Journal of General Microbiology (1979), 115, 377-384.
Printed in Great Britain
377
Competition in the Chemostat between an Obligately and a
Facultatively Chemolithotrophic Thiobacillus
By A L I S O N L. S M I T H A N D D. P. K E L L Y
Department of Environmental Sciences, University of Warwick, Coventry CV4 7AL
(Received 6 March 1979; revised 4 May 1979)
The outcome of competition in thiosulphate-limited chemostat culture between the obligate
chemolithotroph Thiobacillus neapolitanus and the versatile facultative autotroph Thiobacillus A2 was in part a function of pH. In pure culture T. neapolitanus grew faster than
Thiobacillus A2 at pH values up to pH 7.6, but in competition Thiobacillus A2 dominated
at pH 7-35and 7.6, At pH 7.1, T. neapolitanus dominated, although a significant steady state
population of Thiobacillus A2 persisted, apparently growing on organic nutrients excreted
by T. neapolitanus. Coexistence of both organisms occurred under all chemolithotrophic
growth conditions tested with the dominant organism comprising 85 to 99 yo of the population, indicating that competition was not the sole interaction between the species. At pH 7.1,
the inclusion of glucose in the thiosulphate medium resulted in rapid domination of the
culture by Thiobacillus A2, with the virtual elimination of T. neapolitanus. It is concluded
that the capacity for mixotrophy is a selective advantage to a facultative thiobacillus in
competition with an obligately chemolithotrophic species.
INTRODUCTION
The ability of facultatively heterotrophx (or ‘versatile ’) thiobacilli to compete successfully in natural environments with obligately chemolithotrophic thiobacilli or heterotrophs
was discussed by Rittenberg (1972) and Whittenbury & Kelly (1977). Rittenberg (1972)
postulated that under mixed substrate conditions, the facultative organism might predominate as it can achieve greater reproductive power per unit of energy expenditure growing
mixotrophically (or as a chemolithotrophic heterotroph) than can the obligately autotrophic
organism. The ability of the facultative Thiobacillus A2 to outcompete both the obligate
T. neapolitanus and a heterotrophic spirillum in chemostat culture on mixed substrates was
subsequently demonstrated by Gottschal et al. (1979). They also demonstrated, however,
that in autotrophic cultures subject to thiosdphate limitation, the ‘specialist’ chemolithotroph (T. neapolitanus) dominated over Thiobacillus A2 at all dilution rates above 0.025 h-l
and suggested that, as a general principle, ‘specialist ’ organisms would dominate over
‘versatile’ ones by virtue of the higher growth rates attainable by the former under specialist
growth conditions. This principle has been the subject of several theoretical and practical
treatments (Meers, 1971 ; Veldkamp & Jannasch, 1972; Taylor &Williams, 1974; Veldkamp,
1976; Kuenen et a/., 1977; Laanbroek et al., 1979).
In this paper we report competitive and commensal interactions between the facultative
Thiobacillus A2 (Taylor & Hoare, 1969) and the obligate T. neapolitanus (Kelly, 1967, 1970)
showing that under some conditions the former can outgrow the latter even under the
specialist conditions favourable to T. neapolitanus.
0022-1287/79/OooO-8641 $02.00 @ 1979 SGM
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378
A. L. SMITH A N D D. P. K E L L Y
METHODS
Organisms and culture conditions. Thiosulphate agar media were used for culture maintenance as p r e
viously described for Thiobacillus A2 (Wood & Kelly, 1977) and T. neapolitanus strain C (Kelly, 1969;
Tuovinen & Kelly, 1973). Liquid cultures (in shake-flasks and the chemostat) were grown in a medium
containing (g 1-l) :Na2S,0,. 5H@, 12-5;MgS04.7H20,O.l; NH,Cl, 0.4; KH2P04,0 to 4.5 ;Na,HPO,. 2H20,
3-95 to 9.86; NaOH as required; trace metal solution (Tuovinen & Kelly, 1973), 10 ml. Medium pH was
adjusted to the desired value by altering the ratio of phosphates (maintaining the same total phosphate
concentration) and amount of NaOH added.
