nate and thiosulfate oxidationby cell suspen- sions, and .... nate at constant pH 2.0, growth ceasing abruptly when all ..... Forrest, W. W., and D. J. Walker. 1971.
Vol. 134, No. 3
JOURNAL OF BACTERIOLOGY, June 1978, p. 718-727 0021-9193/78/0134-0718$02.00/O Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Oxidation Kinetics and Chemostat Growth Kinetics of Thiobacillus ferrooxidans on Tetrathionate and Thiosulfate MARTIN ECCLESTON AND DONOVAN P. KELLY* Department of Environmental Sciences, University of Warwick, Coventry, CV4 7AL, England Received for publication 10 January 1978
Growth of Thiobacillus ferrooxidans in batch culture on 10 mM potassium tetrathionate was optimal at pH 2.5 (specific growth rate, 0.092 h-1). Oxygen electrode studies on resting cell suspensions showed that the apparent Km for tetrathionate oxidation (0.13 to 8.33 mM) was pH dependent, suggesting higher substrate affinity at higher pH. Conversely, oxidation rates were greatest at low pH. High substrate concentrations (7.7 to 77 mM) did not affect maximum oxidation rates at pH 3.0, but produced substrate inhibition at other pH values. Tetrathionate-grown cell suspensions also oxidized thiosulfate at pH 2.0 to 4.0. Apparent Km values (1.2 to 25 mM) were of the same order as for tetrathionate, but kinetics were complex. Continuous culture on growth-limiting tetrathionate at pH 2.5, followed by continuous.culture on growth-limiting thiosulfate at pH 2.5, indicated true growth yield values (grams [dry weight] per gram-molecule of substrate) of 12.2 and 7.5, and maintenance coefficient values (millimoles of substrate per gram [dry weight] of organisms per hour) of 1.01 and 0.97 for tetrathionate and thiosulfate, respectively. Yield was increased on both media at low dilution rates by increase in CO2 supply. The apparent maintenance coefficient was lowered without affecting YG, suggesting better energy coupling in C02-rich environments. Prolonged continuous cultivation on tetrathionate or thiosulfate did not affect the ability of the organism to grow subsequently in ferrous iron medium.
Thiobacillus ferrooxidans is a chemolithotrophic bacterium which grows at low pH and can obtain energy by oxidizing ferrous iron (3, 21). It can also be adapted to chemolithotrophic growth on a number of inorganic sulfur compounds, including thiosulfate, tetrathionate, trithionate, elemental sulfur, and metal sulfides (2, 6, 15, 17, 23-26). Bounds and Colmer (1) examined the kinetics of thiosulfate and tetrathionate oxidation by T. ferrooxidans, Ferrobacillus ferrooxidans, and F. sulfooxidans as a means of differentiating between these bacteria, and although some differences were apparent in their affinity for thiosulfate, all three are now regarded as strains of T. ferrooxidans (14). No other infonnation is currently available on the oxidation kinetics of reduced sulfur compounds by thiobacilli. T. ferrooxidans plays a major role in the bacterial leaching of sulfide minerals (23), so that its basic physiology and biochemistry have been much studied. More recently, continuousculture studies have been used to provide additional fundamental information on the kinetics of growth and biochemical energy yield on iron (10, 11), but growth behavior on inorganic sulfur compounds such as thiosulfate and tetrathionate
has so far received little attention, and has only
been examined in batch culture (24). We have examined the kinetics of tetrathionate and thiosulfate oxidation by cell suspensions, and the behavior of Thiobacillus ferrooxidans on growth-limiting supplies of tetrathionate or thiosulfate in continuous-flow chemostats, to define the limits of bacterial growth, substrate turnover, and biochemical energy yields during growth on inorganic sulfur compounds.
