Limnol. Oceanogr., 30(5), 1985, 932-943
0 1985, by the American Society of Limnology and Oceanography, Inc.
Diel cycle of metabolism
in Lake Cisb (Spain)’
of phototrophic
purple sulfur bacteria
Hans van Gemerden
Department of Microbiology, University of Groningen,
Kerklaan 30, NL-975 1 NN Haren, The Netherlands
Emilio Montesinos, Jordi Mas, and Ricardo Guerrero
Department of Microbiology and Institute for Fundamental Biology,
Autonomous University of Barcelona, Bellaterra (Barcelona), Spain
Abstract
During a bloom of purple sulfur bacteria in Lake Cis6 (Spain) data were collected on diel changes
in the concentrations of sulfide, sulfur, glycogen, and poly-@-hydroxybutyrate (PHB), the rates of
CO, fixation and H2S oxidation, and the viability of cells along the vertical profile. During the
day, sulfide oxidation resulted in the intracellular accumulation of sulfur and glycogen. At night,
the concentrations of glycogen and sulfur decreased concomitant with the production of sulfide
and PHB. Throughout the day, CO, fixation and H2S oxidation were maximal in the zone of
maximal population density (peak of the layer); however, in the top of the layer the specific rates
were 2-6 times higher. The ratio of CO, fixation to H2S oxidation, the sulfur content of the cells,
and their specific density all indicated that sulfide was predominantly oxidized to sulfur. In the
top and the peak of the layer cell viability was high but decreased rapidly with increasing depth.
It was concluded that the existence of the bloom is the combined result of slow growth at the peak
of the layer and a much faster rate of reproduction at the top of the layer. The cells at the top
stored sulfur extensively and then sank to deeper layers where they could not oxidize the sulfur
because of insufficient light.
In stratified lakes with anaerobic bottom
waters, purple and green sulfur bacteria can
develop profusely, resulting in high productivities
(see Biebl and Pfennig 1979;
Wetzel 1983). Blooms facilitate direct measurements of the activities of these phototrophic bacteria, and such data can then be
interpreted on the basis of observations in
pure cultures. The distribution
of phototrophic bacteria usually varies strongly with
depth. Often defined layers (so-called plates)
are observed, due to opposing gradients of
sulfide and light. Other important environmental parameters are the oxygen concentration, the availability
of small organic
molecules like acetate, and of course, the
presence of sufficiently high concentrations
of macronutrients.
The present study was conducted primarily in Lake Ciso; a few additional data
have been collected in nearby Lake Vilar.
Both lakes are in the karstic Banyoles area
l This research was supported by the Comision Asesora de Investigacidn Cientifica y Tecnica, Spain, and
by the Research Program of the City Council of the
town of Banyoles.
in northern Spain. Their morphometry and
the chemical composition of the lake water
have been described elsewhere (Guerrero et
al. 1980, 1985).
In Lake Ciso, the dominant phototrophic
bacteria were Chromatium spp. and a small
aggregate-forming coccoid organism designated as M-3 (Guerrero et al. 1985), both
purple sulfur bacteria (Chromatiaceae).
Green and brown sulfur bacteria belonging
to the Chlorobiaceae were also present but
contribute only about 4% to the total biomass. Near the surface of the lake algae were
present. There were no cyanobacteria. In the
layer of Chromatiaceae there were few eucaryotic phototrophs, probably only sedimented physiologically inactive individuals
since the light intensity in the plate (< 1
PEinst m-2 s-l) was far too low to support
algal photosynthesis. In contrast to that at
the surface, 14C02 fixation in the bacterial
layer was not inhibited by the addition of
DCMU (Guerrero et al. 1985). The bloom
was not limited by sulfide nor by acetate.
Although concentrations of phosphate in the
lake were low (Guerrero et al. 1980), they
also were not rate-limiting.
It was shown
932
Metabolism of Chromatiaceae
that light is the limiting factor for the development of the phototrophic bacteria in
Lake Cis6 (Guerrero et al. 1985).
The aim of the present study was to estimate the activities of the anoxygenic phototrophic bacterial community
over a full
diel cycle. Sorokin (1970) reported increased concentrations of sulfide at night
and a decrease during daytime for a migrating bloom of Chromatium okenii (Pfennig 1978) in Lake Belovod (USSR) and
showed the importance
of phototrophic
bacteria as prey for planktonic predators (see
van Gemerden and Beeftink 1983). Jorgensen et al. (1979) discussed the role of oxygenic and anoxygenic phototrophic bacteria
(cyanobacteria and Chromatiaceae) in the
sulfur cycle of Solar Lake (Sinai). Parkin and
Brock (198 1) studied the activities of a
bloom of green sulfur bacteria in Knaack
Lake (USA).
