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Chapter IV: Results and Discussion
CHAPTER IV
RESULTS AND DISCUSSION
4.1 Isolates screened from the solid waste and liquid effluent.
Two isolates (one each from solid waste and the other from liquid effluent) were
screened for microorganisms which most chromium tolerant. The two isolates were
further investigated for the toxic effect of chromium by examining the morphological
changes and metal-accumulation by the organisms. The isolate obtained from the solid
waste, named S1, showed yellow colonies (Figure 4.1 (a)), whereas the one isolated from
the liquid effluent, named L4, showed white smooth colonies (Figure 4.1 (b))
Figure 4.1: Chromium microorganism isolated from
(a) solid waste (S1)
(b) liquid effluent (L4)
The biochemical characteristics of both the isolates were determined and listed
in Table 4.1 and were cross-checked with reference from Bergey’s Manual of Systematic
Bacteriology (Volume 2). The information provided is similar to that reported by Pal and
51
Chapter IV: Results and Discussion
Paul (2004). These organisms were then sent for 16S RNA analysis to identify the species
these isolates belong.
Table 4.1 Biochemical characterization of the isolates from solid waste and liquid
effluent.
Characteristics
S1 (solid waste)
L4 (liquid effluent)
Morphological characteristics:
Colony morphology
yellow, round
white, small round
Gram reaction
+ve
+ve
Micro morphology
Rod
Cocoi
Production of catalase
+ve
+ve
Production of oxidase
+ve
+ve
Hydrolysis of starch
-ve
+ve
Gelatin hydrolysis
-ve
-ve
Triple sugar iron (TSI) test
ferments lactose neither glucose nor lactose
and/or sucrose
or sucrose is fermented
Citrate utilization test
-ve
Biochemical characteristics:
-ve
4.2 Effect of chromium toxicity on the growth of isolated organisms
Several reports have been published on the effect of chromium on the growth of
microorganisms.
There are reports where the growth of organism is retarded when
exposed to chromium. Here in this report, effect of chromium when added during the
52
Chapter IV: Results and Discussion
exponential phase is reported. No paper has been reported so far that determine the cells’
behavior when metal is added in their exponential phase. Generally papers are published
where authors have reported the addition of chromium at start of the experiment. Here, the
cell growth during incomplete availability of the nutrients (during log phase) was
examined. It was interesting to know the cell behavior in presence of enzymes produced
during growth phase and then chromium was added. The cell adaptability against the
chromium toxicity was determined when Cr is added from initial 0 ppm to 40 ppm (as
K2Cr2O7). Figure 4.2 shows the growth of the organisms in a batch culture.
3
OD (600nm)
2.5
2
1.5
S1
1
L4
0.5
0
0
10
20
30
40
50
time (hrs)
Figure 4.2: Batch growth of S1 and L4 (in the absence of chromium)
From the Figure 4.2, it can be seen that the exponential phase occurs after 8 hours,
with the stationary phase occurring after 24 hours. The following figures show the effect
of increasing concentration of chromium on the growth of microorganisms. Figure 4.3(a)
shows the effect of chromium (1 ppm) on the metal-unexposed cells, with chromium
added during the exponential phase. An OD of 0.70 and 0.65 was attained by S1 and L4
respectively during the exponential phase. Figure 4.3 (a) showed that S1 couldn’t grow in
the presence of chromium, whereas L4 was able to tolerate the chromium presence.
53
Chapter IV: Results and Discussion
Hence, the adaptability towards chromium was slower in case of S1 when compared to
L4.
