THE EFFECT OF pH ON THE LACTIC ACID FERMENTATION

BY I. C. GUNSALUS AND CHARLES F. NIVEN, JR.

(From the Laboratory of Bacteriology, College of Agriculture, Cornell 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 98
per cent of the sugar fermented as lactic acid (l-3). Traces of volatile
acids, largely acetic, have been reported (2). Some workers have sug-
gested that this may arise from a secondary fermentation or oxidation of

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lactic 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 quantities
of formic and acetic acids and ethyl alcohol in the molecular proportion of
2 : 1: 1. Several earlier workers have reported the presence of formic acid
among the fermentation products of streptococci (7, 8), some having sug-
gested that the formic acid is formed from lactic acid, especially under
alkaline conditions (9). More recently Barron and Jacobs (10) have re-
ported the production of formic and acetic acids from pyruvic acid by
resting cells of hemolytic streptococci.
In view of the suggestion (9) that formic acid was formed in an alkaline
medium, the present study was undertaken in order to determine whether
the pH of the fermentation medium would affect the yields of lactic and
volatile acids. A member of the enterococcus group of streptococci was
selected 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 to
all lactic acid organisms, a partial explanation for the differences found by
various investigations is at hand. In addition to the greater yields of
volatile acids and ethyl alcohol the formation of a polysaccharide at alka-
line reaction, causing the fermentation medium to become viscous, is
reported.

Methods
Bacteriological-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 Group
D (11). Resting cell suspensions of this organism have been previously
studied and found to produce in excess of 90 per cent of the glucose fer-
mented as lactic acid (12).
131
132 13H AND LACTIC ACID FERMENTATION

The 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 to
give a final concentration of 1 per cent. The fermentation flasks were
inoculated 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 to
bring the pH below 3. In those experiments in which the reaction was
held 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-thymol
blue 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

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(13)) the sugar according to the Folin method (14) and the lactic acid by the
method 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 sample
available. The two methods for lactic acid gave similar results when run
on a given sample. The volatile acids were recovered by steam distilla-
tion, an aliquot was titrated, and formic acid determined by the reduction
of mercuric chloride to calomel (17). The acetic acid was determined by
difference after suitable correction for blanks and recoveries. The
identities of the acids present were established by the method of Osburn,
Wood, and Werkman (18). The alcohol was identified as ethyl by the
same method after oxidation to acetic acid by K2Cr0, in acid solution.
The alcohol was determined quantitatively by the method of Friedemann
and Klaas (19).

Results
Three 500 ml. Erlenmeyer flasks containing 300 ml. of medium were
inoculated, 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 was
accompanied 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 acetic
acid, as suggested by Friedemann. In all cases the ethyl alcohol was
slightly 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 fermentation
owing to the difficulty involved in keeping anaerobic conditions while
alkali was being added. The oxidation-reduction balances would support
this contention or indicate that some other reduced product occurs and
has not been detect,ed. The low carbon recoveries at more alkaline re-
actions may be partially accounted for by the formation of a polysaccharide
 I. C. GUNSALUS AND C. F. NIVEN, JR. 133

which accumulated as the fermentation progressed, leaving the medium

very 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 by
addition of N NaOH, similar results were obtained but growth was not so
good, possibly because overneutralization was difficult to avoid. Similar
results were also obtained with a culture of Streptococcus faecalis,
Strain 1OCl.
In a second set of experiments 800 ml. of medium in 1 liter Erlenmeyer
flasks were used and 100 ml. samples were taken at intervals as the fer-
mentation progressed in order to determine the rate of formation of the
various products. In this case 1 per cent K*HPOk was added to the

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TABLE I
E$ect of pH on Products
The mediumconsistedof 1 per cent tryptone, 0.2 per cent yeast extract, 0.1 per
cent KsHPOI, 1 per cent glucose. The entire fermentation was carried out at the
pH indicated. The products are measuredin mM per 100mM of CS(glucoseX 2)
fermented.
pH 5.0 PHI.0 pH 9.0

Lactic acid................................. 87 73 61
Acetic “ .,,__..__.___..__..___.__.,...,_, 6.1 9.4 15.6
Ethyl alcohol.. 3.5 7.3 11.2
Formic acid.. . . .. 7.7 16.8 26.4
Carbon recovered, ye.. . . . . . 95 90 88
Oxidation-reduction balance... .. . . 1.02 1.18 1.18
Ratio, formic to acetic + ethyl.. 2:2.7 2:1.96 2:2.03
GlucoseX 2 fermentedper liter, mM.. . 63.6 112
L 112

