ELSEVIER
FEMS Microbiology Ecology I9 (1996) 14I- I5 1
Coexistence of aerobic chemotrophic and anaerobic phototrophic
sulfur bacteria under oxygen limitation
Frank P. van den Ende *, Anniet M. Laverman, Hans van Gemerden
Department of Microbiolog!. Linkersi& of Groningen, Kerklaan 30. 9751 NN Hot-en. The Netherlands
Received 11August 1995: revised 2 November
1995; accepted 3 November
I995
Abstract
The aerobic chemotrophic sulfur bacterium Thiobacillus fhioparus T5 and the anaerobic phototrophic sulfur bacterium
Thiocupsa roseopersicina M 1 were co-cultured in continuously illuminated chemostats at a dilution rate of 0.05 h ‘. Sulfide
was the only externally supplied electron donor, and oxygen and carbon dioxide served as electron acceptor and carbon
source, respectively. Steady states were obtained with oxygen supplies ranging from non-limiting amounts (1.6 mol O2 per
mol sulfide, resulting in sulfide limitation) to severe limitation (0.65 mol O2 per mol sulfide). Under sulfide limitation
Thiocapsa was competitively excluded by Thiobacillus and washed out. Oxygen/sulfide
ratios between 0.65 and 1.6
resulted in stable coexistence. It could be deduced that virtually all sulfide was oxidized by Thiohacillus. The present
experiments showed that Thiocupsa is able to grow phototrophically on the partially oxidized products of Thiobacilltts. In
pure Thiohucillus cultures in steady state extracellular zerovalent sulfur accumulated, in contrast to mixed cultures. This
suggests that a soluble form of sulfur at the oxidation state of elemental sulfur is formed by Thiobacillus as intermediate. As
a result, under oxygen limitation colorless sulfur bacteria and purple sulfur bacteria do not competitively exclude each other
but can coexist. It was shown that its ability to use partially oxidized sulfur compounds, formed under oxygen limiting
conditions by Thiohucillus, helps explain the bloom formation of Thiocupsu in marine microbial mats.
Kemmds:
Thiobacillus;
Thiocapsa:
Oxygen
limitation:
Sulfide oxidation;
1. Introduction
In microbial
sulfidic
layers,
Simultaneously,
dantly present
reduced sulfur
petition would
low substrate
Jergensen and
mats,
purple
provided
light
sulfur bacteria
penetrates
to
often bloom.
colorless sulfur bacteria are abunin these systems. Both groups exploit
compounds and, consequently,
combe expected to occur, particularly at
concentrations.
Jorgensen
[I], and
Des Marais [2] evaluated the competi-
* Corresponding author
016%6496/96/$15.00
0 1996 Federation
SDf 0168-6496(95)00082-S
of European
Microbiological
Marine microbial
mat
tion between colorless sulfur bacteria and purple
sulfur bacteria in marine microbial mats (Kal@ Vig,
Denmark and Guerrero Negro, Mexico) in relation to
environmental
parameters.
The mats which were
studied, situated in areas without tidal movements,
were permanently overlain by a thin layer of water
and dominated by the purple sulfur bacterium C/zromatium
and the colorless sulfur bacterium Beggiatna. These organisms
exhibited diurnal migration
patterns, and positioned themselves in narrow bands
where the prevailing conditions
were’ optimal for
their development.
As a result of their different
nutritional preferences, particularly with-’ respect to
Societies.
AH rights reserved
142
F.P. c’an den Ende et al./FEMS
Microbiology
oxygen, Chromatium and Beggiatoa were often spatially separated, and direct competition for reduced
sulfur compounds
thus appears to be of less
paramount importance.
In microbial mats developing on sandy intertidal
sediments in temperate zones the dominant purple
sulfur bacterium often is Thiocapsa roseopersicina.
This has been reported for the North Sea barrier
islands of Mellum [3], Schiermonnikoog
[4], and
Texel [5], as well as for salt marshes in Cape Cod,
Massachusetts [6], and sandy beaches on the Orkney
Islands [7]. The type of aerobic sulfide-oxidizing
bacteria which dominate these systems is less well
documented.
Data collected on North Sea barrier
islands showed small motile thiobacilli to develop
profusely, whereas only a few Beggiatoa filaments
were found ([5], unpublished observations).
