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THE EFFECT OF pH ON THE LACTIC ACID FERMENTATIONBY I. C. GUNSALUS AND CHARLES F. NIVEN, JR.

(From the Laboratory of Bacteriology, College of Agricu lture, Corne ll University,Ithaca)

(Received for publication, June 1, 1942)In the fermentation of glucose, homofermentative lactic acid bacteria

(streptococci and lactobacilli) are generally considered to yield 85 to 98per cent of the sugar fermented as lactic acid (l-3). Traces of volatileacids, largely acetic, have been reported (2). Some workers have sug-gested that this may arise from a secondary fermentation or oxidation oflactic acid (4).

In contrast to this, Friedemann (5, 6), in studies of a number of strepto-cocci and pneumococci, has reported the presence of appreciable quantitiesof formic and acetic acids and ethyl alcohol in the molecular proportion of2 : 1: 1. Several earlier workers have reported the presence of formic acidamong the fermentation products of streptococci (7, 8), some having sug-gested that the formic acid is formed from lactic acid, especially underalkaline conditions (9). More recently Barron and Jacobs (10) have re-ported the production of formic and acetic acids from pyruvic acid byresting cells of hemolytic streptococci.

In view of the suggestion (9) that formic acid was formed in an alkalinemedium, the present study was undertaken in order to determine whetherthe pH of the fermentation medium would affect the yields of lactic andvolatile acids. A member of the enterococcus group of streptococci wasselected for study because of the wide range of pH under which these or-ganisms will grow and ferment. While the results found may not apply toal l lactic acid organisms, a partial explanation for the differences found byvarious investigations is at hand. In addition to the greater yields ofvolatile acids and ethyl alcohol the formation of a polysaccharide at alka-line reaction, causing the fermentation medium to become viscous, isreported.

MethodsBacteriological-The culture used, Streptococcus lipuejaciens, Strain 815,was from the departmental culture collection. It conformed to the cul-

tural and serological characteristics of an enterococcus, Lancefield GroupD (11). Resting cell suspensions of this organism have been previouslystudied and found to produce in excess of 90 per cent of the glucose fer-mented as lactic acid (12).

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132 13H AND LACTIC ACID FERM ENTATIONThe medium used in this study contained 1 per cent tryptone, 0.2 per

cent yeast extract, and 0.1 per cent K2HPOd. (In those experiments de-scribed in Figs. 1 and 2, 1 per cent KzHP04 was used.) Glu-cose was sterilized separately and added to the medium aseptically togive a final concentration of 1 per cent. The fermentation flasks wereinoculated with 0.1 per cent of a 12 hour culture and incubated at 37.At the end of the fermentation period sufficient N HzS04 was added tobring the pH below 3. In those experiments in which the reaction washeld at a constant pH, 2 M Na2C03 was added as required. The indicators,added in aqueous solution, were brom-cresol green for pH 5.0, brom-thymolblue for pH 7.0, thymol blue for pH 9.0, and phenol red for pH 7.5f.

Chemical-Residual sugar and lactic acid were run on Somogyi filtrates(13)) the sugar according to the Folin method (14) and the lactic acid by themethod of Friedemann and Graeser (15) or by that of Barker and Summer-son (16)) depending on the amount of acid present and the size of the sampleavailable. The two methods for lactic acid gave similar results when runon a given sample. The volatile acids were recovered by steam distilla-tion, an aliquot was titrated, and formic acid determined by the reductionof mercuric chloride to calomel (17). The acetic acid was determined bydifference after suitable correction for blanks and recoveries. Theidentities of the acids present were established by the method of Osburn,Wood, and Werkman (18). The alcohol was identified as ethyl by thesame method after oxidation to acetic acid by K2Cr0, in acid solution.The alcohol was determined quantitatively by the method of Friedemannand Klaas (19).

ResultsThree 500 ml. Erlenmeyer flasks containing 300 ml. of medium wereinoculated, adjusted respectively to pH 5, 7, 9, and incubated 24 hours.During the growth and fermentation the pH was held at the starting value.The yield of lactic acid decreased with increased pH (Table I). This wasaccompanied by an increase in the volatile acids and alcohol in the ap-proximate ratio of two of formic acid to one each of ethyl alcohol and aceticacid, as suggested by Friedemann. In al l cases the ethyl alcohol wasslightly lower than was expected and the acetic acid correspondingly higher.Since it is known that these organisms oxidize ethyl alcohol to acetic acid(20), itJ is possible that some oxidation occurred during the fermentationowing to the difficulty involved in keeping anaerobic conditions whilealkali was being added. The oxidation-reduction balances would supportthis contention or indicate that some other reduced product occurs andhas not been detect,ed. The low carbon recoveries at more alkaline re-actions may be partially accounted for by the formation of a polysaccharide

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I. C. GUNSALUS AND C. F. NIVEN, JR. 133which accumulated as the fermentation progressed, leaving the mediumvery viscous. Due to difficulties encountered in the separation and puri-fication of this material, no quantitative estimations were made.

