highest between pH 7.0 and 8.0; the generation time increased by 15% at pH 6.0 andby 75% at pH 8.5, and no growth occurred at pH 9.0. In light of these results ...
JOURNAL OF BACTERIOLOGY, May 1980, p. 633-638 0021-9193/80/05-0633/06$02.00/0
Vol. 142, No. 2
Growth of Thiobacillus novellus on Mixed Substrates (Mixotrophic Growth) RACHEL C. PEREZ AND ABDUL MATIN*
Department ofMedical Microbiology, Stanford University School of Medicine, Stanford, California 94305
In a mixotrophic environment, Thiobacillus novellus concurrently utilized glucose and thiosulfate but showed no stimulation of growth rate or yield. In most mixotrophic environments examined, the growth rate was lower than the heterotrophic growth rate, the extent of tbe decrease depending on the concentration and relative proportion of thiosulfate and glucose in the medium. Both thiosulfate and glucose were degraded to their most oxidized products in mixotrophic medium, yet the biomass production in this medium was comparable to that found in heterotrophic medium containing glucose alone at the corresponding concentration. It was postulated that in mixotrophic medium the oxidation of thiosulfate, glucose, or partially that of both was uncoupled from energy generation. These results differ in many respects from those reported earlier by LeJohn et al. (J. Bacteriol. 94: 1484-1491, 1967); experiments designed to exactly duplicate some of the growth conditions employed by these workers did not resolve the discrepancy. that previously described for T. novellus (6) except that FeCl3.6H20 was omitted, and a different trace elements solution (16), which contained a higher amount of iron, was used. There is no agreement on the pH for the cultivation of T. novellus, values ranging between 6.8 and 9.0 having been used (2, 6, 13, 15). Our control experiments, in which the culture pH was maintained constant during growth by means of a pH stat (see below), showed that the growth rate was highest between pH 7.0 and 8.0; the generation time increased by 15% at pH 6.0 and by 75% at pH 8.5, and no growth occurred at pH 9.0. In light of these results, we cultivated the organism at pH 7.0 in this study. The basal medium was supplemented with glucose, Na2S203, or both, at specified concentrations, to obtain the heterotrophic, autotrophic, and mixotrophic media, respectively; control experiments showed that the carbon source(s), the energy source(s), or both were the limiting nutrients in all media used in studies described in this and the two accompanying papers (1, 7). Stock cultures were maintained on heterotrophic and autotrophic solid media; those maintained on heterotrophic media were periodically checked for their ability to grow autotrophically. Unless otherwise stated, the organism was grown at 29 ± 1°C in a C-30 Bioflo unit (New Brunswick Scientific Co., New Brunswick, N.J.), which was used for batch culture operation (culture volume, 450 ml). The culture pH was maintained at 7.0 ± 0.1 by means of a New Brunswick pH stat, which automatically pumped 5% (wt/vol) sterile Na2CO3 solution into the culture to neutralize the acid produced; as expected, little carbonate addition occurred during heterotrophic growth, Organism and growth procedures. The ATCC but relatively large amounts were added during autotype strain of T. novellus (no. 8093) was used through- trophic and mixotrophic growth. The amount of tiout; the experiment presented in Fig. 2 was also carried trant added was recorded and used to calculate the out by using our laboratory strain (6), with very similar amount of S042- formed. Inocula were grown either results. The basal medium employed was similar to heterotrophically (for heterotrophic and mixotrophic 633