2.5
OD (600nm)
2
1.5
S1
L4
1
0.5
0
0
10
20
40
50
time (hrs) 30
Figure 4.3 (a): Toxicity effect of Cr (1 ppm) on metal unexposed organisms
There are reports of decreased microbial activity due to exposure/stress of metals
(Maliszewska et. al., 1985). Cr (VI) was found to inhibit soil biological properties, such as
phosphatase and sulfatase activities and to decrease microbial biomass (Speir et. al.,
1995). Although the extent of inhibition caused by Cr (VI) diminished with time, the
differences were generally much smaller than the observed decline in extractability of Cr
(VI) (Speir et. al., 1995). The increase in adaptability towards chromium is shown in this
study. S1 initially when exposed to Cr (VI) showed decreased growth, but further it was
observed when these 1 ppm exposed cells were exposed to higher concentration of Cr
(VI), it show enhanced resistance. Figure 4.3(b) shows the behavior of these isolates when
2 ppm is added at exponential phase to freshly grown 1 ppm exposed cells.
54
Chapter IV: Results and Discussion
3.5
OD (600nm)
3
2.5
S1
2
L4
1.5
1
0
5
10
15
20
25
30
35
40
time (hrs)
Figure 4.3 (b): Toxicity effect of Cr VI (2 ppm) on 1 ppm Cr exposed cells.
From the above Figure 4.3 (b), it could be interpreted that the bacteria develop a
mechanism where it is able to tolerate higher concentration of metal. S1 is able to grow
better in the presence of toxic chromium when compared to previous batch i.e. unexposed
batch in Figure 4.3(a). Also it was observed that L4 has also gain resistance when
compared to previous batch (figure 4.3(b)). Turpeinen et. al., (2004) has demonstrated a
clear relationship between bioavailability of arsenic and As (III) resistance, indicating that
there had been a selection for As (III) resistant bacteria in the contaminated soils due to
high toxicity of As (III). In this present study, step-wise adaptation of the microorganisms
to gain resistance towards chromium is demonstrated. The effect of chromium on the
growth characteristics and its slow resistance development when metal is added in
exponential phase is reported. The following Figures 4.3 (c-f) show how these two isolates
have developed resistance from Cr concentration ranging from 1 ppm concentration of Cr
to 40 ppm concentration.
55
Chapter IV: Results and Discussion
4.5
OD (600nm)
4
3.5
3
S1
2.5
L4
2
1.5
0
5
10
15
20
25
30
35
time (hrs)
Figure 4.3 (c): Toxicity effect of Cr VI (4 ppm) on 2 ppm Cr exposed cells.
From Figure 4.3(c), it can be seen that the cells have adapted resistance towards
chromium very rapidly. From the absorbance, it can be seen that cells growing in 5 hours
is more when compared to Figure (4.3-b) where cells were only exposed to 2 ppm
chromium. This clearly shows that the cells are developing resistance towards chromium
subsequently.
5
4.5
OD (600nm)
4
3.5
3
2.5
S1
2
L4
1.5
1
0.5
0
0
10
20
30
40
50
60
70
time (hrs)
Figure 4.3 (d): Toxicity effect of Cr VI (10 ppm) on 4 ppm Cr exposed cells.
56
Chapter IV: Results and Discussion
When increasing concentration from 4 ppm to 10 ppm, a slow growth pattern of
the cells was evident (Figure 4.3-d). L4 shows adaption at this concentration as well
(though over a slightly longer period), but S1 had slow activity at this concentration
(Figure 4.3-d).
2.9
OD (600nm)
2.7
2.5
2.3
S1
2.1
L4
1.9
1.7
1.5
0
5
10
15
20
25
30
35
40
45
50
time (hrs)
Figure 4.3 (e): Toxicity effect of Cr VI (20 ppm) on 10 ppm Cr exposed cells.
From Figure 4.3(e), it was found that even at a concentration of 20 ppm (doubled
the previous concentration) exposure of chromium, S1 was able to grow in Cr milieu and
has shown maximum growth, though in a slightly longer time. L4, instead showed
constant increasing growth kinetics.
3.5
OD (600nm)
3
2.5
S1
2
L4
1.5
1
0.5
0
10
20
30
40
50
60
time (hrs)
Figure 4.3 (f): Toxicity effect of Cr VI (40 ppm) on 20 ppm Cr exposed cells.
