Arch Microbiol (1988) 149:317- 323

Archives oF

Hicrobielngy �9 Springer-Verlag 1988

Mixotrophic and autotrophic growth of Thiobacillus acidophilus on tetrathionate Julie Mason and Don P. Kelly

Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK

Abstract. Thiobacillus acidophilus can grow in batch and chemostat culture as a heterotroph on glucose, a chemolitho- autotroph on tetrathionate and CO2, or as a mixotroph. Mixotrophically it obtains energy from the simultaneous oxidation of tetrathionate and glucose, and carbon from both glucose and CO2. Mixotrophic cultures contain lower activities of ribulose 1,5-bisphosphate carboxylase and ex- hibit lower specific rates of tetrathionate oxidation than do autotrophic cultures. Mixotrophic cultures with low concen- trations of glucose have growth rates that are intermediate between slow autotrophic growth and fast heterotrophic growth. Slightly more glucose-carbon is assimilated by mixotrophic cultures than by heterotrophic ones provided with the same concentrations of glucose. Mixotrophic yield in the chemostat is also slightly greater than predicted from autotrophic and heterotrophic yields. These observations indicate that there is preferential assimilation of glucose, at the expense of energy from tetrathionate oxidation, during mixotrophy, resulting in an overall "energy saving" that produces enhanced growth yield. These observations are relevant to understanding the regulatory behaviour of T. acidophilus in its acidic, mineral-leaching habitats.

Key words: Thiobacillus acidophilus - Mixotrophy - Chemostat culture - Glucose oxidation - Tetrathionate oxidation - Ribulose bisphosphate carboxylase

Thiobacillus acidophilus is a sulphur-oxidizing facultative autotroph that occurs in acidic environments where sulphide mineral leaching is taking place and has been isolated from cultures of T. ferrooxidans (Markosyan 1973; Guay and Silver 1975; Kelly and Harrison 1988). It has in common with the other thiobacilli the ability to grow autotrophically using inorganic sulphur oxidation as its only source of en- ergy. Although originally described as unable to grow with thiosulphate as the energy source (Guay and Silver 1975), it subsequently proved able to grow well on thiosulphate, trithionate and tetrathionate as well as on elemental sulphur (Norris et al. 1986; Mason et al. 1987). In common with the neutrophilic species, T. novellus and T. versutus, it is a facultative heterotroph, able to grow well on sugars and organic acids (Guay and Silver 1975). A separately described species, T. organoparus (Markosyan 1973), also capable of growth on the substrates used by T. acidophilus, is now

Offprint requests to: J. Mason

regarded as a strain of T. acidophilus because of its physio- logical similarity, identical % G + C content and high DNA-DNA homology with T. acidophilus (Wood and Kelly 1978; Harrison 1983; Kelly and Harrison 1988). While the mixotrophic growth of T. novellus and T. versutus on mixtures of organic substrates and thiosulphate has been quite thoroughly researched (Gottschal and Kuenen 1980; Leefeldt and Matin 1980; Perez and Matin 1980; Smith et al. 1980; Beudeker et al. 1982), there has been no quantitative assessment of the ability of T. acidophilus to exhibit truly mixotrophic growth, in which energy is derived simulta- neously from organic substrate and sulphur oxidation and carbon is acquired both by autotrophic CO2 fixation and from the organic substrate. T. acidophilus differs from the other mixotrophic thiobacilli in being obligately acidophilic, and thus of comparative interest with the kind of mixotrophic growth seen with some iron-oxidizing thermo- acidophiles (Wood and Kelly 1983 a, 1984) and in Sulfolobus (Wood et al. 1987).

Ecologically, T. acidophilus is of some interest as its only known habitats are the mineral leaching environments in which iron- and sulphide mineral-oxidizing bacteria pre- dominate (Markosyan 1973; Guay and Silver 1975; Norris and Kelly 1978). It is believed to be able to scavenge low levels of organic nutrients but is also capable of oxidizing the sulphur present in pyrite when grown in commensal association with Leptospirillum ferrooxidans (Norris and Kelly 1978). It is thus of potential significance as part of the complex microfiora involved in the bacterial leaching of minerals.

