amount of growth and the final ph after 10 days. The values obtained were compared to those obtained in the base medium and in glucose broth. The data in table 1 indicate the quantity of growth obtained and the final ph reached in each case. One TABLE 1 Growth and limiting ph during glucose and glycerolfermentation by group B streptococci Base medium: 1 per cent tryptone; 1 per cent yeast extract 10 DAYS-37 C. CULTURE NO. Base medium +0.5 per cent glycerol +0.5 per cent glucose Growth* Final pht Growth Final ph Growth Final ph 67a b P D G Average a az.1 * Nephelometer reading (1 scale unit = mgm. bacterial nitrogen per 10 ml.) t Sterile base medium gave a ph of 6.7 after incubation at 370C. for 10 days. can observe that, while the base medium supported growth, the addition of glycerol approximately doubled the amount and afforded a drop in ph of about 1 unit. Thus, glycerol served as an energy source and as a substrate for acid production. Glucose supported 3 to 4 times the growth obtained with glycerol and gave a lower ph. The limiting ph, near 4.3, with glucose is characteristic for Streptococcus mastitidis. As will be shown in more detail later, the limiting ph for glycerol fermentation is about 1 ph higher than for glucose. Aerobic versus anaerobic glycerol fermentation Streptococci of groups B and D plus one strain of Staphylococcus aureus were grown under anaerobic and aerobic conditions in the same media used in the previous experiment. The data presented in table 2 indicate the need of oxygen for acid production from glycerol with all strains tested except 10C0 and 26C0.

3 FERMENTATION OF GLYCEROL BY STREPTOCOCCI These are strains of Streptococcus fecalis and Streptococcus zymogenes, respectively. It should be noted that these two strains, which had been previously reported to ferment glycerol, gave the same final ph (5.0) anaerobically as aerobically. The other strains of enterococci, No. 24, Streptococcus fecalis, and 98d, Streptococcus durans, as well as the group B cultures which produced acid only aerobically, had not been. previously reported to ferment glycerol. The Staphylococcus aureus culture grew well in the base medium aerobically causing it to become alkaline. However, anaerobically the growth was poor in the base medium and the ph remained at 7.0. Acid production from glycerol was apparent under aerobic but not under anaerobic conditions, the growth in the latter case being equal to that in the base medium. The need of oxygen for TABLE 2 Aerobic Vs. anaerobic "fermentation" of glycerol Base medium: 1 per cent tryptone; 1 per cent yeast extract FINAL ph-10 DAYS AT 37 C. CULTURES Base medium +0.5 per cent glycerol +0.5 per cent glucose robic A e robic Anaerobic Aerobic Anaerobic Control (medium) Streptococci Group B 70b P Gl Group D locl C d Staph. aureus acid production by these strains must indicate the necessity for an oxidation of glycerol preceding truly fermentative changes. Therefore the term fermentation as applied to the dissimilation of glycerol by these organisms is used rather loosely and should, in this paper, be construed to mean acid production as fermentation tests are ordinarily run. Factors affecting the rate and quantity of acid production from glycerol For those organisms which require oxygen for their metabolism of glycerol one would expect that the rate of oxygen supply should affect the rate of growth and acid production. Thus, the time of incubation and the depth of the medium are factors in acid production. The statements of Edwards (1932) and Simmons and Keogh (1940) that acid production was not apparent until the 3rd to 6th 157

4 158 I. C. GUNSALUS AND J. M. SHERMAN day could be interpreted as due to the slow rate at which oxygen diffuses into the medium. Our results are of similar nature. In ordinary test tubes (10 ml. of medium in 8 x 6-inch tubes) about 10 days at was required for cultures which ferment glycerol only aerobically to reach their limiting ph. During the first 5 days the ph fell to between 5.5 and 6.5 with most cultures. After 10 days' incubation the ph approached 5.0. No adaptation appeared upon continued transfer of cultures in glycerol. Cultures grown in shallow layers of medium reached the final ph sooner than those grown in ordinary test tubes-in most cases by the 3rd to the 5th day. In several cases the final ph was lower under these conditions than in test tubes but it never fell below This was also found to be the limiting ph of glycerol fermentation by those enterococci which did not require oxygen for the process (see table 2). There was considerable variation in the final ph even with cultures grown in shallow layers. This may be in part due to the different sensitivities of the strains to hydrogen peroxide which is formed when glycerol is oxidized by these organisms. In order to insure the presence of sufficient glycerol for the organisms to attain their limiting ph, concentrations of 0.1 to 2 per cent were tried. The final ph was the same for all concentrations from 0.4 to 2 per cent glycerol. The composition of the medium was also varied in an attempt to obtain a lower final ph, but without success. If the cultures were grown in tryptone alone some strains grew more poorly and did not attain so low a final ph as when yeast extract was present. Acid production from glycerol by various streptococci In order to determine which streptococci were able to produce acid from glycerol, either aerobically or anaerobically, a number of strains of the representative groups of streptococci were tested under aerobic conditions as described in this paper. A summary of the data is presented in table 3. None of the 29 group A strains tested produced acid from glycerol under the conditions used. Although there have been but few reports of tests of group A strains upon glycerol since the advent of the Lancefield precipitin method, we know of no report of glycerol fermentation by such strains. Edwards (1932), however, reported 18 of 36 strains of human origin to produce very slight acid from glycerol. Five trehalose +, sorbitol -, strains from animal sources ("human" group C's) were reported to ferment glycerol much more vigorously than cultures from human sources. We, too, found considerable acid production with "human" group C cultures but failed to find acid production with group A strains. The difference with group A strains may lie in methods. Edwards used an infusion casein-digest medium with initial ph of 7.6 and Andrade's indicator to detect acid production, whereas we used a tryptone yeast extract medium with original ph of 7.0 and determined final ph. The 37 group B cultures tested were all found to ferment glycerol. Thirtythree of the strains tested gave a final ph of whereas the remaining 4 reduced the ph only to as compared to 6.6 for the control medium.