Thiobacillus novellus, like other facultative chemolithotrophs, possesses the unique potential for autotrophic as well as heterotrophic growth. Since in nature these bacteria are likely to encounter the heterotrophic and autotrophic growth substrates simultaneously, it is of considerable interest to determine whether they are capable of mixotrophic growth, i.e., growth during which organic and inorganic substrates are concurrently utilized (4, 11). Mixotrophy in T. novellus has been previously studied by LeJohn et al. (2). These workers showed that the presence of glucose in the medium caused a complete inhibition of thiosulfate utilization by this bacterium and that the rates of growth and glucose utilization in thiosulfate-glucose medium were identical to those found in glucose medium. They also showed that in thiosulfate-glutamate medium, the organism used both substrates concomitantly, but they did not report on whether the growth rate, yield, or both were higher in the mixotrophic medium compared to heterotrophic glutamate medium. Our studies, however, revealed a very different picture, and in this paper we present our findings concerning the comparative growth patterns of T. novellus in autotrophic, heterotrophic, and mixotrophic environments. MATERIALS AND METHODS
634
PEREZ AND MATIN
media) or autotrophically (for autotrophic media). A standard regimen was used to initiate growth. Media were inoculated to a cell density of approximately 0.06 mg of cells (dry weight) per ml of culture, and the airflow rate and the stirring speeds were adjusted to 0.2 liters per min and 200 rpm, respectively. After the culture reached a density of 0.09 mg (dry weight) per ml, these values were increased to 0.5 liters per min and 400 rpm. For experiments in which pH was manually adjusted, 250-ml Erlenmeyer flasks, containing 50 ml of medium, were employed. The medium used in these experiments was supplemented with 0.003% bromothymol blue which served as an internal pH indicator. The flasks were incubated on a rotary shaker (29 ± 1°C), and the acid produced during growth was periodically neutralized by manual addition of sterile 5% Na2CO3 solution. Growth was measured by optical density measurement (1 optical density at 660 nm equalled approximately 0.55 mg of cells [dry weight] per ml of culture). At the end of growth in all cases, culture purity was checked by microscopic examination and streaking on plates containing autotrophic and heterotrophic media. Measurement of residual substrates and accumulated metabolites. At appropriate intervals, 8ml culture samples were collected aseptically on 50 pl of a KCN solution (final KCN concentration in the sample, 5 mM); at this concentration, the inhibitor effectively eliminated respiration of residual or accumulated substrates by the cells but did not interfere with the methods used in their assay. The samples were filtered through 0.45-pm membrane filters (Millipore Corp., Bedford, Mass.), the filtrates being used in assaying the various substrates; the portion of the filtrate used in the sulfite assay was supplemented with 0.5 mM (final concentration) EDTA to prevent autoxidation of sulfite (12). Glucose was measured by the enzymatic method of Schacter (14), thiosulfate was measured by iodometric titration, using formaldehyde to prevent interference by sulfite (12), and tetrathionate and sulfite were measured colorimetrically (12) and by chromatography (10). Extent of glucose degradation. To determine if glucose degradation in heterotrophic and mixotrophic media was complete or partial, cells were grown in media supplemented with [U-'4C]glucose (initial radioactivity, 0.1 pCi/ml of medium), and the ratio between residual glucose and residual radioactivity in culture filtrates was determined at various times during growth; this ratio can be expected to remain constant only if no products of glucose degradation remain in the medium. Cultures were sampled, filtered, and assayed for glucose as described above. To determine residual radioactivity, the filtrates were acidified to approximately pH 3 by the addition of HCI to remove dissolved '4CO2 and counted in a Searle ISOCAP Scintillation Counter, using 0.1 ml in 10 ml of a scintillation fluid (2,5-diphenyloxazole [PPO], 3.2 g; toluene, 0.4 liters; 2-methoxy-ethanol [scintillation grade], 0.2 liters). Miscellaneous. Protein was determined by the method of Lowry et al. (3) using bovine serum albumin as the standard, and cell dry weight was determined as described by Matin and Veldkamp (8). [U-'4C]glu-
J. BACTrERIOL. cose (specific activity, 333 mCi/mmol) was purchased from Amersham Corp., Arlington Heights, Ill.; PPO, from Fisher Scientific Co., Pittsburg, Pa.; 2-methoxyethanol, from Eastman Organic Chemicals, Rochester, N.Y.; and other special chemicals from CalbiochemBehring Corp., La Jolla, Calif.