57
Chapter IV: Results and Discussion
From this Figure 4.3(f), it can be seen that both the isolates have almost reached a
level where is not much further adaption taking place. It appears that the isolates have
reached their maximum chromium tolerance capacity. From the Figure 4.3 (a-f), L4 was
found to be marginally more tolerant to chromium. This has been shown in further
experiments of bioaccumulation, in relation to metal resistance.
4.3 16S rDNA Analysis
4.3.1 16S rDNA analysis of isolate from solid waste (S1)
Both the isolates S1 and L4 were analyzed for 16S rDNA for species
identification. On the basis of nucleotide homology and phylogenetic analysis, S1 was
found to be very similar to Bacillus marisflavi TF 11(AF483624) (Figure 4.4-a). Based on
nucleotide sequence, the percentage homology of this strain was found to be 98% similar
to Bacillus marisflavi TF 11(AF483624) and Bacillus aquimaris (DQ105971) (Table 4.2).
The isolate exhibits catalyse and oxidase activities.
DQ285074 (Bacillus sp.)
AY373362 (Bacillus sp.)
DQ105973 (Bacillus marisflavi)
55
DQ448746 (Bacillus sp.)
AF483625 (Bacillus aquaemaris st. TF-12)
100
AJ315068 (Bacillus sp.)
100
AJ244686 (Bacillus sp.)
AY505499 (Bacillus aquimaris st. GSP18)
DQ105971 (Bacillus aquimaris)
S1
46
AF483624 (Bacillus marisflavi st. TF-11)
Figure 4.4 (a): Phylogenetic Tree of S1 (using neighbor joining method).
58
Chapter IV: Results and Discussion
S. No.
Isolates
1
S1
2
AF483624
3
DQ105971
4
AY505499
5
DQ105973
6
AY373362
7
DQ285074
8
DQ448746
9
AF483625
10
AJ315068
11
AJ244686
PERCENTAGE HOMOLOGY
1
2
3
4
5
6
7
8
9
10
11
*
98
98
98
98
98
98
97
97
97
96
*
100 100 100
99
99
98
98
98
98
100 100
99
99
98
98
98
98
100
99
99
98
98
98
98
*
99
99
98
98
98
98
*
99
98
97
97
97
*
98
98
98
97
*
99
99
98
*
99
99
*
99
*
*
*
Table 4.2: Percentage homology of S1 based on nucleotide sequence
Several Bacillus species are known to remove chromium from contaminated soil
or effluent. However, there is no report on heavy metal removal by Bacillus marisflavi.
Yoon et. al., (2003) isolated Bacillus marisflavi st. TF 11 (AF483624) from sea water of a
tidal flat in Yellow Sea in Korea. Rafidinarivo et. al., (2007) isolated several bacillus
species from marine sediments, out of which Bacillus marisflavi is one of them. Bacillus
marisflavi has also been isolated from water pipeline in Gulf of Mexico (Lopez et. al.,
2006). Narita et. al., (2004) has shown the presence of mercury-resistant transposon
Tn5085 in Bacillus marisflavi. Riis et. al., (2003) has shown Bacillus marisflavi to be a
59
Chapter IV: Results and Discussion
potential hydro-carbon degrader. To the best of this authors’ knowledge, Bacillus
marisflavi is not known for chromium tolerance and bioaccumulation so far. S1 is a
chromium-tolerant bacillus strain which is only 98% similar with the nearest neighbor
(Bacillus marisflavi TF 11 (AF483624) and Bacillus aquimaris (DQ105971)) (Table 4.2).
Hence, S1 is a novel strain, in accordance to 16S rDNA analysis.
4.3.2 16S rDNA analysis of isolate from liquid effluent (L4)
The isolate from liquid effluent was found to be only 96% similar to one
Arthrobacter sp. with accession no. AB248532. From the phylogenetic tree (Figure
4.4(b)) and percent homology (Table 4.3), this isolate showed 94% and 93% similarity to
another Arthrobacter sp. (accession no. AB248526) and Arthrobacter nicotinovorans
(X80743) respectively.
