Effect of Continuously Controlled pH on Lactic Acid Fermentation J
YIELD AND CONVERSION LLOYD L. KEMPE', H. 0. HALVORSON2, AND EDGAR L. PIRET University of Minnesota, Minneapolis, Minn. T h e yield and rate of lactic acid production in wheat grit mashes are functions of the pH of fermentation. U p to an optimum value, the yield varies directly with pH; above the optimum, an inverse relationship exists. The logarithm of the rate of fermentation-varies directly with pH throughout the range studied. When nutrient salts and yeast extract are added to wheat grit mashes, the pH Present address, University of Michigan, Ann Arbor, Mich. Present address, University of Illinois, Urbana, Ill.
of maximum yield is lower than that of the original medium and the yield is slightly higher. The rate of lactic acid production is increased when the yeast extract concentration is changed from 0.0 to 0.7%. In order to obtain a high yield and rate of lactic acid production when the pH is controlled in the calcium carbonate buffer range, i t is necessary to add nutrient salts and accessory growth factors to wheat grit media. However, the same high yield and rate can be obtained without these additions if pH is precisely controlled at optimum value.
Figure 1. Record Chart from pH Recorder-Controller 1852
Ssp(smbsr 1950
INDUSTRIAL AND ENGINEERING CHEMISTRY
LTHOUGH lactic soid could become avaluable raw material for American industry, it is now of minor importance and its status ie not likely to change until more efficient recovery and fermentation processes are developed ( 6 ) . Bemuse reliable automatic pH control equipment is a recent development, little informstion is available concerning the effecte aft^ environmental factor on the unit process of fermentation. Longsworth and Maclnnes ( 8 ) reported different rates of lactic acid production by Lacto6aeillw acidophilus at the controlled p H values of 6.0 and 7.0 in B synthetic medium. Leonard,Peterson, and Jobrrson (4) fermented pretreated waste eulfite liquor to Imtio mid by lnoculstion with a culture medium containing LI high concentration of malt sprouts. They varied the eontrolled pH from about 5.1 to 6.0 and found sn optimum at 5.8 when the pH was not changed during the fermentation. These studies indicate that changes in the hydrogen ion activity influence the coum of the lsctio acid fermentation. This paper p m n t s data to show that the continuously controlled pH level not only alters the yield and average rate of lsctio acid production, but alm aRecta the quantitative requirements of accessory growth fsctors by the organism concerned. APPARATUS
The electrical apparatus uaed for pH control w w of standard .oomercial manufseture. It consisted of a g l w electrodecalomel half-cell system, m electronio amplifier, and B sensitive recording potentiometer. A mechanism in the lstter unit pro-
Figure 2. Gemernl View of-Rlemrding and Control Fquiprnent
1853
vided control within k O . 0 3 H unit bt any desired setting between 3 and 10 by selective$ activating a pum that injected alkali into the fermenting mash 84 required. {he pump wan made entirely of glass and rubber. Fermentations were conduoted at 45" C. in a I-gallon vitrified clay crook. The temperature was eetablided by sn incubstor and deviations were noted an a meording thermometer. On both the pH- and temperature-recording instruments, %-hour charts were used, 84 illustrated in Figure 1 for run 34. A picture of thst part of the a paratus located outside the incubator sppesra in Figure 2; that focated on the inside is shorn in Figure 3.
METHODS
The m h wss prepared by rtirring Mx1 ml. oi cool tap water l n t ~450 g r a m of air-dry whest grite containing approximately 65% starch, and sdding 2 liters of warm ta water while the mixture w m gently heated and stirred. d e n the temperature reached 643" C., 2 grams of mmmeroially repared Aspergillus mywe liquefying diastase were incorporate!. The temperature WSY then raised to 70" C. and mainbined st this value for 30 minutes.
Diseolved gssea were removed before sutoelsving by boiling at reduced pressure after adding 2 drops of oleic mid to prevent fosminn durinc the ~rocess. The mixture wm then autoclaved at
Runs 1 through 10 differkd from suooeeding onee-at-this juncture. In the first ten rum the fermentation proceeded aimultaneaual with sacohsrification bemuse a atarter culture was added d n g with the enzyme; in all others, sacoharific~tionwas canied out as an independent preliminary operation, 80 that the initial mgsr content of the mash could be established. In these later P U ~ Sthe saeehsrifieation was allowed to oontinue for 24
Figure 3.