Chemostat cultures were established in a LH modular type series 500 fermenter (LH Engineering, Slough,
Bucks) with a culture volume of 750 ml, stirred (750 rev. min-l) and aerated (200 ml min-l) with air containing 5 % (v/v) CO,. Temperature was maintained at 30 "Cand pH at desired values by automatic addition
of 1.6 M-K,CO,, using an EIL model 91A or LH Engineering pH control unit. Culture purity was monitored
by examination of growth on nutrient agar.
Discrimination of Thiobacillus A2 and T. neapolitanus in mixed culture, Appropriate serial dilutions of
culture samples were spread on Thiobacillus A2 agar medium, pH 8.4 (Wood & Kelly, 1977) containing
0.1 M-sodium formate instead of thiosulphate. Only Thiobacillus A2 could grow on this medium and it could
be discriminated from T. neapolitanus. Identical numbers of colonies of Thiobacillus A2 were produced on
this medium and on thiosulphate agar, glucose agar or nutrient agar. Dilutions were also spread on to
T. neapolitanus thiosulphate agar, on which both organisms could grow. Thiobacillus neapolitanus produced
u ~and the morphology of colonies of the two species
countable colonies more rapidly than T h i o ~ a c i l ~A2
were quite distinct. Thiobacillus neapolitanus could thus be counted directly on these plates and good agreement was achieved by comparison with the total count and the count of ThiobacillusA2 obtained on formate
agar.
Analytical procedures. Growth was monitored turbidimetrically using a Pye Unicam SP1700 spectrophotometer and absorbance at 440 nm was related to dry weight using calibration curves for pure cultures.
Organism dry weight concentrations and protein contents of cultures were determined in steady state
conditions. For dry weight estimation, bacteria were harvested by centrifuging, washed and dried at 105 "C.
For protein estimation, organisms from 4 ml samples were harvested by centrifuging, washed with distilled
water and dissolved in 2.5 ml 0.5 M-NaOH at 100 "C for 10 min; protein was determined by the Lowry
method.
Thiosulphate and polythionates were determined cyanolytically (Kelly et al., 1969) and glucose by the
method of Somogyi (1945). Thiosulphate oxidation by washed suspensions of organisms harvested from
growing cultures was measured with an oxygen electrode as described previously (Eccleston & Kelly, 1978).
RESULTS
Eflect of p H on growth rates of Thiobacillus A2 and T. neapolitanus
Specific growth rates (p) were determined from exponentially growing pure cultures on
thiosulphate in cultures poised at different pH values. Thiobacillus neapolitanus exhibited a
maximum growth rate (0.22 h-l) at pH 6.6 to 6.8, at which pH Thiobacillus A2 did not grow.
Thiobacillus A2 gave a maximum (0.1 h-l) at pH 7.8 to 8.0, at which pH T. neapolitanus did
not grow (Table 1).
Growth in batch and continuous culture
From measurement of washout kinetics (Pirt, 1975), maximum specific growth rates for
the two strains were calculated to be 0.277 h-l for T. neapolitanus and 0.148 h-l for
Thiobacillus A2. At dilution rates of 0.08 and 0.1 h-l in the chemostat, growth yields
were 4.5 and 4.5 g dry wt (mol thiosulphate oxidized)-l for T. neapolitanus and
6.7 and 6.0g dry wt (mol thiosulphate oxidized)-l for Thiobacillus A2. When grown
in mixed batch culture at pH 7.6, both organisms initially grew faster (p = 0.23 h-l)
than they did alone, but within 10 h viable numbers of Thiobacillus A2 virtually
ceased to increase while T. neapolitanus continued to grow at the same rate for a further
10 h.