MATERIALS AND METHODS Organism. The strains of T. ferrooxidans previously adapted for growth on tetrathionate and thiosulfate (24) were subsequently maintained by weekly transfer in shake-flask cultures (24) at 300C using ironsupplemented medium. Media. The basal salts solution was that of Bounds and Colmer (1) supplemented with FeSO4 7H20 (5 mg/liter). For growth on tetrathionate, K2S406 (microbiological grade, BDH Chemicals Ltd, Poole, England) was supplied at 5 or 10 mM. The initial pH (4.2 to 4.8) was adjusted to the desired growth pH with H2SO4. For batch culture, the medium was sterilized by autoclaving at 10 lb/in2, and the pH was rechecked after cooling. For chemostat cultures, media were sterilized by sequential filtration through coarse (grade 80) and
718
VOL. 134, 1978
KINETICS OF T. FERROOXIDANS ON SULFUR COMPOUNDS
719
fine (grade 03) Whatman Gamma-12 on-line filtration incorporated to check for chemical oxidation of the units (Whatman Lab Sales Ltd., Maidstone, England) substrate, using several concentrations at each test into a sterile 20-liter lightproof receiver. Medium was pH. Apparent Km and Vm.x values for tetrathionate pumped from this to the fermentor at constant rates and thiosulfate oxidation at several pH values were by an MHRE 7 flow inducer (Watson-Marlow Ltd., estimated from plots of the reciprocals of substrate Falmouth, Cornwall, England). Flow rates were deter- concentration against oxidation rate. Determination of biomass. Biomass was estimined at each dilution rate using an on-line burette. For continuous culture on thiosulfate, 1 volume of mated from culture absorbance at 460 nm and by 50 or 100 mM Na2S203 (pH 5.93) and 4 volumes of 5/4- determination of dry weight and protein. Protein was strength basal salts solution adjusted to pH 2.41 were determined after dissolving bacteria in 0.5 or 1 N sterilized separately by on-line filtration and pumped NaOH at 95°C for 15 min and using a modified Lowry separately into the fermentor vessel to give a final method (16) with bovine serum albumin as the staninput concentration of 10 or 20 mM Na2S203 as desired, dard. Because of the small amount of cell material at approximately pH 2.5. Culture pH was maintained present at low dilution rates in the chemostat, dry weight and protein estimations were made from the at pH 2.5 by automatic titration. Growth on iron. Organisms from the fermentor mean of six independent determinations. Plots of abwere tested for their ability to grow in shake flasks at sorbance (at 460 nm) against dry weight and cell 30°C on a ferrous iron medium containing (grams per protein during 31 steady states on tetrathionate or liter): K2HPO4, 0.4; (NH4)2S04, 0.4; MgSO4 7H20, 0.4; thiosulfate were linear over the range studied. We and FeSO4 * 7H20, 27.8, taken to pH 1.4, 1.9, or 2.5 with took 0.1 absorbance unit as equivalent to 11.5 ,jg of protein per ml and 22.0 ug of dry weight per ml, 2 N H2SO4. Batch and continuous cultures. Batch cultures indicating 52.3% of dry weight to be protein. Determination of inorganic sulfur compounds (2-liter) were started with a 5% (vol/vol) shake-flask inoculum and grown at 30°C on 10 mM K2S406 in a and iron. Tetrathionate, thiosulfate, and trithionate CC1500 fermentor (LH Engineering Ltd., Stoke Poges, were assayed spectrophotometrically after selective England) incorporating automatic pH control with 0.5 cyanolysis (9). "Cold cyanolysis" was carried out at M K2CO3. Dissolved oxygen was maintained at 70 to room temperature (18 to 20°C). Thiosulfate was also 100% of air saturation by aeration (100 to 200 ml of air determined by iodometric titration. Ferrous iron was per min) and stirring at 500 rpm. Continuous cultures determined by titration with ceric sulfate using 1,10were started from fully grown batch cultures by pump- phenanthroline-ferrous sulfate complex as the end ing in fresh medium at a constant flow rate and were point indicator. supplemented with 9% (vol/vol) CO2 as required. CulElemental analysis. Duplicate elemental analyses ture volumes were set at approximately 2 liters by of washed and dried organisms derived from four means of an adjustable weir, and actual culture vol- fermentor steady states (D, 0.032 to 0.062 h-') on umes were determined accurately at the end of each tetrathionate medium were carried out using a Perkinrun. Wall growth was almost entirely eliminated by Elmer elemental analyzer. permanently siliconizing the glass fermentor pot by Graphical determination of true growth yield immersing in 5% (vol/vol) dimethyl-dichlor-silane in (YG) and maintenance coefficient (m) from exchloroform. perimental data. Double reciprocal plots of 1/yield Oxygen electrode experiments. Cell suspensions (Y-') against 1/dilution rate (D-') were linear, with for oxygen electrode experiments were prepared from YG = Y at D` = 0, and m = slope. Alternatively, plots 2-liter tetrathionate batch cultures at a given pH by of specific rate of substrate utilization (q) against centrifuging 1.5 to 2.0 liters of stationary-phase culture dilution rate (D) were linear, with YG = 1/slope and at 30,000 x g for 10 min, washing twice, and suspending m = q at D = 0. Best-fit lines were plotted by regression to 0.05 of the original volume in substrate-free basal analysis. medium at the desired pH. Oxidation experiments RESULTS were normally completed within 48 h of harvesting since, although cell suspensions retained oxidative acBatch growth on tetrathionate. T. ferrooxtivity for tetrathionate and thiosulfate during at least idans grew exponentially on 10 mM tetrathio3 weeks of storage at 4 or 20°C, absolute activities nate at constant pH 2.0, growth ceasing abruptly varied between batches or with the same batch aswhen all the tetrathionate had been consumed sayed on different occasions. Oxidation of tetrathionate and thiosulfate by T. (Fig. 1). No thiosulfate or trithionate was ever ferrooxidans cell suspensions was determined in an detected during growth. Specific growth rates oxygen electrode cell (Rank Brothers, Bottisham, Eng- and mean generation times were determined for land) of 3-ml working volume, with constant temper- several controlled pH values (Table 1). Fastest ature control at 30°C. The reaction was started by growth (u = 0.092 h-') occurred at pH 2.5. injecting 0.3 ml of substrate into 2.7 ml of suspension Kinetics of tetrathionate oxidation by (1.6 mg [dry weight] of bacteria). Oxygen consumption suspensions of T. ferrooxidan. The effect of was monitored by a Kipp and Zonen (Delft, Holland) pH on specific rates of tetrathionate oxidation chart recorder, and the maximum sustained linear rates recorded were used in each case to calculate over a range of tetrathionate concentration was nanomoles of 02 consumed per minute per milligram tested using suspensions of T. ferrooxidans of protein. Oxidation rates were directly proportional grown and assayed at various fixed pH values to quantity of bacteria. Controls lacking bacteria were (Fig. 2). At pH 3.0, very low tetrathionate con-