AM409362 (Arthrobacter sp. st. 4C1-b)
AF408952 (Arthrobacter sp.)
AB248529 (Arthrobacter sp.)
AM409361 (Arthrobacter sp. St. 4C1-a)
AY651318 (Arthrobacter sp. st. ADG1)
54
65
AJ785761 (Arthrobacter sp. st. KA4-2)
95
AF102267 (Arthrobacter chlorophenolicus)
100
X80743 (A. nicotinovorans)
99
AB248526 (Arthrobacter sp.)
100
L4
100
AB248532 (Arthrobacter sp.)
Figure 4.4 (b): Phylogenetic Tree of L4 (using neighbor joining method).
60
Chapter IV: Results and Discussion
S. No.
Isolates
1
L4
2
AB248532
3
AB248526
4
X80743
5
AF102267
6
AJ785761
7
AY651318
8
AM409361
9
AB248529
10
AF408952
11
AM409362
PERCENTAGE HOMOLOGY
1
2
3
4
5
6
7
8
9
10
11
*
96
94
93
94
94
94
94
94
94
94
*
96
95
96
96
96
96
96
96
96
*
97
97
98
98
99
99
99
99
*
97
97
96
97
97
97
97
*
98
98
98
98
98
98
*
98
98
98
98
98
*
99
98
98
99
*
99
99
99
*
99
99
*
98
*
Table 4.3: Percentage homology of L4 based on nucleotide sequence
There are several reports on Arthrobacter species on removal or resistance towards
heavy metals, which include chromium. Ren et. al., (2004) isolated Arthrobacter
nicotinovorans that secrete a protease which released cadmium from scallop
hepatopancreas (the main residue after removing the edible parts of scallop) into liquid
medium. Similarly, Horton et al., (2006) has shown reduction of hexavalent chromium by
Arthrobacter aurescens. Arthrobacter viscosus supported on GAC (granulated activated
carbon) has been reported for treatment of chromium (Quintelas et. al., 2009; Quintelas
and Tavares, 2001). Toxicity of hexavalent chromium and its reduction by Arthrobacter
61
Chapter IV: Results and Discussion
sp. isolated from soil contaminated with tannery waste is also known (Megharaj et. al.,
2003). Edgehill (1996) has shown the effects of copper-chrome-arsenate (CCA)
components on pentachlorophenol (PCP) degradation by Arthrobacter strain ATCC
33790. The isolate that is reported in this report is only 96% similar to
Arthrobactersp.(accession no. AB248532). It is only 94% and 93% similar to another
Arthrobacter sp. (accession no. AB248526) and Arthrobacter nicotinovorans (X80743)
respectively. Hence, the strain L4 is novel according to 16S rDNA analysis.
4.4 Chromium removal assay
4.4.1 Chromium removal from synthetic effluent inoculated with isolate from liquid
effluent (L4):
Chromium removal capacity of Arthrobacter sp. (L4) for different initial
concentration of chromium added in logarithmic phase is shown in Figure 4.5(a). In this
experiment, the total chromium (Cr (VI) and Cr (III)) removal from the synthetic effluent
containing different initial concentration of Cr (added during log phase) in the form of
K2Cr2O7 was analyzed. From the Figure 4.5(a), it can be seen that more than 50% of the
chromium is removed during first 3-5 hours and more than 95% is removed from the
synthetic effluent within 24 hours. Several papers have reported the removal of chromium
by Arthrobacter sp. (Megharaj et al., 2003; Horton et al., 2006) but not on the removal of
chromium when added at log phase.
