Table 2.1: Concentration of Cr(VI) ions under different industrial waste water
Industry
Cr(VI) concentration
References
(mg/L)
Electroplating plant
20.7 - 75.4
Tukaram Bai et al., 2005;
Kiptoo et al., 2004
Hardware factory
60.0
Xu et al., 2005
Tannery plant
8.3 – 3,950.0
Esmaeili et al., 2005;
Onyancha et al., 2008
Chrome plating plant
3.7
Gupta et al., 1999
Table 2.2: Electroplating Industry: Wastewater discharge
Parameter
pH
Temperature
Oil and Grease
Suspended solids
Cyanides (as CN)
Ammonical nitrogen (as N)
Total residual chlorine (as Cl2)
Cadmium (as Cd)
Nickel (as Ni)
Zinc (as Zn)
Chromium as Cr
Hexavalent
Total
Copper (as Cu)
Lead (as Pb)
Iron (as Fe)
Total metal
Concentration not to except, mg/l (except for pH
and temperature)
6.0 to 9.0
Should not exceed 5°C above the ambient temperature
of the receiving body
10
100
0.2
50
1.0
2.0
3.0
5.0
0.1
2.0
3.0
0.1
3.0
10.0
Source: EPA Notification [S.O. 393(E), dt 16th April 1987]
Table 2.3 Chemical species of Chromium in the environment (Zayed & Terry, 2003)
Chemical
species
Elemental Cr
Oxidation Examples
state
Cr(0)
-
Remarks
Divalent Cr
Cr(II)
CrBr2 , CrCl2 , CrF2 ,
CrSe, Cr2Si
Relatively unstable and is
readily oxidized to the trivalent
state.
Trivalent Cr
Cr(III)
CrB, CrB2,
CrBr3,CrCl3.6H2O,
CrCl3, CrF3, CrN
Forms unstable compounds
and occurs in nature in ores,
such
as
ferrochromite
(FeCr2O4).
Tetravalent
Cr
Cr(IV)
CrO2, CrF4
Does not occur naturally. The
Cr(VI) ion and its compounds
are not very stable and because
of short half-lives, defy
detection
as
reaction
intermediates between Cr(VI)
and Cr(III).
Pentavalent
Cr
Cr(V)
CrO43-, potassium
perchromate
Does not occur naturally.
Cr(V) species are derived from
the anion CrO43- and are longlived enough to be observed
directly. However, there are
relatively
few
stable
compounds containing Cr(V).
Hexavalent
Cr
Cr(VI)
(NH4)2CrO4, BaCrO4,
CaCrO4, K2CrO4,
K2Cr2O7
The second most stable state of
Cr. However, Cr(VI) rarely
occurs naturally, but is
produced from anthropogenic
sources. It occurs naturally in
the rare mineral crocoites
(PbCrO4).
Does not occur naturally.
Table 2.4: Chromium(VI) reducing bacterial strains
Name of Species
Isolation Conditions/ C-Sources
References
Achromobacter sp. Anaerobic/Luria Broth; glucose- Zhu et al., 2008
StrainCh 1
lactate
Bacillus megatarium Aerobic/ nutrient broth-minimal salt Cheung et al., 2006
TKW3
medium-glucose, maltose, mannitol
Bacillus sp. ES 29
Aerobic/Luria-Bertani (LB) medium
Camargo et al., 2003
Ochrobactrum sp.
Aerobic/glucose
Zhiguo et al., 2009
Providencia sp.
Aerobic-Anaerobic/ Luria
(tryptone-yeast extract)
Pseudomonas
aeruginosa
Aerobic/ nutrient broth or Luria Aguilera et al., 2004
broth
broth Thacker et al., 2006
Pseudomonas putida Anaerobic/Luria- Bertani-citric acid- Park et al., 2000
MK1
Tris-acetic acid
Pseudomonas spp.