We present data here on the distribution
in time and space of sulfide, sulfur, glycogen, and poly-fl-hydroxybutyrate
(PHB),
rates of sulfide oxidation, CO2 fixation, and
acetate incorporation,
and the viability of
the dominant purple sulfur bacteria in Lake
Ciso. The overall metabolism of the organisms was calculated for the early morning,
noon, late afternoon, and night, and the data
are compared with laboratory studies of
similar organisms.
We thank C. Pedros-Alio, I. Esteve, P.
Hofman, and J. Bakker for their contributions to the present work and all of the crew
for maintaining good spirits.
Materials and methods
The methods of sampling, and the analyses of sulfide, elemental sulfur, glycogen,
PHB, acridine
orange direct counting
(AODC), electronic sizing, the estimation
of rates of carbon dioxide fixation and sulfide oxidation have been described elsewhere (Guerrero et al. 1985).
Total biovolumes
were calculated by
multiplying
the cell numbers (AODC) by
the specific volume of the organisms (electronic sizing) (Zimmerman and Meyer-Reil
1974; Montesinos et al. 1983).
Often the cells contained a lot of elemental sulfur. Because sulfur is denser than
structural cell material, a correction for sul-
933
fur must be applied in calculating buoyant
density (Bakken and Olsen 1983). The density of elemental sulfur is about 2 g cm3
(Handbook of chemistry and physics 1974).
However, the sulfur globules inside the cells
of Chromatium spp. apparently do not consist of pure sulfur but of a diluted form,
previously described as hydrated, with a
density estimated to be 1.22 g cm-3 (Guerrero et al. 1984). We converted the volume
of structural cell material (i.e. total biovolume minus the volume of the sulfur
globules) to structural biomass by multiplying by 1.07 g cm- 3. For various bacterial
strains specific densities ranging from 1.04
to 1.09 g crnd3 have been reported (Bakken
and Olsen 1983; Doetsch and Cook 1973).
Guerrero et al. (1984) found the density of
Chromatium warmingii devoid of elemental sulfur to be 1.07 g cm-3.
The total biomass of the population was
the structural biomass plus the weight of
elemental sulfur. Finally, the average specific density was calculated as the quotient
of total biomass and total biovolume.
The cellular content of sulfur is expressed
on a wet weight basis.
Because of the low population density of
Chlorobiaceae, the percentage of sulfur in
the wet weight and the specific density of
the cells can be taken to reflect the average
values for Chromatium spp. and the yet unidentified M-3.
The viability of Chromatiaceae was estimated by the method of van Gemerden
(1980), modified for natural samples as follows. For each depth to be analyzed, two
screwcapped glass tubes were completely
filled with lake water, with a pea-sized air
bubble left to meet pressure changes. Samples with low initial sulfide concentrations
were diluted 1: 1 with 0.2-pm membranefiltered bottom water containing 0.7 mM
sulfide. Both tubes were incubated in dim
sunlight to ensure that all cells capable of
intracellular
sulfur storage would contain
microscopically
visible sulfur globules. After 1-2 h, tube 1 was placed on ice in the
dark. Tube 2 remained in the light for an
extended period to ensure that all cells capable of growth (i.e. to oxidize their internal
sulfur to sulfate) had done so. In the laboratory, all tubes were analyzed for the pro-
934
van Gemerden et al.
portion of cells containing sulfur globules
(fi, $2) and the concentration of cells (N1,
N2, estimated as cell number or BChl concentration). In the initial l-2-h incubation
period, growth is negligible and the concentration of cells in tube 1 thus reflects the
situation in the lake (diluted samples are
multiplied accordingly). Cells in tube 1 devoid of intracellular sulfur globules are considered nonviable; their absolute number
equals N1 X (1 - fi) ml-l.
Cells still containing
sulfur after prolonged incubation in the light are also considered nonviable. Their absolute number
equals N2 X fi ml-‘. The assumption is
made that no death occurs during the prolonged incubation in the light. Viability is
then calculated according to
In the laboratory, with known mixtures
of viable and nonviable cells of Chromatium vinosum, very few cells die during the
second incubation period. We assume that
cells from the lake behave similarly; if not,
our viability values are underestimates. The
fact that viabilities close to 100% have been
found may therefore serve as circumstantial
evidence that the method is valid.
Results
Dieljluctuations in the concentrations of
su@de, sulfur, glycogen, and PHB -Samples for estimating different parameters were
taken at 3-h intervals for 30 h at 1.25, 1.5,
1.75,2,2.25, 2.5, 3,4, and 5 m. There were
no pronounced fluctuations at 3, 4, and 5
m: the concentration
of sulfide at these
depths remained about 0.7 mmol liter-l (24
mg liter- l) throughout the sampling period.
The concentration of sulfur at 3 m increased
slightly during the day, from 0.02 to 0.05
mmol liter-l ; concentrations at 4 and 5 m
were constant. Neither sulfide nor sulfur was
detected at any time at 1.5 m or above; at
1.75 m the highest concentration of sulfide
observed was 0.005 mmol liter-’ (0.17 mg
liter-‘); sulfur concentrations at these depths
were below the limits of detection.