RESULTS Growth parameters and substrate utilization in various environments. In autotrophic medium supplied with 1% Na2S203, T. novellus exhibited a generation time of approximately 20 h (Fig. IA), and the growth roughly jiaralleled thiosulfate consumption (Fig. 1B). In heterotrophic glucose (0.4%) medium growth occurred at an 8-h generation time and ceased upon glucose exhaustion. In contrast, regardless of whether growth was followed by optical density measurement or plate count, a triphasic growth pattern was observed in miixotrophic medium containing both glucose (0.4%) and Na2S203 (1%). For the first three generations, the growth rate was similar to that in the heterotrophic medium; a slower growth rate was then established (13-h generation time), followed by another period of faster growth (Fig. 1A). The disappearance of both glucose and thiosulfate was coincident with growth, but the kinetics of this disappearance differed from those observed in heterotrophic and autotrophic media (Fig. 1B). Glucose was consumed at some 75% of the rate of heterotrophic culture, and thiosulfate utilization was not only slower than autotrophic cultures for a substantial part of the growth period (approximately 45% of the autotrophic rate), but also exhibited a triphasic pattern as opposed to the biphasic pattern observed under autotrophic conditions. Although concurrent and complete disappearance of thiosulfate and glucose occurred from the medium, the final biomass in mixotrophic medium, as measured by optical density, viable count, or protein determination, was somewhat lower than that found in heterotrophic medium (Fig. 1); thus, the utilization of the additional substrate had no salutary effect on the amount of cell material synthesized. One reason for this could be that in mixotrophic medium, as opposed to other media, glucose, thiosulfate, or both were only partially degraded, thus generating less energy. Therefore, we investigated the fate of these substrates in different environments. To determine the fate of glucose, T. novellus was grown in the presence of [ U-"4C]glucose and, at suitable intervals, culture filtrates were obtained and analyzed for residual glucose and radioactivity, as described above. In heterotrophic culture, the amount of radioactivity re-
MIXOTROPHY IN T. NOVELLUS
VOL. 142, 1980
moved from the medium precisely equalled that predicted from the amount of glucose that had disappeared (Table 1), indicating that no product of partial degradation of glucose accumulated in the medium at any time; had such accumulation occurred, there would be more residual radioactivity than could be accounted for by the remaining glucose. Slightly more radioactivity remained in the mixotrophic medium than that ascribable to residual glucose up to 47 h, indicating buildup of traces of some product of glucose degradation. However, even this small amount of excess radioactivity subsequently disappeared (Table 1, Fig. 1). Thus, glucose was essentially completely degraded at all times dur-
635
ing growth in both heterotrophic and mixotrophic media. The fate of thiosulfate was investigated by estimating the amount of sulfate formed in the culture at various times during growth. This estimation was made from the amount of Na2CO3 solution that was automatically pumped into the medium to maintain the culture pH at 7.0 ± 0.1 (see above). Except for the first approximately 30 h, the amount of sulfate found in mixotrophic medium was, on a mole-to-mole basis, nearly twice the amount of thiosulfate removed from the medium (Table 2), i.e., the amount expected had all the thiosulfate removed been converted to sulfate (9). Direct anal-
HOURS
HOURS
FIG. 1. Growth parameters of T. novelus in various media. The culture pH was maintained at 7.0 ± 0.1 by the use of a pH stat. (A) Kinetics of growth in 0.4% glucose medium (0), 1% Na2S203 medium (A), and 1% Na2S2030.4% glucose medium (0). One optical density at 660 nm about equals 0.6 mg of cells (dry weight)/ml of culture. (B) Kinetics of substrate consumption: (-), glucose consumption in glucose medium; (A), thiosulfate consumption in thiosulfate medium; glucose (V) and thiosulfate (a) consumption in thiosulfate-glucose medium.
