PHOTOSYNTHETIC PROPERTIES AND GROWTH OF
PHOTOSYNTHETIC SULFUR BACTERIA IN LAKES
Masayuki
Takahushi’
and Shun-ei Ichimura
Botanical Institute, Faculty of Science, Tokyo Kyoiku University, Otsuka
ABsTRAcr
The photosynthesis-light curve for purple sulfur bacteria had a steeper inclination than
that for green sulfur bacteria at low light intensities. Light saturation occurred at intensities of S-7 klux in the former, in the latter at 10-30 klux. Light inhibition was observed
in purple sulfur bacteria but was negligible in green sulfur bacteria. The optimal temperature for photosynthesis of these bacteria is considerably higher than that of most phytoplankton or green plants. Photosynthetic sulfur bacteria appear ordinarily in the contact
layer between oxidative and reductive zones of meromictic or stagnant holomictic lakes;
the light intensity in this contact layer is usually less than 10% of that at the surface.
On the assmuption that the photosynthetic rate of these bacteria is limited mainly by the
interaction of hydrogen sulfide concentration with light intensity, their growth was analyzed
with a mathematical model. The properties of the growth phase observed in lakes were
similar to those calculated. The main factors determining the growth of photosynthetic
sulfur bacteria in lakes are the II23 concentration in the upper layer and the light conditions
in the deeper layer.
INTRODUCCION
In meromictic
or stagnant holomictic
lakes a dense population of photosynthetic
bacteria appears frequently in the contact
layer between oxidative
and reductive
zones, coloring the water pink or green
(Kondrat’eva
1965; Pfennig 1967). Light
penetration to the depth of this layer is
generally less than 10% of the surface illumination (Takahashi and Ichimura 1968;
Czeczuga 190&r, b, c) . Since photosynthetic
sulfur bacteria normally grow autotrophitally, it is interesting that an enormous
population of photosynthetic
bacteria occurs in a layer near the bottom of the
photic zone. This may be attributed to the
presence of H2S in the habitat and to the
characteristic
photosynthetic
response of
these bacteria to the light intensity and
quality. Pfennig (1967) discussed the pigment system of photosynthetic bacteria in
relation to the quality of light in the habitat. Recently, Triiper and Genovese (1968)
examined the ecological significance of selective light absorption by the pigments
of photosynthetic bacteria with respect to
their vertical distribution in natural waters.
The role of photosynthetic sulfur bacteria as primary producers in the aquatic
l Present address: FRBC
Nanaimo, British Columbia.
Biological
Station,
ecosystem has recently been emphasized
(Kuznetsov 1959; Sorokin 1904, 1966; Czeczuga 196&z, b, c; Takahashi and Ichimura
1968). The amount of organic matter produced by bacterial photosynthesis is too
high to be overlooked in waters containing
H&
At present, however, there is little
information on photosynthetic
bacteria in
the natural environment.
The purpose of
this study is to clarify the ecological frmction of sulfur bacteria and to analyze their
growth under field conditions.
We wish to thank Dr. T. R. Parsons for
critical reading of the manuscript
and
Mr. T. Oikawa for assistance in computer
programming.
METHODS
Our methods were similar to those described previously
(Takahashi and Ichimura 1968). Water samples were taken
with a 3-liter plastic Van Dom sampler.
Immediately
after sampling, oxidation-reduction potential was determined with a
platinum
electrode redox meter (Redox
meter Model RM-1, TOA Elect&s
Ltd.,
Tokyo), and HSS was measured by colorimetry (Amer. Public Health Ass. 1960).
Oxygen fixed in the field was measured
by Winkler titration, temperature with a
thermistor, and light attenuation in water
929
930
MASAYUKI
TAKAIIASHI
with a selenium underwater
photometer
fitted with a neutral glass filter and glass
color filters (Hoya Glass Works, Tokyo,
types B43, G58, and R60). The maximum
transmittance for each color filter was 440,
530, and 630 nm, respectively.
A water
sample of 500 ml or less was filtered
through an HA Millipore filter for the determination of pigments and bacterial cells
were counted from a O.l- to IO-ml aliquot.
The Millipore
filters were refrigerated
and the analysis was made 4-5 hr after
filtration.
Chlorophyll a was measured according to the method recommended by
UNESCO ( 1966). The abbreviations proposed by Jensen, Aasmundrud, and Einhjellen (1964)-BChl
a, BChl c, and BChl
d-were used for bacteriochlorophyll,
chlorobium chlorophyll-660,
and chlorobium
chlorophyll-650.
BChl c and BChl d were
determined from the following equations:
BChl c (mg/m3) = 10.8 X DG02X v/V; BChl
CE(mg/mZ) = 10.2 X DGaJX v/V. Both equations wcrc obtained using the specific absorption coefficients of 92.1 ml (mg cm)-r
for BChl c and 98.0 ml (mg cm)-l for BChl
d in acetone (Stanier and Smith 1960).
Smith and Bcnitez (1955) gave a specific
absorption coefficient of 46.2 ml (mg cm)-’
for BChl a in methanol; this value could
not be used directly in our study because
pigments were extracted with 90% acetone.
The factor was recalculated from a direct
comparison of absorbance in 90% acctonc
and in methanol, and the following proBChl a
visional equation was derived:
(mg/m”) = 25.2 X DT72 X v/V. D is the OPtical density determined in a l-cm cell, v
is the volume of the 90% acetone extract
in ml, and V is the volume of water sample
filtered in liters. Cells were counted on a
Millipore
filter under a microscope after
adding a few drops of cedar oil.
