Estimation of Growth Parameters for Some Oral Bacteria Grown in Continuous Culture under Glucose-Limiting Conditions

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1 INFECTION AND IMMUNITY, June 1986, p /86/ $02.00/0 Copyright C 1986, American Society for Microbiology Vol. 52, No. 3 Estimation of Growth Parameters for Some Oral Bacteria Grown in Continuous Culture under GlucoseLimiting Conditions A. H. ROGERS,2* M. H. DE JONG,1 P. S. ZILM,2 AND J. S. VAN DER HOEVEN' Microbiology Laboratory, Department of Dentistry, The University of Adelaide, South Australia,2 and Department of Preventive and Community Dentistry, University of Nijmegen, 6500 HB Nijmegen, The Netherlands' Received 25 November 1985/Accepted 19 February 1986 The coexistence of bacteria in natural environments can often be explained in terms of competition for a growthlimiting substrate(s), and the outcome of such competition depends upon relevant growth parameters such as substrate affinity and yield. Dental plaque bacteria are frequently carbon and energy limited. Growth parameters for seven oral Sreptococcus species and one Actinomyces viscosus strain were estimated under glucoselimited conditions in continuous culture. In all strains, mixedacid fermentation occurred at low growth rates, while amounts of lactate increased at higher growth rates. Two important growth parameters, ulmax and Ygi,cose, were very similar in the two serotype c Streptococcus mutans strains (T8 and Ingbritt), one of the serotype d/g Streptococcus mutans strains (OMZ65), and the two Streptococcus milledi strains (699B3 and B448). Two other serotype d/g S. mutans strains (KIR and B13) were divergent from this group and had lower ILmax values and a lower YglU,O,e. The maintenance energy coefficients were lower in the S. mutans serotype c strains, and the highest values were observed in the S. milledi strains. While A. viscosus had a lower I'max9 its lower maintenance rate and signfficantly higher yield indicate that it deals much more efficiently with glucose than do the streptococci. The most striking feature of amino acid utilization was that arginine was completely consumed by S. millei strains; similarly, A. viscosus used up all available asparagine as did one of the S. milledi strains at faster growth rates. It is suggested that the ability of strains of S. milledi and S. sanguis to utilize arginine in addition to carbohydrate as a source of energy may explain why such organisms increase in proportion in the plaque of subjects consuming diets almost devoid of fermentable carbohydrate. Dental plaque, the complex microbial community firmly bound to the tooth surface, is intimately involved in the etiology of dental caries and periodontal disease, two widespread diseases in humans. No in vitro model that sufficiently mimics natural dental plaque has been developed, but attention has been drawn to the utility of the chemostat as an environmentally relevant tool in studying the ecology of microbial communities such as those existing in dental plaque (8, 11, 36). In dental plaque, many physiologically related bacteria appear to coexist, a phenomenon common to most natural habitats. Since most natural environments are nutrient limited (40), coexistence has been explained, particularly in the case of aquatic ecosystems, in terms of competition for growthlimiting substrates. According to the principle of growthlimitingsubstrate competition, the outcome of competition depends upon growth characteristics such as Ks, a measure of substrate affinity, maximum specific growth rate (p,max), and also upon the molar growth yields of cells on the limiting substrate(s) (23, 40). By applying this concept to the commonly found dental plaque species Streptococcus mutans and Actinomyces viscosus, preliminary mixed continuous culture studies have been reported (37). However, as a first step to explain the coexistence of these predominant species in dental plaque, it is pertinent to determine growth parameters of the relevant organisms in pure cultures. Such an approach has been used to study interactions among rumen bacteria and the results used to develop a useful computer model (30). Available evidence suggests that the growth of facultative anaerobic bacteria in dental plaque is frequently carbon and energy limited. For example, S. mutans normally exhibits * Corresponding author. 