Fermentation Vessel and Contml Equipment
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
1854
Vol. 42,
No. 9
TABLE I. LACTICACIDYIELDAND SUGARCONVERSION FOR HOMOFERMENTATIVE FERMENTATIONS OF UNFORTIFIED WEEATGRIT MEDIUMSAT 45 ' C.
Run NO.
pH of Run
Final Volume of Mash, Ml.
Time of Run, Hours
4.4 4.75 4.75 5.00 5.60 11 6.00 12 6.20 13 6.20 14 6.10 15 4,186 16 4.40 17 4.60 18 4.80 19 4.80 Initial sugar estimated. 1 2 3 4 10
36:O 46.0 60.0 41.0 25.0 23.5 19.6 54.0 46:O 23.0 27.5
.*
Sugar Initial Gram'
Sugar Final, Grams
230' 230° 230° 230* 230" 233 233 227 283 186 217 241 232 256
218 222 192 49 86 82 33 33 37
...
...
... ..,
...
Lactic Acid Final, Grams 8
Sugar Used, Grams
... ...
49 47 168 121 164 163 138 182 16 11 52 53 24
Sugar Cqnversion, %
Lactic Acid Yield, %
... ...
ii
77 71 74
... ... ... ... ...
.. . I
..
.. ..
Neutralizing Agent
%
3 21 20 73 53 70 65 61 64 8 5 22 23
..*
201 200 194 246
Residual Sugar, 95 96 83 21 37 14 14 15 13
.. .. .... 86
9
pH inactivation point or minimum pH of fermentation.
b
TABLE 11. LACTICACID YIELD AND SUGARCONVERSION FOR HETEROFERMENTATIVE FERMENTATIONS OF UNFORTIFIED WHEAT GRITMEDIUMS AT 45" C. (Initial sugar estimated, 230 grams. Neutralizing agent, 8% ",OH) Final Laotic Lactic Volume Time Sugar Acid Acid Run p H of of Mash, of Run, Final, Final, Yield, No. Run M1. Hours Grama Grams % 6 7
8 9
5.75 5.05 4.75 5.55
3220 3050 2930 3085
35 37 34 32
26 134 132 114
113 61 21 114
tively to lactic acid while the llttter produce volatile acids, alcohol, and carbon dioxide in addition t o lactic acid. The first four fermentations were made with a homofermentative, sporeforming lactic acid organism of the type described by Pnri,
49 26 9
80
49
5 60 w
hours a t 50" C. before the mixture was reautoclaved. When the mash cooled, a sample was withdrawn for sugar analysis before the starter culture was added. Normally about 230 grams of reducing sugara were developed by 450 grams of air-dried wheat grits. The first runs were made with wheat grit mash alone; later mineral salts were added, and in some of the h a 1 runs, both mineral salts and Difco dehydrated yeast extract were incorporated with the wheat grits. Mineral salts were kept in two stock solutions that were prepared and used as indicated in Table 111. Varying amounts of yeast extract were added, as shown in Table IV; the small amount in run 26a determined whether or not this material would change the fermentation, while larger amounts added a t the beginning of later runs served to determine quantitative effects of the substance. Fermentations were conducted with three different kinds of bacteria; two were homofermentative and one w m heterofermentative. The former ferment glucoee almost quantitn-
40
#
20
-
n
5.0
4.0
6.0
PH Figure 4. Lactic Acid Yields Obtained by Ferm3ntation of Wheat Grit Mashes A t controlledpA values and 45' C. with homofermentatlve end heterofermentative lactic acid breteri.
Points
0 8
(3 0
Series Description I and I11 Plain wheat grit mashes fermented by homofernientatirc lactic acid bacteria I1 Plain wheat grjt mash? fermented by heterofermentetive lactic acid bacteria IV Wheat grit mashes containing added mineral salts fermented by homoferrqentative lactic acid bacteria V Wheat grit maahes containing edded mineral salts and yeast extract, fermented by homofermentative lactic acid bacteria
FOR Lactobacillus delbriickii FERMENTATIONS OF WHEATGRIT MEDIUMS TABLE 111. LACTICACIDYIELDAND SUGARCONVERSION FORTIFIED WITH MINERAL SALTS AT 45' c.'
Run NO.
198
20
21 22 23 24 26 34 35 36 37 38 a
b C
pH of Run 4.80 5.00 6.20 5.40 5.60 5.80 5.80 6.20 6.00 3.85s 5.60 5.30
Final Volume of Mash, M1.