The rate of photosynthesis was measured
by the 14C method. In field experiments
the water samples containing photosynthetic bacteria were poured carefully into
100-ml glass bottles which were exposed in
titu or wcrc placed in transparent plastic
tubes rolled with black nylon net to give
AND
SHUN-E1
ICHIMURA
light intensities inside the tubes of 1, 10,
25, 50, and 100% natural sunlight.
The
tubes were held horizontally just under the
surface on a floating framework and exposed for 3 ‘hr in daytime (from 1000 to
1300). According to preliminary
cxperiments, photosynthesis in the water samples
was active and linearly correlated with time
during this period.
In laboratory experiments stock cultures
were used. The green sulfur bacterium,
Chtorobium sp., was isolated from Yumoto
hot sulfur spring, Nikko, by the method of
van Niel ( 1931) and grown in Larsen’s
medium (Larsen 1953). The purple sulfur bacterium, Chromatium strain D, was
grown in van Niel’s medium (van Niel
1931). Stock cultures were centrifuged at
1,500 6 and the bacteria were resuspended
in new medium for each experiment. Glass
bottles (50 ml) were filled with a suspension of bacteria and placed in a temperature-controlled
water bath.
The light
source was daylight fluorescence lamps or
special incandescent lamps (eye lamp, Iwasaki Electrics Co., PSR lOOv-5OOw, 3,200”
K), and the illumination
was regulated by
the distance of the bottles from the light
source. The light intensity was measured
with a sclcnium photocell and also with a
Gorczynski solarimcter. Total carbon dioxide in the water samples and the medium
was determined by Conway’s microdiffusion method. When the water contained
much H2S, a correction factor was used,
determined
expcrimcntally
by adding a
given amount of H2S to a known carbonate
solution.
RESULTS
Photosynthesis
AND
DISCUSSION
by sulfur
bacteria
Response to light intensity
The photosynthetic response of bacteria
to light intensity was examined using cultures grown in Roux’s culture flasks at 3OC
under an illumination
of 10 klux from a
500-w incandescent lamp. The shape of
photosynthesis-light
curves of C hromutium
strain D and ChZorobium sp. differed significantly with respect to both the initial
PHOTOSYNTHESI!3
AND
GROWTH
OF
SULFUR
931
BACTERIA
2-
E
n
<
Y
I-
Chloroblum
cn
z
I
0
LIGHT
%
2.
Chromatium
Chromatium
L
0
I
I
I
I
I
I
IO
20
30
40
50
60
LIGHT
INTENSITY
( klux)
Photosynthesis-light curves of Chloromeasured under an incandescent lamp. Cultures from Lake Suigetsu (1, 6),
Yumoto hot sulfur spring (2, 3), and Chromatium
strain D (4, 5). Experimental temperature was
27-3OC for 1, 3, 4, and 6; 21-23C for 2 and 5.
FIG.
( k lux 1
Relative photosynthesis-light curves of
strain D (a) and Chlurobium
sp. (b)
cultured at 2% under about 2 klux.
FIG.
0.5
0
INTENSlTY
1.
bium and Chrcnnutium
slope and the saturating intensity (Fig. 1).
The curves for Chromutium had a steeper
inclination
than those for Chiiwobium at
lower light intensity and showed light saturation at intensities of 5-7 klux, compared
with 10 to 30 klux for Chlorobium.
Photosynthesis by Chromutium was inhibited at
light intensities as low as 4 klux, whereas
ChZorobium was not appreciably inhibited
even beyond 50 klux. Larsen ( 1953) found
light saturation of photosynthesis by Chlorobium thiosulfatophilum
in the range of
7.5 to 10 X lo3 erg cm-2 se@, approximately equivalent to 0.20-0.26 klux of the
incandescent lamp we used. Wassink, Katz,
and Dorrestein ( 1942) reported on a fairly
wide range from 0.8 to 3 x 104 erg cm-2
set-l (ca. 0.21-0.79 klux) in Chromutium
sp. and also found no appreciable inhibition
under high light intensities. Lippert and
Pfennig ( 1969) showed that photosynthesis
by pure cultures of Chlorobium
thiosulfatophilum and Chromatium okenii reached
light saturation at 700-1,000 lux and 1,000-
2,900 lux, respectively.
These saturating
light intensities are lower than those we
found. This may have resulted partly from
the difference in light source or from the
past history of the bacteria. The incandescent lamp we used includes more infrared
than does sunlight or white light. Green
sulfur bacteria are able to use longer wavelengths more effectively
than do green
plants, and purple bacteria use not only
visible light but also part of the infrared
region (80&900 nm). Furthermore, when
light intensity is measured by a photometer
with a selenium photocell whose spectral
response is restricted to visible light, the
infrared range effective for photosynthesis
of purple sulfur bacteria is omitted.
The photosynthesis-light
curves of bacteria grown at 25C under a light intensity of
about 2 klux showed that light saturation
occurred at 2 klux for Chromutium strain
D and 5 klux for ChZorobium sp. (Fig. 2)values lower than those for bacteria grown
at 10 klux. They are able to photosynthesize at light intensities of less than 509 lux,
although the light source was an infraredrich incandescent lamp. This light intensity
is considered in general to be the compensation intensity of natural phytoplankton.