We have grown 1". acidophilus in batch and substrate- limited chemostat culture and can now present data relevant to understanding metabolic regulation in this unusual acidophilic mixotroph.

Materials and methods

Organism

Thiobacillus acidophilus (ATCC 27807; DSM 700) had pre- viously been maintained in the Department as a stock culture on elemental sulphur. It was maintained on plates or slopes of medium containing (g/l): potassium tetrathionate (2.3); (NH4)2SO4 (3.0); KH2PO,~ (3.0); M g S O 4 �9 7 /-I20 (0.5); CaCI2 �9 2 H2O (0.25); Difco Bacto agar (10.0). The sterile medium was supplemented with iron [2.5 ml 0.2% (w/v) FeSO4.7 H2O in 0.1 M HC1 per litre of medium] prior to pouring plates. The basal salts medium, lacking

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tetrathionate, FeSO, and agar, was the same as that used by Bounds and Colmer (1972) for T.ferrooxidans.

Culture methods

Batch cultures were grown in 250 ml Erlenmeyer flasks using 50 or 100 ml basal salts medium plus iron, supplemented with tetrathionate and/or glucose at various concentrations. For measurement of 14CO2 fixation or [U-14C] glucose assimilation during growth, cultures (50 ml) in 250 ml "Quickfit" flasks were sealed with "Suba-seal" vaccine stoppers and labelled or unlabelled glucose and NaHCO3 solutions injected as required. [U-14C] glucose and NaH14CO3 were provided at specific activities of about 105 cpm/lamol and 4 x 104 cpm/gmol respectively; bicarbonate was added at an initial concentration of 5 mM in the sealed flasks. All cultures were shaken (150 rpm) on an LH Engi- neering orbital shaker at 30~ Chemostat cultures were established in a water-jacketed Quickfit culture vessel (1 1 culture volume), fitted with a stainless steel lid (LH Fermentation plc), and maintained at 30~ by means of a thermo-circulator (Conair Churchill Ltd). Cultures were stirred at 600 rpm, gassed with 1% (v/v) CO2 in air, and maintained at pH 3.0 + 0.2 by automatic titration with 2 M Na2CO3. Basal salts plus iron and substrates (tetrathionate and/or glucose) were separately metered into the culture using Watson Marlow flow inducers. A single dilution rate of 0.032 h - 1 was used for all experiments.

Culture analysis

Samples were removed from cultures for measurement of absorbance at 440 nm, which was related to biomass concen- tration using a calibration of absorbance against organism dry wt. Direct measurements of the concentrations of total cellular organic carbon (TOC) and protein were made by centrifuging 4 ml samples, resuspending in appropriate volumes, and assaying. Protein was determined by the Lowry method after dissolving the cells in 0.5 M NaOH a t 80~ for 15min. TOC was determined in aqueous suspensions using a Beckman model 905B Total Organic Carbon Analyser. Tetrathionate in culture samples was assayed cyanolytically (Kelly et al. 1969). For determination of the assimilation of 14C-substrates, replicate culture samples (1 ml) were filtered through Sartorius SM11306 membrane filters (0.45 gm pore size) and washed with 0.5 mM HzSO4, pH 3.0. Air-dried membranes in scintilla- tion vials were immersed in 10ml Optiphase "Safe" scintillant (LKB Instruments Ltd). Total 14C in cultures was measured by mixing samples (0.1 ml) with 1 ml 5% (v/v) acetic acid in ethanol and heated to dryness to expel all CO2 and bicarbonate. The residue was dissolved in 1 ml water and mixed with 10 ml scintillant. Solutions of NaH14CO3 and [U-14C] glucose were assayed by mixing aliquots (0.01 ml) with 0.I ml of ethanolamine +methoxyethanol (3 + 7, by vol) and 10 ml scintillant. All 14C samples were counted in a Beckman LS-7000 liquid scintillation spectrometer.

