Nutrient-Limited Mixotrophic Conditions - Journal of Bacteriology

JouRNAL OF BACTERIOLOGY, May 1980, p. 645-650 0021-9193/80/05-0645/06$02.00/0

Vol. 142, No. 2

Growth and Physiology of Thiobacillus novellus Under Nutrient-Limited Mixotrophic Conditions R. H. LEEFELDT AND A. MATIN*

Department of Medical Microbiology, Stanford University School ofMedicine, Stanford, California 94305

Thiobacillus novellus was cultivated in a chemostat under the individual limitations of thiosulfate, glucose, and thiosulfate plus glucose. At dilution rate (D) of 0.05 h-1 or lower, the steady-state biomass concentration in mixotrophic medium was additive of the heterotrophic and autotrophic biomass at corresponding D values. The ambient concentrations of thiosulfate, glucose, or both in the various cultures were low and were very similar in mixotrophic, heterotrophic, and autotrophic environments at a given D value. At D = 0.05 h-1, mixotrophic cells possessed higher activities of sulfite oxidase and thiosulfate oxidation compared to autotrophic cells, as well as higher activities of glucose enzymes and glucose oxidation than heterotrophic cells. Thus, in contrast to nutrient-excess conditions, in nutrient-limited mixotrophic environments at these D values, T. novellus did not exhibit characteristics of uncoupled substrate oxidation, inhibition of substrate utilization, and repression of enzymes of energy metabolism. It is concluded that T. novellus responds to mixotrophic growth conditions differently in environments of different nutritional status, and the ecological and physiological significance of this finding is discussed. In the accompanying two papers (9, 12), we have dealt with mixotrophy in Thiobacillus novellus under nutrient-excess conditions of batch culture. These studies showed that during growth in such environments T. novellus employs regulatory mechanisms that appear to be designed to minimize energy generation. The rates of thiosulfate and glucose utilization are reduced in this environment, and substrate oxidation appears to be partly uncoupled from energy generation. Such a strategy, although suitable in a nutrient-excess environment, would as a rule be harmful to the organism in nature where microbial growth for the most part occurs under conditions of extreme nutrient scarcity (1, 10, 16). This consideration led us to predict (9) that during growth in a nutrient-limited mixotrophic environment, T. novellus would employ a strategy different from that revealed in the previous studies (9, 12). This paper is concemed with testing this prediction. To attain growth under nutrient-limited conditions, a chemostat was employed and growth parameters were examined at several dilution rates (D) in autotrophic, heterotrophic, and mixotrophic media.

0.15% (8 mM) glucose, 0.64% (40 mM) Na2S203, or both, respectively; as mentioned already (12), the limiting nutrient(s) in these media was the carbon source, energy source, or both. Bacteria were grown at 29 ± PC in a C-30 Bioflo chemostat (New Brunswick Scientific Co., New Brunswick, N.J.) with a working volume of 520 ml. pH was automatically maintained at 7.0 ± 0.1 by the addition of 5% (wt/vol) of sterile Na2CO3 solution as previously described (12). At least five volume changes (approximately seven generations) were allowed at a given D before the cultures were considered to be in a steady state. To nimize the chances of selection of mutants adapted to a particular D, fresh chemostat cultures were frequently started from stock cultures, and the difference in D for two successive steady states was kept large. At the time of harvest, all cultures were checked for purity and possession of autotrophic potential by streaking on plates containing heterotrophic and autotrophic media. All the data presented pertain to steady-state cultures. The methods used in various measurements (sampling and filtering of cultures [12], assay of residual substrates [12], oxygen uptake measurements [12], preparation of cell-free extracts and enzyme assays [6, 11], and determination of culture dry weight [12], protein [12], and poly-f?-hydroxybutyric acid content [10]) have been previously specified. Maximal growth rate (IL..) during chemostat growth was measMATERIALS AND METHODS ured by the method of Pirt (13). The culture D was The ATCC type strain of T. novellus (no. 8073) set at a value believed to be greater than the imax used in the work described in the accompanying two under a given set of conditions, and the washout rate papers (9, 12) was employed. The heterotrophic, au- was measured. IL. was calculated from the slope of a totrophic, and mixotrophic inflow media used were logarithmic plot of the washout rate, which equals max- D (13). obtained by supplementing the basal medium (8) with 645

