International Journal of Marine Science 2015, Vol.5, No.50: 1-5
2
bifermentans
is a motile, gram-positive anaerobic
bacterium.
Cochin estuary, situated along the south west coast of
India, is highly vulnerable to environmental pollution
due to discharge from industrial, agricultural and
domestic effluents. Around 0.104M m
3
/d of waste
containing organic load are being discharged into
Cochin estuary by 16 nearby industries (Balachandran
et al., 2002). Total heavy metal content of sediments
of Cochin estuary was higher than the average values
reported from other Indian rivers (Table 1) (Mohan et
al., 2012). The present study aims to determine the
resistance to selected heavy metals (Pb, Cd and Cu)
and growth pattern of an anaerobic bacterium
Clostridium
bifermentans
isolated from Cochin estuary, Kerala.
Table 1 Comparison of concentration of Cd, Pb, Cu in
Vembanad Lake with permissible limits (Mohan et al., 2012)
Heavy
metal
Concentration of
heavy metals in
Vembanad Lake
Background
concentration
Cadmium
3.79 - 17.64 µg/g
2 µg/g, USPHS (1997)
Lead
26–630 µg/g
0.5 µg/g, USPHS (1997)
Copper
9.92–504.67 µg/g 30 µg/g, USPHS (1997)
2 Materials and Methods
2.1 Culture media and isolation of
Clostridium
bifermentans
SRB associated anaerobic bacterium
Clostridium
bifermentans
was isolated from sediments of Cochin
estuary (Figure 1) by using Sulphate API medium. 5 g
sediment was directly added to 95 mL Sulphate API
broth and incubated anaerobically for 7 days at 37°C.
Individual colonies were isolated using pour plate
method by using 0.1 ml of incubated culture in
Sulphate API agar plate. Single colonies again
transferred to 30 mL of Sulphate API broth and again
pour plated for obtaining pure culture.
Pure culture was maintained in API broth; Yeast
extract, 1 g/L., Magnesium sulphate, 0.2 mg/L., Dipot-
assium phosphate, 0.1 mg/L., Ferrous ammonium
sulphate, 0.1 mg/L., Sodium chloride, 10 mg/L., Ascorbic
acid, 0.1 mg/L supplemented with sodium lactate
(Himedia laboratories Ltd., India). Pure cultures were
maintained in 100ml serum bottle containing API
broth and made anaerobic by flushing 100% nitrogen
gas for seven days incubation.
Figure 1 Map showing sampling location
About 1000 µg/mL stock solutions for selected heavy
metal salts were prepared from Analytical grade metal
salts (CuSO
4
.5H
2
O, PbN
2
O
6
, Cd (NO
3
)
2
.4H
2
O) in
ultrapure water, sterilized by autoclaving and stored at 4
°
C.
2.2 Growth studies
Growth pattern of
Clostridium bifermentans
was
studied in 30 mL API broth supplemented with
different concentrations (2 to 14 µg/mL) of heavy
metals (Pb, Cd, Cu). pH 7 was maintained. Medium
were inoculated with 0.3 mL young culture (48hr) of
Clostridium bifermentans.
Each bottle was incubated
for 54 hours at 37°C. Control was maintained without
adding metal in media containing bacterial culture.
Anaerobic condition was maintained in all stages.
Growth of
Clostridium bifermentans
was monitored
by measuring optical density at 595 nm using UV-VIS
Spectophotometer (UV MINI-1240, Shimadzu).
2.3 Heavy metal tolerance
Clostridium bifermentans
susceptibilities to heavy
metals (Cu, Cd, and Pb) were determined by minimal
inhibitory concentration (MIC). The MIC is defined as
the lowest concentration of metal that inhibited
growth (Nieto, et al., 1989). 0.3mL of 48 hr incubated
cultures was added to serum bottle containing 30mL
broth. Different concentrations (2 to 14 µg/mL) of
heavy metal salt solutions were added to each bottle
containing Sulphate API medium and anaerobically
incubated for 54 hours at 37°C. A positive control
consisting of a metal deficient medium inoculated with
Clostridium bifermentans
was maintained. A negative
control consisted of broth medium without adding
microorganism.