720
J. BACTERIOL.
ECCLESTON AND KELLY
lo
00
s0 60
40
i ' 20
6
6
at
I
2M. 0
u
10 8
i _ °
4
6 4
0
* 2
y o
2 0 0
20
40
60
80
inhibition (7% reduction of the maximum observed rate), which became more apparent at 80 mM. At pH 1.5 (data not shown), tetrathionate oxidation rates by organisms grown at pH 2.5 increased linearly over the concentration range 0.1 to 5 mM, but high concentrations were inhibitory. Thus 50 mM tetrathionate gave a rate of only 64% of the maximum rate obtained at 5 mM. Controls showed that no chemical decomposition of tetrathionate occurred in the oxygen electrode during these studies. Double reciprocal plots of tetrathionate concentration against specific oxidation rate (Fig. 3) were linear at low substrate concentrations and were used to estimate apparent Km and V,,, values (Table 2). Apparent Km values for tetrathionate oxidation were pH dependent (Table 2) and suggested a greater substrate affity at higher pH. Conversely, oxidation rates were greatest at low pH. In one experiment using a cell suspension grown at pH 2.5, specific oxidation rates for oxidation of 1 mM tetrathionate
100
TIME ( h )
1000
1. Growth of T. ferrooxidans on 10 mM tetrathionate in 2-liter batch cultures with pH control at pH 2.0. 0, Growth (dry weight of organisms, pg/ml); 0, S4062- concentration (mM); [I S2032- and S3062-concentrations (m,n). FIG.
800
*af
TABLE 1. Growth of T. ferrooxidans in batch culture on 10 mM K2S406 Culture pH'
Mean generation time
(h)
Specific growth rate i
15.4 7.5 9.0 22.5
600
~I
400
(h-')
0.045 0.092 0.077 0.031 aMaintained by automatic titration with K2C03. 2.0 2.5 3.0 4.0
a
200 centrations allowed very high oxidation rates, but concentrations above 5 mM did not cause any further increase of rates and were not inhibitory. At pH 2.0 and 4.0 (Fig. 2), specific oxidation 0 rates increased linearly with tetrathionate concentration over the range 0.1 to 7.5 mM. Con40 60 80 100 0 20 centrations above 10 mM, however, produced a very severe substrate inhibition effect. l5S4b 1 (mM) At pH 2.5, the optimal growth pH, oxidation rates increased linearly with substrate concenFIG. 2. Effect ofpH on specific oxidation rates for tration up to 10 mM, whereas 10 to 40 mM gave a range of tetrathionate concentrations by suspensimilar oxidation rates (data not shown). Tetra- sions of T. ferrooxidans. 0, pH 2.0; *, pH 3.0; C, pH thionate at 50 mM produced slight substrate 4.0.
KINETICS OF T. FERROOXIDANS ON SULFUR COMPOUNDS
VOL. 134, 1978 140
721
l I
( b)
0O 4,
120
10
2 9
100
a
so
~1
7
.3
6
-1
S
60
4
40
3 2
20 0
0 0
0
0
2
4
6
S
10
0
lSI NA-. I
1
4
2
3
I
(mm)
5
6
-1
FIG. 3. Double reciprocal plots of specific rates of tetrathionate oxidation (v-) against tetrathionate concentration (s-') by suspensions of T. ferrooxidans cultured and assayed at (a) pH 2.5 or (b) pH 4.0.