The of Run Hours'
Sugar Initial, Grams
1i:o
21.5 23:o 29.0
Sugar Final, Grams 85 51 61 41 64 62 40 55 60 172 49 51
Lactic Acid Final, Gram
Sugar Used Ora&
Sugar Conversion,
%
Lactic Acid Yield,
%
191 156 76 133 140 28 135 151
%
89
131 210
196 199
Residual Sugar,
.. 60
66 64 48 64 14 59 65
Mineral salts were wed in two stock solutions, A and B: Solution A
Solution B
40 grams of MgSO4.1OHzO 2 grams of FeS04.7HnO 2 grams of MnSO,.lHzO Make up to 200 ml. Use 7.5 ml. for 3000 ml. of mash
50 g r a m of sodium acetate 60 gram8 of sodium hexametaphos hate Make up t o 400 ml. Use 20 ml. g r 3000 ml. of maah
See section on methods. pH inactivation point, or minimum pH of fermentation.
Neutralizing Agent
TABLE Iv. LACTIC
0
less
INDUSTRIAL AND ENGINEERING CHEMISTRY
September 1950
CONVERSION FOR LaCbbaCilluS delbriickii FERMENTATIONS O F WHEIT GRIT MEDICUS FORTIFIED WITH YEASTEXTRAOF AND MINERAL SALTS AT 45' C.
ACID YIELD AND SUGAR
Run
pH of
NO.
Run
Final Volume of Mash, M1.
268 27 28 29 30 32 39 40
5.80 5.60 4.80 4.60 6.60 6.00 3.50" 3.90
3240 3565 2980 2830 3318 3190 2905 2830
(Xeutralizingapent, 2.5 N carbonate. See seotion on Methods and Table 111) Yeast Lactic Extract Time Sugar Sugar Acid Sugar Added, of Run, Initial, Final, Final, Used, Grams Hours Grama Grams Grama Grams 3 7 10 10 20 20 20 20
23 15 12 14 15 27 140 70
116 129 106 124 222 246 197 161
13:8 42.0 60.0 21.5 31.8
65:O
78 91 74 99 138 200 56 91
93 114 94 110 197 219 57 91
Sugar Copversion,
%
Lactic Acid Yield, %
M 80 79 90 69 91 98 100
Residual Sur, 20 12 11 11 7 11 71 43
pH inactivation point, or minimuin pH of fermentation.
TABLEV. OVER-ALLRATE OF IACTIC ACID PRODI.CTION OBTAINED BY FERMENTATION O F UNFORTIFIED WHEAT GRITS USIKGHOMOFERMENTATIVE LACTICACID ORGANISMS AT 45" C;. Total Time of
Run No.
pH
HouA
Run
Lactic Acid Produced, Gram
2 3 10 11 12 13 14 16 17 18
4.75 4.76 5.60 6.00 6.20 6.20 6.10 4.45 4.60 4.80
36.0 46.0 41 .O 25.0 23.5 19.5 54.0 46.0 23.0 27.5
49 47 121 173 153 138 182 11 42" 435
Final Mash Volume, MI.
Lactic Acid Produced, Grama 100 M1. Ma&Hour 0.051 0.032 0.100 0.216 0.189 0.206 0.101 0.008 0.062 0.051
a Rune 17 and 18 develo ed 52 and 53 rams of laotio acid, respectivqly, but extr+ CaCOa waa addeb)at begnnrng run, so a flat 10-gram corrertion was applied.
07
TABLE VI. OVER-ALL RATE nB LACPICACID PRODVCTION ORTAINED BY FERMENTATION OF WHEATGRITS P ~ r s MINERAL SALTS A N D YEASTEXTRACT USING Luctohacillile delhdekii AT
c.
Go
Run No.
pH
Total Time of Run, Hours
27 28 29 30 32
6.6 4.8 4.6 5.6 5.0 3.9
13.8 42.0 60.0 21.6 31.8 65.0
40
Lactic Acid Produced,
Mash
Grains
Volume, M1.
Final
Laotic Acid Produced, Grama 100 Ml. Mas&our
90.6 74.0 09.2 138 200 48"
3666 2980 2830 3318 3190 2830
0.184 0.059 0.059 0.193 0,198 0.026
91.6 grama of lactic acid actually produced. but only 48 grams made at
pH 3.9.