The diel cycles of sulfide and sulfur at
depths of 2, 2.5, and 3 m are shown in Fig.
1. At 2 and 2.5 m the concentration of sulfide decreased by day and increased at night;
the reverse was observed for sulfur. At 3 m
no significant changes were observed in
either sulfide or sulfur. The method used to
estimate elemental sulfur does not differentiate between intracellular sulfur as found
in Chromatiaceae and extracellular sulfur as
produced by Chlorobiaceae. The simultaneous presence of sulfide and elemental sulfur may result in the abiotic formation of
polysulfides. On the basis of pigment analysis and size frequency, the contribution
of
Chlorobium sp. was 5% (Guerrero et al.
1985). The dominant anoxygenic phototrophic bacteria in the lake are Chromatium
spp. and M-3. Their contribution
to the
phototrophic community at 2 m was 7 1 and
20%, and in the top and the peak of the
layer virtually all cells are viable. Thus the
greatest part of the elemental sulfur is intracellular, and therefore the fluctuations
observed reflect the combined activities of
Chromatium spp. and M-3 and not an
abiotically induced shift (e.g. between sulfur
and polysulfides).
The diel fluctuations in the concentrations of glycogen and PHB at 2 and 2.5 m
are shown in Fig. 2. The concentration of
glycogen increases by day and decreases at
night, with the reverse for PHB. No such
changes were observed at 3, 4, and 5 m. As
for the changes in the concentration of sulfur, these fluctuations can be taken to reflect
the activities of the Chromatiaceae.
Dielfluctuations in the rates of sulJide oxidation and carbon dioxide fixation -The
diel fluctuations observed at a given depth
cannot be attributed only to the activities
of the phototrophic bacteria at that depth,
due to exchange phenomena between water
masses. This is particularly relevant in lakes
with very steep gradients of sulfide and light.
We therefore estimated the rates of sulfide
oxidation and CO2 fixation in isolated samples. Because the organisms were not at all
evenly distributed over the water column
(Guerrero et al. 1985), the specific activity
(i.e. activity per unit biomass) has to be calculated to determine the time and depth at
which the organisms are most active.
Phototrophic
sulfur bacteria are not the
only organisms that can affect the concen-
Metabolism of Chromatiaceae
0.80
I---
1 ’ I-
l ULFIDE
GULFUR
0.60
6
12 18 24 6
6
12 18 24 6
6
12 18 24 6
TIME ( hours 1
Fin. 1. Diel fluctuations in the concentrations of sulfide and sulfur in Lake Cis6 on 6-7 July 1982. a-2 m;
b-55 m; c-3 m.
tration of sulfide. Sulfide oxidation in the
light by cyanobacteria has been reported under natural conditions (Cohen et al. 1975;
Jorgensen et al. 1979). However, in Lake
Ciso the oxidation of sulfide could bc virtually exclusively attributed to the Chromatiaceae. The concentration
of Chl a at
depths where anoxygenic phototrophs were
found was very low; but, more important,
the Chl a came from algae rather than cyanobacteria. Specific pigments of cyanobacteria (i.e. phycobiliproteins)
could not be
detected by either in vivo absorption spectra or thin-layer chromatography (Guerrero
et al. 1985).
The algae present with the bloom of phototrophic bacteria were inactive or may even
have been dead. A year later, in July 1983,
during a similar bloom of phototrophic bacteria in Lake Ciso, the addition of DCMU
did not significantly reduce the rate of 14COZ
fixation in samples taken from the bacterial
bloom, but did in samples taken from the
algal bloom at 1 m (Guerrero et al. 1985).
We did not try to evaluate the number or
activities of colorless sulfide-oxidizing
bacteria (Thiobacillus-like organisms) or sulfide-producing
bacteria (Desulfovibrio- or
Desulfuromonas-like bacteria). The combined activities of these groups were estimated in dark bottles: in all cases the production of sulfide exceeded its oxidation. At
all depths the production of sulfide did not
change significantly over a full diel cycle.
The maximal rate of sulfide production, at
2 m was 9.7 pmol liter-l h-l, at 2.5 m the
rate was 6.7, but rapidly decreased below
that to 0.5 at 5 m. Interestingly, the net rate
of sulfide production at 1.75 m was 2.7 pmol
liter-l h-l ; no oxygen is present at that
depth.
At 2 m the concentration of sulfide was
high enough to permit direct estimation of
the specific rate of sulfide oxidation. Such
an approach could not be followed for organisms at 1.75 m and above. At these
depths, sulfide had to be added to prevent
depletion during the incubation. To facilitate comparison between the data collected
with and without the addition of sulfide, we
treated samples from 2 m in both ways and
found that the addition of sulfide from a
concentration of 0.2 mmol liter-’ to 0.8,
resulted in a decrease in the rate of sulfide
oxidation from 47 ,umol liter- 1 h- l to 16.