Contrary to Chromatium. Thiocapsa is an immobile organism. Cells are known to produce a slime
layer [8] and have been reported to cement sand
grains together thus forming small clumps [9,10]. In
mats developing on the barrier island of Schiermonnikoog aggregates up to 0.2 mm were observed
(unpublished).
In intertidal environments.
aggregate
formation is an advantage, since it effectively prevents the organism from being washed away with the
tidal currents [9,1 I]. A disadvantage, however, is that
immobile organisms cannot actively position themselves towards layers with optimal conditions. Microelectrode measurements have shown that strongly
fluctuating conditions occur at the depth horizons
where the organisms were found [4,12]. As a consequence. in these ecosystems the organisms were not
concentrated in a narrow band, but rather were distributed over a layer of several millimeters thickness
[4]. In these microbial mats purple sulfur bacteria
and colorless sulfur bacteria thrive in the same depth
layers and thus will compete for mutual substrates
under a wide range of conditions. However, data
collected on the depth distributions of these organisms unambiguously
show that their coexistence is a
common phenomenon.
Previously,
experiments
were carried out with
pure cultures of the obligate aerobic chemotrophic
sulfide oxidizer Thiobacillus thioparus TS and the
facultatively anaerobic phototrophic sulfide oxidizer
Thiocapsa roseopersicina
Ml. These studies have
shown that Thiobacillus has a higher affinity for
Ecalog~
19 llYY61
i-f-15/
thiosulfate [5], and the same is true for sulfide ([ 131,
M.T.J. van der Meer. unpublished
observations).
Therefore it was expected that in the presence of
oxygen
Thiocapsa
would be outcompeted
by
Thiobacillus. However, at oxygen-sulfide
interfaces
the activity of aerobic sulfide-oxidizing
bacteria is
restricted by the limited availability of oxygen. Sulfide oxidation particularly takes place at the oxygen
sulfide interface [12,14-161. For that reason it was
anticipated that oxygen limitation could play an important role in the interactions between chemotrophic
and phototrophic sulfide-oxidizing
bacteria. Oxygen
limitation experiments carried out with pure cultures
of Thiobacillus thioparus T5 have shown that this
organism is able to lower the concentration of sulfide
to undetectable levels, even when oxygen is severely
limiting [16]. However, under the latter conditions
sulfide is not fully oxidized to sulfate, thus yielding
reduced sulfur compounds other than sulfide, which
are potential electron donors for purple sulfur bacteria [16].
In this study, experiments were performed in continuous
culture with mixed populations
of the
chemotrophic sulfur bacterium T. thioparus T5 and
the phototrophic sulfur bacterium T. roseopersicina
MI. In these experiments
the rates of supply of
oxygen and sulfide were varied in order to obtain
different oxygen/sulfide
ratios. The experiments
were carried out to find a clue to the better understanding of the coexistence of colorless sulfur bacteria and purple sulfur bacteria in microbial mats.
2. Materials
and methods
2.1. Bacterial strains
Experiments
were performed with the colorless
sulfur bacterium Thiobacillus thioparus strain T5,
isolated from a marine microbial mat on the island of
Texel, The Netherlands [5], and the purple sulfur
bacterium Thiocapsa roseopersicina strain Ml, isolated from a marine microbial mat on the island of
Mellum, Germany [ 171.
2.2. Culture conditions
The organisms were grown in continuous culture
at a constant dilution rate of 0.05 h-’ with sulfide as
F.P. fan den Ende et al. / FEMS Microbiology
the only externally supplied electron donor, and oxygen and carbon dioxide as electron acceptor and
carbon source, respectively. The culture was continuously illuminated at an intensity of 40 PE m-’ s-‘.
The composition
of the mineral medium was as
described before [16], except for the sulfide concentration (see Table 2). The liquid culture volume was
1000 ml, with a headspace of 500 ml. The headspace
was flushed
with an adjustable
water-saturated
air/nitrogen
gas mixture at a rate of 40 1 hh’. The
medium was pumped from two reservoir bottles at
equal rates. One bottle contained a double-strength
solution of sulfide and carbonate at pH 11, the other
contained a double-strength
solution of the remaining constituents at pH 4.5. Temperature of the culture was kept constant at 25 f O.l”C, and the pH was
maintained at 8 f 0.04. Stirring speed and gas flow
rate were kept constant.
