Marine Biology 110, 161-163 (~991)
Marine
Purple photosynthetic bacteria from a tropical mangrove environment
R.R. Vethanayagam
Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai 608 502, Tamil Nadu, India
Date of final manuscript acceptance: Aprii 9, 1991. Communicated by O. Kinne, Oldendorf/Luhe
Abstract. Purple photosynthetic bacterial strains were
isolated from m u d samples collected from the mudflats of
Pichavaram mangroves (Tamil Nadu, India) in 1989 and
1990. The presence of two m a j o r groups of photosyn-
thetic purple bacteria was recorded, viz., G r o u p 1: purple
sulphur bacteria (family Chromatiaceae, strains belong-
ing to Chromatium sp.); and G r o u p 2: purple nonsulphur
bacteria (family Rhodospirillaceae, strains apparently
belonging to Rhodopseudomonas sp.).
Introduction
The presence of purple bacteria in a "reducing environ-
m e n t " rich in sulphide, which is generally unfavourable
for healthy growth, is very interesting. Such bacteria are
capable of using light to grow, fix nitrogen and produce
hydrogen gas in this hostile environment. Earlier studies
have revealed that strains belonging to Chromatium sp.
(family Chromatiaceae - purple sulphur bacteria) and
Chloroflexus sp. (family Chloroflexaceae - microfilamen-
tous green photosynthetic bacteria) occur in this man-
grove environment (Krishnamurthy et al. 1986).
Purple sulphur bacteria (PSB) range in colour from
pink to purple and contain bacteriochlorophyll a as their
major pigment. These phototrophic anaerobes require
sulphide, which they oxidise to sulphate for growth; CO 2
is the usual source of cell carbon, but they can also utilize
the various organic acids as carbon sources. Purple sul-
phur bacteria are widely distributed in sulphide-rich re-
ducing environments such as mangroves, mudflats, pol-
luted waters, etc.
Purple nonsulphur bacteria (PNB) range in colour
from brown to red and also contain bacteriochlorophyll
a as their major photosynthetic pigment. They have the
ability to utilize a remarkably wide spectrum of reducing
carbon compounds, like malate or succinate, as electron
donors and as carbon sources for growth (Mitsui et al.
1980, K u m a z a w a and Mitsui ]982). They are also capa-
ble of aerobic growth in the dark, as well as anaerobic
===Biology
@ Springer-Verlag 1991
growth in the light. They can also utilize sulphide, at even
lower concentrations than those required by PSB.
Marine purple photosynthetic bacteria from tropical
environments have earlier been successfully isolated (Tru-
per ]970, Ohta et al. 1981, I m h o f f 1983, 1988 a, b). In the
present study, an anaerobic technique was successfully
used to isolate tropical strains belonging to the two dom-
inant groups of purple phototrophic bacteria. The strains
were identified on the basis of cell m o r p h o l o g y and bio-
chemical and physiological analysis (Vethanayagam
1990). In addition, in vivo light absorption spectra of cell
suspensions were recorded to identify the predominant
photosynthetic pigment and the nature of carotenoids.
Materials and methods
Mud samples were collected in 1989 and 1990 from the mudflats of
Pichavaram mangroves (Tamil Nadu, India). Four sites (Madai,
Periyakkadavu, Karithurai, and Chinnavaikkal) were selected for
the study.
From each of these areas, a few grams of wet mud with an
adhering film of water were brought in presterilized MacCartney
bottles to the laboratory within 3 h (to minimize changes in the
microflora). The mud was inserted into screw-cap test tubes (35 ml
capacity) with the help of a sterile spatula. The tubes were then filled
to the brim with media and sealed so that they were air tight. The
medium for PSB (a modification of Pfennig's medium) contained
1.0 g NH4C1, 1.0 g KH2PO4, 1.0 g Na2S (added after autoclaving),
0.2g MgC12.6H20, 0.2g CaClz-2H20, 1.0g NaHCO3, 0.5g
ascorbic acid, 2.82 g sodium acetate, 1.0 g yeast extract, and I 1aged
sea water (ca. 3 mo old). The pH was adjusted to between 7 and 7.5.
The medium for PNB (also a modification of Pfennig's medium)
contained 1.5 g NH4C1, 1.0 g KHzPO4, 0.2 g MgC12-6 H20, 1.0 g
NaHCO 3, 0.2 g C a C 1 2 - 2 H20, 1.0 g yeast extract, 1.0 g malic acid,
0.5 g ascorbic acid, and i 1 aged sea water (ca. 3 mo old). The pH
was adjusted to between 6.5 and 7.
The mud samples were also subjected to Winogradsky's cylinder
technique as described by Carr (1969) for isolating photosynthetic
bacteria. The screw-cap tubes and the Winogradsky column were
placed at a radius of ca. 50 cm around a 60 W bulb.
The photosynthetic bacteria were enriched by transfer every
week into fresh medium, using sterile pasteur pipettes. They were
then purified by the agar-shake culture method described by Pfen-
nig and Truper (1981), a convenient method for creating anaerobic
162
2.0
[ 418.4
1.8 ~ 478.8
, 1.6
"0
586.8
o o
L
0 1.4
854.0
799.2
1.2
J
1.0 r-----
4OO 500 600 700 800
Wavelength (nm)
Fig. 1. Chromatium sp. Absorption spectrum
conditions for optimum culture growth. Isolated organisms were
then examined for cell morphological characters under a phase
contrast microscope (Nikon). The absorption spectra of these bac-
teria were also recorded as recommendedby Haskins and Kihara
(1967), to determinethe nature of photosyntheticpigmentspresent.