This points to severe inhibition
by sulfide
93.6
van Gemerden et al.
r
,
I
1
,
I
, , ,
I .
W
m
12
E
&
8
6
12
18
24
6
6
12
18
24
-
6
TIME (hours)
Fig. 2. As Fig. 1, but of intracellular glycogen and PHB. a-2 m; b-2.5 m.
and indicates that the data obtained with
the addition method are to be interpreted
as underestimates. The production of sulfide in the dark was not affected by the addition of sulfide.
The diel cycle of the total rates of lightdependent sulfide oxidation and CO, fixation observed in the 2-m layer is shown in
Fig. 3b. Both the oxidation of sulfide and
the assimilation of CO2 show maxima in
the middle of the day, being 47 and 2 1 pmol
liter- l h- I . Similar measurements were
made at 1.75, 2.25, 2.5, and 3 m; data for
1.75 m (sulfide only) and 2.5 m are shown
in Fig. 3a and c. The diel fluctuations at
these depths are comparable to those in the
2-m layer, but less pronounced.
Nevertheless, the bacteria at 1.7 5 m seem
to be the most active. The assimilation of
CO, and the oxidation of HZS in the samples
from 1.7 5 m result from the activities of far
fewer organisms than at 2 m. Biomasses at
1.75,2, and 2.5 m were 0.77,7.58, and 4.02
mg wet wt liter- I. The maximal specific rates
of sulfide oxidation at a depth of 2 and 2.5
m were 6.2 and 4.0 pmol mg-l wet wt h-l,
whereas that at 1.7 5 m was 13.3. The data
from 1.75 m were obtained after sulfide had
been added to the samples. Assuming that
the cells are inhibited by sulfide to the same
extent as those at 2 m, the specific rate of
sulfide oxidation at 1.75 m might even be
six times that at 2 m. However, the rates
obtained with the addition method reflect
the potential rate; it is not certain that
enough sulfide is supplied to these cellseither by means of replenishment
from
deeper layers or by production of sulfide in
situ-to
enable them to realize their potential rate of sulfide oxidation.
Purple sulfur bacteria can assimilate some
organic compounds of low molecular weight,
such as acetate. Conceivably-the
uptake of
acetate in situ also reflects the activity of
the organisms, but the situation is not entirely clear. We tried to assay the acetate
concentration in the water as well as to estimate the rate of incorporation
of 3H-labeled acetate. The presence of acetate could
be demonstrated in the deeper parts of the
lake but not at the depth of the bloom. This
might suggest an active assimilation of acetate by the phototrophic bacteria; however,
the incorporation of labeled acetate did not
show this. Tentatively, we conclude that the
growth of the phototrophic
sulfur bacteria
in Lake Cis6 is predominantly
by CO2 fixation and H,S oxidation.
Metabolism of Chromatiaceae
6
12 18 24
6
I
12 18 24
6
6
12 18 24 ’
TIME (hours)
Fig. 3. Diel fluctuations in the total rates of sulfide oxidation and carbon dioxide fixation in Lake Cis6 on
6-7 July 1982. a- 1.75 m (after addition of sulfide, see text); b-2 m; c-2.5 m.
The fixation of CO2 and
H2S are stoichiometrically
can be partially oxidized to
or fully oxidized to sulfate,
1 and 3.
CO, + 2H2S + (CH20)
the oxidation of
linked. Sulfide
elemental sulfur
as shown in Eq.
+ 2s + H,O; (1)
3COZ + 2s + 5H20 --f 3(CH20)
+ 2H,SO,;
4C02 + 2H2S + 4H20 --f 4(CH20)
+ 2H,S04.
(2)
(3)
The ratio between COZ fixation and H2S
oxidation is 0.5 in reaction 1, but 2.0 in
reaction 3. The fact that cell material is
somewhat more reduced than (CH,O) has
little effect on the ratio in Eq. 1 and 3. If we
adopt the overall composition of (CSHs02N)
for C. vinosum (van Gemerden 1968a), the
ratio between COZ fixation and HZS oxidation is 0.48 in reaction 1 and 1.90 in reaction 3. This ratio in lake water can thus
be used to assess the extent to which sulfide
is oxidized to sulfur or sulfate. For the 2-m
layer, the ratio was 0.44 pmol CO2 pmol-l
H,S in the middle of the day and appeared
to be constant throughout the light period
(Fig. 3b). For the layer at 2.5 m the ratio
was 0.26 pmol CO2 pmol-l HZS (Fig. 3~).
These observations suggest that sulfide is
oxidized in the bloom to elemental sulfur
rather than to sulfate. We did not determine
sulfate as extensively as sulfide or sulfur; the
demonstration of small fluctuations is hampered by the high background concentration, about 10 mmol liter- I.