In our experiments, Chromutium
strain
D grew photosynthetically
under continuous illumination
of 100 lux. Cohen-Bazire
(1963) cultured some strains of Chloro-
MASAYUKI
L. HARUNA
(28 Ott 1966)
TAKAHASHI
L. HARUNA
AND
SHUN-E1
ICHIMURA
(14Octl964)
I
1’
KISARATSU
R. ( I Sap 1965 1
L. WAKUIKE
( 4 Aug 196!
I
Im
g2
si
ze’
i
4O
a
0
IO
0
13m
0
0
INTENSITY
I?IG. 3. Photosynthetic
capacity and rate of natural photosynthetic
sulfur bacteria and phytoplankton. Dominant species of phytoplankton
were As&rioneZla Formosa and MeZo&cl italica in Lake Haruna,
Nitzschia sp. in Kisaratsu Reservoir, and Anabmna ci~cu~~ti and Oscilla~toria planktonica
in Lake Wakuike. Photosynthetic
bacteria were purple sulfur bacteria in Lake Hanma, Chromatium
sp. in Kisaratsu Reservoir, and ChZmobiwn
sp. in Lake Wakuike.
Experiment
was done under sunlight at the
surface water temperature.
bium limicola and Clzlorobium thiosulfatophilurn under an intensity of less than 430
lux. Saturating light intensities reported by
different investigators fluctuate noticeably
even for the same species; the shape of
the photosynthesis-light
curve can be modified over a wide range by experimental
conditions as well as by the previous photic
history of the bacteria (see Kondrat’eva
1965). This is also true in phytoplankton
populations, but physiologic adaptation to
low light seems to occur more rapidly in
photosynthetic
bacteria than in phytoplankton.
The photosynthetic response of bacteria
and phytoplankton
to light intensity was
also examined in natural samples taken
from the surface and the contact layer
between oxidative and reductive zones,
where the light intensity was 10 to 0.1%
of surface light. Photosynthesis was measured under various intensities of sunlight
at in situ temperatures
(Fig. 3). The
shapes of the curves from the deeper water
coincided fairly well with those of cultured
bacteria, but the saturating light intensity
was much higher. This may have resulted
from a difference
in the energy distribution
of sunlight and the incandescent lamp.
The photosynthetic capacity of water samples indicated a wide range, according to
the population density, but the assimilation
rate per unit amount of chlorophyll had a
generally narrow range (Table 1). These
assimilation rates were about 10-E% those
of cultured bacteria.
E-Iydrogen sulfide
concentration
Baas-Becking and Kaplan (1956) found
that the purple bacteria (Chromatium)
occurred in an estuary where the Eh value
was -200 to 320 mv, and the green sulfur
bacteria from -300 to -100 mv. In aquatic
environments the increase in the concentration of reductants such as H2S is accompanied by a decrease in Eh. We have
observed successive changes in the flora
of photosynthetic
sulfur bacteria on the
streambed of a hot sulfur spring, where
the concentration of H$ decreases gradually with distance from the head of the
spring. The green sulfur bacteria are predominant near the point of origin and the
purple sulfur bacteria downstream.
To estimate the effect of the reductant
on bacterial photosynthesis, we considered
the interrelation
between HZS concentration and the photosynthetic rates from the
PHOTOSYNTHESIS
TABLE 1.
AND
GROWTII
OF SULFUR
933
I3ACTERIA
The photosynthetic
capacity and the maximum photosynthetic
phytoplankton
or photosynthetic
bacteria, respectively,
rate of wuter
in lakes
dominated
by
-Photosynthesis
capacity
SClgilig
Date
I-Iaruna
Suigetsu
Kisarntsu
Wakuike
Haruna
Suigetsu
Kisaratsu
Wakuikc
14
28
20
1
18
4
b-4
act
Ott
64
66
Jul
06
max rate
Water temp (“C)
in
situ
expt
(23
( ,zzL.
2
1
1
1
1
1
14.0
13.5
27.8
26.0
28.9
28.5
18
18
28.5
22-26
28.9
10-31
6.0
19.5
5.0
8.9
10.5
13.0
1.54
2.28
2.01
3,.9G
6.40
0.66
14 Ott 64
28 Ott 66
20 Jul 66
14
13
8.5
7.5
8.0
16.4
18
18
28.5
5.6
6.0
45.3
0.2G
0.30
0.25
1 Sep 65
18 Aug 66
5
6.2
25.5
21.6’
22-26
28.9
61.7
172
2.14
0.17
2.5
16.1
10-31.5
13.5
0.38
Sep 65
Aug 66
Aug 65
4 Aug
65
empirical data of the field cxpcrimcnts.
The values of photosynthetic
rate mcasured under the same conditions of light
and temperature wcrc plotted against the
concentration of HgS in the water samples
(Fig, 4). At low concentrations of HZS,
the increase was linear with the maximum
at nearly 1.5 mg S/liter. In a side cxpcriment, WC found that the rate of photosynthesis of Chromatium sp. increased with
increasing concentration of Na2S and lcveled off at about 40 mg S/liter.
Temperature
The effect of temperature on the photosynthesis of Chromatium and Chlorobium
appeared at low light intensity; photosynthesis was accelerated considerably with
increasing tcmpcraturc ( Fig. 5). The optimal temperature was 40C for Chromatium
and 30-3X for Chlorobium; light saturation was observed at 2-10 klux for the
former and 5-15 klux for the latter. Similar results were reported by Wassink et al.
(1942) for purple sulfur bacteria.
The
highest rate of photosynthesis of natural
phytoplankton is generally obtained at the
temperature of the habitat (Aruga 1965).