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 the
fermentation period lactic and formic acids increased at approximately
equal rates (Fig. 1). At 5 hours the lactic acid accounted for about 40 per
cent of the sugar fermented and the volatile products for about an equal
quantity. At this time the pH had fallen from an initial value of 7.8
to 6.5. From the 5th hour on (pH 6.5), the curve for lactic acid production
is parallel to the curve for sugar fermented, during which time the increase
in volatile products is negligible. After the 6th hour the rate of fermenta-
tion decreased as the limiting pH for the culture was approached but lactic
acid continued to accumulate slowly until it accounted for 71 per cent of
the sugar fermented in 24 hours, at which time the fermentation was
stopped. These data indicate that the rate of production of volatile acids
and alcohol was appreciable only at a pH greater than 6.5.
134 PH AND LACTIC ACID FERMENTATION

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0
HOURS

FIG. 1. Streptococcus liquefaciens, Strain 815. Amount of glucose used and

products formed during fermentation when the pH is allowed to fall. Medium, 0.2
per cent yeast extract; 1 per cent each of tryptone, glucose, and K2HP04.

i I OLACTIC

4 8 12 16 20 24
HOURS
FIG. 2. Streptococcus liquejaciens, Strain 815. Amount of glucose used and prod-
ucts formed during fermentation when the pH is held above 7.0. Medium, 0.2 per
cent yeast extract; 1 per cent each of tryptone, glucose, and KzHPO+
A duplicate flask of the medium used in Fig. 1 was inoculated and held
at a pH between 7.5 and 8.0 throughout the fermentation period by the
 I. C. GUNSALUS AND C. F. NIVEN, JR. 135

addition of 2 M Na&03 as required. 100 ml. samples were likewise taken

at intervals for analysis. As shown in Fig. 2, all the products continued
to accumulate until the sugar was exhausted. In this experiment the vola-
tile acids and alcohol reached about twice the concentration reached in the
experiment shown in Fig. 1. At 24 hours the lactic acid accounted for 56
per cent of the sugar fermented, as compared with the 71 per cent in the
previous experiment. The carbon recoveries were better than 90 per cent
in the early part of the experiment but fell to 82 per cent at 24 hours.
Whether this could be completely accounted for by the accumulation of
slime was not determined for reasons previously mentioned. In this ex-
periment the analyses indicated a slight decrease in lactic acid from the
7th to the 24th hour.

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The slime which forms in alkaline fermentations causes the medium to
become very viscous. It can be precipitated by acidification or by the
addition of 1 volume of alcohol. Once precipitated, it does not dissolve
readily except in normal alkali, from which it can be reprecipitated with
acid or alcohol. Due to the limited solubility, its rotation could not be
determined. The material which is slowly hydrolyzed by boiling 1 N
H&SO4 yields some reducing sugar, but not a test for ketose by the Seliwanoff
method. The purification and study of this material are being continued.
DISCUSSION

That the fermentation products of such organisms as Clostridium aceto-

butylicum and Aerobacter aerogenes are affected by the reaction of the
fermentation mixture is well known. The data here presented indicate a
similar alteration in the yield of various products among certain strains of
streptococci. The data of Friedemann (5, 6) are interpreted to indicate
that the effect is present among a number of other strains which he ran
under conditions analogous to those used in this work.
The suggestion is offered that the effect of pH is one of the important
factors involved in the difference in results reported by such workers as
Friedemann with highly buffered media and Langwell (8) with cultures
neutralized daily, and the workers who have run their fermentations under
the acid conditions optimum for lactic acid production (1,3).
It should be noted that the ratio of volatile products, formic and acetic
acids, and ethyl alcohol is similar among enteric bacteria lacking hydro-
genlyase (Eberthella typhosa), some pneumococci (5), and Streptococcus
liquefaciens 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 be
formed from sucroseby various streptococci (22), we are not aware of pre-
136 PH AND LACTIC ACID FERMENTATION

vious report,s of the formation of such substances from glucose by these

organisms. The significance of this substance should be investigated
further.
SUMMARY

A homofermentative lactic acid organism, Streptococcus liquefaeiens, has

been 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 buffered
protein-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 important
factor in the production of these substances.

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When the reaction is held at or above pH 6.5, the combined yields of
formic and acetic acids and ethyl alcohol may account for 25 to 40 per cent
of the sugar fermented with the yield of lactic acid falling to 60 per cent
or less.
The formation of a polysaccharide under alkaline conditions of fermenta-
tion is reported.
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