In nearby Lake Vilar, the peak of the layer
of phototrophic
bacteria was at 4.2 m
(Guerrero et al. 198 5). As in Lake Ciso, the
maximal rates of carbon dioxide fixation
and sulfide oxidation were found at the depth
of maximal population density, being 11.2
and 30.0 pmol liter-’ h-l. However, the
specific rate of sulfide oxidation at 4.1 m
was almost nine times that at 4.2 m, and
the specific rate of CO, fixation at 4.1 m
was eight times that at the depth of maximal
population density. The ratios of CO2 fixation to HzS oxidation at 4.1, 4.2 and 4.3
m were 0.43, 0.34, and 0.53 bmol CO,
PrnoP H2S. Thus, in Lake Vilar-as in Lake
Cisd-the
bacteria were most active at the
upper part of the plate, and grow only by
the oxidation of sulfide to sulfur.
938
van Gemerden et al.
Table 1. Sulfur content (% wt/wet w-t) and specific
density (mg mm-3) of Chromatiaceae in Lake Cis6
samples collected on 6-7 July 1982.
Depth
(m)
Time
(hours)
1.75
0600
1200
1800
2400
0600
1200
1800
2400
0900
0600
1200
1800
2400
0900
0600
1200
1800
2400
0900
0600
1200
1800
2400
0600
1200
1800
2400
0900
2
2.5
3
4
5
Sulfur
33.6
46.3
43.0
24.5
27.3
41.6
29.8
37.9
25.9
28.8
26.0
25.1
31.7
46.1
32.1
18.1
39.7
27.4
16.5
0
13.7
24.0
10.8
8.8
(5:::)
9.4
19.2
Specific
density
1.14
1.18
1.14
1.11
1.11
1.14
1.12
1.13
1.11
1.11
1.11
1.11
1.12
1.15
1.12
1.10
1.13
1.11
1.09
1.07
1.09
1.11
1.09
1.08
1.08
(1.17)
1.08
1.10
Specific density of purple sulfur bacteria
in relation to the content of intracellular elemental sulfur -The specific density of cells
devoid of elemental sulfur is 1.07 g cm-3;
the specific density of the sulfur globules is
1.22 g cm-3 (Guerrero et al. 1984; see materials and methods). Consequently, the accumulation
of sulfur inside the cells increases their specific density, which in turn
affects their rate of sinking. Motile organisms may be able to compensate for this if
they have a suitable source of energy. For
the phototrophic bacteria in Lake Ciso experiencing light limitation
(Guerrero et al.
198 5), the increased rate of sinking may be
fatal. The content of intracellular sulfur in
Chromatiaceae is inversely related to the
extent to which sulfide is oxidized to sulfate,
as a consequence of the fact that the oxidation of sulfide is stoichiometrically
linked
to the reduction of CO2 (Eq. 1, 2, and 3).
The complete oxidation of sulfide to sulfate
will result in no sulfur in the cells, which
then have a specific density of 1.07 g cm-3.
If all sulfide is oxidized to sulfur and none
to sulfate (i.e. Eq. 1 only), the maximal sulfur content is reached. Based on a water
content of 80% (Rose 1976), 150 mg of wet
structural cell material is produced in the
oxidation of 2 mmol of sulfide to sulfur (Eq.
1). These cells contain 2 mmol of sulfur (i.e.
64 mg). The sulfur will then be 30% of the
wet weight and cannot be higher on theoretical grounds. The volume of structural
cell material produced in the oxidation of
2 mmol sulfide to sulfur will be 140 mm3
and that of the sulfur globules 52 mm3, resulting in a total volume of 192 mm3. The
maximal specific density of the cells is thus
1.11 mg mm3, (150 + 64):(140 + 52). If
we assume that the average elemental composition of the Chromatiaceae in Lake Ciso
is like that of C. vinosum, i.e. CSHs02N
(van Gemerden 1968a) rather than CH20,
the theoretically maximal percentage of sulfur in the wet weight is 37%, and the maximal specific density of the cells is 1.12 mg
mrne3. The values for Lake Ciso are given
in Table 1.
The average content of sulfur in the samples from 2 and 2.5 m was 32% (wt/wet wt),
resulting in a specific density of 1.12 mg
mme3. With increasing depth the content
of sulfur decreased, to about 10% (wt/wet
wt) at 5 m. Values for the samples from 1.7 5
m appear to be somewhat higher (average
37%) but are less accurate due to the much
lower population
density at that depth.
These data again indicate that the extent of
oxidation of sulfur to sulfate (Eq. 2) is extremely low in Lake Ciso. Since growth on
acetate or other organic substrates tends to
lower the intracellular content of sulfur, the
data indicate not only that little sulfur is
oxidized to sulfate, but also that the extent
to which the cells grow on such organic substrates is marginal. The latter observation
is in agreement with observations
(not
shown) on the incorporation of labeled acetate.