>
Dz2:t
Asterionella
Melosira
Nitzschia
Microcystis
Anabaena
Oscillatoria
Chromatium
Purple and green
sulfur bacteria
Chromatium
Chromatium
and
green sulfur
bacteria
C hloro bium
This is not always true for free-living photo,synthetic sulfur bacteria. They are distributed widely in aquatic environments,
from muddy soils to hot sulfur springs,
whcrc the tcmpcrature
varies widely.
Therefore, it is of intcrcst to study their
photosynthetic
adaptation to varying tcmpcraturc.
We took samples from a depth of 13 m
in Lake Haruna and from the bed of
Yumoto hot sulfur spring. The predominant species in both habitats was a purple
sulfur bacterium, presumably Chromatium
at the sampling
SP* The tempcraturc
depth in Lake IIaruna was about 8C; it
was 30C in the bed of the hot spring. The
H2S concentration was <0.2 mg S/liter in
the lake and 15 mg S/liter in the hot spring.
The optimal temperature was about 32C
for the samples from the hot spring, while
the bacteria from the lake showed a plateau
from S-30C (Fig. 6). The optimal tcmperature for photosynthesis is appreciably
higher for the purple bacteria than for
phytoplankton or green plants, although it
shows some variation.
The photosynthetic Qlo of the sulfur bac-
934
MASAYUKI
TAKAIIASHI
AND
SHUN-E1
ICI-IIMURA
Growth
of photosynthetic
bacteria in lakes
distribution
Vertical
0
I
015
HYDROGEN
SULFIDE
I.‘5
( H2S mg/ I 1
FIG. 4. Relation between H& concentration
and
photosynthesis
of a purple bacterium.
The data
of French ( 1937 ) were used for the computation.
tcria grown at 30C was about 2.5 in Chroand 2.1 in Chlorobium within the
range of 10-3OC. The photosynthetic
Qlo
for purple sulfur bacteria within the range
of 16.3~2SC, calculated from the data of
Wassink et al. (1942), was 2.1, and 2.5
was calculated for the nonsulfur purple
bacterium
Rhodopseudomonas
palustris
over the range of 20-3OC from the data of
Uemura et al. ( 1961).
mtium
Chromat ium
sulfur
Photosynthetic sulfur bacteria occur normally in holomictic lakes during summer
stratification,
when H2S appears in the
anoxic zone. They grow with the progress
of the season and show a characteristic
pattern of vertical distribution
(Takahashi
and Ichimura 1968). Two typical patterns
are shown in Fig. 7, with population dcnsity expressed as the amount of chlorophyll.
In pattern I there is a gradual increase of
bacterial mass with depth with its maximum near the bottom. In pattern II, predominant biomass is in a limited narrow
layer at the boundary between oxidative
and reductive zones. Both patterns develop
markedly in the later phase of the population maximum.
Reduced compounds such as H$ and
molecular hydrogen are necessary for bacterial photosynthesis, as clcctron donors,
and their changes with depth stem to
determine the pattern of vertical distribution of the ‘bacteria in lakes. We studied
the growth and vertical distribution
of
7oo ,
,
Chlorobium
600-
0
LIGHT
FIG. 5.
Chlorohizcm
INTENSITY
Photosynthesis-light
sp. were cultured
(klux 1
curves measured at various
under 2 klux at 3OC.
tcmperaturcs
30
20
IO
LIGHT
INTENSITY
( “C).
Ch~omntium
(klux)
D and
PIIOTOSYNTIIESIS
AND
GROWTII
photosynthetic sulfur bacteria in two water
bodies.
Lake Haruna is a holomictic lake 8 km2
in area with a maximum depth of 11.3 m
in the central part. It circulates in spring
and fall and is covered with ice from January to March. During summer it is strongly
stratified and oxygen disappears from the
deeper water. Hydrogen sulfide is detectable from 11 m down and reaches 2 mg
S/liter just above the bottom. The boundary layer, containing both oxygen and H2S,
is about 3 m thick. The light intensity in
the boundary layer is only 1 to 0.5% of
that at the surface. The vertical distribution of photosynthetic bacteria follows pattern I, and
_^ the
_ predominant spccics is a
---...-
1: --r--
on
the
eastern
shore
of
Tokyo
Bay. The surface area is about 2,000 m2
and the maximum depth is 10 m. The
SULFUR
935
BACTERIA
- . IOOT
Y
m
ci
W
F
z
50-
2
F
:
0
IO
0
30
20
40
~:;%a;
-.
Photosynthesis-temperature
fvnq
-..r cn“1’. icnlahd
~“.,AU~VU
&*“I,
IJWI
50
(“Cl
TEMPERATURE
YII--V*-UAII.
Kisaratsu Reservoir was built in spring
1965 on a sand bank near the city of
Kisaratsu
OF
L&e
curves
HaTuna
of
(a).
Chromatium
sp. from Yumoto hot sulfur Sp&i
(b),
Chhdum
SP. from YumotO hot sulhr
spring (c), and cultured
Chromatium
strain D
(d).
The curves were obtained using an incnndescent light at 7 klux.
,o,
,
,
,
,500
mgChl
/ m3
I
I+--
0
HYDROGEN
FIG. 7. Two typical patterns
Lake Hamana, 30 August 1965.
5
IO
SULFIDE
of vertical distribution
of photosynthetic
sulfur
II. Kisaratsu Reservoir, 18 August 1966.
15
40
(mgS/I
)
bacteria
in lakes.