TABLE 2. Kinetic constants for tetrathionate and thiosulfate oxidationa Apparent Substrate
Tetrathionate
Thiosulfate
Assay pH
1.5 2.0 2.5 3.0 4.0
(jmol of 0Q2
Apparent (MM)
per min per
8.3
1.7
4.0 8.3 0.4 0.1
mg of protein)
1.5
1.0 1.4 0.7
2.0
2.4
1.6
3.0 4.0
1.2,25.0 2.4
2.2, 1.2 1.3
For details of preparation of organism suspensions and measurements of substrate oxidation in oxygen electrode cells, see the text. a
were (nanomoles of 02 per minute per milligram of protein) 252 at pH 1.5, 68 at pH 2.5, and only 40 at pH 3.5, 4.5, and 5.5
Kinetics of thiosulfate oxidation by tetrathionate-grown T. ferrooxidanx Tuovinen and Kelly (24) observed previously that T. fer-
rooxidans would not grow in batch culture on thiosulfate at pH values below pH 3.5, but in this study organisms grown on tetrathionate at pH 2.5 were able to oxidize thiosulfate at pH 2.0, 3.0, and 4.0. Kinetic curves were complex, consisting of three phases (Fig. 4a). At low substrate concentrations (0.5 to 4.0 mM, pH 2.0; 0.5 to 7.0 mM, pH 3.0; 0.5 to 10.0 mM, pH 4.0), oxidation rates increased linearly with increasing substrate concentration. During a second phase (4.0 to 30 mM, pH 2.0; 7.0 to 90.0 mM, pH 3.0; 10.0 to 80.0 mM, pH 4.0), rates increased more slowly with increasing substrate concentration up to a maximum observed rate; this was followed by a third phase of substrate inhibition at very high thiosulfate concentrations. Substrate inhibition was greatest at pH 2.0, and 60 mM thiosulfate reduced the maximum observed oxidation rate (with 30 mM S2032-) by 86%. Inhibition was less severe at pH 4.0, and 200 mM thiosulfate reduced the maxiimum observed rate (at 80 mM) by 39%. There was little evidence for substrate inhibition at pH 3.0. Double reciprocal plots (Fig. 4b) indicated Vmax values for thiosulfate oxidation to be of the order as for tetrathionate (Table 2). Km
same
722
ECCLESTON AND KELLY
J. BACTERIOL.
I0
1600 04
1200 04 I
a
S00 04
400
0-2
0
0
20
0
40
60
(S,O,I
80
100
(mM
0
005
O0S
010
020
025
(1mM)
-
FIG. 4. Kinetics of thiosulfate oxidation by suspensions of T. ferrooxidans grown on 10 mM tetrathionate at the assay pH. (a) Specific oxidation rate against thiosulfate concentration, pH 2.0. (b) Double reciprocal plot of specific oxidation rate against thiosulfate concentration, pH 3.0.
values for thiosulfate were not easily calculated because of the complex nature of the curves, but best estimates were in the region of 2 to 25 mM over the range of pH 2 to 4 (Table 2). At pH 3.0, odd kinetics were apparent (Fig. 4b), with a possibility of two Km values corresponding to low substrate concentrations up to 20 mM (Km 1.2 mM) and to substrate concentrations above 20 mM (Km 25.0 mM). Controls established that no significant chemical oxidation of thiosulfate (10 to 100 mM) occurred at pH 3.0 or pH 4.0, although a very low rate of chemical thiosulfate oxidation was observed using 70 mM, but not 10 mM, at pH 2.0. Chemostat cultivation of T. ferrooxidans -
-
on tetrathionate-limited
media. A chemostat culture of T. ferrooxidans was established at pH 2.5 on a growth-limiting input of 10 mM tetrathionate and maintained for 23 steady states dering two separate runs. Residual tetrathionate and biomass were determined for all steady states at dilution rates (D) between 0.021 and 0.062 h-' (Fig. 5a). Washout was preceded by elemental sulfur precipitation and occurred at dilution rates above 0.067 h-'. Essentially complete oxidation of tetrathionate occurred in all steady states, as described in the fundamental
growth equation K2S406 + 3.5 02 + 3H20 K2SO4 + 3H2SO4, and no intermediate reducedsulfur compounds were ever detected (Fig. 5). Most steady states were established using C02 supplied only from air and from the K2CO3 pHcontrol titrant. In one series, the gas phase was 9% (vol/vol) C02 in air. The rate of steady-state titrant consumption was recorded and was in agreement with that predicted by the fundamental growth equation, i.e., 3K2CO3 per mole of K2S406 oxidized. Steady-state biomass concentration increased with increasing dilution rate (Fig. 5a). In cultures supplemented with 9% C02, biomass was greater at low D and increased less markedly (Fig. 5a). Plots of the reciprocals of yield and dilution rate (Y-' x D-') were linear and indicated true growth yields ( YG; grams [dry weight] per gram-molecule of tetrathionate oxidized) of 12.40 and 12.36 and maintenance coefficients (m; nillimoles of tetrathionate per gram [dry weight] per hour) of 1.02 and 0.55, respectively, for cultures grown with air or with 9% C02 in air. These values were verified by calculating the specific rate of tetrathionate oxidation (qs4o,,; millimoles per gram per hour) and plotting q against D. The plot was linear and indicated YG, 12.50, and m, 0.99. Again, additional C02 had little influence on YG (11.67), but con--
120
(a)
0
8
100 0(sulfur precipiatlonl
80
6
E 4'
20'
t.
