Ing to Unpublished Data of Douglas and Vaughn.) Arena (1936)

THE pH REQUIREMENTS OF SOME HETEROFERMEN- TATIVE SPECIES OF LACTOBACILLUS JOHN C. M. FORNACHON,1 HOWARD C. DOUGLAS AND REESE H. VAUGHN Division of Fruit Products, University of California, Berkeley, California Received for publication May 6, 1940 I. INTRODUCTION The lactobacilli are noted for their ability to grow in acid media. Although media with reactions between pH 3.5 and 5.0 have been recommended for their isolation, the media used in physiological studies by Pederson (1929, 1938), Nelson and Werkman (1935, 1936), Weinstein and Rettger (1932) and others had values ranging from pH 6.2 to 7.0. Shimwell (1935) found the optimum pH for the growth of LactobaciUus pastorianus to be approximately 8.0. It has been observed however that species of Lactobacillus isolated from wines and other similar products thrive best when the medium is appreciably acid. Otani (1936) pointed out that homofermen- tative lactobacilli isolated from sake grew most vigorously between pH 4.0 and 5.0 Fornachon (1936) found the optimum pH for the growth of a heterofermentative species of Lactobacillus isolated from Australian wine to be between pH 4.0 and 5.0. Douglas and McClung (1937) reported a bacterium from Califor- nia wine with an optimum pH between 4.1 and 4.3. (This organism is a heterofermentative species of LactobaciUus accord- ing to unpublished data of Douglas and Vaughn.) Arena (1936) described several lactic acid bacteria isolated from Argentinian wine and found the optimum pH for growth to vary between pH 4.3 and 6.7. 1 Research Officer, Australian Wine Board, Waite Agricultural Research In- stitute, University of Adelaide, South Australia. Formerly Associate in the California Experiment Station. 649 650 J. c. M. FORNACHON, H. C. DOUGLAS AND R. H. VAUGHN Morishita (1929) working with aciduric organisms from dental caries found the best growth between pH 5.0 and 6.8. Longworth and MacInnes (1935) reported the greatest acid production by Lactobacillus acidophilus at pH 5.5 to 6.0. Weiss and Rettger (1934) determined the pH range for best growth of Lactobacillus bifidus as 5.4 to 6.5, and for LactobaciUus acidophilus as 5.8 to 6.4. In the investigations cited above the media were not all the same. It is known that the nature of buffers and other con- stituents, as well as the individual characteristics of the bacteria studied, may influence the response to pH and it is not surprising that variations have appeared. The following study was undertaken to determine more closely the pH range for maximum growth and metabolism of certain gas-forming lactobacilli, and to point out the effect of pH on growth and fermentation reactions of these bacteria.

II. THE ORGANISMS The bacteria used included three cultures of Lactobacillus brevis and four cultures of Lactobacillus hilgardii isolated from California wines together with the following species: Lactobacillus fructovorans and Lactobacillus gracilis from Dr. C. S. Pederson and Lactobacillus pertoaceticus from the University of Wisconsin collection.2 III. EXPERIMENTAL a. Optimum pH for growth Cell counts were used in determining the optimum pH for cell multiplication. Preliminary experiments having shown the optimum pH to be approximately 5.5, equal quantities of the test organisms were sown in a series of flasks of yeast water medium containing 2.0 per cent glucose. The pH was adjusted to approximately 4.5, 5.5 and 7.0 with M/4 phosphate-acetate buffers and measured before inoculation and after completion of ' L. pentoaceticu is now considered a synonym for Lactobacillu brevis. The exact taxonomic position of L. fructovorans, L. hilgardii and L. gracilis is doubt- ful. See Pederson in Bergey's Manual of Determinative Bacteriology, 5th edition, 1939. Lactobacillw hilgardii has been redescribed by the authors and the results will be published. pH REQUIREMSMRS OF LACTOBACILLUS 651 the experiment with the quinhydrone electrode. The culture flasks were incubated at 300C. Cell counts were made at intervals by use of a Petroff-Hausser bacteria counter. Typical growth curves are shown in figure 1. The beneficial effect of an acid reaction is shown. More cells were produced at pH 4.65 and 5.45 than at pH 7.0. b. Optimum pH for dissimilation of glucose The optimum pH for the dissimilation of glucose was determined with suspensions of cells which were prepared as follows: Ten liters of yeast water containing 2.0 per cent glucose were inoculated with one liter of a 24-hour culture grown in glucose