The data for Lake Vilar on the percentage
of sulfur in the wet weight and the density
of cells point in the same direction as those
for Lake Ciso, but are somewhat more scat-
Metabolism of Chromatiaceae
tered. This may be in part because in Lake
Vilar biomass and sulfur assays were performed on different samples, but it also illustrates the technical difficulties in the accurate sampling of such sharp gradients. For
example, in Lake Vilar the biovolumes at
4.1, 4.2, and 4.3 m were 4.3, 31.9, and 2.7
mm3 liter-l. Sampling devices that enable
accurate sampling at l-cm intervals are under construction.
Viability of purple sulfur bacteria across
view of the lower
the vertical projle-In
specific activities observed in samples from
deeper layers, it was of interest to estimate
the viability of the bacteria across the vertical profile. This we did in summer 1983.
The situation in the lake was similar to that
in 1982 except that the layer of maximal
population density was at 2.75 m and the
population of M-3 was slightly higher and
that of Chromatium spp. somewhat lower.
No significant differences were observed in
the viabilities of the different purple sulfur
bacteria. Viability was high, up to lOO%, at
the top and the peak of the layer (2.5 to 3.25
m); below this viabilities dropped to X5%
at 7 m.
Discussion
The oxidation of sulfide to sulfur, sulfate,
and possibly other intermediates is linked
to the reduction of carbon dioxide and may
result in any combination of the following
phenomena: growth, here defined as an increase in structural cell material; synthesis
of glycogen; and excretion of organic compounds. The assimilation of organic molecules of low molecular weight (with acetate
the most likely) could also result in growth
or excretion, but possibly as well in the synthesis of PHB. However, the assimilation
of acetate appears to be of minor importance in Lake Ciso. A comparison of the
rates of sulfide oxidation and CO2 incorporation shows that the product of sulfide
oxidation must have been primarily
elemental sulfur (see Fig. 3). The content of
sulfur in the fresh weight points in the same
direction (Table 1).
On the basis of the ratio between carbon
dioxide fixation and sulfide oxidation, Parkin and Brock (198 1) concluded that sulfide
was completely oxidized to sulfate in a
939
bloom of green sulfur bacteria in meromictic Knaack Lake. The dominant
phototrophic bacteria were species of Pelodictyon
and Clathrochloris.
Chlorobiaceae
are
known to deposit sulfur outside the cells;
however, this remains somehow attached to
the cells (Beeftink et al. unpubl.) and will
therefore affect their buoyant density. Although the organisms in Knaack Lake have
gas vacuoles which reduce their rate of sinking (Clark and Walsby 1978), it is clear that
the absence of extracellular elemental sulfur
attached to the cells is an additional advantage for these nonmotile organisms. In general, green and brown sulfur bacteria (Chlorobiaceae) have low light requirements; their
maintenance rate coefficient (p,) is about
0.001 h-l, compared to the 0.010 h-l generally found for purple sulfur bacteria (van
Gemerden 1980). The light intensities at the
depth of the bloom in Knaack Lake, used
to measure sulfide oxidation by the Chlorobiaceae (0.7 PEinst m-2 s-l: Parkin and
Brock 1980a,b, 198 l), can hardly be expected to support growth of purple sulfur
bacteria in general. The low light requirement of the green sulfur bacteria is even
better illustrated by the fact that the bloom
in Knaack Lake is not even light-limited,
but sulfide-limited (Parkin and Brock 198 1).
This may explain why no sulfur is deposited, but instead all sulfide is oxidized to
sulfate. Although the affinity for sulfide has
not yet been estimated experimentally
for
Pelodictyon spp. or Clathrochloris spp., it
can be deduced from their position in the
lake in combination with the observations
on the kinetics of sulfide oxidation that their
affinity for sulfide must be as good as that
of other Chlorobiaceae (see van Gemerden
1984).
TO assess the kinetic relations between
the electron-donating
reactions and the formation of products in Lake Cis6 over a full
diel cycle, we made calculations for the following periods on 6-7 July 1982: 07001000, 1300-l 600, 1800-2 100, and 22000600. In the 2-m layer, the average rate of
sulfide oxidation between 0700 and 1000
hours on 6 July 1982 was 30 hmol liter-l
h-l. The net production in the layer was 11
bmol liter- ’ h- ’ ; the actual decrease in the
sulfide concentration in the layer accounted
940
van Gemerden et al.
for 4 pmol liter-l h-l, indicating that sulfide
must have been replenished from deeper
layers.
The bloom of purple sulfur bacteria in
Lake Ciso showed a temporary accumulation of glycogen (see Fig. 2a). Glycogen is
either synthesized de novo from CO2 or produced from PHB. The latter compound may
also give rise to growth. For the balance of
reducing power it is irrelevant which process dominates. In the following calculations it is assumed that PHB is converted
into glycogen according to
C4Hs03 + 2C02 + 6(H) --) C6H1206
+ HZ0
(4)
and that the remaining glycogen is synthesized de novo according to
6CO2 + 24(H) + CbH1206 + 6H2O.
