Photosynthetic characteristics of a purple sulfur bacterium, Chromatium buderi, cultured under different ranges of pH, temperature, light intensities and ammonium chloride concentrations were examined. Maximum bacteriochlorophyll a synthesis was observed at pH 6.5 whereas the optimum growth was at pH 8.0. In general, higher temperature tended to inhibit the chlorophyll a synthesis and growth. 30°C is the optimum temperature both for chlorophyll a synthesis and growth. At 25µE mˉ²Sˉ¹ the bacteriochlorophyll a content and growth attained maximum level. The response to this low light intensity is an adaptation that ensures a high photosynthetic rate for the purple sulfur bacterium that usually occurs in dimly lit environment. Besides these, ammonium chloride at low concentration enhances both chlorophyll a synthesis and growth. Above 0.5% of it causes the nitrogen-chlorosis and also retards the growth of the bacterium. Possible chemical and structural mechanisms involved are discussed

Dhevendaran, K.

Aquatic Commons

Studies on CE - Itural Conditions f Mann Chrorr-5_ 
i Truper and Jannasch 
K. DHEVENDARAN* 
Department of Marine Biology, University College of Northwales, Menai Bridge, U.K. 
Photosynthetic characteristics of a purple sulfur bacterium, Chromataun buderi, 
cultured under different ranges of pH, temperature, light intensities and ammonium 
chloride concentrations were examined. Maximum bacteriochlorophyll a synthesis 
was observed at pH 6.5 whereas the optimum growth was at pH 8.0. In general, higher 
temperature tended to inhibit the chlorophyll a synthesis and growth. 30°C is the opti-
mum temperature both for chlorophyll a synthesis and growth. At 25 tIE m —25 the 
bacteriochlorophyll a content and growth attained maximum level. The response to 
this low light intensity is an adaptation that ensure a high photosynthetic rate for the 
purple sulfur bacterium that usually occurs in dimly lit environment. Besides these, 
ammonium chloride at low concentration enhances both chlorophyll a sy thesis and 
growth. Above 0.5 % of it cause the nitrogen-chlorosis and also retard the growth of 
the bacterium. Possible chemical and structural mechanisms involved are discussed. 
The ability of photosynthetic bacteria to 
grow in anaerobic environment and their 
dependance on the light, pH, temperature, 
hydrogen donor and nutrients were well 
documented by Van Niel (1944). They 
have an important role in the production 
of organic matter as well as the purification 
of polluted sewage waters in the aquatic 
environment (Takahashi & Ichimura, 1968). 
The photosynthetic response of the purple 
sulfur bacteria to the environmental condi-
tions both in quantity and quality must be 
considered for the ecology of photosynthetic 
bacteria (Parson et al., 1969). Bergstein 
et al. (1979) reported the temperature range 
in Lake Kinneret, Israel, in which they 
observed the bloom forming, Chlorobiurn 
phacobacteriodes and noted that the tem-
perature had a specific implication over the 
formation of bloom in the stratified lake. 
Besides, the role of nitrogen as one of the 
limiting factors for productivity has relatively 
been neglected. Thus, because of increasing 
process of eutrophication, it is becoming 
important to study the physiological pro-
perties of the photosynthetic bacteria under 
varying levels of nitrogen sources. Recently 
"- Present Address: Department of Aquatic BiologY 
and Fisheries, University of Kerala, Trivandrum -
694 007. 
Shilo and Rhimon (1982) observed that 
ammonia was the toxic inhibitory meta-
bolite in the intensive fish breeding ponds 
and they utilized the bloom - forming cyano-
bacteria to breakdown the inhibitors leading 
to the build up of organic matter. Subse-
quently Dhevendaran (1984) studied the 
utilization of sulfur, as well as other organic 
compounds over the growth of a selected 
Chromatium sp. strain in Porto-Novo waters. 
Differential utilization of organic compounds 
under anerobic conditions resulted in the 
change of pH of the growth medium. It 
is conceivable that the fermentative path-
ways are functional in cells growing anero-
bically (Uffen&Wolfe, 1970). The occurrence 
and the functional significance of photo-
synthetic bacteria in Menai Bridge waters 
have not been studied previously. Truper & 
Pfennig (1981) reported the characteristic 
features of the anoxygenic photosynthetic 
bacteria. Following their techniques in the 
present study an attempt has been made to 
optimize the different cultural conditions 
of Chromatium buderi in the laboratory. 
Materials and Methods 
Sediment samples were collected using 
Peterson grab and transferred aseptically 
Vol. 25, 1988 
10o 100 
	