7.0

.4~~~~~~~~~~~~H54

/0 20 30 40 50 60 70 HOURS INCUBaTED Ar 3o0 C. [FIG. 1. EFFECT OF pH ON GROWTH OF LACTOBACILLUS HILGARDII No. 5 yeast water. After 24 hours incubation at 300C, the bacteria were separated from the culture aseptically by means of a Sharples super-centrifuge and suspended in 250 ml. of 0.9 per cent NaCl solution. Ten ml. of this cell suspension were placed in each of a series of large sterile tubes containing 2.5 ml. of sterile 20.0 per cent glucose solution, 5.0 ml. of sterile 0.9 per cent NaCl solutions and 7.5 ml. of sterile phosphate-acetate buffer of the desired pH. The buffer solutions were so prepared that the final dilutions in the tubes were approximately M/4 with respect to both phosphate and acetate. The number of cells in the mixture was approximately 2 X 1010 per cubic centimeter. Under these conditions a temperature of 370C. was best for the dissimilation of glucose 652 J. C. M. FORNACHON, Af. C. DOUGLAS AND R. H. VAtTGHN and all experiments with cell suspensions were conducted at this temperature. The glucose in the fermenting liquid was determined after various intervals of time by the cerric sulfate method as described by Hassid (1936, 1937) and results expressed in terms of grams of glucose decomposed per 100 ml. Table 1 shows the rates of glucose dissimilation by suspensions of L. hilgardii strain 7. Under the conditions of this experiment the optimum pH for glucose destruction was about 5.5.

TABLE 1 Effect of pH on dissimilation of glucose by suspension. of Lactobacillus hilgardii culture 7

GLUC0SU* UTISUD In GaAMS/100 ML. AgoUR TuD xuseB= nwIAL pH -=AL pH 2 hourst 4 hours 6.5 hours 8 hours 1 4.05 0.040 0.115 0.145 3.92 2 4.46 0.200 0.335 0.460 0.658 4.28 3 4.96 0.280 0.615 0.860 1.095 4.58 4 5.46 0.480 1.022 1.334 1.475 4.88 5 5.96 0.250 0.750 1.048 1.238 5.29 6 6.43 0.420 0.667 0.910 1.045 5.94 7 6.86 0.370 0.680 0.904 0.960 6.44 8 7.60 0.200 0.380 0.528 0.650 7.07 * Initial concentration of glucose 2 grams per 100 ml. t Incubation temperature 870C. c. Optimum pHfor decomposition of 1-malic acid Table 2 shows the effect of pH on the decomposition of 1-malic acid by growing cultures. None of the organisms attacked the acid at pH 7.0. The optimum pH range appeared to be between 4.0 and 5.5. d. Effect of pH on the utilization of sugars Table 3 shows the effect of pH on acid production from various sugars important for taxonomic purposes. The medium was yeast water containing 1.0 per cent of test carbohydrate. The basal medium and 10.0 per cent sugar solutions were sterilized separately and the sugar added aseptically TABLE 2 Effect of pH on decomposition of 1-malic acid DUICBUASU IX TITRATABLU ACIDITY RXPRZBSU As w. N/10 3ASE/10 ML. OGAISM* pH 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 L. gracilis...... 2.3 2.6 2.3 1.7 0.3 t L. pentoaceticus .... 2.6 3.4 3.0 2.9 1.6 0.7 L.fructovorans. 0.4 0.3 L. brevi4t...... 0.1 0.4 0.3 L. hilgardii .0.5 0.3 L. hilgardii§ ...... 2.5 2.6 2.6 1.8 0.4 0.2 * Incubated for 35 days at 300C. t Blank indicates no change in titratable acidity. $ Figures represent average of three cultures. § Figures represent average of two cultures. The medium: 1.0 per cent Bacto-tryptone, 0.5 per cent Bacto yeast extract, 0.1 per cent K2HPO4, 0.5 per cent 1-malic acid dissolved in distilled water. Sterilized by filtration through Berkfeld candle.

TABLE 3 Effect of pH on utilization of various sugars Increase in titratable acidity as ml. N/10 base/10 ml. -ARBADINOS | D-XTLOBZ GL|UCOB U ISUGARX pH 6.8 5.5 6.8 5.5 6.8 5.5 6.8 5.5 L. pentoaceticus ...... 11.70 10.00 11.74 10.90 2.70 2.60 6.20 4.64 L.fructovorans ...... t 0.30 5.24 5.00 4.40 L. brevisl ...... 9.10 8.50 12.90 11.10 4.54 4.62 2.92 L. hilgardii. 7.70 11.64 7.12 5.90 6.00 L. hilgardi. 9.00 8.60 4.42 4.80 4.64