I:
936
MASAYUKI
TAKAHASHI
AND
I
___ ____--- ------
a
I-
O
0
1
5
LIGHT
I
I
IO
15
INTENSITY
I
20
I
1
30 40
( k Iux )
1
50
f
FIG. 8. Basic photosynthesis-light
curves used
for calculation
of rate of daily photosynthesis;
a an d a’ rcprcsent observed and calculated curves
( a = 0.70, h I 1.52 ) for ChZm~&iunz, and 11 and
11’ are for Chromutium
(a = 0.153).
water mass is stagnated throughout
the
year, with marked development of thcrmoand chemoclincs. During summer, oxygen
disappears at about 6 m, and the H2S-containing zone rises to 5.5 m. There is a
large amount of HZS in the upper layer of
the reductive zone, reaching 20 mg S/liter
just above bottom. The thickness of the
contact layer is about 1 m and it receives
5-15% of the surface light. The predominant species are purple and green sulfur
bacteria, and the distribution
follows pattern II.
Mathematical expression for daily
photosynthesis of bacteria
WC constructed a mathematical model as
a first approach to an interpretation of the
growth of photosynthetic
sulfur bacteria
in both lakes.
The cxprcssion used for the photosynthcsis-light curve without light inhibition is a
rectangular hyperbola:
‘=
bI
l+aI’
(1)
where p is the photosynthetic rate [mg C
(mg Chl)-1 hr-I], I is the light intensity,
and a and b arc constants* This formula
was first proposed by Tamiya ct al. ( 1953)
SHUN-E1
ICHIMURA
to relate photosynthetic rate and light intcnsity of a ChZoreZZa suspension and was
later applied to photosynthesis of terrestrial
plants (Monsi and Saeki 1954), phytoplankton (Ichimura
1956), and aquatic plants
(Ikushima 1967). The basic curve that we
used to obtain the constants a and b is 3
in Fig. 1 for Chlorobizcm. From this the
values of a and b were cstimatcd, and the
shape of the curve obtained by combining
these values with equation ( 1) agreed well
with the observed curve (Fig. 8).
For the curve showing light inhibition at
higher light intensities, we used the cquation of Stcclc ( 1962) :
p = a P,,,Ie’
-nT,
(2)
where p is the photosynthetic rate, I is the
light intensity, a is a constant, and PmnXis
the maximum photosynthetic
rate. From
curve 4 in Fig. 1, the value of a was cstimated to bc 0.153, and P,,, was 2 mg C
(mg Chl)--’ hr-l.
When the percentage transmission of
light at various depths was plotted on a
semilogarithmic
scale, a fairly linear rclation was found between the semilog plots
of light transmission and depth. WC therefort expressed light attenuation in natural
waters empirically as :
I, = lee. lL2,
(3)
where I, is the relative light intensity at
depth x (in m), I0 is the surface light intensity, and 7cis considered as the attcnuation coefficient of the light flux in water
(l/m>.
The daily course of incident light intcnsity on a horizontal plane can be given
roughly by ( cf. Ikushima 1967) :
I, = I,,,, sin” (V/D) t,
(4)
where T1 is the light intensity at any given
time on a bright day and I,,,, is that at
the highest altitude of the sun. D is determined by the measurement of daylength.
From equations (I), (2), (3), and (4),
the mean hourly rate of photosynthesis of
bacteria at any depth, at any time, will bc
given by the following equations.
PHOTOSYNTHESIS
AND
GROWTH
For Chlorobium:
Ph =
OF
SULFUR
BACTERIA
DAILY PHOTOSYNTHESIS
l t2Te-kQin3
937
(mgC/mgChl/doy 1
(5)
(dD)t
0 max Ckz sin3 (n/D)t’
For C hromatium :
Ph = aPmaxI0 max e-lcxsin3 (T/D)
&-a10 nlare~= sin* (T/D) t .
t x
(6)
Integrating
Ph over the entire daytime
[t = (O+ D/2) x 21 gives the daily net photosynthesis Pd at depth x.
The P&epth
profiles in Fig. 9 were
constructed by introducing
the constants
(a = 0.70, b = 1.52 for Chlorobium; a =
0.153 for Chrowatium;
3c= 0.075) into
equations ( 5) and ( 6)) respectively, where
lornax was assumed to be 100 klux and D
was 12 hr. The attenuation coefficient 0.075
(l/m)
was that taken from the data for
440 nm obtained at a station in the Kuroshio
FIG. 9. Vertical
changes of calculated
daily
(Japan Current).
The calculation of Pd photosynthesis of photosynthetic
sulfur bacteria.
was done by computer (HITAC 5020, Com- a. Chlurobium:
Q = 0.70, b = 1.52. b. Chro= 100 klux; D = 12.
mutium:
a = 0.153.
puter Center, Univ. Tokyo).
The daily
photosynthetic rate of ChZorobium showed
maximum did not ala maximum at the surface of the water and the photosynthetic
that of Chromutium at the depth of 20% ways agree, with some discrepancy also in
light. There was better agreement in the the compensation depth. This may be attrend of both profiles in the deeper zone tributed to a difference in the light enviwhere the light intensity is lower than 1.5% ronment in deeper zones; in this calculation
of that at the surface.
we did not consider the changes resulting
from selective light absorption by seawater.
The P,depth profile was also examined
We assumed above that bacteria and H2S
by an in situ experiment made on a clear
are distributed homogeneously in the whole
day (Iomax = 120 klux) at a fixed station
in the Kuroshio. In this experiment we water body. In natural waters, however,
used Chromatium strain D cultured at 23C H$ is normally detected in the disphotic
under a light intensity of 4 MUX. The bac- or aphotic zone ( Fig. 11). When the calteria were suspended in bottles at various
culation includes the photosynthesis-HzS
relationship as well as the vertical distribudepths during the daytime ( 1100-1300).