 Bacteriochlorophyll o 
--- -0 Growth curve 
80-z 
6og 
E 
20 
;8O 
-§- 0 
o 60 
7-5 
.0 
E 40 
E 
"T‹ 
20 
7 
100 
E 
40 
0 
_0 
E 40 
E 
Tc 
E 
20 20 
	 o Bacteriochlorophyll\ a 
o 	 ® Growth curve 
80 
2 
Cr" 
E 
0 P 
T.< 
20 	 30 	 40 
Temperature (°C) 
Fig. 2. Effect of temperature on bacteriochlorophyll a 
synthesis and growth of C. buderi 
100 
2, 80 
2 0 
.2 60 
88 	 K. D EVENDARAN 
to sterile polythene bags. One gram 
of wet sediment was aseptically removed 
and transferred into 100 ml of the sterile 
enrichment medium. The medium con-
tained per litre of aged seawater; 1.0 g 
MgS• 4, 0.6 g K2 HPO4 , 3.0 g NH4 Cl, 
1.0 g yeast extract, 2.58 g sodium malate, 
vitamin 2 solution as described by Pfennig 
and Lippert (1966) and 0.5 g Na 2 S. The 
pH of the medium was adjusted to 6.8. 
The medium with sample was taken in 
glass stoppered bottle and incubated with 
continuous illumination at 20 tiEm 2 S —1 
at room temperature (25 + 2°C) for 10 days. 
After the incubation period, 0.1 ml of ali-
quot was taken and poured into the sterile 
off 
Fig. 1. Effect of pH on bacteriochiorophyll a 
synthesis and growth of C. buderi 
petridishes following the method of Siefet 
et al. (1978) and mixed the above enrichment 
medium with 2 % agar and incubated again 
under continuous illumination as before. 
Single pure colonies appeared were identified 
according to the method of Pfennig & Truper 
(1974) and Truper & Pfennig (198]). 
Protein content of the bacterial culture was 
estimated by the method of Lowry et al. 
(1951). 
Bacteriochlorophyll a was extracted from 
pure culture of purple sulfur bacteria with 
acetone: methanol (7:2). 10 ml of cell 
supension was centrifuged, the pellet was 
resuspended in 1 ml of distilled water and 
9 ml of solvent mixture was added. After 
30 min at 4°C, the mixture was centrifuged  
at 7,000 rpm. The absorbance of the 
supernatant was measured at 775 nm. The 
bacteriochlorophyll a concentration was 
calculated using the extinction coefficient 
E 775 = 75 litres/n mol cm as given 
by Clayton (1963). 
esuits 
	 rscusslon 
Fig. 1 shows the effect of pH on the syn-
Thesis of bacteriochlorophyll a and also on 
the growth of C. buderi. Maximum bacteria-
chlorophyll a synthesis was noticed at pH 
6.5. At pH 8.0, only 50 % of the maximum 
bacteriochlorophyll a synthesis was observed. 
Good growth of C. buderi occurred at pH 
range 6.5 to 9.0 with a maximum at pH 
8.0. close to that of sea water. From this 
investigation it may be inferred that the 
bacteriochlorophyll a synthesis and growth 
of C. buderi are independent and the pH 
is one of the determining factors for the sur-
vival and productivity in the anaerobic con-
dition in the marine environment, thereby 
determining the ecological niche of the iso-
late studied. Similar pattern of observation 
regarding the growth and longivity of bio-
luminescence of the bioluminescent bacteria 
was made by Lakshmanaperumalsamy et al. 
(1982) in tropical seawater. The isolate 
seems to be more sensitive towards the acidic 
rather than alkaline conditions. Recently 
Dhevendaran et al. (1986) studied the cul-
tural conditions on arylsulfatase activity in 
Escherichia coil and observed maximum 
FISHERY TECHNOLOGY 
	  Bacteriochlorophyll a 
0— 	
-o Growth curve 
100 	 100 
40 
E 
20 
o 
BO, 
2 
606 
D\ 
0 6 
20 
Leo 
-5 
.2 60 
CULTURAL CON ITIONS OF MARINE CHROMATIUM BUDERI 
	 89 
enzyme activity at pH 6.6 whereas the opti-
mum growth of the strain occurred at pH 
5.6 thereby confirming our results that the 
growth is independent of the physiological 
activity of the culture. 
C. buderi has been tested further for its 
reaction towards temperature. Fig. 2 
shows that both enhanced bacteriochloro-
phyll a synthesis and optimum growth noticed 
at 30°C. However, they were not affected 
to any considerable extent when it was 
