SPORULATION REQUIREMENTS OF BACILLUS COAGULANS VAR. THERMO- ACIDURANS IN COMPLEX MEDIA'

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1 SPORULATION REQUIREMENTS OF BACILLUS COAGULANS VAR. THERMO- ACIDURANS IN COMPLEX MEDIA' MIKIO AMAHA, Z. JOHN ORDAL, AND ALI TOUBA Technology, University of Illinois, Urbana, Illinois Received for publication December 5, 1955 A survey of several strains of Bacillus coagulans var. thermoacidurans revealed considerable variation in sporulation when the organisms were cultured on a variety of peptone containing media. Our culture of strain 43P was especially lacking in sporulation ability, although it sporulated when cultured on a medium such as tomato juice agar. The general characteristics of B. coagulans var. thermoacidurans have been described by Berry (1933), Stern et al. (1942), and Becker and Pederson (1950). Stern et al. (1942) developed a medium, known as thermoacidurans agar, for the isolation and cultivation of the organism. They also reported that sporulation of B. thermoacidurans on this medium was slow and that up to two weeks incubation was required to produce a satisfactory spore crop. Becker and Pederson (1950), however, reported that B. thermoacidurans produced abundant spores on tryptone glucose yeast extract agar having a reduced glucose content. It is known that certain of the conventional media may be deficient in minerals essential for maximum sporulation (Knaysi, 1945; Foster and Heiligman, 1949). For instance, manganese is an important mineral stimulant for sporulation by a variety of mesophilic and thermophilic aerobic bacilli (Charney et al., 1951; Curran and Evans, 1954; Greenberg and Halvorson, 1955; Weinberg, 1955). However, Curran and Evans (1954) failed to demonstrate any stimulatory effect of manganese on the sporulation of B. coagulans var. thermoacidurans when cultured in skim milk. In the present investigation, sporulation by several strains of B. coagulans var. thermoacidurans was studied in agar slant cultures and shake flasks in an effort to determine conditions which would support abundant sporulation for the production of large spore crops. I Supported in part by funds from Bankhead Jones Act, United States Department of Agriculture. 34 MATERIALS AND METHODS Cultures. Four cultures were used in this investigation: strains 43P-1 and 43P-2 obtained from the National Canners Association (NCA) in 1952 and 1954, respectively; strain 8038, obtained from the American Type Culture Collection (ATCC) in 1951; and strain ib, obtained from Dr. F. M. Clark, University of Illinois, originally isolated by Berry (1933). ATCC strain 8038 is described as being the same strain as 43P of the NCA. However, our cultures showed differences in nutritional requirements and proteolytic activity as well as in sporulation ability. Media. The basal media used in these studies were as follows: thermoacidurans agar (Difco), tryptone glucose extract agar (Difco), nutrient agar (Difco) and tomato juice agar (10 per cent tomato juice and 0.4 per cent K2HPO4). The ph of the medium was adjusted by hydrochloric acid or sodium hydroxide prior to sterilization. Procedure. Sporulation on agar media was determined following 3, 5 and 10 days incubation. Smears were made and stained by Schaeffer and Fulton's method (1933). The vegetative cells and spores were counted in five different fields and the per cent sporulation calculated. Agar slants were made in 16- x 150-mm test tubes containing 8 ml of medium. Erlenmeyer flasks (250-ml) containing 50 ml of medium covered with gauze were shaken on a rotary shaking machine at 350 rpm. All slants and shake culture were inoculated with 0.05 ml and 0.2 ml of an 18-hr thermoacidurans broth culture, respectively. Unless otherwise stated, all cultures were incubated at 45 C. Growth, ph and per cent sporulation in shake cultures wvere determined after incubation for 3 and 5 days. The total cell count was determined b) a Petroff-Hausser chamber and the ph by a Beckman model G ph meter.