Substrate oxidation by suspensions

Cultures grown on 10 mM tetrathionate or tetrathionate plus 1 mM glucose were centrifuged (25,000 x g, 10 rain in a Beckman J2-21 centrifuge), washed twice with dilute H2SO4,

pH 3.0, and resuspended in the dilute acid, and held at 4~ during use. Endogenous respiration was estimated in a working volume of 3 ml in a Clark oxygen electrode cell (Rank Bros Ltd, Bottisham, UK) at 30~ prior to adding tetrathionate or glucose (0.05-0.5 mM) and measuring the rate of substrate-dependent oxygen reduction.

Assay of ribulose 1,5-bisphosphate carboxylase ( RuBPC)

This was done using the permeabilized whole cell procedure (Smith et al. 1980; Wood and Kelly 1984). The organisms (0.8 mg dry wt) were collected on membrane filters and permeabilized with 0.4 ml 10% (v/v) Triton X-100 prior to assay as described previously, with incubation at 30~ for 15 min with and without 1.9 mM ribulose 1,5-bisphosphate. 14C-fixation proceeded at a linear rate during this period in the presence of ribulose bisphosphate.

Materials

Ribulose 1,5-bisphosphate was from Sigma (London) Ltd; Na2CO3 and glucose each labelled with 14C were from Amersham International PLC; K2S406 was supplied by Fluka AG (Switzerland) and British Drug Houses Ltd.

Results

Growth and tetrathionate oxidation of Thiobacillus acidophilus grown in batch culture with or without glucose

Thiobacillus acidophilus was capable of wholly autotrophic, chemolithotrophic growth in mineral medium, using 10 mM tetrathionate as sole energy source. Under such conditions it had a specific growth rate (/~) in flasks open to the atmo- sphere and not supplemented with CO2 of 0.046 h-1 at 30 ~ C. When T. acidophilus was inoculated into medium with 20 mM tetrathionate, growth did not occur, showing this compound to be an inhibitory substrate at high concentra- tions, as is the case for the acidophilic T. ferrooxidans (Eccleston and Kelly 1978). When T. acidophilus previously grown in batch culture on tetrathionate, or taken from an autotrophic tetrathionate-limited chemostat, was inoculated into media containing 10 mM tetrathionate and various con- centrations of glucose, stimulation of growth rate was ob- served. Specific growth rate increased from 0.053 h-1 with 0.5 or l m M glucose to 0.068h-* (5raM), 0.073h -1 (10raM) and 0.077h -1 (20 or 50mM). Heterotrophic growth rate with 1 mM glucose without tetrathionate was 0.077 h - 1.

Decrease in pH of cultures on 10 mM tetrathionate was similar autotrophically and in the presence o f 5 mM glucose (Fig. 1), while tetrathionate oxidation proceeded rapidly in both. Autotrophically, growth and tetrathionate oxidation were clearly interdependent: plotting tetrathionate oxidized versus increase in biomass (e. g. Fig. 2) gave the differential relationship between the two, defined as the "lithotrophic index" (Ixmol tetrathionate oxidized during an increase of 1 mg in organism dry wt). The absolute rate of tetrathionate consumption was greater in the presence of glucose (Fig. 1), resulting in complete oxidation of the tetrathionate supplied by the time that only about half the glucose supplied had been used. Following exhaustion of tetrathionate in the mixotrophic culture (Fig. 1) growth on glucose continued to

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Fig. 1. Growth of Thiobacillus acidophilus in batch culture on tetrathionate (9 mM) with (solid symbols) and without (open sym- bols) glucose (5 raM). Biomass (�9 � 9 tetrathionate oxidation (V, T); pH ([], I ) . Initial pH was 3.2. Inocula for the experiment were taken directly from a tetrathionate-limited autotrophic chemostat culture