897 mixedacid fermentation in the plaque of monoinfected gnotobiotic rats (34), just as it does in chemostat cultures under carbohydrate limitation (3). Moreover, the growth yield of oral streptococci and A. viscosus on the natural substrate, saliva, has been found to be carbohydrate limited in vitro (6). Accordingly, in the present study, a number of plaque organisms were grown under glucose limitation at various growth rates in the chemostat, and relevant growth parameters were determined. MATERIALS AND METHODS Organisms. S. mutans Ingbritt was obtained from B. Krasse, University of Goteborg, Goteborg, Sweden. The origins of S. mutans T8, OMZ65, KIR, and B13 have been described previously (29). Streptococcus milleri B448 was obtained from J. Hardie, London Hospital Medical College, London, United Kingdom; strain 699B3 was a local dental plaque isolate; and A. viscosus Ut2 was a human isolate (5). Stock cultures were maintained in skim milk (20 C) and for shortterm purpose on Trypticasesoy agar plates (BBL Microbiology Systems, Cockeysville, Md.). Medium and culture conditions. In all experiments, a filtersterilized, chemically defined medium was used (35). The medium contained a range of amino acids (total, 2.6 g liter'), vitamins, inorganic components, trace elements, and 10 mm glucose to give glucoselimiting conditions. It supported the batch growth of a wide range of strains of oral streptococci and actinomyces. The cell yield of these strains increased linearly with the amount of glucose added to the medium up to glucose concentrations of at least 50 mm. Organisms were grown in a chemostat with a working capacity of 350 ml; the temperature was maintained at 37 C, and the ph was controlled at ph 7.0, except as otherwise noted in the text, by the automatic addition of 2 M KOH.

2 898 ROGERS ET AL. INFECT. IMMUN. TABLE 1. Effect of dilution rate on some growth parameters and metabolic end products of oral bacteria grown in chemically defined medium containing 10 mmol of glucose liter' as the growthlimiting substrate Organism and Products formed (mol * mol of glucose') C recovery Yg YATPb dilution rate (h)a Formate Acetate Succinate Lactate Ethanol (%) (g of cells * mol1) (g of cells * mol1) A. viscosus Ut S. mutans Ingbritt a Cells were grown in chemically defined medium with 10 mm glucose as the growthlimiting substrate. b YATP was calculated on the assumption that ATP was produced as 2 mol of ATP per mol of acetate formed; 1 mol of ATP per mol of lactate; 1 mol of ATP per mol of ethanol; and 1.33 mol of ATP per mol of succinate formed. The culture was gassed with N2 containing 5% (vollvol) CO2 (flow rate, 200 ml. min'). Medium flow was controlled with a Gilson Minipuls 2 peristaltic pump attached to a voltage stabilizer or with an LKB multiperpex The dilution rate (D) was increased stepwise from 0.05 h1. Cultures were allowed to reach equilibrium for at least 10/D h at each dilution rate before samples were taken for analyses. Culture purity was checked daily by microscopic examination and by plating on Trypticasesoy and mitissalivarius agar plates which were incubated at 37 C in an N2CO2 (95:5) atmosphere. To measure residual glucose and fermentation end products, cells must be rapidly removed from the chemostat (35). Culture samples were therefore quickly passed through membrane filters (0.45,um pore size; Millipore Corp., Bedford, Mass.) such that the total time required to obtain filtersterilized samples did not exceed 15 s. Analytical procedures. Ethanol was measured enzymatically (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). Formate, acetate, and lactate were determined isotachophoretically (38). Volatile products were also analyzed by gasliquid chromatography (18). Amino acids were assayed by highpressure liquid chromatography by a modification of the method of Hill et al. (17). Residual glucose was measured by a cyclic enzymatic method (20). Optical density of the cultures was measured at 560 nm. Bacterial dry weights were determined by taking duplicate 20ml samples, washing them twice in distilled water, and drying the washed deposits to constant weight at 105 C. Calculations. The maximum specific growth rates (Lmax) of some strains were determined by the washout method (19). Plots of log A560 against time (t) were analyzed by linear regression to derive slopes equal to PUmax D. For these strains, the ILmax values agreed with those obtained by the usual method in which cell populations are measured during the exponential growth phase in batch culture. The ILmax, values