62
Chapter IV: Results and Discussion
45.00
40.00
Chromium in solution (ppm)
35.00
2 ppm
30.00
4 ppm
25.00
10 ppm
20.00
20 ppm
15.00
40 ppm
10.00
5.00
0.00
0
20
40
60
80
100
120
140
Time (hrs)
Figure 4.5(a): Removal of chromium by L4 at various initial concentrations (2-40
ppm)
4.4.2 Chromium removal from synthetic effluent inoculated with isolate from solid
waste (S1):
Chromium removal capacity of Bacillus sp. (S1) isolated from solid waste at
different initial concentration of chromium added in logarithmic phase is shown in Figure
4.5(b). In this experiment, total chromium (Cr (VI) and Cr (III)) removal from the
synthetic effluent containing different initial concentration of Cr (added during log phase)
in the form of K2Cr2O7 was analyzed. From the Figure 4.5(b), it can seen that more than
63
Chapter IV: Results and Discussion
50% of the chromium is removed during first 3-5 hours and more than 95% is removed
from the synthetic effluent in 24 hours. Several papers report for the removal of chromium
by Bacillus sp. Here, in this report a novel isolate which is 98% similar to Bacillus
marisflavi has been reported. Bacillus marisflavi is only known for degrading hydrocarbon
(Riis et. al., 2003). In this report, chromium tolerance and bioaccumulation capacity by S1
is reported.
45
40
Chromium in solution (ppm)
35
30
25
2 ppm
4 ppm
20
10 ppm
20 ppm
40 ppm
15
10
5
0
0
20
40
60
80
100
120
140
Time (hrs)
Figure 4.5(b): Removal of chromium by S1 at various initial concentrations (2-40
ppm)
64
Chapter IV: Results and Discussion
4.4.3 Comparative study between S1 and L4 for removal of Chromium
A comparison between the growth pattern of S1 and L4 at different initial concentration of
Chromium in solution (ppm)
chromium
added
during
log
phase
is
shown
in
Figures
4.6(a-e).
2.5
2
L4
1.5
S1
1
0.5
0
0
5
10
15
20
25
30
35
40
Time (hrs)
Chromium in solution (ppm)
Figure 4.6(a): Chromium removal at 2 ppm initial concentration of Cr for S1 and L4
4.5
4
3.5
3
L4
2.5
S1
2
1.5
1
0.5
0
0
10
20
30
40
50
60
Time (hrs)
Figure 4.6(b): Chromium removal at 4 ppm initial concentration of Cr for S1 and L4
65
Chapter IV: Results and Discussion
Chromium in solution
(ppm)
12
10
8
L4
6
S1
4
2
0
0
10
20
30
40
50
60
Time (hrs)
Chromium in solution
(ppm)
Figure 4.6(c): Chromium removal at 10 ppm initial concentration of Cr for S1 and
L4
25
20
L4
S1
15
10
5
0
0
5
10
15
20
25
30
35
40
45
50
Time (hrs)
Chromium in solution
(ppm)
Figure 4.6(d): Chromium removal at 20 ppm initial concentration of Cr for S1 and
L4
45
40
35
30
25
20
15
10
5
0
L4
S1
0
10
20
30
40
50
60
Time (hrs)
Figure 4.6(e): Chromium removal at 40 ppm initial concentration of Cr for S1 and
L4
66
Chapter IV: Results and Discussion
From all the above Figures 4.6(a-e), it can be observed that S1 (isolate from solid
waste: Bacillus sp.) was able to sequester more chromium when compared to L4 (isolate
from liquid effluent: Arthrobacter sp.). These results corroborate our earlier toxicity
studies, which shows that Arthrobacter sp. (L4) was more tolerant towards Cr, and was
unable to sequester more Cr compared to Bacillus sp. (S1). S1 was less tolerant to
chromium due to high amount of sequestration when compared to L4. The toxic effect of
Cr, as manifested in cell morphology, is reported later in section 4.10. Scott and Palmer
(1988) have shown that Arthrobacter sp. (A. viscosus) was able to produce more exopolysaccharides which accounts for the higher tolerance of the cells towards heavy metal.
The polysaccharides bind the heavy metal outside the cells, thereby protecting the cell
from lysis. In section 4.9 of this report, it has been shown that L4 produce more
polysaccharides when compared to S1. Hence it can be related that L4, being more
tolerant to Cr (as a result of more secretion of exo-polysaccharide) was unable to sequester
more Cr when compared to S1.