2 1956] S1PORULATION OF BACILLUS COAGULANS 35 RESULTS The effect of the addition of mineral salts to thermoacidurans agar on sporulation and morphology. Our strain of 43P-1 sporulated very poorly on thermoacidurans agar, and after 10 days incubation at 45 C usually less than 0.5 per cent of the total cells were found to be spores. A reduction in the glucose content of the thermoacidurans agar failed to stimulate sporulation. However, when the organism was cultured on a tomato juice agar good sporulation occurred. Likewise, the addition of tomato juice or a soil extract to thermoacidurans agar enhanced sporulation. Tomato juice was then ashed and the sulfuric acid extract of the ash was added to thermoacidurans agar. The positive effect of the ash supported the hypothesis of a mineral ion requirement. A variety of mineral salts were then added to thermoacidurans agar to determine which, if any, would stimulate sporulation. It was found that salts of manganese, nickel and cobalt markedly stimulated sporulation, whereas the other salts tested such as MgSO4 (1 to 100 ppm), CaCl2 (1 to 100 ppm), FeSO4 and ZnSO4 (1 to 10 ppm), and CuS04 (1 ppm) were ineffective. Essentially no sporulation occurred in the absence of added manganese, nickel or cobalt even after 10 days of incubation. A positive effect due to manganese was noted when as little as 0.01 ppm.imnso4 was added to the medium. Sporulation wvas inecreased as the concentration of MInSO4 was increased, the optimum concentration being about 1 ppm. The stimulatory effect of manganese substantiates the findings of Charney et al. (1951), Curran and Evans (1954), Greenberg and Halvorson (1955), and Weinberg (1955). However, these authors failed to or did not demonstrate stimulation by nickel or cobalt. The addition of manganese, nickel or cobalt ions to thermoacidurans agar also stimulated growth and affected the morphology of the cells grown on such media. On media lacking either of these ions growth was reduced and the individual cells were filamentous. When manganese, nickel or cobalt ion was added to various media the amount of growth was increased and the individual cells were normal rods. Table 1 presents data for three strains of B. coagulans var. thermoacidurans cultured on three different media with and without added manganese. The data indicate that in cultures which were filamentous little or no sporulation occurred. Likewise cultures which consisted of normal rods produced a high percentage of spores. The effect of added manganese on cell morphology and sporulation was even more striking when the cultures were incubated at 55 C. At this incubation temperature and on thermoacidurans agar, all strains grew primarily as filamentous cells and no sporulation was apparent. However, when manganese was added to the medium the growth was more abundant, TABLE 1 the cells were normal rods and sporulation was nearly complete after incubation for 3 days. Prickett (1928) reported that the cells from cultures of thermophilic bacilli incubated at 56 C tended to be more filamentous than did the cells A comparison of the sporulation abilities of three strains of Bacillus coagulans var. thermnoacidurans on Agar Media (ph 7.0) agar media with and without added manganese 43P B Per cent Growth Per cent GrPwthperes Growth Grwh spores spores Groth rent Thermoacidurans agar... M-f <0.1 M-rf 15 M-rf 15 Thermoacidurans agar + 1\InSO4*... G-r 80 G-r >98 G-r >98 Nutrient agar... M-f <0.1 M-rf 15 M-rf 15 Nutrient agar + MnSO4*... G-r 80 G-r >98 G-r >98 Tryptone glucose extract agar... M-f <0.1 M-r 30 M-r 50 Tryptone glucose extract agar + MnSO4*. G-f 85 G-r >98 G-r >98 M, medium growth; G, good growth; f, filamentous cells; r, normal rods. Incubation: 5 days at 45 C. * MnSO4-1 ppm.