The water temperature was 24.7C at the tion of H$ in water, the calculated Pr
depth profile of both bacteria will be closer
surface and 20C at 100 m. The attenuation
to the in siti forms. The next calculation
coefficients ( l/m) obtained from semilog
plots of light transmission with depth were was made using the data on H$ in Kisa0.075 for blue light (max 440 nm), 0.023 ratsu Reservoir and Lake Haruna (cf. Fig.
for green light (530 nm), 0.308 for red 11). The photosynthesis-H2S relation was
light (630 nm), and 0.075 for neutral. The approximated from the data in Fig. 4. The
results are shown in Fig. 10, with the pho- results of the calculation are shown in Fig.
12. In aquatic environments such as reptosynthesis-depth
curve of phytoplankton
resented by Kisaratsu Reservoir, the photofrom the same station inserted for comparison. There was fairly good agreement in synthetic rate of bacteria increases rapidly
the shape of the curves obtained by calcu- with the occurrence of HZS and subselation and experiment, but the depths of quently decreases. In contrast, an aquatic
IO,.,
MASAYUKI
TAKAHASIII
PHOTOSYNTHESISt
AND
SHUN-E1
rel.)
ICIIIMURA
HYDROGEN
0 ,
1004
”
“I
”
”
SULFIDE
‘$I
”
(mWI)
”
ip
”
6
FIG.
0.011
’
’
’
’
1
1
’
1
I
I I
Vertical
changes of calculated
and
experimental
photosynthesis
in water.
a. Photosynthesis of natural phytoplankton
at 1300-1600
hours at each samphng depth in the Kuroshio
( 32” 29’ N, 136” 30’ E ) . In situ tem~rature
was
20-2X,
30 October 1967. b. Photosynthesis
of
cultured Chromatium
strain D determined by suspending at each depth in natural seawater (as in
expt a). c. Calculated
daily photosynthesis
of
Chbrobium.
d. Calculated
for Chromatium.
FIG.
10.
environment having a low concentration of
I&S, such as Lake Haruna, shows a slight
and gradual increase of photosynthesis by
bacteria in the upper zone of the contact
layer, with no remarkable change near the
bottom where H$ is relatively abundant.
The characteristic features of these vertical
changes suggest that the cardinal factors
determining the photosynthetic rate are the
concentration
of H$ in the upper layer
and light conditions in the deeper layer.
Growth of photosynthetic bacteria
under field conditions
In the foregoing discussion the biomass
bacteria was indicated
of photosynthetic
H&
11.
in natural
Vertical
changes
waters.
in
the amount
of
as the amount of chlorophyll and the rate
of photosynthesis was expressed per unit
amount of chlorophyll.
As a measure of
cellular material, however, it seems desirable to use cellular carbon. The ratio of
cellular carbon to chlorophyll
in bacteria
must be found to obtain a conversion factor
for deducing the amount of carbon from
the amount of chlorophyll.
The amount of chlorophyll
in bacteria
varies widely according to their physiological state and to their history of illumination
and nutrients. We measured organic carbon with a carbon-analyzer
(Yanagimoto
CHN Corder MT-l) after the sample was
burned at 65OC. The ratio of organic carbon to BChl a in Chromatium
strain D
grown under 20 klux was 52.7 during the
log phase of growth and 29.7 during the
of
stationary phase. Under an illumination
2 klux thcsc values wcrc 19.9 and 15.1,
giving an averaged ratio of 36 at log phase
and 22 at stationary phase. Kondrat’eva
(1965) reported that 1 mg dry wt of Chro-
PIIOTOSYNTIIESIS
DAILY
AND
GROWTII
PHOTOSYNTHESIS
OF
Pd
SULFUR
939
BACTERIA
( mgC/mgChl/day)
5
0
0
L. HARUNA
FIG. 12,. Theoretical
vertical changes of daily photosynthesis
(P,) of photosynthetic
sulfur bacteria
Number of each curve indicates bacterial dencalculated
for Kisaratsu Reservoir and Lake Haruna.
sity as chlorophyll
( mg/m3).
The photosynthetic
layer limited by I&S is shown by the curve S. For
calculation,
the absorption
coefficient
0.0115 was used for Chlorobium
and 0.0084 for Chromdum.
was comparable to 0.02 mg of
BChl a at log phase, although she did not
give details of the light conditions, and
van Niel (cited in Kondrat’eva 1965) gave
the ratio of organic carbon to dry weight
of purple sulfur bacteria as 0.55 to 0.56.
Using the ratio of van Niel and the results
of Kondrat’eva, we find that 1 mg BChl a
corresponds to 28 mg org-C.
For green sulfur bacteria, Cohen-Bazire
( 1963) reported that 6 strains of Chlorobium grown under light intensities lower
than 430 lux contain 100 to 190 pg Chl/mg
protein. Assuming the ratio of protein to
dry weight to be 0.35 to 0.38 and that of
cellular carbon to dry weight to be as
above, we can calculate that the ratio of
protein to carbon is 0.63 to 0.68, and carbon
to chlorophyll is 3.5 to 6.8. B,ut her bacteria were grown under extremely low light
conditions. When we used our data, we
obtained a range of carbon to chlorophyll
ratios from 25 to 13 (avg, 19).