Peterson, and Johnson (7) arid designated as P.S.F.L. by them. A heterofermentative isolate was used for runs 6 through 9. This organism grew well at the usual temperature of 45" C. and produced considerable lactic acid. It was examined by morphological and biochemical tests and found to classify most nearly as Lactobacillus buchnen'. Beginning with run 10, all fermentations were conducted with a United States Department of Agriculture strain of hctobacill ~ L Sdelbrackii NRRL R-445.
equimolar mixture of sodium, potassium, and ammonium earbonate for a11 remaining runs. The latter mixture also contained 4.8 grams of sodium hexametaphosphate per liter. Apparently these solutions were equivalent, because no evident changes developed from substitution of one in place of another. When alkali utilization stopped, the lactic acid fermentation was considered complete. The terminal mash volume was then measured and duplicate sugar and lactic acid samples were taken. The sugar sample was immediately frozeii and maintained in this condition until analyzed; the lactic acid sample was preserved in 0.1 N sulfuric acid and stored in a refrigerator. Sugar was determined by the method of Shaffer and Somogyi (8) on the clear supernatant liquid obtained by centrifuging the sample that had previously been melted and diluted with distilled water. The Somogyi method is not entirely specific for reducing sugars, and reducing sugars are not necessarily fermentable; nevertheless the Somogyi values obtained by routine analyses of similar mashes are useful in evaluating the efficiency of similar fermentations. Lactic acid was determined by the method of F n e d e m n n and Graeser (S), which utilized the manganese dioxide oxidizing agent instead of the potassium permanganate solution previously advocated by Friedemann. Sugar interference was minimized by a preliminary purging with copper sulfate and lime. Freeman and Morrison ($) found this technique satisfactory for the determination of lactic acid in synthetic fermentation mixtures containing low sugar concentrations. Fermentation efficiency was expresaed in t e r m of lactic acid yield and sugar conversion. The former was obtained by dividing the amount of Somogyi sugar initially present into the quantity of lactic acid produced, the latter from the weight ratio of lactic acid developed to that of sugar disappeared; these were expressed on a percentage basis. The per cent reeidud sugar was obtained from the ratio of sugar remaining a t the end to thst present at the beginning of the fermentation. RESULTS
Fermentations were carried out in the following general inanner: About 2500 ml. of prepared mash were heated to 50" C., sampled for sugar, poured into a crock and placed in the incubator. Next the equipment illustrated in Figure 3 was emplaced, special nutrients were added, and fermentation was initiated by incorporation of 100 ml. of starter culture that had been grown in a liquid medium containing 0,5'?& glucose and 2% yeast extract.
Series I I1 111 IV
The experimental technique was not designed to prevent incidental contamination after fermentation began. Rrather, the usual industrial precautions for averting such contamination were followed. These included sterilization of the mashes, heavy inoculation with the dtsired organism, and fermentation at high temperature and relatively low pH values. Three different alkali sohitions were used to neutralize the lnrtic acid as i t formed: 8% m ~ m o n i afor runs 1 through 16, 8% ammonium rarbonrtte for ruiis 17 through 19a, and a 2.5 .V
In Figure 4, lactic acid yield is plotted against fermentation pH for the various series of runs. When homofermentative organisms were used, the optimum p H of fermentation was reduced from 0.0 in plain wheat grit mashes to 5.2 when nutrient salts were added and then to 4.6 when both nutrient salts and yeast extract were used together. The maximum yield of 70% in plain wheat grit mashes was not changed by the addition of nutrient salts, but the combination of salts and yeast estrwt raised the value slightly.
Altogether five series of runs were made.
V
Runs 14
6-9
10-19 19a-26 and 84-38 268-32 and 89-40
Organism P.S.F.L. L. bwhnsri.. L. ddbtzlcky L. delbrtlcktc L. d e l M c k i i
Mash Compoaition Wheat grita Wheat grita W h a t prilta mineral salts Wheat gnts tnrneral salts Wheat grits f mineral salts yeaat extract
++
+
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
1856
Vol. 42, No. 9
OF DATA AND CALCULATED RESULTSFOR HOMOFERMENTATIVE LACTIC-4crD FERMBNTATIONS OF WHEATGRIT TABLE VII. SUMMARY MEDIUMS AT OPTIMUM pH VALUESAND 45" C.
Run No. 11
+ + + + +
21
29 30 a
Optimum PH 6.00
Mash Composition Wheat grits Wheat grits mineral salts Wheat grits mineral salts yeast extract Wheat grits mineral salts yeast extract
Time of Fermentation, Houra 25
5.20
30a
4.60
60
5.60C
21.5
Av. Rate pf Fermentation. G r a m Lactic Acid/100 MI. Mash/Hour 0 216
Lactic Acid Produced Grams
Lactic Acid Yield, %
Sugar Conversion,
Residual Sugar.