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produce a further doubling in biomass. While the absolute rate of tetrathionate oxidation was greater in the mixotrophic culture, the specific rate per unit biomass was lower than by the autotrophic culture. This was illustrated by determining the lithotrophic index for cultures growing on tetrathionate with various concentrations of glucose (Fig. 2). The index declined from 200 ~tmol tetrathionate oxidized (rag increase in organism dry wt) -1 for the autotrophic culture to 110 (0.5 mM glucose), 96 (1 raM), 60 (5 mM), 56 (10 raM), 44 (20 mM) and 40 (50 mM glucose). The possibility that these differences could have been pro- duced by the presence of a heterotrophic contaminant was disproved by demonstrating that only T. acidophilus was observed by microscopic examination; and the fact that equal numbers of colonies were produced on agar plates containing glucose or tetrathionate, when dilutions of the culture were spread on them. In these batch cultures, bio- mass production at the end of growth was 72 mg dry wt 1-1 (42 mg cell-protein 1-i) autotrophically (10 mM tetrathio- hate); 86 mg (55 mg protein) heterotrophically (1 mM glu- cose); and 135 mg (87 mg protein) mixotrophically (both substrates), making the mixotrophic yield 8 5 - 92% of that of the sum of the cultures on the substrates separately.

The rates at which T. acidophilus could oxidize tetra- thionate or glucose were measured using cell suspensions

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Fig. 2. The effect of glucose concentration on tetrathionate oxi- dation in mixotrophic batch cultures of T. acidophilus. Tetrathionate oxidized is plotted against increase in biomass for cultures growing on tetrathionate (10 raM) and glucose at: 0 mM ((3); 1 mM (&); 5 mM ( �9 and 20 mM (V)

obtained from cultures growing autotrophically on tetrathionate or mixotrophically on tetrathionate and glucose (1 raM), and harvested from culture when 50% of the tetrathionate had been oxidized. Rates of oxidation (nmol O2/min/mg dry wt) of tetrathionate or glucose were 88 or 19 (autotrophic culture) and 55 or 44 (mixotrophic culture), respectively. The constitutive ability to metabolize glucose was further demonstrated by the immediate response of growth and tetrathionate oxidation to 5 mM glucose added to a culture growing autotrophically on 10 mM tetrathionate (Fig. 3). Prior to the addition of glucose, the rate of tetrathionate consumption was similar in both cultures, but the lithotrophic index was more than halved from 200 gmol tetrathionate used (mg increase in organism dry wt) - 1 in the autotrophic control to 80 after the addition of glucose (Fig. 3 B).

Assimilation of glucose and carbon dioxide by Thiobacillus acidophilus growing with tetrathionate in batch culture

Thiobacillus acidophilus previously grown on tetrathionate alone was inoculated into sealed flasks of media contain- ing 10 mM tetrathionate and supplemented with 5 mM NaHCO3, or with bicarbonate and 0.5 mM glucose, or with 1 mM glucose alone. Growth was followed by measurement of absorbance, disappearance of tetrathionate, and incorpo- ration of 14C in cultures labelled either with l~C-bicarbonate or [U-14C] glucose. Autotrophic growth on tetrathionate and 14C-bicarbonate, measured either by increase in absorbance or incorporation of 14C proceeded in this exper- iment with a specific growth rate (/~) of 0.062h -1. Tetrathionate oxidation was also exponential and plotting tetrathionate consumed against increase in biornass

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Fig. 3A, B Effect of the addition of glucose to a culture of T. acidophilus during autotrophic growth on tetrathionate (10 raM). Duplicate cultures were grown for 42 h on tetrathionate then one culture (solid symbols) was supplemented with 5 mM glu- cose (arrows). A Biomass (�9 � 9 and tetrathionate disappearance (A, � 9 for the two cultures; B tetrathionate oxidation relative to increase in biomass in both cultures

Table 1. Biomass production and activity of ribulose 1,5-bisphosphate carboxylase in Thiobacittus acidophilus grown in chemostat cultm-e at a dilution rate of 0.032 h-1

Growth limiting- Volume Biomass concentration Yield b RuBPC activity substrate (raM) replacements

(nag dry wt 1- ~) (rag TOC" I-~) (nmol/min (nmol/min �9 dry wt) - I culture)

KzSaO6 (12.0) 3.0 186�9 86 15.6 30.2 5641 K2S406 (9.6) + glucose (1.0) 0.6 250.0 - - 18�9 4555