Thiobacillus was maintained in continuous culture, whereas Thiocapsa was routinely grown in
batch culture. To obtain a mixed culture, half the
volume of the Thiobacillus culture was removed and
replaced by the same volume of a growing batch
culture of Thiocupsa.
2.3. Oxygen /sulfide
ratios
Mixed cultures of Thiobacillus and Thiocapsa
were subjected to different oxygen to sulfide supply
ratios, and were sampled after a steady-state situation
had established.
By applying a fixed dilution rate and a constant
concentration
of sulfide in the reservoir solution
(SR_\u,fide), a constant sulfide supply rate was maintained. The oxygen supply rate was regulated by the
composition of the air/nitrogen
gas mixture in the
headspace. Oxygen consumption was calculated according to (G,o,s,rxru,,
- G,.cu,,u,,)
X K,a X
H, in which O,.,,,
M,XTt,REis the percentage of air
in the gas-mixture. 02_cuLTuRE is the oxygen concentration in the culture fluid as percentage of air saturation, K,a is the gas-transfer coefficient (in min- ’ ),
and H is the conversion factor from air saturation
(%) to oxygen concentration
(in PM). The oxygen
concentration
in the culture was determined with an
oxygen electrode (Ingold, Switzerland).
Since the
presence of Thiocapsa appeared to influence the
gas-transfer coefficient,
K,a was determined sepa-
Ecology
19 (1996) 141-151
143
rately after each steady state, according to Pirt [ 181.
H was determined using the Winkler titration [19].
2.4. Analytical
procedures
Sulfide was measured calorimetrically
using the
methylene-blue
method of Pachmayr [20]. Sulfate
was measured calorimetrically
as chloranilate
according to Bertolacini and Barney [21]. Zerovalent
sulfur and bacteriochlorophyll
a (BChla) were determined spectrophotometrically
in methanol extracts
[22]. Thiosulfate, tetrathionate and polysulfides were
measured calorimetrically
after cyanolysis
of 0.2
pm filtered samples [23,24]. Incubations
were as
described previously [16]. The same method was
used to check for the presence of non-pelletable
zerovalent sulfur, using 60 min incubation at 90°C at
pH 8.7. Protein was assayed with the Folin phenol
reagent [25] on cell pellets solubilized in 1 M NaOH,
after removal of zerovalent sulfur by methanol extraction. Glycogen was measured as total hexose
with the anthrone reagent [26]. Corrections for nonglycogen sugars derived from structural cell material
were made based on protein measurements. Conversion factors applied were 0.176 mg sugar mg- ’
protein for Thiocupsa cells [17], and 0.045 mg sugar
cells. Cells were
mgg ’ protein for Thiobacillus
counted using an electronic particle counter (Coulter
Counter, model ZM, equipped with a Chanalyzer
256). Control counts were done with a phase-contrast
microscope in a Biirker-Turk counting chamber for
Thiocapsa, and by epifluorescence
microscopy after
staining with DAPI 1271 for Thiobacillus.
2.5. Redox conversion factors
The following conversion factors
construct the redox balances: 8 mol
sulfide, 6 mol e- per mol zerovalent
mol e- per g cell-protein [28], 0.136
glycogen [29].
were used to
e- per mol
sulfur, 0.456
mol e- per g
3. Results
Attempts were made to co-culture Thiobacillus
thioparus T5 and Thiocapsa roseopersicina Ml at
four different oxygen/sulfide
ratios: three of them
resulted in stable coexistence of the two organisms.
I44
F.P. LYW den Ende et al./ FEMS Microbiology
Ecology 19 f 1996) 14/L151
Table 1
Rates of oxygen and sulfide supply applied to mixed cultures of Thiobncillus and Thiocnpsa grown at a dilution rate of 0.05 l-0.053
and the resulting steady state concentrations
of BChln, protein. glycogen. and the number of cells
Ratio
02/S’_
Supply rates
(FMmin-‘)
tmmol/mmolf
02
I .56
1.33
0.91
0.65
13.0
10.6
7.0
4.9
BChlcr
(/Lgl_‘)
Protein
(mg I-‘)
Glycogen
(mg I-‘)
0
166
625
1066
25.5
35.4
440
51.8
0
12.5
28.9
37.9
Supplying
the culture with 100% air in the
headspace, which allowed a maximum
attainable
oxygen supply rate of 16.6 Frnol min- ’ , in combination with a concentration of sulfide in the reservoir
solution of 9.7 mmol l-‘, which gave a sulfide
supply rate of 8.3 pmol min-‘, resulted in complete
wash out of the phototroph. Under these conditions.