Results
Both PSB and PNB took ca. 4 d to make their initial
appearance in the screw-cap test tubes. The Winogradsky
column yielded interesting results, in that all forms of
photosynthetic bacteria appeared as coloured spots on
the mud-glass interface after 10 d of incubation. The
coloured spots (colonies), which were carefully scooped
out and purified by the agar-shake culture method, in-
cluded Chromatium sp. and what appeared to be
Rhodopseudomonas sp.
The pure strains of Chromatium sp., when grown in
the presence of sulphide, formed sulphur globules inside
their cells. The sulphur globules appeared evenly distrib-
uted within the cell. (On storage of the strains for 3 to
4 mo in a refrigerator at 4 to 6 °C, sulphur globules disap-
peared; to restore these globules, cells required feeding
with 0.03% sulphide.) Individual cells were rod-shaped
and motile. They did not contain any gas vacuoles.
The strains of (?)Rhodopseudomonas sp., even when
grown in the presence of sulphide, did not reveal any
sulphur globules inside their cells. Individual cells were
rod-shaped and motile. It was also observed that the cells
did not attain a spherical shape in medium below pH 7.
Cell suspension color of the Chromatium sp. strains
ranged from purple to pink, and in (?)Rhodopseudomonas
R.R. Vethanayagam: Purple photosynthetic bacteria
371,6
1.0-
t
0.8-
848.8
I
._,~ 0,6-
79i. 8
0 0,4-
0.2-
I I i I I
900 400 500 600 700 800 900
Wavelength (rim)
Fig. 2. Rhodopseudornonas sp. Absorption spectrum
sp. was red. The absorption spectrum of Chromatium sp.
(Fig. 1) showed absorption maxima at 854, 799.2, 586.8,
521,478.8, and 418.4 nm. The absorption spectrum of
(?)Rhodopseudomonas sp. (Fig. 2) showed maximal peaks
at 848.8,796.8, 589.6, 486 and 371.6 nm.
Discussion
The main physiological property of purple photosynthet-
ic bacteria is their ability to grow rapidly even under
extreme anaerobic conditions by using only visible light
as their energy source. This characteristic was quite evi-
dent during growth and isolation of our photosynthetic
bacteria. Strains belonging to Chromatium sp. and
(?)Rhodopseudomonas sp. were found in all mud samples
tested and hence were considered to be the predominant
photosynthetic bacterial flora. The Chromatium sp.
strains were earlier identified from this mangrove envi-
ronment by Krishnamurthy et al. (1986).
Medium containing HES is selective for sulphide-oxi-
dising bacteria such as Chromatium sp. Physiologically,
those strains which contain sulphur globules inside their
cells must be able to oxidize sulphur further to sulphate
(Pfennig 1977). In Rhodopseudomonas sp. no elemental
sulphur was formed as an intermediate oxidation prod-
uct.
Chromatium sp. strains comprised rod-shaped, highly
motile cells with no gas vacuoles. These features also
form prominent key characters in C. vinosum, C. violas-
cens, C. gracile, C. minutissimum, C. purpuratum, C.
tepidum, C. minus, C. okenii, C. weissei, C. warmingii and
R.R. Vethanayagam: Purple photosynthetic bacteria
C. buderi ( h n h o f f 1988a). A rod-like cell shape is also
characteristic of strains belonging to the genus Rho-
dopseudomonas ( l m h o f f 1988 a); furthermore, these bac-
teria do not become spherical below p H 7. These have
been found to be key features in R. palustris, R. viridis,
and R. acidophila (Pfennig and Truper 1974).
The colour of the Chromatium sp. strains varied from
purple to pink. The same colour variation pattern has
also been observed in cultures of C. buderi, C. minus and
other Chromatium spp. (Truper and Pfennig 1981). The
colour of Rhodopseudomonas sp. cultures was found to
vary between brownish-red and red. This is in conformity
with results obtained for cultures of R. palustris and
Rhodobacter sulfidophilus by Truper and Pfennig (1981).
The absorption spectrum of Chromatium sp. (Fig. 1)
revealed the presence of bacteriochlorophyll a. The peak
at 521 nm would indicate the presence of the okenone
series of carotenoids, which give a pink to purple colour
(Truper and Pfennig 1981). This is a characteristic feature
of C. purpuratum, C. minus, C. okenii and C. weissei
(Imhoff, 1988 a). The absorption spectrum of Rhodopseu-
domonas sp. (Fig. 2) also revealed bacteriochlorophyll a
to be the principal photosynthetic pigment. The peak at
486 nm indicates the presence of carotenoid spirilloxan-
thin (in the normal spirilloxanthin series), which pro-
duces coloration in the culture (Truper and Pfennig
198l); this feature is unique to the species R. palustris,
R. sulfoviridis, R. rutila and R. marina ( I m h o f f 1988a).
The absorption spectra of PSB and PNB displayed
c o m m o n peak values at, respectively, 854 and 849, 799
and 797, and 587 and 590 nm. These peak values confirm
the presence of bacteriochlorophyll a in the two groups.
Further electron microscope studies will be helpful in
determining these groups' correct systematic status.
This study demonstrates that photosynthetic bacteria
can be easily isolated from a mangrove environment.
Such bacteria could contribute significantly to primary
productivity of coastal seaboards and to food-web dy-
namics of various tropical coastline ecosystems.
Acknowledgements. I am indebted to Prof. K. Krishnamurthy for
his supervision and encouragement of this research. I thank the
Director of the Centre of Advanced Study in Marine Biology, and
the authorities of Annamalai University for providing necessary
facilities to carry out this work. I also thank Prof. C. Lakshmi-
narasimhan, Department of Botany, A.V.V.M. Sri Pushpam Col-
lege (Autonomous), India, for providing facilities in his laboratory
for use of the Nikon microscope in this study.
163
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