for most strains were therefore measured by the latter method. The maintenance energy coefficient (m) and the true growth yield (Yg) for glucoselimited cultures were calculated by the methods of Pirt (27) and Herbert and Kornberg (16), using the specific rate of glucose consumption (qs) in glucoselimiting chemostat cultures: q, = D(Sr S)lx, where q, is the rate of glucose utilization (millimoles of glucose. milligrams (dry weight) of cells' * hour'); D is the dilution rate (hour'); Sr is the glucose concentration in the inflowing medium (millimoles liter1); S is the glucose concentration in the outflowing culture (millimoles liter'); and x is the dry weight of cells (milligrams liter'). As compared with Sr (1,800 mg * liter'), the measured glucose concentration in the outflowing culture (1.8 to 3.6 mg. liter') was extremely low up to,u values near Rwmax and neglected for the calculation of q, A plot of qs against D gives the maintenance energy m (millimoles of glucose milligrams of cells' * hour') as the intercept and the reciprocal of the gradients equivalent to the true growth yield Yg (milligrams of cells millimoles of glucose') (26): q, = (DlYg) + m. Bestfit lines were plotted by regression analysis (r > 0.9) for all plots. RESULTS Effect of growth rate on metabolic products and growth yields. Representative data for two strains are shown in Table 1, from which it can be seen that, at low dilution (growth) rates, the fermentation end products for (all five) streptococcal strains were mainly formate, acetate, and ethanol in a 2:1:1 ratio with small amounts of lactate. However, at higher dilution rates the pattern was reversed, and lactate became the major end product. Gasliquid chromatography revealed no traces of other volatile end products. A. viscosus fermented glucose to formate, acetate, and succinate rather than ethanol, in a ratio of 1:1:1. At higher dilution rates increasing amounts of lactate were found. The measured amounts of residual glucose did not exceed 20,uM up to p. values near maximum. It can be seen that the production of cells from glucose, the molar growth yield, Ygiucose, was strikingly higher for A. viscosus than for the streptococci. Interestingly, for S. mutans Ingbritt, Ygluco,e decreased at high growth rates when lactate became a major fermentation product. In contrast,

3 VOL. 52, 1986 YATP showed a slight increase (Table 1). At very low growth rates the cell yields of all organisms were lower. With respect to amino acid utilization (Table 2), no significant trends between dilution rates were observed in any of the eight organisms studied. In general, the S. mutans strains used no more than 30% of any single amino acid. The most striking feature of the two S. milleri strains (B448 and 699B3) was their utilization of asparagine and arginine. Arginine was completely consumed by both strains and converted to ornithine (Table 2). It is worth noting that in a separate experiment (data not shown), the level of arginine in the medium was raised from the normal 1.1 mm to 7.5 mm, and under these conditions S. milleri B448 also metabolized all of the arginine to ornithine. In general, A. viscosus Ut2 used more of the amino acids than the streptococci; however, only asparagine was used up completely. In a separate experiment, no relation between the growth of A. viscosus Ut2 and the concentration of asparagine was observed. Growth parameters. The two serotype c S. mutans strains Ingbritt and T8 and the serotype d/g strain OMZ65 possessed almost identical growth parameters, although strain OMZ65 had a higher maintenance energy coefficient (Table 3). The serotype d/g S. mutans strains KIR and B13 produced lower yields and lower Pmax values than the other S. mutans strains. Both strains often grew rather poorly in batch culture, even in complex media such as yeastsupplemented ToddHewitt broth. S. milleri B448 and 699B3 displayed growth characteristics very similar to those of S. mutans Ingbritt and T8. The maintenance energy coefficients for both S. milleri strains TABLE 2. Amino acid utilization by various oral bacteria grown at ph 7.0 in continuous culture in a chemically defined medium containing 10 mmol of glucose liter' as the growthlimiting substratea % Utilized at D (h') of: Amino acid S. mutans S. milleri B448 A. viscosus Ut2 Ingbritt Aspartic acid Hydroxyproline 7 7 NDb ND 6 24 Threonine Serine Asparagine Glutamic acid Proline ND ND Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine +3c Phenylalanine Histidine Lysine Tryptophan Arginine Cysteine Ornithine ND ND a In the chemically defined medium (35), amino acids were present in amounts of ca. 1 to 2 mmol * liter. b ND, Not determined. c Values preceded by + indicate formation; in the case of ornithine, the figures are calculated from the amount of arginine originally present in the medium. GROWTH PARAMETERS OF SOME ORAL BACTERIA 899 TABLE 3. Kinetic parameters of some oral bacteria grown at ph 7.0 in continuous culture in a chemically defined medium containing 10 mmol of glucose * liter' as the growthlimiting substrate m SEb y b cells' mo1) h'1) Organism IL... (h1) (mmol g S. mutans Ingbritt ± a (ph 6.5) 0.44 ± S. mutans T ± S. mutans OMZ ± (ph 6.0) 0.88 ± S. mutans KIR ± S. mutans B ± S. milleri 699B ± (ph 5.5) 1.01 ± S. milleri B ± A. viscosus Ut ± a Calculated from data given in reference 10. b These values were obtained from linear regression equations of plots of q, versus D; m, the maintenance energy coefficient, is the intercept, and the reciprocal of the gradient is equivalent to Yg, the true growth yield. were higher than those observed for S. mutans. In contrast, A. viscosus had a lower maintenance energy coefficient, a lower p.max, and a significantly higher cell yield from glucose than any of the streptococci. DISCUSSION End product analysis of the five S. mutans strains presently studied confirmed previous findings (3, 10) that, under glucoselimiting conditions, the organism is heterofermentative, although increasing amounts of lactate are produced as the growth rate is increased. The same was found for the two strains of S. milleri. This, coupled with the fact that the phenomenon also occurs in Streptococcus sanguis (3), lactic streptococci (33), and Streptococcus bovis (31), confirms that it is common in facultative anaerobic streptococci. The observation that A. viscosus generates substantial amounts of succinate as well as formate and acetate, especially at slower growth rates, is in agreement with previous studies (2, 14). It has been suggested that additional energy can be generated by the formation of succinate (2, 13). The high cell yield of A. viscosus observed in our study can only be explained if substantial energy is generated by the production of succinate. At all dilution rates A. viscosus Ut2 consumed all available asparagine (Table 2), as was observed before (14). However, asparagine was not a growthlimiting substrate since the growth of A. viscosus Ut2 was not restricted in a medium without asparagine and no rise in yield was obtained by increasing its concentration in the growth medium (unpublished data). The molar growth yield (Table 1) shows a complex dependency on the growth rate. The low yield at the lower values of D is accounted for by maintenance demands as expressed by the equation of Pirt (26). This equation adequately describes the relationship between Y and,u in carbonenergylimited cultures (provided that,. is not lower than 0.02 h1). The growth yield of S. mutans further appeared to be influenced by the fermentation pathway and the generation of ATP from glucose. This is reflected by the drop in yield at high values of D, which can be explained by the shift from mixedacid fermentation (3 mol of ATP. mol of glucose') to homolactic fermentation (2 mol of ATP. mol of glucose'). The concomitant increase in YATP might be

4 900 ROGERS ET AL. INFECT. IMMUN. explained by coupling of the extrusion of lactate with the generation of a proton motive force, as was observed for Streptococcus cremoris (25). The molar growth yield of A. viscosus was strikingly higher than that of S. mutans or S. milleri, indicating that this organism deals more efficiently with glucose. Assuming that 1.33 mol of ATP are formed per mol of succinate the YATP of A. viscosus is near the theoretical maximal value (32). Values of saturation constants (Ks) for growthlimiting carbohydrate sources are about 105 mol liter' for most microorganisms (27). This makes a direct estimation of Ks values from,us plots often difficult since the concentration of residual substrates in the culture is too low to be measured with sufficient accuracy. In quickly filtersterilized samples we measured residual glucose concentration varying between 5 and 20 ixm. No satisfactory,us plots and Ks values could, however, be obtained from these data. This failure was apparently due to reasons such as: (i) substrate utilization