Competition in mixed chemostat culture on limiting thiosulphate
The outcome of competition between the two species on limiting thiosulphate was dependent on the pH of the culture. Commencing with equal numbers of the two species growing
together in batch culture at pH 7.6 in the chemostat vessel, Thiobacillus A2 was outcompeted
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379
Competition between thiobacilli
Table 1. SpeciJc growth rates in autotrophic batch culture on thiosulphate for
Thiobacillus A2 and T. neapolitanus at several p H values
Specific growth rate, p (h-l)
--
A
PH
T. neapolitanus
6.0
6.2
6.4
6.6
6.7
6-8
7.0
7.2
7.4
7.6
7-8
8.0
8.2
8.4
8.7
9.0
0.116
0.213
0.220
0.224
0.193
0.182
0.139
0
-
3
ThiobacillusA2
-
-
0
0.002
-
0.074
0.087
0.089
0.096
0.105
0.099
0.086
0.088
04035
0
-
-
-, Not determined.
90
fi
E
$
5
60
a)
.-
Y
2
30
C
0
1
3
I
7
I
I
I
I
I
I
I
L
23
31
39 41
Culture volume changes
15
A
43
45
Fig. 1. Effect of pH and dilution rate on the outcome of competition between Thiobacillus A2 (0)
and T.neapolitanus ( 0 )in continuous culture on limiting 50 m~-Na,S,o,. Relative numbers (yoof
total population) of organisms and culture absorbance at 440 nm (A)were determined following
the imposition of the following series of conditions at the times indicated by the arrows: (a) pH 7-6,
batch culture; (b) pH 7-6, D = 0.02 h-l; (c) pH 7.6, D = 0.08 h-l; ( d ) pH 7.1, D = 0.08 h-l;
(e) pH 7.1, D = 0.02h-l. Absorbance values are proportional to dry weights of organisms ;a value
of 1.0 is equivalent to 300 mg Thiobacillus A2 1-1 or 360 mg T. neapolitanus 1-1 when applied to
pure cultures. Media at pH 7.6 or 7.1 contained (g 1-l): Na,HP04. 2H20, 7.9 or 6.0; KH2P04,1-5
or 2.94; adjusted with NaOH to the precise values.
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380
A. L. SMITH A N D D. P. K E L L Y
n
90
%
rn
26
60
1
u
.-
5
30
p:
0
0
1
60
0
~
4
120
"
8
180
~
~
300
240
Time (h)
, ,
~
'
12
16
20
Culture volume changes
24
360
~
28
420
~
32
"
"
'
~
Fig. 2. Outcome of competition between Tkiobadus A2 ( 0 )and T. neapditanus ( 0 )at pH 7.35
and 7.1. Relative numbers and culture absorbance ( A ) were monitored as for Fig. 1 : (a) pH 7-35,
batch culture; (&) pH 7.35, D = 0.08 h-l; (c) pH 7.1, D = 0.08 h-l.
and fell to 7 yo of the population (Fig. 1). A dilution rate of 0.02 h-l was then commenced
and Thiobacillus A2 rapidly dominated and continued to constitute more than 95% of the
total population after 10 volume changes at D = 0.08 h-l (Fig. 1). During this period T.
neapolitanus was not eliminated, but remained at 1 to 6 % of the population. A subsequent
decrease in the steady state culture pH to 7.1 at D = 0.08 h-l (Fig. 1) resulted in takeover
of the culture by T. neapolitanus, although Thiobacillus A2 persisted at about 5 % of the
total population for 19 volume changes. Decreasing the dilution rate to 0.02 h-l at pH 7.1
enabled Thiobacillus A2 to increase rapidly to about 14%, although T. neapolitanus remained dominant over a further 6 volume changes (Fig. 1). The higher absolute growth
yield achieved by Thiobacillus A2 was reflected in the changes in mixed culture absorbance
during these transitions (Fig. 1).