(5)
In situ, the average increase in the concentration of glycogen in the 2-m layer between
0700 and 1000 hours accounted for 3.9 pmol
liter-’ h-l. In the same period, the concentration of PHB decreased from 6.7 to 2.9
pmol liter-‘, which would result in the synthesis of glycogen at a rate of 1.3 pmol liter- ’
h-l. According to Eq. 4 this requires the
input of reducing power at a rate of 7.8 pmol
liter-l h-l. The synthesis of the remaining
glycogen from CO2 requires the input of reducing power at a rate of 62.4 (Eq. 5), the
total being 70.2 pmol liter-l h-l. The rate
of sulfide oxidation was estimated to be 30
pmol liter-l h- I. Since in the oxidation of
sulfide to sulfur two electrons are involved,
the rate of supply of reducing power is 60
pmol liter-l h-l. It appears, therefore, that
in the morning all reducing power released
in the oxidation of sulfide to sulfur is channeled into the synthesis of glycogen, and
none is used for growth. Similar phenomena
have been observed in pure cultures of C.
vinosum and Chromatium weissei. Cells exposed to light after being incubated in the
dark do not start to grow within a few hours;
however, sulfide oxidation starts immediately, resulting in deposition of glycogen inside the cells (van Gemerden 1968a, 1974).
In the lake, the rates of sulfide oxidation
and CO2 fixation showed maxima in the
middle of the day (Fig. 3), whereas at that
time less glycogen was deposited. This in-
dicates either that the excess reducing power
is used for the synthesis of structural cell
material or that excretion takes place. Excretion has been reported to occur in blooms
of phototrophic
bacteria (Czeczuga and
Gradzki 1973; Abella 1980), and the phenomenon is of ecological importance (Pfennig 1978). In a bloom of Chromatium in
Lake Ciso in 1979 similar to that we studied, excretion of 14C-labeled organic compounds was substantial: 49% of the 14C02
fixed was not retained on the filter (Abella
1980). This reflects the combined effect of
excretion and lysis, and the intensity of these
processes can be expected to increase with
depth. An indication of the occurrence of
lysis is the lower sulfur content of the cells
in the deeper layers of the lake (Table 1).
The inclusion of high amounts of sulfur
in the cells increases their specific density
(Table 1) and sedimentation
seems to be
inevitable. Preliminary
experiments have
shown that killed cells sink faster when sulfur has been allowed to deposit before the
addition of formaldehyde. It remains to be
investigated whether living cells incubated
in a light gradient can compensate for the
sinking to some extent by means of the
movement of their flagella. Sedimentation
means that the cells very soon experience a
light intensity not high enough to meet their
maintenance requirements.
The minimal
light intensity at which C. vinosum can
maintain its cell integrity, i.e. no growth but
no death either, has been reported to be
about 1.5-4 PEinst m - 2 s- l (van Gemerden
1980). The light intensities encountered at
the depth of maximal population density in
the lake are of the same order of magnitude
(Guerrero et al. 1984, 1985). Thus, the
hoarding of substrate which has been shown
to improve the competitive
position of
Chromatiaceae in well illuminated habitats
(van Gemerden 1974) appears to be a drawback under light-limited
conditions.
Below 3 m, the cells are completely depleted of endogenous energy sources, as can
be deduced from the fact that the total sugar
concentrations
show no diel fluctuations.
Glycogen at these depths is not to be expected either, since it takes only one night
to reach the zero glycogen level (cf. Fig. 2a
and b). Elemental sulfur can be considered
941
Metabolism of Chromatiaceae
an endogenous storage product in the sense
that it can be used as an electron donor but
not as a source of energy. Once the cells have
reached a depth at which insufficient light
penetrates to fulfil their maintenance requirements, lysis will begin, and sooner or
later the elemental sulfur will be released
into the water. Guerrero et al. (1985) reported that light limitation was demonstrated by the fact that cells from the 2-m layer,
when incubated closer to the surface, exhibited increased rates of sulfide oxidation.
No such increased rates were observed when
samples from 3 m or deeper were incubated
at 1 m; this suggests a low viability at these
depths.
In the viability studies done in Lake Ciso
in summer 1983, despite the lower activity
in the peak of the layer than in the top, the
cells in the peak were fully alive. Their inability to move back again to the layers with
higher light intensities apparently can be explained by lack of a suitable source of energy.
It is usually difficult to assess the actual
increase in structural cell material of one or
two species of bacteria in natural habitats.
Blooms facilitate more accurate field observations. It should be realized, however,
that such processes as carbon dioxide fixation and sulfide oxidation reflect photosynthetic activity, which results not only in
growth but also in the formation of storage
polymers and the excretion of organic molecules. With phototrophic
organisms, pigment analyses have been used to measure
the development of bacterial plates. The
generation times calculated from such observations invariably are much longer than
one would expect from the rate of CO2 fixation. From integrated biomass data, the
net doubling times for Chromatium spp. in
Lake Ciso have been calculated to be 500
h or more (Abella 1980; Montesinos and
Esteve 1984).