Using the conversion factor 36 for Chromatium and 19 for Chlorobium,
we can
approximate the relative growth rate of
bacteria from the daily photosynthetic pro-
m&urn
duction obtained by equations ( 5 ) and ( 6 ) .
For Chromatium:
k Chr = ?4f3x Pd.
For Chlorobium:
k Chl= %9 x Pd.
Here k is the relative growth rate and Pd
is the daily photosynthesis.
In the calculation of Pd ( cf. Fig. 9) we
did not include the effect of self-shading
because we considered only dilute suspensions, in which the light was absorbed
primarily by bacteria. When the bacteria
become dense, however, attention should
be given to self-shading. Here, we examined the light absorption by bacteria themselves using cultures of Chronzatium and
Chlorobium.
Measurements of light absorption were made by the ospal glass
method (Shibata, Benson, and Calvin 1954).
Suspensions of bacteria were poured into
a glass cuvette having a l-cm light-path
and diffusion filters, and the absorption
was measured with a photometer (Hitachi
FPW)
equipped
with an incandescent
lamp. The absorption coefficient was 0.049
(mg Chl/m2) for Chromatium and 0.056
for Chlorobium in the log phase, and 0.025
940
MASAYUKL
CHLOROPHYLL
AMOUNT
TAKAIIASIII
AND
SHUN-E1
( mg Chl /m2 1
60, 0
ICIIIMURA
BACTERIA
I
2
(x104wC/m3)
4
5
$i
Chromatium
7
I
I
’
0,oy1?3438?O(x103
I ?34
n
w
Gh1orobiun-t
1
L.HARUN4
Chromatium
and 0.048 respectively in the stationary
phase. The high values in young cultures
arc probably due to the presence of molecular sulfur which is produced either inside
or outside of the cells.
The absorption cocfficicnt was also determined with sunlight as the light source. In
this cxperimcnt, the samples were old cultures of Chromatium,
one grown under
1-2 klux and the other under 20 klux. The
light transmission was measured with sclcnium photocells (Tominaga and Ichimura
1966). The results are presented in Fig.
13. The samples did not show any appreciablc difference in absorption coefficients.
The cocfficicnt of 0.0084 (mg Chl/m”) lies
between the values for Chlorella and Skeletonemn measured by the same technique.
The values of the absorption coefficients
obtained in the two experiments differed
greatly, possibly because of the difference
in light sources and light rcccptors used.
The value of 0.0084 seems to be reasonable
for Chromntium from an ecological viewpoint. ChZorobiu,m cultured under lo-20
3
234
1,
-6.0°+
FIG. 13#. Relation between
chlorophyll
amount
and light transmittance
for three photosynthetic
sulfur bacteria
and two algae (from Tominaga
and Ichimura
1966). a. Ch&&
susnension. ‘b.
Skeletonemu suspension. c. Chromat&m
strain D
suspension cultured under 20 klux (0)
and about
500 klux (@).
d. Chlorobium
suspension (+)
cultured under IO-20 klux.
BIOMASS
I
I
L HARUNA
(Norobturq
FIG. 14. Theoretical
increasing ..*natterns
of bac.
terial biomass as cell carbon calculated
for Kisaratsu Reservoir
and Lake Haruna.
Number
on
each curve shows the number
of days after
inoculation.
klux sho,wed a value of O.OllEi-similar
to
that for SkeZetonemu.
The effect of self-shading on the photosynthetic rate of the bacterial population
was estimated in an environment having
the characteristics of Kisaratsu Rcscrvoir
and of Lake Haruna (14 October) in Fig.
11. In Kisaratsu Reservoir, H2S appears
in large amounts from a depth of 6 m,
where light intensity is reduced to 10%
of that at the surface, On the other hand,
the concentration of H2S in Lake Haruna
is low, appearing at 10 m where light intensity is only I.% of surface light. The calculation of photosynthetic rate was pcrformcd
from equations (5) and (6) on the assumption that the bacteria occurred at 6 and
10 m, respectively. The highest light intensity in a day at these depths was 10 klux
PIIOTOSYNTIIIBIS
AND
GHOWTII
FIG.
Haruna
KISARATSU
SULFUlX
941
URCTEltIA
NUMBERS
6cells/mlI
CELL
I
OP
(x 105cells/ml)
R.
L. HARUNA
15. Increasing patterns of bacterial cell numbers observed in Kisaratsu
(1964).
Dominant
bacterium in both waters wns Chromatium
sp.
in the former and 1 klux in the latter (see
Fig, 12). The photosynthetic
rate is diminished ( relatively)
with an increase in
the density of bacteria and the effect of
self-shading cannot be overlooked, even in
low density populations.
The growth of organisms is, in general,
given by:
N s = ~o(zp(~L-to),
(7)
in which No and N1 rcprescnt the biomass
or number of organisms at the beginning
Reservoir
and in Lake
(to) and at time tL, and k is the relative
growth rate. Assuming that Chromatium is
distributed homogeneously at a density of
1 mg C/m3 at the beginning, then the
growth of bacteria can be calculated as an
increase of cellular carbon from ( 5)) ( 6))
and (7) with consideration for self-shading.
In the calculation, I0 ,,lnS is 100 klux, D is
12, k is ‘%ctfor Chromntium
and Mo for
Chlorohium,
and it is assumed that the
“E
=z
r-
”
0
2-
KISARATSU
R.
x
z
mw
4 I-
. .’
c-
, ,/’
z
D A YS
16.
Theoretical
increasing
patterns
of
bacterial
biomass as cell carbon calculated
for
Kisaratsu Reservoir
and Lake Haruna.