5 FERMENTATION OF GLYCEROL BY STREPTOCOCCI This report of glycerol fermentation by Streptococcus mastitidis is contrary to the results generally reported for this organism (Sherman, 1937). Gunnison, et al. (1940b) reported that 17 of some 250 group B cultures "irregularly produced a slight amount of acid from glycerine" in a 7-day incubation period. Simmons and Keogh (1940) have reported all of 100 group B strains of human origin to ferment glycerol within a 2-week period. While about one third of the strains tested by us were of human origin, no difference was noticed in their fermentative ability. Only 13 group C strains were available but of the 6 "animal" cultures (trehalose -, sorbitol +) none fermented glycerol and all 7 of the "human" cultures (trehalose +, sorbitol -) fermented this substrate. The fermentation of glycerol by "human" C strains is in agreement with the recent report of Simmons TABLE 3 Acid production from glycerol by streptococci Base medium: 1 per cent tryptone; 1 per cent yeast extract ENAL ph-10 DAYS AT 371C. CULnTUJlRES NO. STRNS Base medi per cent CULTURZS lrztese glyeeiumrol glucose Range Average Range Average Range Average Group A Group B Group C "animal" "human" Group D.[... Group E Group G "minute" "non-minute" S. lactis S. salivarius & Keogh (1940) who studied 169 strains, and with the previous reports of Edwards (1932) and Sherman (1937). Gunnison, et al. (1940a), however, did not observe glycerol fermentation with "human" group C strains under their test conditions. Of 29 enterococci tested all fermented glycerol, though two Streptococcus durans cultures reduced the reaction by only about 0.5 ph unit. The remaining 7 S. durar& cultures reduced the ph to or 1 to 1.5 units. This organism, which has not previously been reported to ferment glycerol, does so only aerobically. Glycerol fermentation has heretofore been generally reported as a variable characteristic for Streptococcusfecalis, (Sherman, Mauer and Stark, 1937). Of 9 strains here tested all produced acid but several strains did so only aerobically. The remaining enterococci, Streptococcus zymogenes and Streptococcus 159

6 160 I. C. GUNSALUS AND J. M. SHERMAN liquefacies, are generally reported to ferment glycerol -and in our studies most strains were found to do so anaerobically. Of the 4 strains of S. zymogenes previously reported by Smith and Sherman (1938) not to ferment glycerol, 3 were tested and found to produce acid aerobically under the test conditions used, although 1 strain reduced the ph only to 6.1. Of three group E strains available all produced acid though one only weakly, giving a final ph 6.2. Forty group G strains were tested. Twelve of these which were of the "minute" type did not ferment glycerol. The remaining 28 strains, "non-minutes", all fermented glycerol. This result is in agreement with those of Simmons and Keogh (1940). Several strains each of Streptococcus lactis, Streptococcus salivarius and Streptococcus bovis and one of Streptococcus thermophilus were tested but none of them produced acid under the conditions used. In one test with Streptococcus lactis (no. 125) in a shallow layer of medium, feeble acid production was encountered. It is possible that different conditions might allow detection of glycerol fermentation by this and other strains. Several lactobacilli cultures and one pneumococcus culture were tested for acid production from glycerol. Lactobacillus acidophilis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus delbreuckii and Lactobacillus lactis produced acid aerobically. Of these only Lactobacillus lactis produced acid from glycerol anaerobically. (Lactobacillus bulgaricus was unable to grow in the glycerol test medium.) The one pneumococcus strain tested formed acid from glycerol aerobically but not anaerobically. DISCUSSION With many lactic acid bacteria, acid production from glycerol takes place only under aerobic conditions, relatively few being able to ferment it anaerobically. On the other hand, a number of species of streptococci do not produce acid from glycerol under either condition. In the fermentation of glycerol the limiting ph is somewhat higher than with most substrates. In the case of those organisms which ferment glycerol anaerobically, oxygen not being a factor, the limiting ph is near 5, as compared with about 4 with glucose. Another factor contributing to a higher limiting ph with glycerol for those organisms which "ferment" only aerobically is their sensitivity to hydrogen peroxide which accumulates during the oxidation and fermentation of this substrate (MacLeod, 1938; Gunsalus and Umbreit, 1942). Other factors involved in acid production from glycerol may need clarifying. When the fermentation of glycerol is observed under conditions allowing sufficient oxygen to be present, it appears to be possible to draw useful lines between "animal" and "human" strains in group C and "minute" and "nonminute" members of group G. Our apparent separation of the group A streptococcus from those strains of groups C and G most likely to be confused with it by the inability of group A strains to ferment glycerol should be checked under similar conditions with a