3 36 AMAHA, ORDAL AND TOUBA [VOL. 72 from cultures incubated at 37 C. He did not relate the morphology of the cells to the composition of the medium. Webb (1949, 1953) and Shankar and Bard (1955) have demonstrated that the magnesium content of the culture media affects the morphology of the resulting cells. In magnesium deficient media the organisms grew as long filaments. Webb (1953) also reported that the magnesium requirement could be replaced by manganese. In our experiments, however, the addition of up to 100 ppm magnesium sulfate failed to produce a demonstrable effect either on the morphology of the cells or on the ability of the cells to sporulate. The effect of the initial ph on sporulation. The effect of various initial ph levels of thermoacidurans agar on subsequent sporulation were examined. The range investigated was ph 4.5 to 7.5. In addition, a comparison was made on the sporulation abilities of strains 43P-1 and 43P-2. While sporulation was determined on all cultures incubated for 3, 5, and 10 days, onlv the data for the 10-day incubation period are presented (figure 1). In the presence of added manganese, sporulation is stimulated and is relatively constant for both strains over the ph range investigated. In the absence of added manganese, while the sporulating ability of both strains was markedly reduced, strain 43P-2 showed superior sporulating ability at the lower <,, 100 < -< 8O a 40 0~ 20 F o--o--o 43P1 x w x 43p2I,* X~~~ ph values with an apparent optimum at about ph 5.5. It is interesting to note that in the presence of added manganese the initial ph of the medium had little or no effect on the per cent sporulation. Degeneration in sporulation ability following serial transfer. The difference in sporulation ability of the two strains 43P-1 and 43P-2 as described above, suggested a degeneration in sporulation ability because of the transplant history. Winogradsky (12) found that his Clostridium pasteurianum ceased to form spores in subcultures, and that growth was mainly in the form of long chains of vegetative cells. A similar degenerative process in consecutive subcultures of Clostridium butylicus, and Bacillus sphaericus has been reported by Kutzenok and Aschner (1952) and Powell and Hunter (1955) respectively. In order to investigate the degenerative process with our cultures and to determine whether or not the addition of manganese would cause recovery to normal sporulation, several experiments were performed using strains 43P-1 and 43P-2. The two strains were serially transferred in thermoacidurans broth (ph 7.0) at intervals of 2 or 3 days. The sporulation ability of the transfers was checked by subculturing on thermoacidurans agar, with and without added manganese. This procedure caused a degeneration in the sporulation ability of both p0. % WITH Ml, S0 (1 ppm) WITHOUTrM,SO Figure 1. Effect of initial 43P-2 at 45 C INITIAL ph OF THERMOACIDURANS AGAR ph levels of thermoacidurans agar on sporulation of the strains 43P-1 and

4 1956] SPORULATION OF BACILLUS COAGULANS 37 strains. For example, strain 43P-1, which originally formed about 2.5 per cent spores on thermoacidurans agar and per cent on the same medium fortified with manganese, produced less than 0.1 per cent and 15 per cent, respectively, when sporulation was checked after 20 transfers in thermoacidurans broth. Likewise strain 43P-2, which originally produced 20 and per cent under identical conditions on the same two media, produced only 0.5 and 50 per cent when similarly checked after 20 transfers in thermoacidurans broth. It is therefore apparent that subculturing on a medium containing adequate manganese does not fully restore the sporulating abilities of strains which have degenerated because of the nature of their previous transplant history. Sporulation requirements in shake culture. In parallel with the experiments on agar slants, the sporulation of the same cultures of B. coagulans var. thermoacidurans in shake flasks was also investigated. Table 2 presents a comparison of the growth and the sporulation ability of the three strains, 43P-1, 8038 and IB, in shake culture. Data for strain 43P-2 are very comparable to that presented for strain 43P-1. Strains 8038 and 1B produced significant amounts of spores in all the media tested. Sporulation by these two strains was also stimulated by the presence of added manganese and in this respect the results paralleled those obtained on agar slant culture. However, strain 