Chromatium in the reservoir
( 1) and in the lake ( 3 ) ;
Chlorobium
in the reservoir (2,) and in the lake (4).
FIG.
OJ0
,V
,
10
20I
I
30
I
40
I
50
I
60
I
70
DAYS
FIG. 17. Increasing
patterns of bacterial
cell
numbers ( Chromatium
sp. ) observed in Kisaratsu
Reservoir (1965) and in Lake Haruna (1964).
942
MASAYUKI
TOTAL
BACTERIAL
( mgChl/
TAKAIIASIII
CHLOROPHYLL
m2)
0
f
1416
FIG. 18. Relation hetween
rophyll and the depth of the
reducing environment
during
of photosynthetic
bacteria in
total bacterial chloupper limit of the
the growing period
lakes.
vertical distribution
of I-1$ is constant
through the season. The results are shown
in Fig. 14. The bacterial biomass increases
rapidly in water high in I-1$ and its maximum shifts upward with time. On the
contrary, the growth of bacteria in water
with a low level of I-1$ is slow and the
effect of self-shading slight until late in
AND
SHUN-ET
ICHIMURA
growth. The seasonal changes in the vertical distribution
of bacteria measured in
situ in the waters of Kisaratsu Reservoir
and Lake Haruna arc illustrated in Fig. 15.
The propcrtics of the observed growth
phase were very similar to those calculated.
The total amount of bacterial carbon per
unit surface area of water was approximated by planimctry from Fig. 14, and the
values obtained were plotted against time
(Fig. 16). The increasing phase of bacterial biomass again coincided with that of
the number of bacterial cells measured in
both waters ( Fig. 17).
In natural waters ,the growth phase and
vertical distribution
pattern of photosynthetic sulfur bacteria are apparently determined by the light conditions and by the
concentration of HZS.
Optimum amount of chlorophyll in
natural populations of photosynthetic
sulfur bacteria
Steemann Nielsen (1962) deduced theoretically the maximum total amount of
phytoplankton
chlorophyll to be expected
in the cuphotic layer of natural waters; the
amount and caldated
chlorophyll
maximum under a unit surface
TABLE 2. Obsewecl chlorophyll
area in l&es.
Chlorophyll
existing from surface to the clepth of O.l’% of the surface light intensity
was summed
--Chlorophyll
Lake
Sinsei
Yunoko
Harutori
Hiruga
Wakuikc
Hamana
Haruna
Kisaratsu
Suigctsu
Suga
Naknnuma
* Equivnlent
Chl n
206
56.8
65.1
51.7
86.0
20.4
56.4
98.2
8.5.t
11.4t
37.3
I
24.8
t
to BChl
amount (mg Chl/m2)
BChl a
BChl c
BChl tl
587
S87
352
182
79.9
119
492
235
71.91
t
36.1
2-4.9-t
436
320
128
198
574
235
71.9%
$
36.1
24.91
84.2
138
48.4
79.3
81
c or BChl
d.
+ Not
Total BChl
exact.
:I Unmeasured.
Rel. light (TO) Calculated
BChl max
at depth H$
(mg BChl/ma)
appears
2.5
0.07
5.3
4.3
1.0
10.01
7.0
3.0
1.5
7.0
4.7
100
10
1
Date
8
31
21
9
4
3,l
15
22
1
16
20
17
20
11
280"
0
345"
330"
2759
550"
370"
300*
140"
370"
330”
8256 690”
7900 400"
2759 200"
0 Equivalent
to BChl
(1.
Jul
Aug
Jul
Sep
Aug
Aug
Ott
Ott
Sep
Aug
Jul
Dee
Jul
Ott
66
66
66
65
65
65
65
66
65
66
66
68
66
68
PHOTOSYNTHESIS
AND
GROWTH
result was 400-800 mg/m2. In natural very
eutrophic waters, however, the highest
quantity of chlorophyll is generally about
200 mg/m2. The theoretical maximum thus
appears to be 2-4 times higher than the
observed quantity.
A possible reason for
this difference is the difference in the
extinction coefficients of natural waters and
those used in mathematical models, The
in natural waters is
light attenuation
strongly affected by suspended matter other
than phytoplankton.
We found a similar situation with photosynthetic sulfur bacteria. The maximum
quantity of chlorophyll in a bacterial population can be calculated with the following
equations, with consideration of light and
concentration of H&3 in the habitat.
In the first case, when H2S is distributed
in the whole water body:
where [Chl,,,]
is the maximum quantity
of total chlorophyll ( mg/m2), I0 and Ic are
the light intensities at the surface layer
and the compensation depth, respectively,
and CYis the absorption coefficient of bacterial suspension ( mg Chl/m2 ) .
In the second case, when H2S appears
from depth m:
log WLn
CMnax= [CU&l - o 4342a ,
(9)
where Chl,,, is the maximum quantity of
total chlorophyll
( mg/m2) and I, is the
light intensity at depth m. The results of
the calculation made by considering a =
0.0084 (mg Chl/m2) for Chromutium and
0.0115 for Chlorobium are shown in Table
2 and Fig. 18. The calculated maximum
quantity of total chlorophyll
is a little
higher than that observed. The highest
value measured in natural waters was about
600 mg/m2, whereas the theoretical value
was about 800 mg/m2. If adequate allowance is made for the extinction coefficient
by considering the absorption of light by
detritus, the theoretical maximum value
may approach the level existing in natural
waters.
OF
SULFUR
BACTERIA
943
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