incubated both at 25°C and 37°C. Mila-
zzo & Fitzgerald (1967) recorded similar 
observation in Proteus rettgeri where maxi-
mum growth and highest arylsulfatase acti-
vity occurred at 28°C. Subsequently Dhe-
vendaran et al. (1986) confirmed it with the 
same pattern of activity and growth of 
Eseherichia coil at 29°C. When the tem-
perature was raised above 40°C, there was 
a declining trend both in growth as well as 
in chlorophyll a synthesis ( ergstein et al., 
1979). 
Photosynthesis and growth were deter-
mined in growing cells at different light inten-
sities, ranging 10-50 p.E m —2 S —1 at 30°C 
(Fig. 3). The results i dicated that light 
intensity exerted its most pronounced effects 
10 	 20 	 30 	 40 	 50 
Light intensity (Al E m-251 ) 
Fig. 3. Effect of light intensity on bacteriochloro-
phyll a synthesis and growth of C. buderi 
on the bacteriochlorophyll a synthesis and 
also on growth of C. buderi. As the light in-
tensity increased from 10 to 15 ILE m — 2 S 
there was an increase of 15 % chlorophyll a 
synthesis. However, with further increase 
in the light intensity upto 25 it E m —2 S —1 , 
there was a sudden rise (about 3 fold) in the 
bacteriochlorophyll a content reaching the 
maximum concentration. Photosynthetic 
rate was related to the amount of bacterio-
chlorophyll a and growth of C. buderi was 
also attained the maximum level. From 
the present study, it became clear that the 
purple sulfur bacterium, C. buderi attained 
the maximum chlorophyll a synthesis per mg 
protein at a light intensity of 25 p-Em — 2 S —1 
and showed a higher photosynthetic rate 
similar to the report of Takahashi et al. 
(1972). The relationship between chloro-
phyll content and light intensity have already 
been reported by Sistrom (1962), Holt & 
Tvlarr (1965), Takahashi a al. (1972) and 
hevendaran ' (1984) for purple bacteria, 
and Nielsen and Jorgensen (1968) for green 
algae. Their results showed continuous 
increase of chlorophyll per mg protein as 
zero light intensity was approached. In the 
present data, however, samples grown in 
very low light (10 and 15p-Em — 1)showed 
lower chlorophyll levels than those at 25 
(.1E m -2 S —1 . 
Further, Fuller et al. (1963) and Cohen-
Bazire (1963) have reported that the plastids 
of photosynthetic bacteria change morpho-
logically depending on light intensities : 
samples grown in 'high' light had low num-
bers of chromatophores and sometimes 
fewer lamellae than corresponding samples 
grown in 'low' light. However, cells grown 
under anerobic dark condition (Uffen 
Wolfe, 1970) have less membrane system 
of characteristic chromatophore than in 
anaerobic light condition. Further, Rho-
dospirillum rubrum which was grown anero-
bically, in the dark, was exposed to an actinic 
light beam at 874 nm, the cytochromes 
become oxidized as characterized by a 
decrease in absorption, and the absorption 
capacity was regained by cytochrome and 
became reduced when the actinic light was 
shutt off. These data indicate that cells of 
R. rubrum growing under anerobic dark 
condition continued to synthesize a com-
plete light responsive pyotosynthetic appara-
tus. These changes in plastids and chro-
matophores might also influence the photo-
synthetic rates. 
The uptake of ammonia by the photo-
synthetic bacteria plays an important role 
Vol. 25, 1988 
40 
0 
20 
03 0-01 0.05 	 0.1 	 02 
o-o Bocteriochlorophyll 
o------o Growth curve 
Ammonium chloride concentration (% w./y) 
00 
80, 
60 6 
100 
at 
= so 
60 
g 40 
E 
E 
6 20 
90 	 K. DHEVENDARAN 
in the reduction of toxicity of ammonia, 
which is one of the major inhibitory meta-
bolites of the fish and is considered to be a 
powerful fish poison. It is also produced 
by the ammonification process of the organic 
nitrogenous matter anerobically in the sedi-
ments (Ram et al., 1980). esides this, the 
sediment serves both as a source from which 