In a series of experiments in which neutralization was accomplished byaddition of N NaOH, similar results were obtained but growth was not sogood, possibly because overneutralization was difficult to avoid. Similarresults were also obtained with a culture of Streptococcus faecalis,Strain 1OCl.In a second set of experiments 800 ml. of medium in 1 liter Erlenmeyerflasks were used and 100 ml. samples were taken at intervals as the fer-mentation progressed in order to determine the rate of formation of thevarious products. In this case 1 per cent K*HPOk was added to the

TABLE IE$ect of pH on ProductsThe mediumconsistedof 1 per cent tryptone, 0.2 per cent yeast extract, 0.1 percent KsHPOI, 1 per cent glucose. The entire fermentation was carried out at thepH indicated. The products are measuredn mM per 100mM of CS(glucose 2)fermented.

pH 5.0 PHI.0Lactic acid................................. 87 73Acetic .,,__..__.___..__..___.__.,...,_, 6.1 9.4Ethyl alcohol.. 3.5 7.3Formic acid.. . . . . 7.7 16.8Carbon recovered, ye.. . . . . . 95 90Oxidation-reduction balance.. . . . . 1.02 1.18Ratio, formic to acetic + ethyl.. 2:2.7 2:1.96Glucose 2 fermentedper liter, mM.. . 63.6 112 L

pH 9.06115.611.226.488

1.182:2.03112

medium instead of 0.1 per cent in order that the conditions would be analo-gous to those used by Friedemann (5, 6). During the early part of thefermentation period lactic and formic acids increased at approximatelyequal rates (Fig. 1). At 5 hours the lactic acid accounted for about 40 percent of the sugar fermented and the volatile products for about an equalquantity. At this time the pH had fallen from an initial value of 7.8to 6.5. From the 5th hour on (pH 6.5), the curve for lactic acid productionis parallel to the curve for sugar fermented, during which time the increasein volatile products is negligible. After the 6th hour the rate of fermenta-tion decreased as the limiting pH for the culture was approached but lacticacid continued to accumulate slowly until it accounted for 71 per cent ofthe sugar fermented in 24 hours, at which time the fermentation wasstopped. These data indicate that the rate of production of volatile acidsand alcohol was appreciable only at a pH greater than 6.5.

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134 PH AND LACTIC ACID FER MENTATION

0HOURS

FIG. 1. Streptococcus liquefaciens, Strain 815. Amount of glucose used andproducts formed during fermentation when the pH is allowed to fall. Medium, 0.2per cent yeast extract; 1 per cent each of tryptone, glucose, and K2HP04.

i I OLACTIC

4 8 12 16 20 24HOURSFIG. 2. Streptococcus liquejacien s, Strain 815. Amount of glucose used and prod-ucts formed during fermentation when the pH is held above 7.0. Medium, 0.2 percent yeast extract; 1 per cent each of tryptone, glucose, and KzHPO+A duplicate flask of the medium used in Fig. 1 was inoculated and heldat a pH between 7.5 and 8.0 throughout the fermentation period by the

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I. C. GUNSALUS AND C. F. NIVEN, JR. 135addition of 2 M Na&03 as required. 100 ml. samples were likewise takenat intervals for analysis. As shown in Fig. 2, al l the products continuedto accumulate until the sugar was exhausted. In this experiment the vola-tile acids and alcohol reached about twice the concentration reached in theexperiment shown in Fig. 1. At 24 hours the lactic acid accounted for 56per cent of the sugar fermented, as compared with the 71 per cent in theprevious experiment. The carbon recoveries were better than 90 per centin the early part of the experiment but fel l to 82 per cent at 24 hours.Whether this could be completely accounted for by the accumulation ofslime was not determined for reasons previously mentioned. In this ex-periment the analyses indicated a slight decrease in lactic acid from the7th to the 24th hour.