164 176
70
69
81 90
14 24
80
90
11
0.059
62
69
7
0.193
99b 138
%
%
0.2'
Eetimated. g r a m of wheat g r i t s used for thirr run' other rum shown based on 450 gram8 of wheat grits. Not optimum pH but rather average pH balue assumed for calcium carbonate buffer control.
b 225 0
then continued for 2 hours. More dextrose was then added; this time only 3 grams were used. The fermentation began again and lasted 1 hour. As a final test, a few grams of sucrose were added. The fermentation revived, but proceeded at a much slower rate. Bergey ( 1 ) states that sucrose is only slowly fermented by Lactobacillus delbriickii.
100
0 80 w
60
%0 40 20 aQ
Figure 5 shows that the conversion of sugar to lactic acid varied inversely with the pH of fermentation but was not affected by changes in mash composition. Figure 6 shows that not only was the minimum sugar value lowest where the mediums were enriched with both mineral salts and yeast extract, but also the residual sugar value remained at the minimum as the fermentation pH was increased. This indicates that approximately 11% of the reducing substances present in enzymatically hydrolyzed wheat grits are unfermentable. It further demonstrates that all the fermentable sugar was utilized in those fermentations carried out above the optimum pH in mediums containing added nutrient salts and yeast extract. To substantiate this latter conclusion, 6 grama of dextrose were added to the mash of run 27 after alkali utilization had completely stopped. The fermentation immediately revived and
Further examination of the residual sugar plots in Figure 6 shows that the optimum fermentation pH values obtained from the minimum points on these curves correspond with the optima derived from Figure 4. The average rate of fermentation was expressed as grams of lactic acid produced per 100 ml. of mash per hour. As indicated in Figure 7, the logarithm of the average rate of fermentation varied directly with the pH of fermentation. The addition of yeast extract increased the rate of fermentation at a given pH value, but did not alter the correlation. Although the maximum yield of lactic acid occurred a t pH 4.6 in the media fortified with yeast extract and salts, the rate of fermentation at this pH was only about one third that found in the same medium at pH 5.6. The data upon which the curves in Figures 4 through 7 are based are summarized in Tables I through VI. Table VI1 presents a comparison of the results found at the various pH optima and at pH 5.6. The latter value is included because it is a common pH for fermentations buffered with calcium carbonate.
I
I i
-4E 0.20 0
0 i o . ,0 0
-0.08
2 0.06 0 a 0
F 0.04 0 a A
0.023.0 4.0
5.0
6.0
PH Figure 6.
Residual Sugar in Wheat Grit Mashes Fermented at controlled pH values and 45' C. with
homofermentative lactic acid bacteria. same as in Figure 4
Symbols
4.0
5.0
6.0
7.0
PH Figure 7. Average Rate of Lactic Acid Production Obtained by Fermentation of Wheat Grit Mashes A t controlled pH values and GoC. with homafermentatire lactic acid bacteria. Symbols mame as in Figure 4
September 1950
INDUSTRIAL A N D ENGINEERING CHEMISTRY
At the outset it was thought that wheat grit mashes might supply all the nutrients required by Isctobacilli and thus be suitable without supplement for studying the effects of controlled pH on the lactic acid fermentation. However, analyses of the early runs showed considerable and variable amounts of residual sugar. If the fermentations had been complete, all the residual sugar values should have been low and similar. Because they were not, it appeared that the medium was deficient in some constituent. So when run 19 stopped, 1 gram of solid potassium dihydrogen phosphate was added. Within an hour fermentation revived; within 3 hours normal activity probeeded in this s u p posedly exhausted mash. Based upon this observation, nutrient salts were added to the mashes, but even the addition of minerals did not appreciably lower the minimum residual sugar value. So when run 26 appeared nearly complete, 3 grams of Difco dehydrated yeast extract were added. Within a few minutes the fermentation rate increased and then continued to rise for several hours; previously the reaction rate had decreased rapidlyat this stage of the fermentation. The activity of yeast’ extract indicated the need for organic nutrient supplements in the mash at this pH. The experimental technique just described should demonstrate whether or not other fermentation mashes have a sufficient quantity of growth factors. Large amounts of growth factors are required in present commercial lactic acid fermentations where calcium carbonate is used to neutralize the acid as i t forms. Some of the materials used for the preparation of these commercial mashes originally contain sufficient growth factors. Where they do not, nutrients must be added in the form of malt sprouts, corn steep liquor, etc. The reason for using large amounts of accessorygrowth factors in commercial lactic acid fermentations is apparent from a study of Figures 4 and 7. In plain wheat grit mashes the optimum lactic acid yield occurs a t p H 6.0 but amounts to only 10% at pH 4.5. Likewise, the average fermentation rate at pH 0.0 is about five times more rapid than a t p H 4.5. However, when yeast and nutrient salts are added, the maximum yield occurs