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and heterotrophic biomass at corresponding D values. In contrast, at D = 0.08 h-1, the mixotrophic biomass was lower than the heterotrophic biomass at the same D value. Qualitatively very similar results were obtained when the cell biomass was estimated by measuring protein rather than the dry weight of the organisms, which suggested that the increased mixotrophic biomass at D values of 0.05 h-1 and lower was not due to buildup of a reserve polymer. Direct analyses of cells for poly-.8-hydroxybutyric acid strengthened this inference; only small amounts were found, approximately 0.6% of dry weight in cells grown autotrophically and mixotrophically and approximately 1% of dry weight in heterotrophic celLs, and these amounts were not significantly influenced by culture D value. The steady-state concentration of thiosulfate, glucose, or both in the culture fluid was very low at D = 0.06 h-1 or lower under all growth conditions (Table 1), which is consistent with their being the growth-limiting nutrient. Further, at D values of 0.05 h-1 or lower, the steady-state concentrations of thiosulfate and glucose in mixotrophic culture were very similar to the concentrations of these substrates in autotrophic and heterotrophic cultures, respectively, at corresponding D values. Thus, at these D values there was no inhibition of thiosulfate or glucose utilization in the mixotrophic medium; had inhibition occurred, a higher steady-state level of the ---790 affected substrate would be found in the mixotrophic culture compared to the single-substrate , 80 Icultures at the corresponding D value. The K. for thiosulfate and glucose, which can be calcuE 70 IF lated from these data using the equation, K. = 8 60 l - DID x i (where K. is the affinity constant, i is the steady-state concentration ofthe residual substrate in the culture, and other symbols as -50 specified previously), was in the range of 10-i M. 0~ In contrast, at D = 0.08 h-1 the steady-state E 40 tF concentration of glucose in mixotrophic culture cn was approximately 300-fold higher than in het< 30 lF erotrophic culture grown at the corresponding D value. The thiosulfate concentration was also m -j 20 _ some 90- to 100-fold higher than that found -J w under any other conditions, including the near 10 maximal D value obtainable under autotrophic AI0 conditions (Table 1). It is therefore evident that in contrast to D values of 0.05 h-1 and lower, at 0 0.02 0.04 0.06 0.08 D = 0.08 h-', the utilization of both thiosulfate and glucose is partially inhibited in mixotrophic DILUTION RATE (h-1) FIG. 1. Steady-state ceU biomass of T. novelus in medium compared to the autotrophic and hetnutrient-limited heterotrophic (0), autotrophic (A), erotrophic cultures, respectively, grown at near values. In this respect, as well as in not and mixotrophic (0) environments at specified dilu- p tion rates. See text for substrate concentration in the exhibiting an increased cell biomass compared to the heterotrophic culture (Fig. 1), the D = various media.

RESULTS Maximal growth rate during nutrientlimited growth. Initial studies suggested that the 1 in autotrophic medium was higher under nutrient-limited conditions than was found in batch cultures (12). We therefore determined the p during nutrient-limited growth in all the different media included in this study, as described above. The values were as follows: 0.055 h-1, 0.083 h-1, and 0.085 h-l, in autotrophic, mixotrophic, and heterotrophic media, respectively. It is evident, therefore, that in thiosulfatecontaining media 1.is greater under nutrientlimited conditions than under nutrient-excess conditions of batch culture; apparently, higher thiosulfate concentrations are pardally inhibitory to the growth of the organism. Steady-state cell biomass and residual substrates in various environments at different dilution rates. Under autotrophic and heterotrophic conditions, the steady-state cell biomass increased with increasing D values (Fig. 1), as is customary with chemostat culture of microorganisms (2, 17). However, under mixotrophic conditions, the biomass increased with D up to 0.05 h-1 and then declined. At all D values up to 0.05 h-1, i.e., the highest D at which the organism could be grown in all the three different nutritional environments, the mixotrophic biomass was additive of the autotrophic