SUGAR CTO CTOR MAWTOSN SUCROMS

L. pentoaceticus ...... 4.20 2.50 5.40 2.90 1.10 0.90 L. fructovorans. L. brevs...... 4.00 2.70 4.44 2.50 L. hilgardii 5.70 1.00 5.60 7.20 L. hilgardii 4.40 0.90 4.80 3.00 * Incubated for 14 days at 300C. t Blank indicates no change in titratable acidity. $ Figures represent average of several cultures. L. hilgardii cultures divided on basis of decomposition of 1-malic acid as in table 2. Correction was made for acid production in control cultures containing no sugar. 653 654 J. C. M. FORNACHON, H. C. DOUGLAS AND R. H. VAUGHN to the basal medium. Inoculations consisted of three standard loops (4 mm.) of young liver-infusion broth cultures. The effect of pH is particularly striking with respect to the utilization of glucose-and certain of the disaccharides. Glucose, galactose, lactose, maltose and sucrose were actively fermented in yeast water at pH 5.5. At pH 6.8 these sugars were not attacked. Similar results were obtained in tryptone yeast- extract medium. Anvexperiment to test the possible chemical inversion of the disaccharides at pH 5.5 showed no inversion of lactose, maltose or sucrose, in the yeast juice medium after 7 days at 30'C. All of the cultures showing acid production for these sugars at pH 5.5 were growing 3 days after inoculation. Furthermore, if inversion occurred at pH 5.5 it would be expected that L. fructovorar& and L. pentoaceticus would show acid production from all of the disaccharides.

IV. DISCUSSION AND CONCLUSIONS The optimum pH for growth did not differ greatly from that for metabolism of~carbon compounds for the organisms investi- gated, both being greatest in the range of -pH 5.0 to 6.0. This fact may be of little taxonomic importance so far as the vigorously growing species are concerned, but it should be given due con- sideration when carrying out studies on those cultures more exacting in their requirements for growth and metabolism; under such conditions the determination of optimum pH is quite as important as the determination of optimum temperature range. When the gas-forming lactobacilli are grown in unbuffered media containing carbohydrates which are readily attacked, the adverse effect of a neutral reaction is quickly obscured, but with carbon sources less readily decomposed or whose decomposition iswnot'accompanied by acid production causing a fall in pH, (as in the case of malic acid) the pH of the medium may be the limit- ingtfactorlinrdetermining whether they are decomposed. At- tention shouldlbe~givenlto the optimum pH for growth and metabolism when investigating the heterofermentative species of Lactobacillus. pH REQUIREMENTS OF LACTOBACILLUS 655 The non-technical assistance of Federal Works Progress Administration Project numbers 11589-B5, 10481-B5, Northern California, Area 8, is acknowledged. REFERENCES ARENA, A. 1936 Alteraciones bacterianas de vinos argentinos. Rev. facultad agr. vet. (University Buenos Aires), 8, 155-318. DOUGLAS, H. C., AND MCCLUNG, L. S. 1937 Characteristics of an organism causing spoilage in fortified sweet wine. Food Research, 2,-471-475. FORNACHON, J. C. M. 1936 A bacterium causing diseases in fortified wines. Australian J. Exptl. Biol. Med. Sci., 14, 215-222. HASSID, W. Z. 1936 Determination of reducing sugars and sucrose in plant material. Ind. Eng. Chem., 8, 138-140. HASSID, W. Z. 1937 Determination of sugars in plants. Ind. Eng. Chem., 9, 228-229. LONGWORTH, L. D., AND MACINNES, D. A. 1935 Bacterial growth with auto- matic pH control. A. An apparatus. B. Some tests on the acid production of Lactobacillus acidophilus. J. Bact., 29, 595-607. MORISHTA, T. 1929 Studies on dental caries, with special reference to aciduric organisms associated with the process. I. Isolation and description of organisms. J. Bact., 18, 181-198. NELSON, M. E., AND WERKMAN, C. H. 1935 Dissimilation of glucose by heterofermentative lactic acid bacteria. J. Bact., 30, 547-557. NELSON, M. E., AND WERKMAN, C. H. 1936 Diversion of the normal hetero- lactic dissimilation by addition of hydrogen acceptors. J. Bact., 31, 603-610. OTANI, Y. 1936 Untersuchungen ueber die Hyochi Bazillen im Sake. J. Faculty Agr. Hokkaido Imp. Univ., 39, part 2, 51-142. PEDERSON, C. S. 1929 The types of organisms found in spoiled tomato products. Tech. Bull. 150, N. Y. Agr. Expt. Sta., Geneva. PEDERSON, C. S. 1938 The gas-producing species of the genus Lactobacillus. J. Bact., 35, 95-108. SEamWELL, J. L. 1935 The cultural characteristics of Saccharobacillus pastorianus. J. Inst. Brewing, 41, 481-487. WEINSTEIN, L., AND RETrGER, L. F. 1932 Biological and chemical studies of the Lactobacillus genus with special reference to xylose fermentation by L. pentoaceticu. J. Bact., 24, 1-28. WEIsS, J. E., AND RETTGER, L. F. 1934 Lactobacillus bifidus. J. Bact., 28, 501-517.