43P-1 failed to sporulate in shake culture even when manganese was added. Higher concentrations of manganese, reduced glucose content of the media, an increase in the surface: volume ratio of the liquid medium in the shake flask, and culturing on nutrient broth or tryptone glucose extract broth with or without added manganese were without effect. Brain heart infusion broth when fortified with 1 ppm MnSO4 stimulated some sporulation. Greenberg and Halvorson (1955) also noted that added manganese was necessary for good sporulation by B. polymyxa in this medium. Since strain 43P-1 produced some spores in a medium consisting of 10 per cent tomato serum it was decided to investigate the effects of some of the known constituents of tomatoes on sporulation when such constituents were ad(led to thermoacidurans broth containing I ppm MnSO4. According to Rice and Pederson (1954), citric and malic acids are the principal organic acids in tomatoes and are present in concentrations of about 0.5 and 0.05 per cent, respectively. When these two acids were added to the basal medium either alone or in combination in a concentration equivalent to that furnished by 10 per cent tomato serum, the results were again negative. However, when the concentration of malic acid was increased 10-fold (0.05 per cent) abundant sporulation occurired. This positive effect of malic acid suggested that perhaps this strain was deficient in one or more TABLE 2 Growth and sporulation of the three strains of Bacillus coagulans var. thermoacidurans in shake cuilture at 45 C Kind of Medium Thermoacidurans broth... Thermoacidurans broth + MnSO4*... Thermoacidurans broth... Thermoacidurans broth + MnSO4* per cent tomato serum... Thermoacidurans broth + 1 per cent tomato serum... * 1 ppm MnSO4 H20. Initial ph of Media ph Strain 43P-1 No. of Per cent cells spores X 107/ ml Strain 8038 After 5 days shaking ph 8.20 No. of cells X 107/ ml Per cent spores ph Strain 1 B No. of cells X 1O7/ ml ( Per cent spores

5 38 AMAHA, ORDAL AND TOUBA [VOL. 72 enzymes involved in the citric acid cycle, and hence additional acids related to this cycle were investigated for their possible effect on sporulation. The acids tested and the results obtained are listed in table 3. The data indicate that TABLE 3 Effect of the addition of various organic acids to the basal medium on the sporulation of 4SP-i in shake culture Additive* None... Malic acid... Citric acid... Fumaric acid.. Succinic acid... Isocitric acid... a-ketoglutaric acid. Oxalacetic acidf... Pyruvic acid... L-Alanine... D-Alanine... ph After 5 Days Shaking No. of cells X 107/ml Per cent spores * Each additive tested at a concentration of 0.05 per cent. t Sodium ethyl oxalacetate; sterilized by Seitz filtration. Basal medium: Thermoacidurans broth fortified with 1 ppm of MnSO4H2O. After addition of the acid, the ph of medium, if necessary, was adjusted to ph 7.0 with sodium hydroxide. WITH t4,s% ( P. _wrnithour M,SO% malic and a-ketoglutaric acids markedly stimulated sporulation, succinic acid showed a moderate effect and that isocitric and L-alanine showed a slight positive effect. The ineffectiveness of sodium ethyl oxaloacetate may have been due to its instability when the culture was shaken. None of the acids showed any demonstrable effect on sporulation if the manganese was omitted from the basal medium. Powell and Hunter (1955) reported that the addition of 0.04 M bicarbonate or ketoglutaric acid to potato-ccy medium strongly stimulated sporulation by B. sphaericus strain NCTC With our test organism the addition of sodium bicarbonate (0.05 per cent) to thermoacidurans broth with and without added manganese failed to stimulate sporulation in shake culture. The effect of manganese on the growth and metabolism of strain 4SP-1 in shake culture. In order to further investigate the effect of manganese on growth and sporulation by strain 43P-1, the amount of growth, the ph change, and the rate of glucose consumption were followed in thermoacidurans broth shake culture with and without the addition of manganese (1 ppm MnSO4. H20) or manganese and malic acid (0.05 per cent). Growth was followed by determining the optical density of the cultures at 630 mg in a Coleman Junior spectrophotometer (Model ' - TURBI DITY wd~~~~ - - -, O TIME OF SHAKING IN HOURS Figure 2. The growth, ph, and glucose consumption of strain 43 P-1 in thermoacidurans broth shake culture with and without added manganese at 45 C.