TABLE 1. Disappearance of glucose and radioactivity during growth of T. novellus in heterotrophic and Hos Glucose Hours Glucoseconsumed from start of expt g/1jOml % of ini-
conmedb
13 18 24 30 44 47 51 54 57
mixotrophic media" containing [U-`4C]glucose Radioactivity conGlucose consumed' sumed_Glucoseconsume
sumedd
Ratio'
cpm/ml
%ofini-
Ratio'
g/100
0.021 0.040 0.085 0.150 0.40
5 10 21 38 100
3,800 7,650 18,000 29,000 75,000
5 10 24 38 100
1.0 1.0 0.9 1.0 1.0
-
-
-
-
-
-
aConcentrations: glucose, 0.4%; Na2S203, 1%. b In glucose medium. Ratio of glucose disappeared/radioactivity disappeared. d medium. -I1, glucose-thiosulfate Not determined. '
Radioactivity con-
% of ini-
/ I % of ini-
e
-
0.040 0.064 0.160 0.200 0.250 0.320 0.400
-
10 16 40 50 62 80 100
-
-
-
-
6,000 9,200 25,000 30,000 40,000 51,000 69,000
8 13 36 43 57 73 99
1.2 1.2 1.1 1.2 1.0 1.1 1.0
636
PEREZ AND MATIN
TABLE 2. Disappearance of thiosulfate and production of sulfate and sulfite in mixotrophic mediuma by T. novellus
Thiosulfate diamppeared
Sulfate Ratio of Sulfite found in found in sulfate culture (mmol/100 start of from cultureb found/thioculture' sulfate dis- (mmol/100 ept° (mmol/100 expt ml) ml) appeared ml) Hours from
1.64 1.18 1.39 0.09 1.39 2.18 1.57 0.10 1.66 1.71 2.84 0.08 2.45 4.42 1.80 0.05 2.97 5.84 1.97 0.01 6.72 3.64 1.85 NDd 6.63 12.70 1.92 ND a 0.4% 1% Glucose, (22 mM); Na2S203, (65 mM). b Calculated from the amount of Na2CO3 used to maintain culture at pH 7.0 ± 0.1. eSulfite concentration was measured colorimetrically. d ND, Not detected.
26 29 32 40 44 47 54
yses for tetrathionate in the culture filtrates obtained at various times gave consistently negative results. Sulfite was detected, however, especially during the first 30 h of growth, but the amount was small and by 44 h it was barely detectable; the second phase of rapid growth in mixotrophic medium (Fig. 1) appeared to coincide with the disappearance of sulfite from the culture. All the added thiosulfate could be accounted for by the sulfate found at the end of growth in mixotrophic medium and autotrophic medium (data not shown). Thus, thiosulfate was eventually completely converted to sulfate in both mixotrophic and autotrophic environments.
Effect of initial substrate concentration on the expression of the mixotrophic potential. The findings reported above are in sharp conflict with those of LeJohn et al. (2; see above). This discrepancy could be related to differences in the concentration of substrates used in the two studies. In contrast to the above experiments, the substrate concentrations employed in most of the work of LeJohn et al. (2) were 0.75% Na2S203 and 0.9% glucose. When we used these concentrations, the growth rate of T. novellus in mixotrophic medium was identical to that in heterotrophic medium containing 0.9% glucose alone, which agreed with the finding of LeJohn et al.; however, in contrast to their finding of a complete inhibition of thiosulfate utilization m such mixotrophic media, we again found a concurrent disappearance of both thiosulfate and glucose during growth, the rate of glucose disappearance in mixotrophic medium being lower than that in heterotrophic medium. Another discrepancy between the results was that LeJohn et al. (2) found a generation time of 2 h
J. BACTERIOL.
in heterotrophic medium, whereas we found it to be 8 h. Although the substrate concentration difference clearly cannot explain the discrepancy between our results and those of LWJohn et al. (2), the data show that the expression of mixotrophic potential of T. novellus is influenced by the initial concentration of thiosulfate and glucose in the medium. We therefore examined the effect of change in the proportion of these substrates on mixotrophic growth, using 0.1% glucose and various thiosulfate concentrations. In contrast to mixotrophic media containing 0.4% glucose (Fig. 1A), growth was monophasic in mixotrophic media containing 0.1% glucose, but its relation to heterotrophic growth was similar to that described above for the higher glucose concentration (i.e., 0.4%; Fig. 1). Thus, compared to the heterotrophic 0.1% glucose medium, the presence of thiosulfate, at any concentration tested, decreased the rate of growth as well as glucose utilization by the organism (Table 3), and a direct correlation seemed to exist between the initial thiosulfate concentration and the decrease in growth rate. The two substrates were concurrently utilized, but yet there was no significant difference in the final cell biomass between the heterotrophic medium and the various mixotrophic media. Effect of intermittent pH adljustment on the expression of the mixotrophic potential. In contrast to our studies in which culture pH was maintained constant at 7.0 ± 0.1 by the use of a pH stat, LUJohn et al. employed manual addition of base at various times during growth to control the pH of their cultures. To determine if this difference had a bearing on the conflicting findings, we repeated the experiments, using the manual method of pH control. In the mixotrophic mecium, growth occurred at the heterTABLE 3. Growth parameters of T. novellus in mixotrophic media containing 0.1% glucose and different concentrations of thiosulfate Final
cnn concn (%)
0.00 0.25 0.50 1.00
ttion(h) time 8.0 9.0 10.0 11.2
Glucose consumption
Halving concna timeb (mg) (h)
61.5 65.6 61.5 67.4
Ratec (W)
Relative
rated
4.7
0.15
-
-e
-
-
9.0 10.0
0.08 0.53 0.07 0.47 a of cells ml of Milligrams culture. (dry weight)/100 b Time interval for doubling the amount of glucose consumed at a given time. 'Rate = log e/halving timne. (Log e = 0.693.) dRatio of consumption rate in mixotrophic medium/consumption rate in heterotrophic medium. -, Not determined.