the limiting factor for T~ziobacillus was sulfide
(steady-state concentration
< 1.5 PM), rather than
oxygen (steady-state concentration 47 PM). The observed rate of oxygen consumption
(13.0 pmol
min-‘)
was lower than the maximum supply rate,
whereas the sulfide uptake rate equalled the supply
rate, resulting in an oxygen/sulfide
ratio of 1.56
mm01 mmol- ’ (Table 1). Most of the sulfide supplied was oxidized to sulfate, a low concentration of
zerovalent sulfur was also encountered, whereas the
concentrations
of polysulfide
(SE-),
thiosulfate
(S,OiP) and tetrathionate (S,Oi-)
were even lower
(Table 2).
At oxygen/sulfide
ratios of 1.33 and lower, resulting in oxygen limitation rather than in sulfide
limitation,
stable coexistence
was observed. With
decreasing oxygen availability. population densities
Table 2
Steady state concentrations
of sulfur compounds
s’-
( PM)
( FM)
St(firno
1.56
1.33
0.91
0.65
9720
9060
8528
8453
<
<
<
<
6
4
2
2
The concentration
1.5
1.5
1.5
1.5
of sulfide in the in-flowing
S I-‘)
medium (S,)
Tiziobaciif~ts
Thioccqxw
12
10
3.8
3.1
0
I .5
2.1
3.3
of Thiobacillus decreased concomitant
with an increasing cell number of Thiocupsa and increasing
concentrations of BChla (Table 1). Under these conditions sulfur speciation changed from almost complete oxidation to sulfate to increasing concentrations
of zerovalent sulfur, reaching a maximum concentration of 1.9 mmol So 1-l at O,/H,S
of 0.65 mmol
mm01 - ’ (Table 21, but sulfate remained the main
product of sulfide oxidation. Concentrations
of sulfide in the cultures were below the detection limit at
all conditions. The recovery data show that no other
sulfur species were present (Table 2).
3. I. Su&r
balances
In order to facilitate a proper comparison between
data collected with slightly different sulfide concentration in the in-flowing medium, sulfur species were
calculated as a percentage of SR_\u,fide (Fig. 1, top
panel). Under sulfide limitation,
resulting in the
competitive
exclusion of Thiocupsa, 96% of the
in-flowing sulfide was oxidized to sulfate, and 4% to
zerovalent sulfur. With decreasing oxygen supply
rates product formation shifted to sulfur, which max-
in mixed cultures of Thiobacillus
Ratio
o?/sz~mmol/mmol)
sR
Cell number
(NX 10” 1-1)
S’8.3
8.0
1.5
1.5
h-' ,
and Thiocapsa at different oxygen/sulfide
supply ratios
s,of( /X)
s,o;( /.LM)
S”
(/*mol S I-‘)
so;( /AM)
S-recovery
(%ofSa)
15
6
9
9
9
5
10
10
348
500
2240
1930
9300
8700
6200
6500
100
102
100
101
and sulfur recovery
balance are also shown.
F.P. can den Ende et al./ FEM.7 Microbiology Ecology 19 (1996) 141-151
145
imally was 26% of SR_ru,fide,whereas sulfur present
in thiosulfate, polysulfide, and tetrathionate together
not exceeded 0.8% of SR__ifide (Fig. 1, top panel).
As shown in Table 1, decreased oxygen availability resulted in increased Thiocapsa cell numbers.
Microscopical
observation
of these co-cultures revealed that virtually all zerovalent sulfur was present
as intracellular sulfur in Thiocapsa (Fig. 2B). Extracellular sulfur globules were not observed, nor did
sulfur precipitate on the wall of the culture vessel, a
well-known nuisance in pure cultures of Thiobacillus. No visible extracellular sulfur was pelleted after
centrifugation of samples, and the negative results of
cyanolysis after 0.2 pm-membrane
filtration showed
that very small granules of sulfur were absent as
well. It thus appears that in co-cultures subjected to
1.5
1.5
1.4
1.4
1.3
1.3
1.2
1.1
1
0.9
0.8
1.2
1.1
1
0.9
0.8
/Sulfide (mmol / mmol)
0.7
0.7
RatioOxygen
Fig. 1. Effect of increasing oxygen limitation on speciation of
sulfur compounds formed during sulfide oxidation in mixed cultures of Thiobacillus and Thiocapsa (top panel). and pure cultures
of Thiobacillus (bottom panel). Values are calculated
from
steady-state concentrations
(Table 2 and [16]) and expressed as a
percentage of the sulfide concentration
in the in-flowing medium
to allow direct comparison. The degree of oxygen limitation is
quantified as the amount of oxygen supplied per sulfide supplied.