1.3 305.9 - - 18.6 5681 3.6 300.0 150 - 15.5 4644

Glucose (1.0) 0.5 199.9 - - 12.9 2573 3.0 112�9 - 112.5 5.6 633

a TOC, total organic carbon in centrifuged organisms b g dry wt/g tool tetrathionate or glucose consumed

(Fig. 4 B) gave a straight line relationship of slope equal to 117 mmol tetrathionate oxidized/g increase in biomass. This indicated a batch culture growth yield of 8.5 g dry wt/mol tetrathionate. Plotting 14CO2-incorporation against increase in biomass (Fig. 4 A) also gave a linear relationship, with autotrophically grown organisms containing 44% (w/w) carbon relative to dry wt. A parallel culture grown on tetrathionate with I mM glucose, but without bicarbonate, exhibited a/~ of 0.116 h -1 (measured both by increase in absorbance and incorporation of 14C from glucose). Carbon incorporation from glucose paralleled increase in biomass and gave a line that was identical to that obtained for autotrophic ~4COz-incorporation (Fig. 4A). This indicated that virtually all the cell carbon was derived from glucose under these conditions, with little contribution from CO2 fixation�9 Growth ceased when glucose was exhausted, at which time about 60% (0.57 mM) of the initial 0.92 mM glucose had been assimilated. The balance of the added 14C- glucose was converted to acid-volatile product (presumed to be CO2) at a constant differential rate, relative to growth, during glucose assimilation. Tetrathionate oxidation did not occur during the first quarter of exponential growth (Fig. 4 B), but the specific rate of oxidation then increased progressively until growth ceased. Although some 14C-as- similation could have resulted from autotrophic or anaplerotic CO2-fixation in these cultures, growth did ap- pear to be predominantly heterotrophic under these con- ditions. When inoculated into medium with tetrathionate,

glucose and bicarbonate, T. acidophilus grew with a p of 0.058 h-1 and showed similar kinetics of tetrathionate con- sumption to the cultures with 1 mM glucose without added bicarbonate (Fig. 4B). The kinetics of 14C-incorporation were, however, different (Fig. 4A). During the first half of the growth period (biomass increasing by about 50 mg dry wt 1-1; Fig. 4A), glucose and CO2 both provided carbon for growth and were consumed in about a 2:1 ratio. There was, however, an increase in the differential rate of glucose assimilation, paralleled by a decreasing rate of CO2-fixation (Fig. 4A). The maximum carbon assimilation values ob- served were about 20 mg of carbon 1-1 from glucose and 10 mg of carbon 1-1 from CO2, suggesting the production of only about 70 mg biomass dry wt 1-1 rather than the l l 0 - 1 1 5 m g dry wt 1-1 that was indicated by culture absorbance (Fig. 4A). Cessation of glucose assimilation re- sulted from its complete consumption by the culture, and the subsequent increase in culture absorbance probably arose from production of sulphur from the excess tetrathionate by the bacteria. The earlier part of the growth did, however, show mixotrophic growth in that glucose, carbon dioxide and tetrathionate were metabolized concurrently. The cultures incorporated about 76% (0.29 tool) of the initial glucose (0.38 mM) supplied, the remainder being respired from the cultures. This suggested that the small amount of tetrathionate metabolized must have provided some energy for glucose assimilation as well as for CO2 fixation.

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Fig. 4 A, B. Oxidation of tetrathionate (10 mM) and incorporation of Igc from labelled CO2 and glucose during mixotrophic growth of T. acidophilus under different conditions in batch culture. A Autotrophic cultures (�9 receiving 5 mM NaH14CO3 only; mixotrophic culture with [U-14C] glucose and no NaHCO3 (V); mixotrophic cultures with [U-14C] glucose +NaHCO3 (~') and NaH14CO3 + unlabelled glucose (O). B Tetrathionate oxidation relative to increase in biomass by autotrophie cultures (�9 mixotrophic cultures on glucose with NaHCO3 (A) and without NaHCO3 (&)