MIXOTROPHY IN T. NOVELLUS
VOL. 142, 1980
otrophic rate for approximately the first three generations and then came to an abrupt halt (Fig. 2); about 50% of the added thiosulfate and 70% of the added glucose still remained in the medium at this time. This general pattern was observed at any initial thiosulfate concentration above 0.5%. At thiosulfate concentrations below 0.5%, such as 0.3%, for which data are presented (Fig. 2), only transient growth inhibition was observed; the onset of growth inhibition coincided with the complete disappearance of thiosulfate from the medium, and this was followed by relief of inhibition. The physiological basis of these responses was not investigated, but it is evident that the method of pH control cannot explain the discrepancy between our results and those of LeJohn et al. DISCUSSION As opposed to the findings of LeJohn et al. (2), T. novellus is clearly able to express its mixotrophic potential since in all the mixotrophic environments examined in this study, the organism consumed glucose and thiosulfate concurrently. However, neither the mixotrophic growth rate nor yield was higher than that observed under heterotrophic conditions. Thus, although T. novellus can express its mixotrophic potential, its growth is not enhanced in a mixotrophic environment; as such, it constitutes the first documented case of a facultative chemolithotroph using substrates gratuitously under mixotrophic conditions. Several questions are raised by the findings presented in this paper. One of these concerns the lack of enhanced growth yield in a mixotrophic environment despite the concurrent and C
o
O
. 3.0
l
l
40 HOURS
60
i
zw W ~-aEo P
1.0 0
0.5
al I E O (n 0
0.1
0
, 20
80
FIG. 2. Growth of T. novellus in mixotrophic media with manual adjustment ofpH to approximately 7 by intermittent addition of sterile 5% Na2CO3 solution. Open symbols, growth; closed symbols, thiosulfate utilization. (0, 0), 1% Na2S203-0.4% glucose medium; (A, A), 0.3% Na2S203-0.4% glucose medium. Growth in 0.4% glucose medium under these conditions was essentially identical to that shown in Fig. 1.
637
complete dissimilation of thiosulfate and glucose. The growth yield in 0.4% glucose-1% thiosulfate medium was some 25% lower than would be the case if thiosulfate and glucose oxidations were coupled to energy generation with the same efficiency as in autotrophic and heterotrophic media, respectively. It is not known whether this uncoupling affected the oxidation of thiosulfate, glucose, or both, or what its mechanism is. Some of the obvious possibilities as regards the latter are an increased ATPase activity, an increased membrane proton permeability, or a decrease in the number of proton-translocating sites in the respiratory chain during mixotrophic growth. Another question relates to the physiological basis of the generally observed decreased growth rate in mixotrophic media. It is conceivable that generation of sulfite during mixotrophic growth plays a role in it. This compound, even at relatively low concentration (-l10- M), inhibited the growth rate of T. novellus when added to glucose medium (D. Drake and A. Matin, unpublished data), and there appeared to be a correlation between slow growth in 0.4% glucose-1% Na2S203 medium and the accumulation of sulfite in the culture (Fig. 1; Table 2). Sulfite could exert its effect by inhibiting glucose transport (7) or, since its metabolism in T. novellus is catalyzed by sulfite-cytochrome c oxidoreductase (9), by competing with NADH (generated from glucose oxidation) for the available cytochrome c. Other questions concern the physiological basis of the inhibition of thiosulfate and glucose utilization in mixotrophic media compared to autotrophic and heterotrophic media, respectively, and