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TABLE 2. Amount of cell material synthesized in 0.08 h-' mixotrophic culture resembles the nurelation to the amount of glucose consumed at trient-excess mixotrophic cultures studied previously (12). However, it differs from the latter various D values in heterotrophic and mixotrophic media in that there is evidently no uncoupled oxidation % Gluof the substrates. This is indicated by the fact cose' carthat if the decrease in the amount of substrates Dilution rate bon not terl ma- Glucose utilized under these conditions is taken into consumed accounted (h) account (Table 1), the observed biomass concenfor by cell material tration (61 mg of cells [dry weight]/100 ml of culture) is very simlar to that expected (65 mg Heterotrophic of cells [dry weight]/100 ml of culture) if the medium oxidized substrates were coupled to biomass for0.02 39 134 63 0.03 48 150 60 mation with the same efficiency as during

theaized

growth

at

near

I.

values in the

single-substrate

0.04

47

149

60

0.05 67 149 44 media (i.e., autotrophic and heterotrophic me0.06 67 149 44 dia). In other words, the decrease in biomass 0.08 74 144 36 formation under these conditions is completely accounted for by the decrease in the amount of Mixotrophic mesubstrates utilized. dium Role of glucose. It is evident from the above 0.02 61 154 50 that in the mixotrophic media both thiosulfate 0.03 68 150 43 0.04 81 149 32 and glucose contribute to cell growth. The role 0.05 83 149 30 of glucose under these conditions could be to 0.06 83 149 30 provide only cell carbon, or to provide both 0.08 61 90 15 carbon and energy. Information on this point was obtained by comparing the amount of glua Based on the assumption that carbon constitutes cose carbon used up with the amount of carbon 50% of cell dry weight. in the cell material synthesized under these conditions (Table 2). At various D values in the ergy is derived from thiosulfate oxidation, and mixotrophic environment, some 15 to 50% more glucose serves only as carbon source (3). glucose disappeared from the medium than As might be expected from the fact that glucould be accounted for by the cell material syn- cose was the only energy source under heterothesized; this unaccounted glucose must have trophic conditions, more glucose remained unbeen used in energy generation along with thio- accounted for by the cell material synthesized in sulfate. It is noteworthy that at D = 0.08 h'1, this medium compared to the mixotrophic mecomparatively little glucose was used in energy dium (Table 2). generation; thus, it appears that under these Regulation of enzyme synthesis in varconditions, the mixotrophic growth of T. novel- ious energy-limited environments. As dislus is analogous to that of T. intermedius in cussed above, in mixotrophic cultures at D = nutrient-excess environments, during which en- 0.06 h-' or lower there is no inhibition of thiosulfate or glucose utilization. This situation is different from the nutrient-excess mixotrophic TABLE 1. Steady-state residual substrate cultures which exhibit marked decrease in the concentrations in cultures of T. novellus at various rate of substrate utilization (12). One of the dilution rates underlying mechanisms of this decrease is enResidual substrate concn various cultures zyme repression in the latter cultures (9), and (UM) the question therefore arose as to whether mixDilution otrophic cultures grown at D = 0.06 h-' or lower Autorate MisoHeteroMixowould also exhibit such repression. To obtain trophic trophic trophic trophic information on this point, we grew the organisms glucose fate glucose thiosulfate to a steady state at D = 0.05 h-1 in different nutritional environments and compared their 11 56 11 60 0.02 respiratory and enzymatic activities as described 76 19 9 78 0.03 44 44 0.04 (12). The mixotrophic celLs possessed a higher 44 115 44 78 0.05 respiratory capacity for thiosulfate, as well as a 44 46 75 0.06 higher sulfite oxidase activity compared to au11 0.08 8,500 3,000 totrophic cells (Table 3); moreover, they exa hibited a higher glucose-oxidizing ability and Not determined. in