As pH 7.6 was at the upper limit for normal growth of T. neapolitanus (Table l), the
experiment was repeated at pH 7.35 commencing with a mixture of 25% Thiobacillus A2
and 75 % T. neapolitanus allowed to grow as a batch culture for 16 h before imposing a
dilution rate of 0.08 h-l (Fig. 2). During batch growth T. neapolitanus outcompeted Thiobacillus A2 to reach 97% of the total population, but following the switch to continuous
flow culture it was rapidly outgrown by Thiobacillus A2 until after 6 to 7 volume changes the
ratio of Thiobacillus A2 to T. neapolitanus was 94: 6 (Fig. 2). This ratio did not vary significantly over a further 16 volume changes. The culture pH was then lowered from 7-35 to
7. I , which resulted in a slow but progressive takeover by T. n~apolitanus(Fig. 2). Thiosulphate and polythionates were undetectable in these experiments within 1 (at D = 0.08 h-1)
to 3 (at D = 0.02 h-l) volume changes after commencing continuous flow cultures. It is
possible that in both series (Figs 1 and 2) the steady states attained, in which both organisms
always coexisted, were not perfect, as some indication of regular oscillations of relative
numbers is apparent.
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'
'
38 1
Competition between thiobacilli
80
60
40
/
20
20
0
1
1
0
I
I
2
40
1
60
I
4
80
Time (h)
I
I
I
120
100
I
I
I
6
8
10
Culture volume changes
140
I
240
I
12
d
19
Fig. 3. Effect of glucose on the outcome of competition between ThiobacillusA2 ( 0 )and T. neapolitanus ( 0 )in chemostat culture on limiting 50 m~-Na,S,0,at pH 7.1, D = 0.08 h-l. ( a ) Na,S20,
alone; (6) Na2S203plus 2.3 mM-glucose; (c) Na2S,03alone. See Table 2 for comparative total viable
numbers of the two species.
Eflect of glucose on competition between Thiobacillus A2 and T. neapolitanus in
thiosu~hate-limitedculture at p H 7.1
Commencing with a mixture of Thiobacillus A2 and T. neapolitanus in a ratio of 80:20
(Fig. 3), continuous culture at p H 7.1 on limiting thiosulphate alone at a dilution rate of
0.08 h-l led to rapid dominance by T. neapolitanus. After 4 volume changes, the culture
contained about 1.3 x lo8 Thiobacillus A2 ml-l and 9.7 x lo8 T. neapolitanus ml-l (i.e. ca 13%
Thiobacillus A2). At this time the 50 mwthiosulphate medium feed was supplemented with
2.3 mM-glucose, which resulted in an extremely rapid takeover of the culture by Thiobacillus
A2 to reach 60 to 70 yo of the total population within 2 volume changes and about 99.9 yo
of the total population within 4 volume changes (Table 2, Fig. 3). Between 80 and 100 h
the viable numbers of T. neapolitanus fell from 3 x lo8 (at 80 h) to lo6 (at 100 to 105 h).
Since washout of non-growing organisms would only have reduced the numbers to about
4 x lo7, T. neapolitanus must actually have lost viability during the takeover of the culture
by Thiobacillus A2. This observation has not been investigated further. Subsequently reverting to a glucose-free medium resulted in a recovery of T. neapolitanus to reach 3576 of the
total population after 9 volume changes even though it had fallen to 0.05 yo of the total in
the presence of glucose.
Thiosulphate oxidation by washed suspensions of Thiobacillus A2 and T. neapolitanus
The growth rates exhibited in batch culture at pH 7.6 by the two species (Table 1) would
suggest that T. neapolitanus rather than Thiobacillus A2 might have dominated in mixed
continuous culture at this pH. As the reverse was observed (Fig. l), the oxidation of thiosulphate by non-growing suspensions was examined to see if differences in apparent K,
values for thiosulphate oxidation might reveal some advantage enjoyed by ~ ~ i o b a c ~A2.
l~us
Values for apparent Km were calculated from double reciprocal plots of thiosuIphate concentration (14 concentrations between 0.1 and 10 mM) and oxidation rate for both strains
at p H 7.6 and 7.1. The relation was biphasic for T. neapolitanus at both p H values, but
Thiobacillus A2 gave a linear plot for all thiosulphate concentrations between 0.1 and
10 mM. Apparent mean K, values (mM-thiosulphate) from three determinations for oxidation by suspensions of Thiobacillus A2 were 0.24 k 0.02 (pH 7.1) and 0-14& 0.02 (pH 7.6).