6 1956] SPORULATION OF BACILLUS COAGULANS 39 6 A). The glucose content of the medium was determined by the method of Somogyi (1945). Typical data are presented in figure 2. The results for the culture containing both manganese and malic acid are not presented, as these results were almost identical to those obtained with the culture containing only added manganese. The curves representing the optical density of the cultures indicate that in media containing added manganese growth is more rapid and that the maximum growth is attained about 1 } hr earlier than in cultures without the added manganese. The rate of glucose consumption was very similar in both cultures, the slight difference being undoubtedly due to the difference in growth rates. Likewise the ph changes were similar, and the lag in the culture lacking added manganese was a reflection of the reduced growth rate. The microscopic determination of the total number of cells by the Petroff-Hausser chamber showed that the maximum population in cultures with added manganese was about twice that of cultures without added manganese (table 2). However, as shown in figure 2, the turbidity curves showed a minor difference in the maximum density obtained in these two types of culture. This may be attributed to the difference in cell size of the organisms in the two cultures. Although the cells grown in thermoacidurans broth shake culture without added manganese were not so long as the filamentous cells obtained on agar medium without manganese, the length of the cells was about twice that of the cells grown with manganese in the shake culture. DISCUSSION The experiments reported here have demonstrated that the addition of manganese to peptone containing media, such as thermoacidurans agar, nutrient agar or tryptone glucose extract agar, markedly stimulated sporulation by the three strains of B. coagulans var. thermoacidurans. As the addition of manganese to such media also increased the amount of growth and affected the cellular morphology of the resulting cells, it may also be concluded that such media are deficient in manganese for optimum girowth as well as sporulation. According to the analytical data on the manganese content of the main constituents of the thermoacidurans medium. yeast extract (Difco), proteose peptone (Difco), and agar (Difco) have average manganese concentrations of 7, 5, and 70 ppm, respectively. Thus it may be readily calculated that the manganese content of thermoacidurans agar would be about 1.5 ppm. That this concentration is inadequate for strain 43P-1 in the peptonecontaining media used in this study is evidenced by the fact that sporulation is increased by the addition of to 3 ppm of manganese (0.01 to 10 ppm as MnSO4 H20). As information is lacking on the manganese compounds in the thermoacidurans medium, it is reasonable to assume that only a portion of the total manganese content (1.5 ppm) is available for use by the organism, and probably the remainder is strongly combined with peptide or protein molecules. In this respect, it is interesting to note that strains 8038 and IB, which were able to form some spores on thermoacidurans agar without added manganese, also exhibited a proteolytic activity against casein and gelatin, whereas such proteolytic activity was not demonstrable with strain 43P-1. It is possible that this difference in proteolytic activity is related to the difference in sporulation ability of these strains on thermoacidurans agar. Stockton and Wyss (1946) found that manganese is required for proteinase production by B. subtilis in peptone media. Their data indicate that manganese is essential for the elaboration of the proteinase enzyme and is not required only as an activator for the proteinase system. Charney et al. (1951) considered that the role of manganese in proteinase formation was correlated with the manganese requirement for sporulation. Curran and Evans (1954) were of the same opinion and quoted the theory of sporogenesis developed by Hardwick and Foster (1952) to support this opinion, namely that "Since manganese seems to be essential for substantial proteinase production... this element may promote the formation of spores by contributing to the reserve of intracellular enzyme proteins believed by Hardwick and Foster to be the basic material for spore synthesis." In order to obtain presumptive evidence for the conjectured relationships between proteinase activity and sporulation a simple experiment was performed using strain 43P-1. The culture was streaked on nutrient agar plates containing gelatin or skim milk, witlh or without