4.5 Intracellular compartmentalization of the chromium uptake:
Table 4.4 shows the relative distribution of chromium in the different compartments of S1
and L4, after metal uptake over 48 hours.
Cellular fraction
Periplasmic fraction
Cytoplasmic fraction
Membrane fraction
Chromium
Concentration
(ppm)
S1
L4
10.02
12.02
14.54
10.24
9.68
8.34
Table 4.4: Relative distribution of
accumulated Cr (initial conc: 40 ppm)
after 48 hours incubation.
67
Chapter IV: Results and Discussion
The values in the table were calculated taking average values obtained from
duplicate experiments. It can be inferred that out of 40 ppm, a total of 34.24 ppm and
32.60 ppm has been accumulated by S1 and L4 respectively. A mass balance obtained by
summing up the metal uptake and the chromium remaining in the solution in Figure 4.6
(e) shows that almost all the 40 ppm chromium was accounted. From Table 4.4, it can be
determined that periplasmic accumulation was more in the case of L4 when compared to
S1. The total intracellular accumulation was higher for Bacillus sp. when compared to
Arthrobacter one. The reason behind this less accumulation in the case of Arthrobacter sp.
could be production of more amount of EPS. Scott and Palmer (1988) have showed that
Arthrobacter is able to produce more amount of EPS and hence more resistant and less
intracellular uptaking.
4.6 FTIR analysis
The infrared spectra of the control biomass (i.e. in the absence of chromium) and
treated biomass (at 40 ppm exposed chromium) of L4 and S1 are shown in Figures 4.7(a)
and 4.7(b) respectively. The absorption peaks between 3400-3600cm-1 corresponds to -OH
and -NH stretching of the protein and acetamido group (Bai and Abraham, 2002; Deng
and Ting, 2005). The strong absorption peak ranging from 1640-1650 cm-1 is attributed to
amide I band of amide bond in N-acetyl glucosamine polymer or of the protein peptide
bond. It also relate to the C=O chelate stretching of -COOH functional group (Bai and
Abraham, 2002; Yee et al., 2004; Park et al., 2005). Another characteristic peak found in
68
Chapter IV: Results and Discussion
both the isolates relates to amide II band at the proximity of 1554 cm-1 (Kapoor and
Viraraghavan, 1997; Yee et al., 2004; Park et al., 2005). The peak at the proximity of 1400
cm-1 corresponds to the symmetric vibrations of the C=O of COO- (Yee et al., 2004). The
peaks at 1200-1250 cm-1 are related to the asymmetric stretching of phosphodiesters, free
phosphates, or monoester phosphate functional groups (Yee et al., 2004). The absorption
peak between 1050-1100 cm-1 corresponds to –CN stretching.
5 4
3
2
1
Absorbance
6
Treated Biomass
with 40 ppm Cr
Control
0
500
1000
1500
2000
2500
3000
3500
4000
4500
Wave number (cm-1)
Figure 4.7(a): FTIR analysis of L4 (Arthrobacter sp.) before and after chromium
exposure.
In above Figure 4.7 (a) after chromium treatment, six changes of the functional
groups on the biomass have been found. The first change (1) corresponds to the formation
of small peaks between 3200-3600 cm-1. These peaks may be due to overlapping of –OH
and –NH stretching. It is in consistent with the result given by Deng and Ting (2005).