in the time required to sterilize the sample, especially at high dilution rates; (ii) at least two uptake systems for glucose in oral streptococci, operative at different dilution rates (10); (iii) fluctuations in the residual substrate concentration in the culture owing to mechanical reasons such as stirring, dropwise addition of substrates, etc., especially at low dilution rates. Since K, reflects an important ecological property, namely, the capacity of the organism to scavenge substrate at low concentrations, and since it has been shown that the competition between two organisms for a single substrate is solely dependent on their respective substrate affinities, an alternative approach to this question seems indicated. Such an approach has been used in a study in which, under glucoselimiting conditions, the competition between pairs of streptococci was recorded. It appeared that S. mutans strains always lost competition for glucose or sucrose from S. sanguis and S. milleri, indicating that S. mutans had a lower affinity for these substrates (35). The maintenance energy represents the collective energy demands for processes that do not produce a net increase in biomass. There is general agreement that maintenance energy demand in carbonenergylimited cultures is growth rate independent of all,u values higher than about 0.05 h' (27, 28, 39). The significance of the maintenance demand for the competitiveness of a microorganism is probably expressed in its effect on the molar growth yield, especially at very low growth rates. The differences in the maintenance rates of the organisms included in this study are relatively small and unlikely to have any significant effect on the competition in mixed cultures. Theoretical considerations and experimental studies by Gottschal and Thingstad (12) have indicated that the yield, in addition to Ks values, determines the proportions of organisms in mixed cultures under multiplesubstrate limitation. From these studies it could be inferred that a high molar growth yield favors an organism in a complex natural environment. In this regard, the high yield of A. viscosus merits attention. This characteristic might well be related to the abundance of Actinomyces species in the mouth. Comparison of the patterns of amino acid utilization by the streptococci with previous studies is difficult since published data for S. mutans Ingbritt grown under carbon and energy limitation indicate that such patterns are influenced by several factors, including the composition of the growth medium (7, 9, 15). In contrast to the above studies, we used a chemically defined medium. The complete consumption of available asparagine by A. viscosus has already been discussed, and in relation to the streptococci, the most striking feature involves the utilization of this amino acid and arginine by S. milleri. Asparagine was completely consumed at faster growth rates, while arginine was entirely used at all dilution rates (Table 2). Thus, S. milleri would appear to experience both glucose and arginine limitation. The arginine was quantitatively converted to ornithine, presumably via the arginine deiminase pathway with concomitant energy generation in the form of carbamyl phosphate (1). Arginine catabolism has been linked to the formation, by some oral streptococci, of glycogen (21) and is efficiently coupled to growth in Streptococcus lactis, S. milleri, and S. sanguis (4, 33, 36). The effects of arginine might explain the dominance, particularly at slower growth rates, of organisms such as S. milleri and S. sanguis in glucoselimited mixed continuous cultures of oral microorganisms (22, 24). Further, it has been argued that the ability to utilize arginine is related to the clinical observation that the proportions of S. sanguis increase and those of S. mutans decrease in subjects consuming a proteinrich diet devoid of carbohydrate (36). ACKNOWLEDGMENTS A.H.R. gratefully acknowledges the financial Australian Dental Research and Education Trust. support of the LITERATURE CITED 1. Abdelal, A. T Arginine catabolism by microorganisms. Annu. Rev. Microbiol. 33: Buchanan, B. B., and L. Pine Path of glucose breakdown and cell yields of a facultative anaerobe, Actinomyces naeslundii. J. Gen. Microbiol. 46: Carlsson, J., and C. J. Griffith Fermentation products and bacterial yields in glucoselimited and nitrogenlimited cultures of streptococci. Arch. 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