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382
A. L:SMITH
A N D D. P. K E L L Y
Table 2. Changes in numbers of Thiobacillus A2 and T. neapolitanus during continuous mixed
culture at p H 7-1, D = 0.08 h-I, on limiting thiosulphate in the absence or presence of glucose
-
The initial increase in total numbers reflects the use of excess thiosulphate present at the time of
switching from exponential batch growth to continuous flow culture. No significant levels of
thiosulphate, polythionates or glucose were detected in any steady states.
Limiting substrate(s)
in medium input (mM)
+
Na2S203(50)
Glucose (2.3)
Na2S203 (50)
Time
(h>
1
5-1 1
33-48
52-56
61-72
78-85
96
100-1 34
144-149
242
Culture volume
changes
0.1
040.8
2.7-3.9
4-2-45
4.9-5.8
6.2-6.8
7.7
8.0-10-7
11.6-1 1.9
19.4
x No. of viable organisms ml-l
Thiobacillus A2
210
195
125
157
41 5
1220
1050
908
1050
510
7
T. neapolitanus
54
260
970
867
540
3 40
22
0.8
5.3
270
With 0.2 to 2 mM-thiosulphate, the values for T. neapolitanus were 0.10 f.0.05 (pH 7.1) and
0.12 & 0.03 (pH 7.6); and 5.9 k 3.5 and 1-00& 0-3 with 2 to 10 mxl-thiosulphate. No firm
conclusions can be drawn from these results, especially as the steady state cultures contained
no detectable thiosulphate, so that the concentration to which the organisms were exposed
was below these apparent K, values. Apparent Vmxvalues were very low since T. neapolitanus in particular rapidly lost activity in non-growing suspensions.
DISCUSSION
These experiments serve to demonstrate that a facultative thiobacillus like Thiobacillus
A2 is likely to exhibit a marked survival advantage over obligately lithotrophic strains such
as T. neapolitanus and that interactions other than solely competition can exist between such
species. As expected, and as separately demonstrated by Gottschal et al. (1 979), T. neapolitanus dominated over ThiobaciZZzis A2 in thiosulphate-limited culture in our experiments at
pH 7.1. However, in contrast to the view expressed by Gottschal et al. (1969) that the
specialist organism would generally outcompete the versatile one under such conditions,
we have shown that the outcome of competition was pH-dependent. Thus at pH 7-35 and
7.6, Thiobacillus A2 was the dominant organism even though separate batch culture of the
two species showed T. neapolitanus to grow faster than Thiobacillus A2. At pH 7.35 or 7.6,
T. neapolitanus fell to about 5 % of the total number of organisms (Figs 1 and 2) but was
never completely eliminated. Similarly, at pH 7.1 at which T. neapolitanus dominated,
Thiobacillus A2 persisted at 5 to 14 yo of the total population (Fig. l), being more abundant
at a lower dilution rate than a high one. These observations of the establishment of seemingly
stable mixed populations on a single limiting substrate are not consistent with the theory of
competition between two species under such conditions. The organism of faster growth rate
would be expected to exclude the second one completely (Jost et aZ., 1973; Veldkamp, 1976;
Kelly, 1978). The key to explaining these phenomena is given in the fact that Thiobacillus
A2 dominates over T. neapolitanus even at pH 7.1 when a small amount of glucose is added
to the medium (Fig. 3). Under such conditions Thiobacillus A2 consumes all the available
glucose, as T. neapolitanus strain C cannot incorporate glucose (Kelly, 1968), which would
allow a steady state concentration of about 3.6 x los viable organisms ml-l in the absence
of thiosulphate, compared with about lo9on 50 mM-thiosulphate alone. A large population
could thus be produced that could compete with T.neapolitanus for the available thiosulphate.