7 FERMENTATION OF GLYCEROL BY STREPTOCOCCI 161 larger number of cultures. It is too much to believe that such a separation would be possible by this simple means. Against this possibility are the data of Edwards (1932) in which 18 of 36 hemolytic strains from human sources, not tested by the Lancefield precipitin method but in all probability including group A cultures, fermented glycerol. To our knowledge, the necessity for the oxidation of a substrate preceding its fermentation in order to supply energy for growth has not previously been reported for organisms which contain only the flavin type of respiration. Staphylococcus aureus which contains iron catalysts produces acid from glycerol only aerobically. This confirms the observations or Colwell (1939) who has reported staphylococci to produce acid only aerobically from mannitol as well as glycerol. SUMMARY Oxygen is necessary for acid production from glycerol by the majority of lactic acid bacteria. These organisms, which lack iron catalysts, require oxygen to utilize glycerol as an energy source. Of the organisms tested only some "enterococci" and one strain of lactobacillus produced acid from glycerol anaerobically. Whether acid is produced aerobically or anaerobically the limiting ph is about 5.0. When acid production under aerobic conditions is used as a criterion of fermentation, suggestive results are obtained: the group C streptococcus of the so-called "human" variety ferments glycerol whereas the "animal" variety does not. In group G the "minute" type does not ferment glycerol whereas the other members of the group do. All members of groups B and D tested ferment glycerol. None of the members of group A tested nor members of the "lactic" and "viridans" groups of streptococci produce acid from glycerol under the conditions used. REFERENCES COLWELL, E. V The relation of aerobiosis to the fermentation of mannitol by staphylococci. J. Bact., 37: EDWARDS, P. R The biochemical characters of human and animal strains of hemolytic streptococci. J. Bact., 23: FRED, E. B., PETERSON, W. H., AND DAVENPORT, A Fermentation characteristics of certain pentose destroying bacteria. J. Biol. Chem., 42: GUNNISON, J. B., LUXEN, M. P., CUMMINGS, J. R., AND MARSHALL, M. S. 1940a The differentiation of hemolytic streptococci from various sources by the group precipitin reaction and by biochemical tests. J. Bact., 39: GUNNISON, J. B., LUXEN, M. P., MARSHALL, M. S., AND ENGLE, B. Q. 1940b Hemolytic streptococci in raw market milk. J. Dairy Sci., 23: GUNSALUS, I. C., AND SHERMAN, J. M Glycerol oxidation and fermentation by streptococci. J. Bact., 43: 22. GUNSALUS, I. C., AND UMBREIT, W. WV Unpublished data. GUNSALUS, I. C., AND WOOD, A. J The dehydrogenation of alcohols by streptococci of Group B. J. Bact. (in press). MAcLEOD, C. M Metabolism of "sulfapyridine-fast" andl parent strains of pneumococcus type I. Proc. Soc. Exptl. Biol. Med., 41:

8 162 I. C. GUNSALUS AND J. M. SHERMAN ORLA-JENSEN, S The Lactic Acid Bacteria. Copenhagen. RUDERT, F. J A study of Lactobacillus casei and related organisms. Thesis, Cornell University. SHERMAN, J. M The streptococci. Bact. Revs., 1: SHERMAN, J. M., MAUER, J. C., AND STARK, PAULINE Streptococcus fecalis. J. Bact., 33: SIMMONS, R. T., AND KEOGH, E. V Physiological characters and serological types of haemolytic streptococci of groups B, C and G from human sources. Austral. J. Exptl. Biol. Med. Sci., 18: SMITH, F. R., AND SHERMAN, J. M The hemolytic streptococci in human feces. J. Infectious Diseases, 62: STIER, T. J. B., ARNOLD, W., AND STANNARD, J. N A photoelectric densitometer for use with suspensions. J. Gen. Physiol., 17: Downloaded from on November 15, 2018 by guest