Fig. 2. Light micrographs
of sulfide oxidizing cultures grown
under oxygen limitation. (A). Pure culture of Thiobacillus. Amorphous clumps of extracellular zerovalent sulfur can be seen between cells. (B). Mixed culture of Thiobacillus and Thiocapsa.
Intracellular zerovalent sulfur can be seen as black globules inside
Thiocapsa cells, whereas extracellular zerovalent sulfur is absent
(bar = 10 Km).
oxygen limitation, Thiocupsa was growing, at least
in part, on sulfur species that resulted in intracellular
sulfur formation. The lower panel of Fig. 1 shows
data collected in pure cultures of Thiobacillus subjected to similar conditions. These data show that
with decreasing
oxygen
availability,
increasing
amounts of sulfide were not oxidized beyond the
level of zerovalent sulfur, which in this case was
exclusively present as extracellular sulfur (Fig. 2A).
Maximally 15-20% of SR_su,fidewas oxidized to thiosulfate. As was the case in the co-cultures, sulfide
was below the detection limit at all times. It remains
to be elucidated which sulfur species resulted in the
growth of and glycogen formation
when co-cultured with Thiobacillus.
by
Thiocapsa
3.2. Redox balances
When the oxygen supply rate was reduced to
values below 1.6 mol oxygen per mol sulfide, the
oxygen concentration
in the culture was below the
detection limit. Thus, all oxygen supplied was completely consumed. Under these conditions a stable
coexistence of Thiobacillus and Thiocapsa was observed. Like Thiobacillus, Thiocapsa can use oxygen
for sulfide oxidation [ 17,301. However, when the
specific content of BChla is sufficiently high. Thiocapsa will grow fully phototrophically
even in the
presence of oxygen [28]. At light saturation the
threshold value lies between 1.9 and 4 pug BChla
mg-’
protein [31,32]. Even if all protein in the
oxygen-limited
steady states were accounted to Thiocapsa, the BChl u/protein ratio would never be lower
than 4.7 ,ug mg-’ (Table l), thus indicating that
BChla concentrations
are high enough for full phototrophic growth of Thiocupsa. It thus could be
deduced that all oxygen was consumed by Thiobacillus.
In order to establish the fate of reducing equivalents in co-cultures of Thiobacillus and Thiocapsa,
redox balances were calculated (Fig. 3). The protein
assay does not discriminate between Thiocapsa and
Thiobacillus material. Cell numbers (Table 1) cannot
easily be transferred to biomass due to differences in
cell size (Fig. 4). Direct conversion of biovolume to
biomass is hampered by inaccuracy of volume determination of the small Thiobacillus cells. However,
the contribution of Thiobacillus to total protein can
be calculated from the measured oxygen consumption rates (Table 1). In the sulfide-limited
steady
states, Thiocapsa was washed out completely, and
the protein formed (25.5 mg 1-l > thus originated
exclusively from Thiobacillus. The oxygen consumption rate was 13.0 pmol mini ’ (780 pmol hh ’ ).
Dividing this figure by the dilution rate, the oxygen
consumption per liter culture medium (15.3 mmol O2
1-l > was obtained. Since four reducing equivalents
are needed to reduce molecular oxygen, the yield of
Thiobacillus was calculated to be 0.42 mg protein
per mol reducing equivalents transferred to oxygen
(25.5/(4 x 15.3)). P revious pure culture experiments
2 90
.k
60
1
”-
1.5
1.4
1.3
1.2
1.1
Ratio Oxygen /Sulfide
1
0.9
0.8
(mmol I mmol)
0.7
Fig. 3. Fate of reducing equivalents available from sulfide oxidation with increasing
oxygen limitation in mixed cultures of
Tbiobucillus and Thiocapsa. 100% (solid circles) represents full
oxidation of all sulfide supplied to sulfate. Upper panel: reducing
equivalents associated with Thiobacillus. Values are presented
cumulative for biomass and respiration. Solid squares represent
reducing equivalents used for respiration and biomass in pure
cultures of Thiobacillus [16]. Lower panel: reducing equivalents
associated with Thiocapsa. Values are presented cumulative for
biomass, glycogen and intracellular zerovalent sulfur. Solid squares
represent reducing equivalents in extracellular zerovalent sulfur
and thiosulfate in pure cultures of Thiobacillus [ 161.