60
1,_
Urw
4
6-
3
40 0 D
20 2 0
0
0o08
0-06
004
002
DILUTION RATE
(h)' 6
s
0-12 4 0 0
I IIl
-m
0o1 £
3
I
0-10
I 4e
2
0.09
006 0 0
10
30
20
40
so
0 0
I
0-02
004
006
(hE DILUTIN PATE (N") DLUTION RAEE FIG. 5. Steady-state data from two chemostat runs of T. ferrooxidans on 10 mM tetrathionate at pH 2.5, with and without supplementary CO2. (0, 0) Biomass; (5) biomass using 9% (vol/vol) CO2 in air; (0) residual tetrathionate (mM). (a) Biomass and residual tetrathionate concentration against dilution rate. (b) Double reciprocal plot of Y x D. (c) Specific rates of oxidaton of tetrathionate (q) by steady-state cultures at several dilation rates. Best-fit lines were determined by regression analysis. 723
J. BACTERIOL.
ECCLESTON AND KELLY
724
siderably reduced m to 0.34. The overall mean h-' on growth-limiting supplies of 10 mM thiovalue for YG from the four determinations was sulfate with 9% (vol/vol) CO2 in air. A further established 20 mM 12.23, with m as 1.01 and 0.45 with low and high six steady states thiosulfate at pH 2.5 at dilution rates between CO2 supply, respectively. The effect of CO2 supplementation and tetra- 0.026 and 0.104 h-1. Increasing the thiosulfate thionate limitation on steady-state biomass was concentration from 10 to 20 mM resulted in a further investigated during four additional doubling of culture absorbance (at 460 nm) from steady states, A to D (Table 3), at constant 0.178 to 0.236 at D = 0.026 h-'. Corresponding dilution rate (D = 0.045 h-1) and pH (2.5). increases were observed in true dry weight and Taking the mean values of absorbance, dry protein, confirming thiosulfate to be the limiting weight, and protein as measures of biomass, nutrient. Washout occurred at D 0.129 h'I and was biomass was reduced by 17.6% on limitig 10 mM tetrathionate when the supply of additional preceded by sulfur precipitation. Sulfur precipiCO2 was cut off (steady states A and B, Table tation also occurred at D = 0.093 h-' on one 3). A corresponding increase in biomass of 18.3% occasion. All thiosulfate supplied was completely utilized in all steady states, and no tetwas observed on limiting 5 mM tetrathionate when extra CO% was supplied (steady states C rathionate or trithionate was ever detected in and D, Table 3). In contrast, halving the tetra- the culture supematant. A plot of the specific rate of thiosulfate oxithionate concentration from 10 to 5 mM caused dation (qo,) against the dilution rate was linear a 53.2% drop in biomass without extra C02 (steady states B and C, Table 3) and an apparent (Fig. 6) and allowed YG (gramS [dry weight] per 52.9% drop in biomass with supplementary CO2 gram-molecule of thiosulfate oxidized) and m (millimoles of thiosulfate per gram per hour) to (steady states A and D). Steady states on limiting tetrathionate were be determined as 7.33 and 0.83, respectively. A also obtained at pH 2.0, 3.0, and 4.0 with little corresponding plot (not shown) of 1/yield effect on growth yield, except at pH 4.0, where against 1/dilution rate showed some scatter of points, but a best-fit line plotted by regression a drop of about 14% compared to pH 2.5 was observed. Cell clumping was often observed in analysis indicated values of 7.62 and 1.10 for YG and m, respectively, supporting the previous valresponse to a change of culture pH. ues. Mean values by the two procedures were Elemetl composition of T. ferroaxidan 7.48 and 0.97. grown on tetrathionate. No significant differAs with tetrathionate, additional CO2 inence in composition of organisms from four steady states was seen. Mean values (+ standard creased steady-state biomass on thiosulfate. At D = 0.026 h-', two steady states could be estaberror of the mean) from eight analyse were (percent dry weight): carbon, 48.72 (±1.07); hy- lished, with and without C02, in which culture drogen, 7.07 (±0.13); and nitrogen, 11.05 (±0.81). absorbance at 460 nm decreased by 23.4% from Chemostat cultivation of T. ferrooxidans 0.419 to 0.321 when additional CO% was no longer on thiosulfate with CO3 supplementation. supplied. Corresponding reductions of 27.9 and After 3,884 h of continuous cultivation of 10 mM 23.7% were also observed in dry weight and tetrathionate, the medium supply was switched protein. Batch growth of T. ferrooxrdrn on iron to thiosulfate for a further 938 h. In contrast to batch culture, a steady state was easily estab- after prolonged continuous cultivation on 0.026 lished (within 100 h) at pH 2.5 and D inorganic sulfur compounds. T. ferrooxidans on
were
=
=
TABLE 3. Tetrathionate and CO2 limitation of yield for steady-state T. ferrooxidans cultures' Steady-state biomas Length of steady Steadyb state condiio
state ob-
Gas phase
Se,
here(h)
Cultureabsorbance'
weight
Proti
(mg/ liter ~~~~~~~~~~~~~~~~~~~~~~~~~ ltr
51.4 10 0.386 :t 0.004 (7) 104.1 31 Air + 9% CO2 42.8 Air 0.314 ±0.009 (16) 86.1 10 123 21.6 41.7 C Air 32 5 0.130 ± 0.003 (8) 25.8 5 D 100 50.1 0.166 ± 0.)05 (8) Air + 9% C02 'Cultures were maintained at pH 2.5 and a dilution rate of 0.045 h-'. Tetrathionate was undetectable in states at both input subate concentrations. steady b SR, InpUt substrate (MM in inpUt mediUm). Measured at 460 nm, ± standard error. Parentheses indicate number of determinations.