the advantage gained by the organism by this inhibition and the partial uncoupling of substrate utilization from energy generation. In the accompanying paper (7), we examine the physiological basis of decreased glucose utilization as well as discuss the question of the advantage gained. The marked differences between our findings and those of LeJohn et al. (2) also deserve comment. In addition to the mode of substrate utilization in mixotrophic media, our results are at variance with theirs in several other respects. We were unable to grow T. novellus at pH 9, whereas their entire study was carried out using this pH value; we found that the organism requires biotin in mineral salts media (5), but evidently they did not; and we found a generation time of 8 h in glucose heterotrophic medium, whereas they report it to be 2 h. The pronounced nature of these differences suggests that the strain used by LeJohn et al. (2) is different from the ATCC strain used in our studies; indeed, since there are many instances
638
PEREZ AND MATIN
of conflicting observations in the literature on T. novellus (4), it is possible that several different strains of this bacterium have been in use. ACKNOWLEDGMENTS This work was supported in part by the Stanford University School of Medicine C.S.R.B. General Equipment Joint Teaching and Research Fund and the Petricciani Foundation. R.C.P. held a predoctoral fellowship from the Ford Foundation.
LITERATURE CITED 1. Leefeldt, R H., and A. Matin. 1980. Growth and physiology of Thiobacilus novelus under nutrient-limited mixotrophic conditions. J. Bacteriol. 142:645-650.
2. LWohn, H. B., L. Van Caeseele, and H. Lees. 1967. Catabolite repression in the facultative chemoautotroph ThiobaciUus novelus. J. Bacteriol. 94:1484-1491. 3. Lowry, 0. H., N. J. Rosebrough, A. L Farr, and R. J. Randall. 1951. Protein measuremnt with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 4. Matln, A. 1978. Organic nutrition of chemolithotrophic bacteria. Annu. Rev. Microbiol. 32:433-468. 5. Matin, A., F. J. Kahan, and R. H. Leefeldt. 1979. Growth factor requirement of ThiobaciUu8 novelUw. Arch. Microbiol. 124:91-95. 6. Matin, A., and S. C. Rittenberg. 1971. Enzymes of carbohydrate metabolism in ThiobaciUus species. J. Bacteriol. 107:179-186. 7. Matin, A., . Schleies, and R. C. Perez. 1980. Regula-
J. BACTERIOL. tion of glucose transport and metabolism in Thiobacdillus novelus. J. Bacteriol. 142:639-644. 8. Matin, A., and H. Veldkamp. 1978. The selective advantage of a Spirillnm sp. in a carbon-limited environment. J. Gen. Microbiol. 105:187-197. 9. Oh, J. K., and L Suzuki. 1977. Isolation and characterization of a membrane-associated thiosulfate-oxidizing system of Thiobacillus novellus. J. Gen. Microbiol. 99: 397-412. 10. Okuzumi, K., and I. Kazutani. 1975. Studies on the biochemistry of thiobacilli. (v). Physiological studies of T. thiooxidans. Hakko Kogaku Zasshi (J. Fennent. Technol.) 43:10-17. 11. Rittenberg, S. C. 1969. The roles of exogenous organic matter in the physiology of chemolithotrophic bacteria. Adv. Microbiol. Physiol. 3:159-196. 12. Roy, A. B., and P. A. Trudlnger. 1970. The biochemistry of inorganic compounds ofsulfur. Cambridge University Press, London. 13. Santer, KL, J. Boyer, and U. Santer. 1959. Thiobacilu novellus. I. Growth on organic and inorganic media. J. Bacteriol. 78:197-202. 14. Schacter, H. 1975. Enzymic microassays for D-mannose, D-glucose, D-galactose, L-fructose, and D-glucosamine. Methods Enzymol. 41:3-10. 15. Taylor, B. F., and D. S. Hoare. 1969. A new facultative ThiobaciUus and a reevaluation of the heterotrophic potential of Thiobacillus novelus. J. Bacteriol. 100: 487-497. 16. Vishniac, W., and M. Santer. 1957. The thiobacilli. Bacteriol. Rev. 21:195-213.