with T. thioparus T5 under oxygen limitation have
shown that this yield is independent of the degree of
oxygen limitation [16]. In combination with the measured oxygen consumption rates, this yield allowed
for the calculation of the Thiobacillus protein in the
oxygen-limited
mixed cultures. The remainder of the
protein was attributed to Thiocapsa. Thiocapsa protein figures obtained in this way agreed well with
those based on the concentration of BChl a (Table I >
using the average BChla content in light-saturated
anoxic sulfide-limited
cultures of T. roseopersicina
Ml (21.4 pg BChla mgg’ protein) ([13], supplemented with unpublished BChla data). A conversion
F.P. mn den Ende et al./FEMS
Microbiology
factor of 0.456 mmol e- mgg ’ protein was applied
to calculate the amount of reducing power used for
biomass formation in Thiocupsa [28].
The results (Fig. 3) show that all reducing equivalents that could not be used by Thiobacillus due to
electron acceptor (oxygen) limitation are found associated with Thiocapsa. As shown in the top panel of
Fig. 3, most of the reducing equivalents that allow
for the synthesis of biopolymers
of Thiocapsa are
the result of reduced respiration rates of Thiobacillus
and to a far lesser extent the result of a lower
biomass of the colorless sulfur bacterium. The in-
CELL NUMBER
Ecology 19 (1996) 141-151
147
creased availability of reducing equivalents for Thiocapsa not only results in increased concentrations
of
structural cell material, but as well in increased
storage of elemental sulfur and glycogen, as is observed in other anoxygenic phototrophs [33].
Thiobacillus and Thiocapsa are sufficiently different in size to allow for electronic sizing. As shown in
the left panels of Fig. 4, a decrease in the cell
number of Thiobacillus with decreasing O,/H,S
ratios was accompanied by an increase in Thiocapsa
cell numbers. The average cell size of Thiobacillus
reduced with increasing oxygen limitation, whereas
::
;:...,
T
(N*lO%
:o;:;*s;i11;6
:::.
..,,
::::
::::
::
:
i
j
jj;;
:
:
: : : :
:
:
:
: ::::
; jjjj
jiiii
:
:
:.::::
:j::::
;
jjijii
j
j
;jj;
:
j
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:
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:
: : : :
::::
::::
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::::
::::
: :::
::::
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: :::::::
“’
:::
:::
:
::
::::
::::
\
::::
,I
; 02 HzS=0.65
:::
::.:
:
::;j: :: :::::
::::
::::
j ; ::j:;;
jjjj
::::
.::
:::::
::.:
:: : : : : : :
i : :::::
i:ji
; : :::,
j:ji
::::
: j ii;;
::::
__,,
jj::
_jibTr
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:::
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,,.
..:
:::
:::
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1
CELL SIZE (urn?
10
k
0.1
CELL SIZE f.um9)
Fig. 4. Cell number and biovolume of Thiobacillus and Thiocapsa in mixed cultures at different ratios of oxygen supplied per sulfide. Left
panels: distribution of cell numbers (N lo9 I- ‘) over 138 size classes. Data presented in left panels were used to calculate biovolume ( ~1
I- ’ ) shown in right panels.
148
F.P. ran den Ende et al./FEMS
Microbiology
Thiocupsa cell size increased. Taking the cellular
volume into account, the total biovolume of each
population in the co-cultures can be calculated (Fig.
4, right panels). Changes in the total Thiocupsa
biovolume
agreed with the calculated
biomass
changes (Fig. 3). Although the biovolume determination of Thiobacillus cells is somewhat less accurate
due to the small individual
cell size of these
chemotrophs, the data clearly show that the increase
in Thiocupsu biomass is much larger than the decrease in Thiobucillus biomass, again reflecting the
large proportion of reducing equivalents
used by
Thiobucillus for respiration.