Such calculations are relevant when the
lake is considered as a whole. However, for
the estimation of growth at different depths
the following points should be taken into
account. First, the growth of phototrophic
bacteria does not proceed continuously, but
is confined to the light period. In view of
the fact that glycogen rather than structural
81’1’111””
20
40
60
80
100
VIA 6 I L I TY (percentage)
Fig. 4. Average viability of Chromatium spp. and
strain M-3 in Lake Cis6 in samples taken on 12 July
1983. The location of the three zones of the bacterial
layer is also indicated.
cell material is produced in the morning, it
seems reasonable to assume actual growing
periods of no more than 10 h. Furthermore,
it is to be expected that the rate of growth
is not constant over the entire period. This
can be deduced as well from the curves for
CO, fixation and H2S oxidation, which show
maxima in the middle of the day (Figs. 3
and 4). Second, and more important, the
observed increase in biomass is not the result of growth of the entire population, but
rather of a small fraction. In the light-limited bloom in Lake Ciso, only those organisms multiply which are close enough to the
surface to receive enough light. Therefore,
the specific rate of CO, fixation and the specific rate of H2S oxidation are more appropriate to locate the sites of active growth
than the population density. In Lake Ciso,
only about 5% of the population is located
between 1.75 and 2 m, but between 50 and
65% of the activity is found there. Consequently, the actual growth rate could be very
much higher than that calculated on the basis of the entire population.
In the laboratory it has been shown for
942
van Gemerden et al.
,
various species of bacteria, including phototrophs isolated from lakes in the Banyoles
area, that cell size increases with increasing
growth rate. Analyses of the size distribution of phototrophic
bacteria in Lake Ciso
and other lakes in the Banyoles area revealed that the largest individuals
are indeed found at the upper side of the plate
(Montesinos 1982).
During the night glycogen is virtually
completely degraded, and at the same time
some PHB is produced. The concentration
of sulfide in the 2-m layer increases, that of
sulfur decreases. These processes all correspond nicely to laboratory observations on
the dark metabolism of purple sulfur bacteria. Cultures of C. vinosum, incubated in
the dark, convert glycogen to PHB, a process accompanied by the reduction of sulfur
to sulfide (van Gemerden 1968b):
1 monomer
of glycogen + 1 monomer of
PHB + 6(H),
(6)
and
3 sulfur + 6(H) + 3 sulfide.
(7)
In other words, purple sulfur bacteria produce some sulfide during the night by reduction of intracellular sulfur. From an ecological point of view, the production
of
sulfide is in itself less important than the
fact that this process is accompanied by the
production of ATP. In the dark, the latter
compound is required for maintenance purposes and also for active swimming. An additional advantage of the reduction of intracellular sulfur could be that the specific
weight of the cells is decreased, which probably results in a lower rate of sinking. On
the other hand, the breakdown of glycogen
results in the synthesis of PHB which is
stored intracellularly.
In the 2-m layer in Lake Ciso, the concentration of glycogen decreased during the
night at an average rate of 2.6 pmol liter-’
h-l, and the rate of PHB formation was
between 1.6 and 3.1 pmol liter-’ h-l. Simultaneously the concentration
of sulfide
increased, at an average rate of 10.8 pmol
liter- l h- I, whereas sulfur decreased at 9.8.
These values agree well with the ratios shown
in Eq. 6 and 7. It thus appears that the
changes observed at night can be attributed
to the dark metabolism of the dominant
organisms, Chromatium spp. and strain
M-3.
The development of a layer of phototrophic bacteria in stratified lakes can be
described as follows. Actively growing cells
are located at the top of the layer. Cells unable to maintain their position accumulate
in a zone of maximal abundance but of low
activity due to insufficient light. These cells,
loaded with elemental sulfur, glycogen, and
perhaps PHB from their previous photosynthetic metabolism, have buoyant densities as high as 1.18 g cm-3 (Table 1). Consequently, they slowly sink and reach depths
where light conditions are even less favorable. During this process, they remain viable (Fig. 4) due to the breakdown of glycogen (Fig. 2). The glycogen content,
however, is depleted in < 12 h (Fig. 2), and
the PHB formed from glycogen is deposited
inside the cells, resulting in still high buoyant densities. Thus, as the population grows
during spring and summer, more and more
cells are trapped deeper in the water, resulting in both a thickening of the layer and
an increase in cell numbers. These cells
eventually will reach the bottom sediments,
where the remaining organic matter will be
mineralized. In this way phototrophic bacteria contribute a significant portion of the
organic matter which is needed to complete
the anaerobic carbon and sulfur cycles of
the lake.
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Submitted: 27 August 1984
Accepted: 27 March 1985