A B
concentrtion
KS405
KINETICS OF T. FERROOXIDANS ON SULFUR COMPOUNDS
VOL. 134, 1978 16 14
10 .3
a 6
4
2
0'1 0
002 0-04 006 00 010 DUWTION
ATE (h')
FIG. 6. Chemostat culture of T. ferrooxidans on 20 mM thiosulfate at pH 2.5. Plot of the specific rate of thiosulfate oxidation (q) against dilution rate. Bestfit line was determined by regression analysis.
retained its ability to oxidize and grow on ferrous iron even after many hours of continuous cultivation on tetrathionate or thiosulfate. After 2,468 h on tetrathionate medium at pH 2.5, organisms from the fermentor were used to inoculate a series of shake-flasks containing ferrous iron medium at pH 1.4, 1.9, and 2.5 (2% [vol/vol] inoculum containing 92.5 ,ug [dry weight] of organisms per ml). Growth at 300C and complete iron oxidation occurred in flasks with starting pH 2.5 and pH 1.9 within 140 h, and with starting pH 1.4 after 168 h. Similarly, after 628 h of continuous cultivation on thiosulfate at pH 2.5, organisms taken from the fennentor and inoculated directly into ferrous iron medium (5%[vol/vol] inoculum containing 105 ,g [dry weight] of organisms per ml) at initial pH 1.4 grew and completely oxidized the ferrous iron within 5 days. No growth or significant iron oxidation occurred in uninoculated controls. Organisms originating from the fermentor and grown on ferrous iron could be maintained indefinitely by further transfers on ferrous iron medium at pH 1.4. DISCUSSION Our results provide for the first time kinetic data on the growth of T. ferrooxidans in both batch and continuous culture on both tetrathionate and thiosulfate. Although pH 2.5 was reported previously as optimal for growth on tetrathionate (24), specific growth rates in this study were generally higher than those previously reported (24), possibly as
725
a result of adding trace amounts of iron to the culture medium, a requirement also found necessary for prolonged trouble-free maintenance of stock cultures on inorganic sulfur compounds. Oxygen electrode studies with suspensions showed the apparent Km for tetrathionate oxidation to be pH dependent, with a greater affinity for substrate at higher pH. But oxidation rates were generally highest at low pH, so that optimal growth at pH 2.5 appeared to represent a balance of submaximal values for Km and oxidation rate with low substrate toxicity at pH 2.5. Changing the pH in continuous culture at constant dilution rate had very little effect on steady-state biomass. Although cell suspensions retained their oxidative activity for tetrathionate and thiosulfate for a considerable period, absolute oxidation activities varied between batches or even with the same batch assayed on different occasions, and because of this we suggest that attempts to differentiate between T. ferrooxidans strains on the basis of their activity towards inorganic sulfur compounds (1) be treated with extreme caution. Hitherto, it has not been possible to grow T. ferrooxidans on thiosulfate medium at pH values below 3.6 (24). Our continuous-culture studies provide the first evidence that the bacteria can in fact oxidize and grow on thiosulfate at lower pH values, under conditions where the thiosulfate is held only briefly at the low pH value before being consumed by the bacteria. Both tetrathionate and thiosulfate could, however, be inhibitory substrates at higher concentrations at pH values away from the optimum for growth: thiosulfate instability at acid pH might give rise to inhibitory products producing the effects observed with higher thiosulfate concentrations. Calculations of the theoretically available free energy from the oxidation of thiosulfate and tetrathionate (AF0, 936 and 1,654 kJ mol', respectively) indicate that thiosulfate might support 56.6% of the growth obtained mole for mole with tetrathionate. In fact, thiosulfate YG is 61.2% of the tetrathionate YG, which is close to the calculated figure. If real, the discrepancy could indicate an energy loss in the initial scission of tetrathionate, but it is probably within the accuracy of the methodology. The enhancement of yield under conditions of tetrathionate and thiosulfate limitation by additional C02, without significant effect on YG, indicates that the apparent maintenance coefficient varies with C02 supply. This probably indicates that, while sulfur compound supply is growth limiting, the efficiency of coupling of its oxidation to growth is a function of availability of C02. The proportion of "growth-uncoupled" oxidation would be