Autotrophic, mixotrophic and heterotrophic growth of T. acidophilus in chemostat culture

A tetrathionate-limited autotrophic culture was established at a dilution rate of 0.032 h - 1 and steady state biomass and specific activity of ribulose 1,5-bisphosphate carboxylase (RuBPC) determined (Table 1). Steady state conditions were defined when the biomass concentration remained constant and no change was observed in the specific activity of ribulose bisphosphate carboxylase. This was normally the case within the passage of three volumes of the culture medium, after which no further change occurred. On switching from autotrophic culture to a mixture of glucose and tetrathionate, a transition was seen, with increase in biomass and decrease of RuBPC activity to a steady state level that was about half that of the autotrophic culture (Table 1). Comparing the steady state mixotrophic biomass (after 3.6 volume replacements) with the autotrophic value, indicated that applying the autotrophic yield of 15,6 g dry wt (tool tetrathionate)-1 to the mixotrophic culture would mean that 149 mg 1-1 of the 300 mg dry wt 1-1 was due to tetrathionate-dependent autotrophy and 151 mg 1-1 to glucose assimilation. The steady state biomass on glucose

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alone (after three volume replacements; Table 1) was only 112.5 mg 1-1, indicating that the mixotrophic steady state biomass production exceeded that expected from the sepa- rate data from autotrophic and heterotrophic cultures by about 14%. More efficient use thus appeared to be made of glucose to support growth under mixotrophic conditions. On switching from mixotrophy to heterotrophy the specific activity of RuBPC fell to about 19% of the autotrophic level during three volume replacements (Table 1). There was, however, no evidence of active destruction of the enzyme as its rate of loss from the culture (Table 1, last column) was consistent with washout kinetics. This washout would have continued, to produce zero specific activity, as no enzyme remains in heterotrophic cultures after prolonged culture on glucose.

Discuss ion

These observations show that Thiobacillus acidophilus grows mixotrophically when provided with mixtures of glucose and tetrathionate, using both substrates simultaneously. This is particularly interesting as some other thiobacilli, such as T. versutus, are known to exhibit diauxic growth on mixtures of substrates such as pairs of sugars or carboxylic acids, or glucose and carboxylic acids, or thiosulphate and sucrose (Wood and Kelly 1977, 1983 b; A. Kraczkiewicz-Dowjat and D.P. Kelly in preparation). Simultaneous use of organic and inorganic energy sources has also been found for T. novellus (Perez and Matin 1980), T. intermedius (London and Rittenberg 1966; Matin and Rittenberg 1970), Alcaligenes eutrophus (DeCicco and Stukas 1968; Rittenberg and Goodman 1969) and Paracoccus denitrificans (Banerjee and Schlegel 1966). In these organisms, diverse effects were seen, some of which can be compared with our results. In T. acidophilus it seems that the rates of growth and of tetrathionate oxidation are a function of the glucose concen- tration supplied. Thus, mixotrophic growth on 1 mM glucose was actually slower than heterotrophic growth on the same concentration of glucose and the specific rate of tetrathionate oxidation was decreased by increasing concen- tration of glucose. These effects are comparable to those seen in thiosulphate-glucose mixotrophy with T. novellus and T. intermedius, and are indicative of interregulatory effects between the autotrophic and heterotrophic modes of metabolism. T. acidophilus clearly exhibits true mixotrophy in that all three substrates (glucose, tetrathionate and CO2) can be used simultaneously and their use is interdependent. Thus the level of RuBPC and the maximum rate of tetrathionate oxidation are lower mixotrophically than autotrophically, and the proportion of glucose incorporated mixotrophically is apparently greater than under hetero- trophic conditions although substantial CO2 fixation also occurs. In glucose-limited chemostat culture the yield was 112,5 mg dry wt mo1-1, indicating that up to 69% of the added glucose-carbon was converted to biomass. This was virtually identical to the yield produced by T. versutus in glucose-limited chemostat culture (Smith et al. 1980).