4. Discussion
4.1. Competition
for sulfide
Thiobucillus and Thiocupsu
in
co-cultures
qf
Both Thiobucillus and Thiocapsa can oxidize sulfide. During steady-state growth in continuous culture the outcome of competition
for the limiting
substrate can be explained by the affinity of the
competing
organisms
to this substrate. Substrate
affinity during balanced growth is determined by the
initial slope of the Monod-equation
/.L,,,/( K, - s),
with s approaching zero. Thiobucillus has a maximum specific growth rate ( /_L,,,) on sulfide of 0.58
hh’, and a saturation constant (K,) of 2.35 PM
(M.T.J. van der Meer, unpublished),
resulting in an
affinity to sulfide of 0.247 hh’ pmol-’
1. Thiocupsu
has a pmax on sulfide of 0.09 hP ‘, and a K, of 8.0
PM [ 131, resulting in an affinity to sulfide of 0.011
hh ’ pmoll’
1. Thiobucillus
thus has a 20-fold
higher affinity for sulfide than Thiocupsn. It is therefore not surprising that Thiobucillus completely outcompeted Thiocupsa under sulfide limiting conditions (O,/H,S
= 1.56, Fig. 4).
Under oxygen limiting conditions a stable coexistence of Thiobacillus and Thiocupsa developed. Sulfide was consumed completely in these mixed cultures (Table 2). Since the affinity for sulfide of
Thiobucillus under oxygen limitation is not exactly
known, a direct comparison of affinities as in the
case of sulfide limitation is difficult. However, sufficient data are available from pure culture studies to
allow an interpretation
of the fate of sulfide in the
Ecology 19 (1996) 141-151
mixed cultures. In sulfide grown pure cultures of
Thiobacillus subjected to oxygen limitation virtually
all sulfide was consumed; however, part of the sulfide was oxidized to sulfur species less oxidized than
sulfate [ 161. Steady-state sulfide concentrations
were
below 1 PM at oxygen/sulfide
ratios exceeding 0.6
mmol mmol- ’ [ 161. In sulfide-limited.
phototrophitally growing cultures of Thiocupsa the steady-state
sulfide concentration
at a dilution rate of 0.05 h-’
was 10 PM [ 131. The concentration
of sulfide was
below the detection limit (1.5 PM) in all steady-states
of the mixed cultures (Table 2). Substituting
pmax
and K, in the Monod-equation
with the values for
Thiocupsu mentioned earlier. it can be calculated
that with a sulfide concentration
of 1.5 PM Thiocupsa maximally can attain a growth rate of 0.0014
hh’, which is insufficient to maintain itself in the
cultures at the dilution rate of 0.05 hh ’ used here.
For kinetic reasons it is therefore expected that in
mixed cultures Thiobucillus was not hindered in the
use of sulfide as a substrate by the presence of
Thiocupsu. Apparently, the observed population of
Thiocupsu is to be explained by the utilization of
other sources of reducing equivalents than sulfide.
However, given its biomass, Thiocupsu will use
some sulfide, especially at strong oxygen limitation.
4.2. Substrates
Thiobucillus
used by Thiocupsu in co-cultures
with
Considering the conclusion that Thiobucillus used
most of the sulfide supplied, it appears that the
Thiocupsu biomass is the result of the consumption
of partial oxidation products formed by Thiobacillus.
If Thiobucillus would form the same products in
mixed cultures as in pure cultures a small population
of Thiocupsu could be expected to develop as the
result of thiosulfate oxidation, and possibly by oxidizing part of the zerovalent sulfur formed. Instead, a
substantial population of Thiocupsu was found, and
most of the sulfide supplied was found to be completely oxidized to sulfate (Fig. 1). As in pure cultures of Thiobucillus, zerovalent sulfur was present
in the mixed culture, however in this case exclusively as intracellular reserve in Thiocupsu. Judged
from the absence of extracellular sulfur and thiosulfate in the mixed culture (Fig. 1, top panel), Thiocups~7 was apparently
able to use all of the remaining
F.P. can den Ende et al./ FEMS Microbiology Ecology 19 (1996) 141-151
reducing equivalents
that could not be used by
Thiobacillus due to shortage of oxygen (Fig. 3). The
utilization of these product(s) by Thiocapsa predominantly resulted in the formation of biomass and
glycogen, but intracellular sulfur was present as well
(Fig. 1, top panel).