More significantly, the maximum specific growth rate of Thiobacillus A2 growing mixoDownloaded from www.microbiologyresearch.org by
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Competition bet ween thiobacilli
383
trophically at pH 7.1 on glucose plus thiosulphate must exceed that of T. neapolitanus leading
to the almost total exclusion of the latter (Table 2, Fig. 1). Dominance of Thiobacillus A2
at pH 7.35 and 7.6 in thiosulphate-limited culture could result at least in part from its
growth rate being increased to a value greater than that of T. neapolitanus by organic nutrients excreted by T. neapolitanus. In pure culture on thiosulphate, T.neapolitanus excretes
up to 27 %of the carbon dioxide it fixes (Kelly, 1969), including considerable amounts of
glycollate (Gottschal et al., 1979), which could enable the survival of small populations of
Thiobacillus A2 in mixed cultures dominated by T. neapolitanus. The higher population at
D = 0-02 h-l than at D = 0.08 h-l (Fig. 1) could reflect greater excretion of metabolites by
T. neapolitanus at what might be a rather unfavourable growth rate (about 7 % of ,urnax).
Certainly cultures subject to growth inhibition, for example by phenylalanine, excreted far
larger amounts of fixed carbon (Kelly, 1969). The persistence of significant numbers of T.
neapolitanus in cultures dominated by Thiobacillus A2 is less obviously explained but may
indicate a complex interaction between the two species. If the advantage of Thiobacillus A2
is a marginally greater growth rate than that of T. neapolitanus at pH 7.35 and 7.6, due to
excreted metabolites from the latter organism, a decline of T. neapolitanus to too low a level
would reduce the growth rate of the Thiobacillus A2, thus allowing T. neapolitanus to increase in numbers again. The stable ratio of the two could thus be a dynamic balance. The
populations of both organisms may in fact show oscillations in the steady states (e.g. Fig. I),
which would be consistent with such dynamic metabolic interrelationships. Stable mixed
cultures of heterotrophs on single limiting substrates in the chemostat can be maintained
(Brunner et al., 1968; Slater & Somerville, 1979), but in many of these one or more members
of the communities are growing on breakdown products of the primary substrate. Persistence of seemingly uncompetitive organisms has also been reported (Godwin & Slater, 1979).
Mutual stimulation of growth rate when the two organisms were grown in mixed batch
culture could indicate stimulation of Thiobacillus A2 by T. neapolitanus metabolites, but
the stimulation of the latter might be consequent on the removal of those metabolites,
since T. neapolitanus is known to be sensitive to some organic metabolites (Kelly, 1969,
1971;Matin, 1978), and it and other chemolithotrophs may exhibit stimulated growth rates
in dialysed cultures (Pan & Umbreit, 1972).
These results demonstrate that although one or other of a facultative or obligate chemolithotrophic autotroph may dominate in mixed cultures depending on substrate supply and
pH, both may coexist under various conditions, showing that, in addition to competition,
there can be synergism, commensalism or mutualism as has indeed been modelled or observed with purely heterotrophic systems (Megee et al., 1972; Taylor & Williams, 1974;
de Freitas & Frederickson, 1978). The results demonstrate very clearly that the facultative
organism Thinbacillus A2 will dominate under mixotrophic nutrient conditions at p H
values at which it would not compete successfully with T. neapolitanus under solely chemolithotrophic and autotrophic growth conditions. Facultative autotrophs exhibiting mixotrophic growth physiology would therefore be expected to be of greater abundance in
natural environments than obligate autotrophs.
We thank the Natural Environment Research Council for supporting this work. We are
indebted to Gijs Kuenen and Jan Gottschal for showing us results of their competition
studies prior to publication while this paper was in preparation.
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