However the hydroxyl group may not be involved in metal accumulation. The second
69
Chapter IV: Results and Discussion
change (2) was the slight disappearance of the peak at 2926 cm-1 . It may be assigned to
the –CH stretching of the functional groups (Kapoor and Viraraghavan, 1997; Park et al.,
2005). The third change (3) is the disappearance of the absorption peak at 1554 cm-1
attributed to the –NH bending after chromium adsorption. As amino group is the major
constituent of the cell wall, its bending may be due to chromium binding (Bai and
Abraham, 2002). Consistent with result given by Han et al. (2006), the fourth change (4)
was interpreted as the disappearance or weakening of peak at1400 cm-1. It could be
attributed to the complexation of carboxylate functional group by coordination with metal
ions. The fifth change (5) is the disappearance of peak at 1238 cm-1, which corresponds to
the involvement of –SO3 in metal removal (Lameiras et al. 2007). The last change (6)
corresponds to the shift of peak at 1078-1060cm-1, that could be due to the involvement of
C-O bond of polysaccharides in chromium attachment (Han et al., 2006)
5
4 3 2
1
Absorbance
6
Treated Biomass with
200mg/L Cr6+
Control
0
500
1000
1500
2000
Wave number
2500
3000
3500
4000
4500
(cm-1)
70
Chapter IV: Results and Discussion
Figure 4.7(b): FTIR analysis of S1 (Bacillus sp.) before and after chromium
exposure.
In Figure 4.7 (b), after Cr (VI) treatment, six changes of the functional groups on
the biomass have been found. The first change (1) was slight disappearance of the peak at
2926 cm-1. It may be assigned to the –CH stretching of the functional groups (Kapoor and
Viraraghavan, 1997; Park et al., 2005). The second change (2) corresponds to
strengthening of the peak at 1650 cm-1, that corresponds to the C=O chelate stretching.
This change is in consistent by Park et al. (2005) and suggests that the carboxyl groups are
involved in the binding of chromium. The third change (3) is the disappearance of the
absorption peak at 1554 cm-1, which is attributed to the -NH bending after chromium
adsorption. As amino group is the major constituent of the cell wall, its bending may be
due to chromium binding (Bai and Abraham, 2002).
The fourth change (4) is the
formation of new peak at 1457 cm-1, which is attributed to the asymmetric bending of –
CH3 of the acetyl moiety (Bai and Abraham, 2002). The fifth change (5) is the
disappearance of peak at 1230 cm-1 which relates to the C-O stretching of COOH group
and hence strengthens that the carboxyl group is involved in chromium binding. The last
change (6) is the shift of peak from 1065 to 1110 cm-1, which could be due to involvement
of the C-O of polysaccharides in chromium attachment. A similar study on C. miniata to
adsorb chromium also suggested that C-O is involved in metal uptake (Han et al., 2006).
4.7 X-ray diffraction (XRD) analysis
Both the isolates from solid waste and liquid effluent were analyzed using X- Ray
diffractometer before and after exposure to 40 ppm chromium. The diffraction pattern
71
Chapter IV: Results and Discussion
were compared with that obtained from JCPDS (Joint Committee for Powder Diffraction
Studies) - International center for diffraction data for peak identification. The peaks were
analyzed using PDF (Powder diffraction file) - version 4. Figures 4.8 (a) and (b) give an
overall scan within the range of 2θ from 10° to 80° for isolate L4 with and without
chromium respectively. Similarly Figure 4.8 (c) and (d) shows the results for isolate S1
with and without chromium treated respectively.