T. acidophilus differs from the acidophilic, moderately thermophilic mixotrophs studied previously (Wood and Kelly 1983a, 1984) in being able to switch readily between autotrophic and heterotrophic growth, as can the neutro- philes, T. novellus and T. versutus. The advantages of this metabolic versatility to survival in natural environments has

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been considered previously (Beudeker et al. 1982; Kelly and Kuenen 1984) and led to the view that the rate o f response to changing conditions is a key factor in successful survival and competition by facultative thiobacilli (Beudeker et al. 1982; Kuenen and Beudeker 1982). It is noteworthy from our data that T. aeidophilus does appear to respond immediately to the various culture conditions imposed on it, but it is also apparent that some responses are relatively slow and could equally indicate ecological advantage gained by a progressive response to changing conditions. For ex- ample, in T. versutus, switching from thiosulphate limited autotrophic growth to heterotrophy on acetate, resulted in active degradation o f RuBPC (Gottschal et al. 1981). This clearly did not occur in T. acidophilus, in which significant RuBPC was retained after three chemostat volume replace- ments on glucose. To an organism likely to find intermittent and irregular supply o f either or both of organic nutrients and oxidizable sulphur compounds in its environment, the retention o f useful levels of key enzymes clearly enables immediate use of substrate as it becomes available. Consequently the pace of regulation-induced major meta- bolic change is probably generally slower in such mixotrophs than in flexible heterotrophs. For comparison, T. versutus, Alcaligenes eutrophus H16 and Pseudomonasfaeilis all re- tained significant levels ( 1 0 - 7 5 % of autotrophic activity) of RuBPC after prolonged heterotrophic culture (Gott- schalk et al. 1964; McFadden and Tu 1967; Smith et al. 1980). The only other study on the assimilation o f organic compounds by T. acidophilus was that of Kingma and Silver (1981), who showed that pyruvate could be metabolized during growth on sulphur. A detailed reanalysis o f their data (Kelly unpublished) showed that cultures on sulphur supplemented with 0.1 m M pyruvate grew at the same rate as autotrophic cultures, but oxidized and assimilated all the pyruvate during early exponential growth to obtain 3 0 - 40% of the cell-carbon from the added pyruvate.

We can thus conclude that T. acidophilus exhibits con- siderable versatility in its response to the availability of organic compounds and in its ability to use soluble reduced sulphur compounds simultaneously. It is surprising that there have been relatively few reports of its natural occur- rence (Markosyan 1973; Guay and Silver 1975). This may reflect that the acidic mineral-leaching environments are relatively stable in nutrient balance and consequently favour specialist chemolithotrophs and heterotrophs rather than mixotrophs.

Acknowledgements. This work was supported by an award from the Natural Environment Research Council. We are grateful to Dr. M. Silver, who originally supplied the culture of T. aeidophilus to D.P.K.

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Smith AL, Kelly DP, Wood AP (1980) Metabolism of Thiobacillus A2 grown under autotrophic, mixotrophic and heterotrophic conditions in chemostat culture. J Gen Microbiol 121:127- 138

Wood AP, Kelly DP (1977) Heterotrophic growth of Thiobaeillus A2 on sugars and organic acids. Arch Microbiol 113:257- 264

Wood AP, Kelly DP (1978) Comparative radiorespirometric studies of glucose oxidation in three facultatively heterotrophic thiobacilli. FEMS Microbiol Lett 4: 283 - 286

323

Wood AP, Kelly DP (1983 a) Autotrophic and mixotrophic growth of three thermoacidophilic iron-oxidizing bacteria. FEMS Microbiol Lett 20:107 -- 112

Wood AP, Kelly DP (1983 b) Use of carboxylic acids by Thiobacillus A2. Microbios 38:15-25

Wood AP, Kelly DP (1984) Growth and sugar metabolism of a thermophilic iron-oxidizing mixotrophic bacterium. J Gen Microbiol 130:1337 - 1349

Wood AP, Kelly DP, Norris PR (1987) Autotrophic growth of four Sulfolobus strains on tetrathionate and the effect of organic nutrients. Arch Microbiol 146: 382- 389

Received June 25, 1987/Accepted October 28, 1987