Environmental Engineering Reference
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microscope (Leica DM 750, Germany). The cell morphology was compared with
the cells grown without arsenic.
Reduction of As(V) to As(III) was studied following Hu et al. ( 2012 ). It was car-
ried out in presence of 0.07 mM, 0.14 mM, and 0.21 mM of As(V) in MB inocu-
lated with 1 % (v/v) of 14 h grown culture. The medium was incubated at 37 °C with
shaking. The cell-free supernatant was used to determine the amount of As(III)
produced through reduction. Due to limitation of the spectrophotometric method of
arsenic species analysis, in this experiment such low concentration was used. The
cell-free supernatants were obtained at intervals of 24 h by centrifugation (6,000× g,
at 4 °C for 10 min). Cell-free extract (30 ml) was then acidifi ed with 1 % HCl and
M/l phosphate. Three parallel sub-samples, 10 ml each, were treated with 1 ml
of an oxidizing reagent (KMnO 4 ), a reducing reagent (NH 4 N 2 S) and deionized (DI)
water (untreated sub-sample), respectively. Sample treated with reducing agent was
incubated at 80 °C for 30 min and cooled to room temperature. After 30 min, 1 ml
colour reagent [a mixture of 10.8 % C 6 H 8 O 6 ; 3 % (NH 4 ) 6 Mo 7 O 24 .4H 2 O; 0.56 %
C 8 H 4 K 2 O 12 Sb 2 .3H 2 O and 13.98 % H 2 SO 4 in a volume ratio of 2:2:1:5] was added to
each sub-samples and the absorbance was measured after 5 min at 880 nm using a
spectrophotometer. The As(III) and As(V) concentrations were calculated using the
equations: Total As = Oxidized - Reduced; As (III) = Oxidized - Untreated; As
(V) = Untreated - Reduced. The percentage of As(V) reduction by the biomass was
calculated using the equation: R (%) = (Co − Ce) × 100/Co; Co and Ce are the initial
and equilibrium concentration of arsenate (mg/l) in the solution, respectively.
To determine arsenic removal effi ciency of KUMAs1, 1 % (v/v) of 14 h grown
culture was inoculated in MB supplemented with 2 mM of either As(V) or As(III)
and incubated for 96 h on a rotary shaker at 37 °C. Control sets were prepared with
same concentration of either form of As but without inoculation to determine the
artifacts might arise due to the metalloid sorption on the glass surface of the con-
tainer. Cell pellet from each treatment regime were harvested by centrifugation at
6000× g for 10 min after 48 h, 72 h and 96 h of incubation. The cell pellets were then
acid-digested and the total arsenic content in the digest was determined by atomic
absorption spectroscopy. The As removal percentage by the biomass was calculated
using the equation: R (%) = Ce × 100/Co; Co and Ce are the initial and equilibrium
concentration of As (mg/l) in the solution and pellet respectively.
Results and Discussion
Soil Analysis
The pH, organic carbon and arsenic concentrations of the experimental soil were
found to be 7.39, 0.83 % and 3.86 ppm (mg/kg soil), respectively. Average total As
content in such kind of agricultural soil irrigated with ground water has earlier been
reported to be between ~1.38 and 12.27 mg/kg (Bhattacharya et al. 2009 ).
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