4500
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Figure 4.8(a): XRD data for 10°[...]... IV: Results and Discussion S No Isolates 1 L4 2 AB 248 532 3 AB 248 526 4 X80 743 5 AF102267 6 AJ785761 7 AY651318 8 AM409361 9 AB 248 529 10 AF408952 11 AM409362 PERCENTAGE HOMOLOGY 1 2 3 4 5 6 7 8 9 10 11 * 96 94 93 94 94 94 94 94 94 94 * 96 95 96 96 96 96 96 96 96 * 97 97 98 98 99 99 99 99 * 97 97 96 97 97 97 97 * 98 98 98 98 98 98 * 98 98 98 98 98 * 99 98 98 99 * 99 99 99 * 99 99 * 98 * Table 4. 3: Percentage... (ppm) 12 10 8 L4 6 S1 4 2 0 0 10 20 30 40 50 60 Time (hrs) Chromium in solution (ppm) Figure 4. 6(c): Chromium removal at 10 ppm initial concentration of Cr for S1 and L4 25 20 L4 S1 15 10 5 0 0 5 10 15 20 25 30 35 40 45 50 Time (hrs) Chromium in solution (ppm) Figure 4. 6(d): Chromium removal at 20 ppm initial concentration of Cr for S1 and L4 45 40 35 30 25 20 15 10 5 0 L4 S1 0 10 20 30 40 50 60 Time... shown in Figures 4. 6(a-e) 2.5 2 L4 1.5 S1 1 0.5 0 0 5 10 15 20 25 30 35 40 Time (hrs) Chromium in solution (ppm) Figure 4. 6(a): Chromium removal at 2 ppm initial concentration of Cr for S1 and L4 4. 5 4 3.5 3 L4 2.5 S1 2 1.5 1 0.5 0 0 10 20 30 40 50 60 Time (hrs) Figure 4. 6(b): Chromium removal at 4 ppm initial concentration of Cr for S1 and L4 65 Chapter IV: Results and Discussion Chromium in solution... (PCP) degradation by Arthrobacter strain ATCC 33790 The isolate that is reported in this report is only 96% similar to Arthrobactersp.(accession no AB 248 532) It is only 94% and 93% similar to another Arthrobacter sp (accession no AB 248 526) and Arthrobacter nicotinovorans (X80 743 ) respectively Hence, the strain L4 is novel according to 16S rDNA analysis 4. 4 Chromium removal assay 4. 4.1 Chromium removal... ppm 20 10 ppm 20 ppm 40 ppm 15 10 5 0 0 20 40 60 80 100 120 140 Time (hrs) Figure 4. 5(b): Removal of chromium by S1 at various initial concentrations (2 -40 ppm) 64 Chapter IV: Results and Discussion 4. 4.3 Comparative study between S1 and L4 for removal of Chromium A comparison between the growth pattern of S1 and L4 at different initial concentration of Chromium in solution (ppm) chromium added during... limitation and/ or by chemical reactions Figure 4. 11(a) and 4. 11(b) show the color difference between control and treated samples of S1 and L4 respectively for protein estimation Figure 4. 12(a) and 4. 12(b) summarize the EPS content for isolates S1 and L4 respectively in absence and presence of Cr (at 40 ppm) 80 Chapter IV: Results and Discussion S1 Cr treated S1 L4 Cr treated L4 Figure 4. 11:: Color difference:... L4 being more tolerant to chromium 4. 10 Atomic force microscopy (AFM) Analysis The morphological changes in the cells before and after Cr exposure were examined using AFM Figures 4. 13 (a, b and c) and Figure 4. 13 (d, e and f) show the cell morphology for L4 (Arthrobacter sp ) and S1 (Bacillus sp.) respectively Figure 4. 13(a): Cell morphology for L4 without chromium exposure 83 Chapter IV: Results and. .. for Arthrobacter sp (L4) without chromium 200 180 Intensity (Counts) 160 140 120 100 80 60 40 20 0 15 20 25 30 35 40 45 50 55 60 2θ Figure 4. 9(b): XRD data for 17° ... no AB 248 526) and Arthrobacter nicotinovorans (X80 743 ) respectively AM409362 (Arthrobacter sp st 4C1-b) AF408952 (Arthrobacter sp.) AB 248 529 (Arthrobacter sp.) AM409361 (Arthrobacter sp St 4C1-a)... (Bacillus sp.) DQ105973 (Bacillus marisflavi) 55 DQ 448 746 (Bacillus sp.) AF483625 (Bacillus aquaemaris st TF-12) 100 AJ315068 (Bacillus sp.) 100 AJ 244 686 (Bacillus sp.) AY50 549 9 (Bacillus aquimaris... AB 248 532 AB 248 526 X80 743 AF102267 AJ785761 AY651318 AM409361 AB 248 529 10 AF408952 11 AM409362 PERCENTAGE HOMOLOGY 10 11 * 96 94 93 94 94 94 94 94 94 94 * 96 95 96 96 96 96 96 96 96 * 97 97 98 98 99