-a

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higher levels of many glucose enzymes than heterotrophic cells (Table 3). Thus, during nutrient-limited growth at this D value there was no repression of enzyme synthesis in mixotrophic environment; indeed, the synthesis of some of the enzymes examined was stimulated. As noted above, the D = 0.08 h-1 mixotrophic cultures exhibit partial inhibition of substrate utilization; measurements showed (Table 3) that these cultures also exhibit the enzyme repression characteristic of mixotrophic nutrient-excess conditions (9); both the rate of glucose oxidation and the activity of glucose 6-phosphate dehydrogenase were decreased some three- to fourfold in these cells compared to the heterotrophic cells grown at corresponding D value. DISCUSSION We postulated (9) that the regulation of mixotrophic growth of T. novellus will be different in a nutrient-limited environment compared to nutrient-excess environments studied previously (9, 12). The data presented in this paper bear out this postulation for chemostat cultures grown under thiosulfate plus glucose limitation at a D value of 0.06 h-1 or lower. As opposed to nutrient-excess mixotrophic conditions (9, 12), there was no inhibition of thiosulfate and glucose utilization at these D values and, as might be expected from this finding, no repression of the metabolic enzymes of these substrates. In addition, there was evidently no decrease in the efficiency of coupling of substrate oxidation to energy generation, since the biomass production under mixotrophic conditions was additive of that under heterotrophic and autotrophic conditions at a given D value. At these D values under mixotrophic conditions, energy was clearly derived from both thiosulfate and glucose oxidations and glucose

probably served as the major carbon source. Additional carbon may have been derived from the small amount of yeast extract added to the basal medium (8) (to satisfy the biotin requirement of the organism [7]), and possibly also from C02. Ribulose diphosphate carboxylase is generally repressed by organic compounds in facultative chemolithotrophs (4, 14), which suggests that the contribution of C02 to cell carbon would be minimal under these conditions. However, this finding applies to saturating concentration of organic material and it is not known whether ribulose diphosphate carboxylase is repressed also at subsaturating concentration of organic compounds, like those employed here. Since many enzymes are derepressed in bacteria grown at subsaturating concentration of organic compounds (5, 6; A. Matin, in P. H. Calcott, ed., Continuous Cultures of Cells, in press), it is possible that in facultative chemolithotrophs ribulose diphosphate carboxylase continues to be synthesized and to function under these conditions. Indeed, the fact that mixotrophic biomass concentrations at D values of 0.05 h-' and below were not more than additive of individual autotrophic and heterotrophic biomass concentrations at corresponding D values indicates that C02 may have continued to be fixed under mixotrophic conditions. Experiments are now in progress to determine directly whether and to what extent C02 serves as a carbon source during the growth of T. novellus in the presence of subsaturating concentrations of organic compounds. A different pattern of mixotrophic growth was exhibited by T. novellus at D = 0.08 h-1, which resembled the pattern observed under nutrientexcess conditions (9, 12). There was partial inhibition of substrate utilization, as well as repression of a key enzyme of glucose metabolism. A

TABLE 3. Effect of nutrient-limited growth environment on respiratory and enzymatic activities of T. novellus grown at dilution rates of 0.05 and 0.08 h-' D=0.05h-1

D=0.08h-1

Activitya

Heterotrophic

Mixotrophic

Autotrophic

Heterotrophic

Mixotrophic

Thiosufate oxidation _b 135 60 Sulfite oxidase (EC 1.8.3.1) 10,930 5,430 Glucose oxidation 11 22 20 5 Glucokinase (EC 2.7.1.2) 170 490 250 270 Glucose 6-phosphate dehy27 210 60 23 drogenase (EC 1.1.1.49) Isocitrate dehydrogenase (EC 4,830 4,220 4,700 4,980 1.1.1.42) Cisaconitase (EC 4.2.1.3) 1,950 2,020 2,000 2,220 NADH oxidase (EC 1.6.99.3) 70 72 270 290 a Oxygen uptake, nanomoles per minute per milligram of protein. Enzyme activity, 10-4 enzyme unit per mg of protein. b _, Not determined.