The slime which forms in alkaline fermentations causes the medium tobecome very viscous. It can be precipitated by acidif ication or by theaddition of 1 volume of alcohol. Once precipitated, it does not dissolvereadily except in normal alkali, from which it can be reprecipitated withacid or alcohol. Due to the limited solubility, its rotation could not bedetermined. The material which is slowly hydrolyzed by boiling 1 NH&SO4 yields some reducing sugar, but not a test for ketose by the Seliwanoffmethod. The purification and study of this material are being continued.

DISCUSSIONThat the fermentation products of such organisms as Clostridium aceto-

butylicum and Aerobacter aerogenes are affected by the reaction of thefermentation mixture is well known. The data here presented indicate asimilar alteration in the yield of various products among certain strains ofstreptococci. The data of Friedemann (5, 6) are interpreted to indicatethat the effect is present among a number of other strains which he ranunder conditions analogous to those used in this work.

The suggestion is offered that the effect of pH is one of the importantfactors involved in the difference in results reported by such workers asFriedemann with highly buffered media and Langwell (8) with culturesneutralized daily, and the workers who have run their fermentations underthe acid conditions optimum for lactic acid production (1,3).

It should be noted that the ratio of volatile products, formic and aceticacids, and ethyl alcohol is similar among enteric bacteria lacking hydro-genlyase (Eberthella typhosa), some pneumococci (5), and Streptococcusliquefaciens grown under alkaline conditions.It is suggested that this factor may also help to explain the results ob-tained by Friedemann (21) and attributed by him to the age of the culture.Although large quantities of polysaccharides have been reported to beformed from sucroseby various streptococci (22), we are not aware of pre-

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136 PH AND LACTIC ACID FERMENTATIONvious report,s of the formation of such substances from glucose by theseorganisms. The significance of this substance should be investigatedfurther.

SUMMARYA homofermentative lactic acid organism, Streptococcus liquefaeiens, hasbeen shown to form large quantities of formic and acetic acids and ethyl al-cohol in the ratio of 2: 1: 1 during the fermentation of glucose in a bufferedprotein-rich medium. This observation confirms the results which Friede-

mann obtained with a number of streptococci.The reaction of the fermentation medium was shown to be an importantfactor in the production of these substances.When the reaction is held at or above pH 6.5, the combined yields offormic and acetic acids and ethyl alcohol may account for 25 to 40 per centof the sugar fermented with the yield of lactic acid falling to 60 per centor less.The formation of a polysaccharide under alkaline conditions of fermenta-tion is reported.

BIBLIOGRAPHY1. Tatum, E. L., and Peterson, W. H., Ind. and Eng. Chem., 27, 1493 (1935).2. Hewitt, L. E., Biochem. J., 26,208 (1932).3. Whittier, E. O., and Rogers, L. H., Ind. and Eng. Chem., 23,532 (1931).4. Peterson, W. H., Fred, E. B., and Anderson, J. A., J. Biol. Chem., 63, 111 (1922).5. Friedemann, T. E., J. Bact., 36, 527 (1938).6. Friedemann, T. E., J. Biol. Chem., 130,757 (1939).7. Foster, L. E., J. Bact., 6, 211 (1921).8. Langwell, B., J. Butt., 9, 79 (1929).9. Walker, E. W. A., and Ryffel , J. H., Brit. Med. J., 2, 659 (1903).10. Barron, E. S. G., and Jacobs, H. R., J. Butt., 36,433 (1938).11. Sherman, J. M., Butt. Rev., 1, 1 (1937).12. Smith, P. A., and Sherman, J. M., J. Butt., in press (1942).13. Somogyi, M., J. Biol. Chem., 86, 655 (1930).14. Folin, O., and Malmros, H., J. Biol. Chem., 83, 115 (1929).15. Friedemann, T. E., and Graeser, J. B., J. Biol. Chem., 160, 291 (1933).16. Barker, S. B., and Summerson, W. H., J. BioZ. Chem., 138, 535 (1941).17. Official and tentative methods of analysis of the Association of Official Agricul-tural Chemists, Washington, 5th edition, 466 (1940).18. Osburn, 0. L., Wood, H. G., and Werkman, C. H., Ind. and Eng. Chem., Anal. Ed.,8, 270 (1936).19. Friedemann, T. E., and Klaas, R., J. Biol. Chem., 116, 47 (1936).20. Greisen, E. C., Thesis, Cornell University (1942).21. Friedemann, T. E., J. Butt., 43, 29 (1942).22. Niven, C. F., Jr., Smiley, K. L., and Sherman, J. M., J. BioZ. Chem., 146, 105(1941).

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