7 40 AMAHA, ORDAL AND TOUBA [VOL. 72 added manganese (Smith et al. 1952). The results, however, were entirely negative as no evidence of proteolytic activity could be detected even though sporulation was markedly enhanced on the plates containing added manganese. It would therefore appear that the role of manganese in stimulating sporulation by strain 43P-1 is not specifically related to its proteinase activity. The fact that the addition of both manganese and malic or some of the other acids related to the citric acid cycle were needed to stimulate sporulation in thermoacidurans broth shake culture seems to suggest that the citric acid cycle is involved in the sporulation process and that the cells of strain 43P-1 when grown in shake culture are deficient in one or more of the enzymes involved in the cycle. Manganese is known to be indispensable for some of the components of the citric acid cycle as well as for the dismutation of pyiuvic acid (Sumner and Sommers, 1953). SUMMARY The requirements for sporulation of three strains of Bacillus coagulans var. thermoacidurans on agar slants and in shake cultures were studied. Considerable variation in the degree of sporulation was encountered when the organisms were cultured on peptone-containing media and incubated at 45 C. Essentially no sporulation occurred when cultures were incubated at 55 C. Sporulation was improved when a natural product, such as tomoto juice or the ash of the natural product, was added to the medium. On peptone-containing agar media, growth as well as sporulation was markedly stimulated by the addition of manganous sulfate, nickel sulfate, or cobalt sulfate. The maximum stimulation was obtained by the addition of 1 ppm of the mineral salts. The effect was particularly striking when the cultures were incubated at 55 C. At this temperature, in the absence of added manganese, the whole culture consisted of filamentous cells which were incapable of producing spores. The addition of manganese also broadened the ph range over which sporulation occurred. Sporulation of both strains of 43P decreased gradually during a series of transplants in thermoacidurans broth. The addition of a manganous salt to thermoacidurans agar on which sporulation was checked did not stimulate full recovery of sporulation even though it markedly increased sporulation of the degenerated cultures. In thermoacidurans broth shake culture, the addition of manganese alone was sufficient to stimulate a high degree of sporulation by strains 8038 and 1B but not for strain 43P-1. It was found that in addition to manganese, it was necessary to add malic or a-ketoglutaric acid to thermoacidurans broth in order to stimulate a high degree of sporulation by strain 43P-1 in shake culture. Succinic acid likewise provided a moderate stimulation, and isocitric and L- alanine provided a slight but definite stimulation. It is presumed that the added acids "triggered" the citric acid cycle to provide additional energy required for the sporulation process. It appeared that the role of manganese in stimulating sporulation is not specifically related to stimulation of proteinase production by this element. A comparison of the growth, ph change, and glucose consumption in cultures with and without added manganese failed to show any marked differences, except that the rate and amount of growth were increased in the presence of added manganese. REFERENCES BECKER, M. E. AND PEDERSON, C. S The physiological characteristics of Bacillus coagulans (Bacillus thernmoacidurans). J. Bacteriol., 59, BERRY, R. N Some new heat resistant, acid tolerant organisms causing spoilage in tomato juice. J. Bacteriol., 25, 72. CIIARNEY, J., FISHER, W. P., AND HEGARTY, C. P Manganese as an essential element for sporulation in the genus Bacillus. J. Bacteriol., 62, CURRAN, H. R. AND EVANS, F. R The influence of iron and manganese upon the formation of spores by mesophilic aerobes in fluid organic media. J. Bacteriol., 67, FOSTER, J. W. AND HEILIGMAN, F Mineral deficiencies in complex organic media as limiting factors in the sporulation of aerobic bacilli. J. Bacteriol., 57, GREENBERG, R. A. AND HALVORSON, H Studies on an autolytic substance produced by an aerobic spore-forming bacterium. J. Bacteriol., 69, HARDWICK, W. A. AND FOSTER, J. W On the nature of sporogenesis in some aerobic bacteria. J. Gen. Physiol., 35,

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