ammonia may leak out and diffuse into the 
water column, as well as a sink and trap for 
the nitrogenous matter from the water-
column. In order to establish the physiolo-
gical role of C. buderi, an attempt has been 
made to know the uptake of ammonia for 
growth and utilization for synthesis of organic 
compound using ammonium chloride. The 
uptake of ammonium chloride is summari-
zed in Fig. 4 and it shows that it is more 
rapid at lower concentration rather than in 
higher concentration. At 0.1 % level the 
rate of bacteriochlorophyll a synthesis was 
Fig. 4. Effect of ammonium chloride on bacterio-
chlorophyll a synthesis and growth of C. 
buderi 
30 % of maximum synthesis whereas between 
0.1 and 0.3 % level the rate of synthesis was 
maximum. It is understood that the uti-
lization at this stage might be due to nitrogen 
assimilation with the carbon skeleton stored 
in the cell during photosynthetic activity. 
This is confirmed by the observation made 
by Shilo & Rimon (1982) with Microcystis 
marginata. With further increase in con-
centration it exerted a declining trend and 
it may inhibit the enzymatic pathways as 
suggested by Losada & Guerrero (1979). 
Subsequently, Flores et al. (1980) observed 
that ammonium at low concentration caused 
a rapid and effective inhibition of nitrate 
utilization in cyanobacterium, Anacystis 
nidulas. The actual factor determining the 
ammonium effect could thus be either the 
entry of ammonium into the cells or its incor-
poration into amino acids. Transport of 
ammonium into the cells might interfere 
with that of nitrate by competing for some 
common transport element or energy source 
that is present in limiting amounts. Evidence 
has been shown by Laane et al. (1980) and 
Jones & Monty (1979) indicating that short-
term depressing effect of ammonia on nitrogen 
fixation in Azotobacter vinclandil and 
Rhodopseudomonas sphacroides. It is noted 
that excess ammonium accumulation resulted 
in the bleaching of the cells thereby causing 
the 'nitrogen chlorosis' as observed by Liere 
et al. (1977) in the blue-green algae, Oscilla-
toria agardhil. 
It seems that there is a close relationship 
existing between the ammonium utilization 
and bacteriochlorophyll a synthesis to the 
specific concentration of ammonium chloride. 
The medium without the ammonium chloride 
showed only a feable concentration of 
bacteriochlorophyll a . It is understood 
that the actual factor for determining the 
ammonium effect for the normal metabolism 
of the bacterial cells is thus imperative. 
Since the role of marine photosynthetic 
bacteria in the nitrogen cycle of the sea is 
yet to be fully established, it could be more 
appropriate to study the metabolic pathways 
with intermediate enzymes. 
The author is thankful to the authorities of Uni-
versity College of Northwales, U.K., for providing 
facilities, to Ministry of Education and Culture, 
India for the award of Indian merit scholarship and 
to Prof. P. Natarajan, Professor and Head of the 
Department of Aquatic Biology and Fisheries, Uni-
versity of Kerala for his encouragement. 
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(1979) Can. J. Microbiol. 25, 999 
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Cohen-Bazire, G. (1963) in Bacterial 
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FISHERY TECHNOLOGY 
CULTURAL CON ITIONS OF MA INE CHROMATIUM BUDERI 
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Dhevendaran, K., Chandramohan, D. & 
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Vol. 25, 1988 


Studies on cultural conditions of marine Chromatium buderi Truper and Jannasch 1968

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Studies on cultural conditions of marine Chromatium buderi Truper and Jannasch 1968

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