726
ECCLESTON AND KELLY
greater under low C02 supply at any growth rate. The YG values may be used to estimate the probable ATP yield of tetrathionate and thiosulfate oxidation. For tetrathionate, YG 12.23 represents the fixation of 5.96 g of carbon or 0.5 mol of C02 per mol of tetrathionate oxidized. Since the principal mechanism of C02 fixation in T. ferrooxidans is the Calvin cycle (23), in which 1 C02 requires 3 ATP and 2 reduced nicotinamide adenine dinucleotide (NADH) molecules for fixation to the level of hexose, this requires 1.5 ATP and 1.0 NADH. Since tetrathionate oxidation, described in the fundamental growth equation above, generates 14 reducing equivalents [(H)] per mol, and since 1.0 NADH requires 2 (H) for formation, only 12 (H) are then available for respiratory transport to oxygen. Consequently, only 0.857 mol of tetrathionate provides (H) for energy-linked transport to oxygen, and the remaining 0.143 provides (H) for C02 reduction. In autotrophic bacteria, about 80% of total ATP available is used by the Calvin cycle, the remainder being required for biosynthesis from the hexose level and attendant metabolic functions (4, 10, 19). Consequently, the fixation of 0.5 mol of CO2 indicates that 1.875 ATP were available for its conversion to cell constituents. Since the reduction of oxidized NAD is known to be energy requiring in T. ferrooxidans (8, 10, 23) and probably requires 2 ATP per NADH (10), a further 2 ATP must be available to produce the 1 NADH required. This is a minimum estimate, since NADH is required in smaller amounts for other biosynthetic steps. In summary, conversion of 0.5 C02 to cell material represents a minimum of 3.875 ATP generated from the oxidation of 0.857 K2S406, or 4.5 ATP per mol of tetrathionate. It thus seems unlikely that more than 5 ATP can be generated during the oxidation of 1 tetrathionate molecule, although seven oxygen atoms are consumed (fundamental growth equation above). This might be consistent with 3 ATP being formed by oxidative phosphorylation during the consumption of three oxygen atoms during sulfite oxidation (P/0 = 1), while two 02 molecules are consumed by the oxygenase described by Suzuki and Silver (20) in the conversion of the sulfanesulfur atoms of tetrathionate to sulfite. The remaining 2 ATP could be formed via APS (adenosine 5'-phosphosulfate, adenylyl sulfate) by the reactions of sulfite originally described by Peck (18). This is summarized in the model in Fig. 7, which shows the possible intermediates in the oxidation of tetrathionate (and of thiosulfate) and likely sites of ATP synthesis. Using the YG for thiosulfate, calculations as for tetrathionate showed that 2.78 ATP could be
J. BACTERIOL.
03S-S-S-SO3 .2.
so5
So;
2S
p2 02t 2 H20
4H+
S0j *^-, 4 so 2
+~~I
Via
4 APS?
2 ATP by subtrate lee
phosphoyyntionJ 4 H20
1
502
36DHI e3P
3
20
3 ADP+3 Pi 3 ATP (by oi%Wotiw
Ion) 2C FIG. 7. Hypothetical mechanism for tetrathionate oxidation and ATP synthesis by T. ferrooxidans, based on chemostat growth yields. Overall, the model is consistent with the oxidation equation S4062- = 4 SO42- + 6 HW and the production of 5ATP per mol of tetrathionate oxidized.
4S5;+ 2H9+
formed per mol of thiosulfate oxidized, indicating a probable maximum of 3 ATP per mol for the consumption of 202 molecules. This is again wholly consistent with the model in Fig. 7, 1 02 being consumed by the oxygenase, 102 supporting synthesis of 2 ATP, and a third ATP appearing from substrate-level phosphorylation (via 2 sulfite and consequently 2 APS molecules). This model is consistent with YG values around 7 for thiosulfate reported for other thiobacilli (7) growing on thiosulfate. It is, however, not consistent with evidence that sulfane-sulfur oxidation supported oxidative phosphorylation (12, 13) or with the high YG reported for T. neapolitanus (5) and for aerobic and anaerobic T. denitrificans (7,22). It does, however, provide a working hypothesis compatible with our yield data. ACKNOWLEDGMENT This work was supported by research grant GR3/2693 from the Natural Environment Research Council.
KINETICS OF T. FERROOXIDANS ON SULFUR COMPOUNDS
VOL. 134, 1978
LITERATURE CITED 1. Bounds, H. C., and A. R. Colmer. 1972. Comparison of the kinetics of thiosulfate oxidation by three iron-sulfur oxidizers. Can. J. Microbiol. 18:735-740. 2. Colmer, A. R. 1962. Relation of the iron oxidizer, Thiobacillus ferrooxidans, to thiosulfate. J. Bacteriol.
15.
