Use of Indigenous Bacteria from Arsenic Contaminated Soil for Arsenic Bioremediation - Management of Natural Resources in a Changing Environment - page 151

Arsenic resistant bacterial strains were isolated from soil sample by serial
dilution and plating on nutrient agar medium (HiMedia, India) supplemented with
various concentration of arsenite (0.5-2 mM) as NaAsO 2 (HiMedia, India). The
isolate which showed maximum level of As resistance (20 mM) was fi nally selected
for further experiment. The isolate was then purifi ed to have a pure culture by cycles
of single colony isolation and liquid culture transfer on minimal broth (MB) supple-
mented with 2 mM As(III) and maintained for further experiment. The obtained
bacterial isolate was designated as KUMAs1.
The isolate was identifi ed based on the morphological and standard
biochemical tests according to Bergey's Manual of Systematic Bacteriology
(Sneath 1986 ). The ~1.309 Kb 16S rDNA fragment was amplifi ed from the
genomic DNA of KUMAs1 using 16S rRNA bacterial specifi c forward and
reverse primer sets 5 ′ -AGAGTYTGATC(AC)TGNCTYAG-3 ′ and 5 ′ -TACGG(CT)

TAYCTTGTNACRACYT-3 respectively (Amnion Bioscience, India). The

sequence data were aligned and analyzed using NCBI Blast function (Altschul et al.
1990 ) and Ribosomal Database Project (Maidack et al. 1997 ). The phylogenetic
lineage of KUMAs1 was built from 16S rDNA sequence using MEGA 4.0
(Tamura et al. 2007 ).
Minimum inhibitory concentration (MIC) values of As(V) as Na 2 HAsO 4 .7H 2 O
(HiMedia, India) and As(III) as NaAsO 2 (HiMedia, India) were determined in both
Nutrient broth (NB) and MB having pH 7.2. Cells of KUMAs1 were grown in NB
and MB initially for 14 h at 37 °C; 1 % culture (v/v) was inoculated in 5 ml of both
media separately having different concentrations of As(III) (0-25 mM) and As(V)
(0-500 mM) and incubated at 37 °C for 48 h. MIC was determined by counting
colony forming units (CFU/ml) by serial dilution plating on respective medium.
MIC values of other heavy metals like cadmium (CdCl 2 .H 2 O), zinc (ZnSO 4 .7H 2 O),
chromium (K 2 CrO 4 ), cobalt (CoCl 2 .6H 2 O) and nickel (NiCl 2 .6H 2 O) against
KUMAs1 were also determined in NB and MB following the same procedure as
mentioned above.
Twelve different antibiotics discs (HiMedia, India) namely, Tetracycline (30

′

ʼ g/

disc), Imipenem (10 ʼ g/disc), Tobramycin (10 ʼ g/disc), Polymyxin B (300 units),

Ticarcillin/Clavulanic acid (75/10 ʼ g/disc), Gentamycin (10 ʼ g/disc), Amikacin

(30 ʼ g/disc), Netillin (30 ʼ g/disc), Colistin (10 ʼ g/disc), Ciprofl oxacin (5 ʼ g/disc),

Kanamycin (50 g/disc) were tested against KUMAs1

to determine the sensitivity on Mueller-Hinton agar medium (HiMedia, India).
Growth response of KUMAs1 under aerobic condition was determined in pres-
ence of As (2 mM) both in NB and MB supplemented with either in the form of
As(V) or As(III) and compared with their respective control sets. The growth
response was determined by counting the CFU/ml at different time intervals. The
effect of pH and temperature on growth was also determined in MB supplemented
with 2 mM of either As(V) or As(III).
For determining the effect of arsenic on cell morphology, cells of KUMAs1 were
grown in NB medium for 14 h and 1 % culture (v/v) was later inoculated in fresh
medium having 2 mM As(V) and 2 mM As(III), respectively and incubated for 4 h.
Microscopic observation of the cells was carried out under a phase contrast

ʼ g/disc) and Ampicillin (50 ʼ