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logical explanation of this finding relates to the it beneficial to shut off the synthesis of enzymes nutrient concentration in the culture vessel at of autotrophic metabolism; this investigation this D value. D = 0.08 h-' is very close to the suggests, on the contrary, that it is precisely the use autotrophic and A-max of the organism under these conditions, and ability to simnultaneously it is well established that at such high D values heterotrophic metabolic capacities that confers growth occurs in the chemostat at near-satura- on them their unique selective advantage, as was ting nutrient concentrations (2, 17). Evidently, proposed by Rittenberg (14, 15). From a physiological point of view, these studthese concentrations are high enough to make excessive energy generation a potential problem, ies bring into focus the differences in the reguand the organism responds by shifting to the lation of bacterial enzyme synthesis under con-

strategy suited to nutrient-excess conditions (9). ditions where carbon and energy sources are liiigand where they are in excess. As we It is noteworthy that under these conditions only one component of this strategy is employed, have seen under nutrient-excess conditions, thii.e., partial inhibition of substrate utilization, osulfate repressed several enzymes of glucose since there was no evidence of uncoupling. The metabolism (9), whereas under nutrient-limited steady-state concentrations of thiosulfate and conditions it even stimnulated their synthesis! As glucose in the medium at this D value were some mentioned already (9), nothing is known about sevenfold less than the initial concentrations employed in most of the batch culture studies (9, 12), and it may be concluded that the use of the additional component of the strategy becomes necessary only at higher substrate concentrations. This is the first illustration among chemolithotrophs of the idea that the inability of bacteria to concurrently utilize a mixture of substrates (as is the case, for instance, during diauxie) applies only to environments of high nutritional status (5). Analogous to the findings reported here, it has been shown that carbon-limited chemostat cultures of bacteria would concurrently utilize mixtures of lactose and glucose, fructose and glucose, or glucose and aspartate at low D values, even though at higher D values the utilization of one of the substrates was inhibited (5; A. Matin, in P. H. Calcott, ed., Continuous Culture of Cells, in press). These bacteria therefore possess the regulatory resilience to make efficient use of nutrients when they are in short supply and yet to minimiz their use and, in the case of T. novellus, circumvent energy generation from them when their oversupply becomes a threat to survival. To the extent that carbon- and energy-limited conditions are the rule in most natural environments, the data suggest that the mixotrophic potential of T. novellus should be of selective advantage over heterotrophs and obligate chemolithotrophs, which are confined to the use of either the organic or the inorganic growth substrates. Since under such conditions T. novellus can couple the concurrent oxidation of organic and inorganic energy substrates to growth, it can probably grow faster than its less versatile competitors. These considerations argue against the contention of Whittenbury and Kelly (18) that facultative chemolithotrophs must exist and compete as heterotrophs in nature and must find

the molecular basis of the interaction that enables thiosulfate and glucose to influence the synthesis of enzymes of each other's metabolism, and the fact that the nature of this interaction changes with the nutritional status of the environment adds another intriguing dimension to this question. Nutrient-limited as well as nutrient-excess growth conditions are probably ecologically pertinent. Because of the great popularity of batch cultures, a great deal has been learned of microbial behavior and regulation under the latter conditions, but relatively little is known of microbial behavior during nutrient-limited growth. These papers provide an illustration of how different microbial behavior can be under the two conditions. ACKNOWLEDGMCENTS 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.

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