1857
at pH 4.6 and then drops slowly as the pH is raised to 6.0. The average rate of fermentation is increased b y the addition of nutrients to the extent that the rate at pH 5.6 in the supplemented mash is equal to that of the plain mash at p H 6.0. Because calcium carbonate usually buffers p H in the range 4.5 to 5.0, i t is apparent that both the yield and rate of lactic acid production are considerably improved in this range when large amounts of growth factors are present. It is also evident that equivalent yields and rates of fermentations can be obtained from plain, unsupplemented mashes when the p H is controlled at their optimum value. ACKNOWLEDGMENT
This study was made possible by a grant of funds from the Graduate School of the University of Minnesota and by equipment obtained from surplus property of the United States Government. The senior author was the recipient of a fellowship from the Standard Oil Company of Indiana during one year of the research. LITERATURE CITED
(1) Berge:
D. H.,“Bergey’s Manual of Determinative Bacteriology, 6th ed., p. 359, Baltimore, Williams and Wilkins Co.,
1948. (2) Freeman, G. G.,and Morrison, R. I., Analyst, 71,511 (1946). (3) Friedemann, T. E., and Graeser, J. B., J . Biol. C h a . , 100,291 (1933). ENQ. (4) Leonard, R. H.,Peterson, W. H., and Johnson, M. J., IND. CHEM.,40,67 (1948). (5) Long, Louis, Jr., “Sugar and Sugar By-Products in the Plastics Industry,” p. 18, New York, Sugar Research Foundation, 1949. _. -.
(6)
Longsworth, L. G., and MacInnes, D. A,, J.Bact., 29, 595 (1935); 31,287 (1936);32,567 (1936).
(7) Pan, S. C., Peterson, W. H., and Johnson, M. J.. IND.ENQ. CHEM.,32,709 (1940). (8) Shaffer, P. A., and Somogyi, M., J. Biol. Chem., 100,695 (1933). R~PCEIVSD June 18,1948. Presented before the Division of Agrioultural and Food Chemistry, Fermentation Section, at the 113th Meeting of the AMSBICAN CHEIIICAL SOCIETY, Chiaago, Ill.
Lactic Acid Fermentation Rate EFFECT OF CONTINUOUSLY CONTROLLED pH R. K. FIN”, H. 0. HALVORSON’,AND EDGAR L. PIRET University of Minnesota, Minneapolis, Minn.
The rate of fermentation of dextrose solutions to lactic acid by Lactobacillus delbruckii has been studied at various pH levels from pH 3.5 to 6.0. The pH was held constant by automatic controls throughout each fermentation, and equipment was devised to continuously record the rate at which acid was produced. The maximum rate of acid production during a fermentation cycle was increased as much as fourfold by raising the pH only 0.5 units. At all levels of pH relatively high concentrations of accessory nutrients were necessary for the rapid production of acid. Increasing the concentration of yeast extract in the medium from 0.3 to 3.0% stimulated the rate of acid production, but higher concentrations did not provide further increases in rate. The rate of fermentation appeared to be independent of the sugar content of the medium in the range of concentrations from 5 to 10%. The effects of pH on the yield of lactic acid and on the residirul swgar content of the broth confirmed earlier work in ’ Present sddreaa, University of Tllinoih. Yrhana, Ill.
which a wheat-grit mash was used. At the pH corresponding to highest yield, the maximum rate of acid production was about 0.13 gram per hour per 100 ml. of broth, and this value was substantially independent of the composition of the mediums tested.
I
T HAS been pointed out (9) that the large scale production of lactic acid by the fermentation of blackstrap molasses is commercially attractive owing to a market for high grade acid in the plastics industry. New methods for the recovery and purification have been proposed ( 1 ) which may make it more economical to use ammonia instead of calcium carbonate to neutralize the acid formed during fermentation. Such a change would necessitate the careful control of p H through the fermentation, and accordingly a study of the proper environmental conditions was undertaken. A previous paper from this laboratory (4) reported the effects of controlled p H on the yield and conversion attained in the fermentation of wheabgrit mashes. Using a variety of lactic acid