In pure cultures of Thiobacillus extracellular zerovalent sulfur was the main product of sulfide oxidation under oxygen limitation. This sulfur was present as globules associated with individual Thiobacillus cells, after some time resulting in large detached
aggregates (Fig. 2A). In mixed cultures of Thiobacillus and Thiocapsa extracellular
zerovalent
sulfur
globules only were observed transiently after reduction of the rate of supply of oxygen and not during
steady states (Fig. 2B). T. roseopersicina is known
to be able to grow on particulate S” [8], however,
growth on this substrate is extremely slow and, more
importantly, does not result in storage of intracellular
sulfur. It thus can be ruled out that Thiocapsa was
using particulate S” as electron donor. In addition, if
formed in mixed cultures, at least some sulfur particles should have been visible, or, when too small to
observe microscopically,
a measurable amount should
have been found in the culture fluid. Since this was
not the case it can be concluded that Thiobacillus in
oxygen-limited
steady states of mixed cultures
formed a soluble reduced sulfur compound, rather
than particulate S”. The nature of this soluble product, formed by Thiobacillus and consecutively
used
by Thiocapsa, remains unknown. In view of their
role as precursor in the formation of elemental sulfur, polysulfides
(H-S,-H)
and thionates (H-S,SO,) are possible candidates (R. Steudel, personal
communication).
A schematic representation
of the
interactions is given in Fig. 5.
4.3. Why is Thiocapsa
bial mats?
successful
in intertidal micro-
Thiocapsa is an immobile organism. Its depth
distribution in a marine microbial mat was studied in
detail by De Wit et al. [4] where it was reported to
occur from 1.5 to 5 mm depth, with 85% of the
population concentrated between 2 and 3 mm depth.
At night oxygen penetration in the sediment was less
than 1 mm. However, when surface irradiance exceeded 200 PE mm2 s- ’ (one hour after sunrise to
149
Fig. 5. Schematic representation
of the interactions
between
Thiobacillus and Thiocapsa under oxygen limitation. See text for
details.
one hour before sunset), oxygen was detected down
to 3-3.5 mm depth. Thus, the majority of the population was facing the presence of oxygen during
most of day, whereas anoxic conditions were mostly
prevailing at night.
Regarding a 24-h period, Thiocapsa is hindered in
one way or another for most of the time. During the
night the uptake of substrates is not feasible. In the
presence of oxygen (daytime) dissimilatory
sulfate
reduction continues,
although at lower rates than
under fully anoxic conditions [35,36]. However, little
of the sulfide produced can be expected to reach the
anoxygenic phototrophs because of their low affinity
compared to that of colorless sulfur bacteria [5,37,38].
This competitive disadvantage is not eliminated before anoxia coincides with the availability of light. In
the intertidal microbial mats studied on the North
Sea barrier islands, such favorable conditions occur
only for short periods just after sunrise and just
before sunset [4,12]. During these periods Thiocapsa
takes full advantage of its hoarding capacity, i.e.
sulfide is oxidized no further than sulfur, which is
stored inside the cells, and CO, fixation results in
the intracellular deposition of glycogen rather than in
the formation of structural biomass. With respect to
the utilization of small organic compounds (i.e. acetate) it is anticipated that anoxygenic phototrophs
are unable to compete successfully with aerobic heterotrophs.
150
F.P. mvz den Ende et al./ FEMS Microbiology
Although during the oxic light period growth of
T&~upsa
on external substrates is restricted it has
the capacity to grow phototrophically
in the presence
of oxygen using intracellularly
stored zerovalent sulfur as electron donor [5]. During the anoxic dark
conditions it has the ability to synthesize BChlcr and
to grow at the expense of stored glycogen [34]. Thus
for most of the day growth of Thiocapsa depends on
intracellular storage compounds rather than external
supply of reducing power. In order to be able to use
these capacities it is fully dependable on the short
favorable periods at sunrise and sunset to replenish
the stock of zerovalent sulfur and glycogen. However, the present experiments
show that not only
anoxic light periods but also the periods of oxygen
limitation can be used to this end. Thus, the period
that can be used to load the cells with reserve
materials is extended. These observations
help to
understand
the blooming of Thiocapsa in marine
microbial mats.
Acknowledgements
The authors would like to thank Bart E.M. Schaub
for cell volume measurements.
This study was carried out as part of the EC MAST II project “Oxicanoxic interfaces as productive sites” (contract no.
MAST2-CT-93-0058).
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