83:761-765. L Temple and M. E. Hinkle. 1950. An iron-oxidizing bacterium from the acid drainage of some bituminous coal mines. J. Bacteriol. 59:317-328. Forrest, W. W., and D. J. Walker. 1971. The generation and utilization of energy during growth. Adv. Microb. Physiol. 5:213-274. Hempfling, W. P., and W. Vishniac. 1967. Yield coefficients of Thiobacilus neapolitanus in continuous culture. J. Bacteriol. 93:874-878. Hutchinson, M., K. I. Johnstone, and D. White. 1966. Taxonomy of the acidophilic thiobacilli. J. Gen. Microbiol. 44:373-381. Justin, P., and D. P. Kelly. 1978. Growth kinetics of Thiobacillus denitrificans in anaerobic and aerobic chemostat culture. J. Gen. Microbiol. 107:49-58. Kelly, D. P. 1978. Bioenergetics of chemolithotrophic bacteria, p. 363-386. In A. T. Bull and P. Meadow (ed.), Companion to microbiology. Longmans, London. Kelly, D. P., L. A. Chambers, and P. A. Trudinger. 1969. Cyanolysis and spectrophotometric estimation of trithionate in a mixture with thiosulfate and tetrathionate. Anal. Chem. 41:898-901. Kelly, D. P., M. Eccleston, and C. A. Jones. 1977. Evaluation of continuous chemostat cultivation of ThiobaciUus ferrooxidans on ferrous iron or tetrathionate, p. 1-7. In W. Schwartz (ed.), Gesellschaft fur Biotechnologische Forschung conference-bacterial leaching. Verlag Chemie, Weinheim, Germany. Kelly, D. P., and C. A. Jones. 1978. Factors affecting metabolism and ferrous iron oxidation in suspensions and batch cultures of Thiobacillus ferrooxidans. In L. E. Murr (ed.), Metallurgical applications of bacterial leaching and related microbiological phenomena. Academic Press Inc. New York. Kelly, D. P., and P. J. Syrett. 1963. Effect of 2:4-dinitriphenol on carbon dioxide fixation by a Thiobacillus. Nature (London) 197:1087-1089. Kelly, D. P., and P. J. Syrett. 1966. Energy coupling during sulphur compound oxidation by Thiobacillus sp. strain C. J. Gen. Microbiol. 43:100-118. Kelly, D. P., and 0. H. Tuovinen. 1972. Recommenda-
3. Colmer, A. R., K.
16.
4.
17.
5. 6.
7. 8. 9.
10.
11.
12. 13. 14.
18. 19.
20. 21. 22.
23.
24.
25.
26.
727
tion that the names Ferrobacillus ferrooxidans Leathan and Braley and Ferrobacillus sulfooxidans Kinsel be regarded as synonyms of Thiobacillus ferrooxidans Temple and Colmer. Int. J. Syst. Bacteriol. 22:170-172. Landesman, J., D. W. Duncan, and C. C. Walden. 1966. Oxidation of inorganic sulfur compounds by washed cell suspensions of Thiobacillus ferrooxidans. Can J. Microbiol. 12:957-963. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Margalith, P., M. Silver, and D. G. Lundgren. 1966. Sulfur oxidation by the iron bacterium Ferrobacillus ferrooxidans. J. Bacteriol. 92:1706-1709. Peck, H. D. 1968. Energy-coupling mechanisms in chemolithotrophic bacteria. Annu. Rev. Microbiol. 22:489-518. Stouthamer, A. H. 1973. A theoretical study of the amount of ATP required for synthesis of microbial cell material. Antonie van Leeuwenhoek J. Microbiol. Serol. 39:545-565. Suzuki, I., and M. Silver. 1966. The initial product and properties of the sulfur-oxidizing enzyme of thiobacilli. Biochim. Biophys. Acta 122:22-33. Temple, K. L., and A. R. Colmer. 1951. The autotrophic oxidation of iron by a new bacterium: Thiobacillus ferrooxidans. J. Bacteriol. 62:605-611. Timmer-ten-Hoor, A. 1976. Energetic aspects of the metabolism of reduced sulfur compounds in Thiobacillus denitrificans. Antonie van Leeuwenhoek J. Microbiol. Serol. 42:483-492. Tuovinen, 0. H., and D. P. Kelly. 1972. Biology of Thiobacillus ferrooxidans in relation to the microbiological leaching of sulphide ores. Z. Allg. Mikrobiol. 12:311-346. Tuovinen, 0. H., and D. P. Kelly. 1974. Studies on the growth of Thiobacillus ferrooxidans. V. Factors affecting growth in liquid culture and development of colonies on solid media containing inorganic sulphur compounds. Arch. Microbiol. 98:351-364. Tuovinen, 0. H., S. I. Niemela, and H. G. Gyllenberg. 1971. Tolerance of Thiobacillus ferrooxidans to some metals. Antonie van Leeuwenhoek J. Microbiol. Serol. 37:489-496. Unz, R. F., and D. G. Lundgren. 1961. A comparative nutritional study of three chemoautotrophic bacteria: Ferrobacillus ferrooxidans, ThiobaciUus ferrooxidans and Thiobacillus thiooxidans. Soil Sci. 92:302-313.