Aerobic/Vogel-Bonner (VB)-Dglucose
Mclean and Beveridge,
2001
Shewanella alga
(BrYMT) ATCC
55627
Aerobic-Anaerobic/ M9 brothglucose
Guha et al., 2001
Shewanella
putrefaciens MR-1
Anaerobic/ lactate-fumarate
Myers et al., 2000
Table 2.5. Properties of purified bacterial chromate reducing enzymes
Bacteria
Enzyme
Substrates
Cofactor/ Mol. Wt
electron
(KDa)
donor
and
subunits
P. ambigua G1 Chr
Chromate,
FMN
65 Dimer
nitrocompounds
NAD(P)
P. putida
Chr
Quinones,chromate, FMN
50 Dimer
2,6 DichloroindoNAD(P)H
phenol,Potassium
ferricyanide
E. coli
Chr
Chromate
FMN
84 Dimer
NAD(P)H
E. coli
YieF
E. coli
NfsA
T. Scotoductus
Chr
Chromate, quinone,
2,6 -Dichloroindo
Phenol, Potassium
Ferricyanide, V (V)
, Mo(VI)
Chromate,
Nitrocompounds
Chromate
R. sphaeroides
Chr
Chromate
V. harveyi
NfsA
Nitrofurazone,
Trinitrotoluene
Chromate
References
Suzukis et
al, 1992
Parked et al,
2000
Bae et al.,
2005
FMN
NAD(P)H
50 Dimer
Ackerley et
al., 2004a
FMN
NADH
FMN
NAD(P)H
NADH
50 Dimer
FMN
NADH
50 Dimer
Ackerley et
al., 2004b
Opperman
et al., 2008
Nepple et
al., 2000
Kwak et al.,
2003
72Dimer
42 Dimer
Table 3.1 : List of Instruments used during the present study
INSTRUMENTS
FUNCTION
MODEL No.
Vertical Autoclave
Sterilization
PI110P6 SANCO
Analytical Balance
Weight measurement
CPA225D
SARTORIUS
Laminar airflow
Aseptic conditions
MICROSIL
pH
Measurement of pH
Digitl pH meter
BOD Incubator
Incubation of cultures
NSW-152 CALTON
Ultra Low Temperature
freezer
Preservation of cultures
RQFV-265 REMI
Double Distillation Unit
Preparation of the stock
solution, throughout the
experiment etc.
MQD2PQ Bhanu
Scientific
Spectrophotometer
(UV/Vis)
Estimation of Biomass and
Cr (VI) degradation
UV-2450 SHIMADZU
BOD Incubator Shaker
Batch degradation kinetics
of Cr (VI)
LETTD ORBITEK
Ultra Centrifuge
Collection of pellet and Cr
(VI) estimation
0191 LOGIC
CONTROLS PVT
Atomic
Absorption
Spectrophotometer
Estimation of total
chromium
Z-6100 HITACHI
Electrophoresis Unit
Protein profile study
Model-192 BIORAD
Table 4.1: Physico-chemical characterization of Electroplating effluent
Parameters
Concentration Standards*
Color
Temperature (0C)
Dull green
18.90±0.06
Shall not exceed 5oC above
ambient temperature of receiving
water body
6.0 to 9.0
10
2100
pH (1:2)
5.66±0.61
Conductivity(millimoh/cm2)(1:2) 59.2
Oil and grease
20
Total dissolved solids
480±0.62
Total suspended solids
58±0.93
Biochemical oxygen demand
58±0.93
30
Chemical oxygen demand
675±0.45
250
Sulfate
199
Phosphate
0.79
Total chromium
48±0.186
2.0
Hexavalent chromium
25±0.823
0.1
Cadmium
16.85±0.914
2.0
Copper
0.354±0.01
3.0
Iron
3.31±0.31
3.0
Nickel
27.713
3.0
Lead
0.66±0.094
0.1
Zinc
40.54 ±0.26
0.5
All values are expressed in mg/L, otherwise stated; values are mean ±SD (n=3)
*Source- Environmental Protection Agency Notification, (1987).
Table4.2: Physico-chemical characterization of soil contaminated with electroplating
waste water
Parameters
Soil organic matter (%)
Total Chromium (mg/g)
Potassium (1:2) (mg/L)
Sodium (1:2) (mg/L)
Values are mean ± SD (n=3)
Concentration
1.85±0.5
6.00±2.5
112.50±5.6
61.60±3.21
Table 4.3: Morphological tests for isolated bacterial strains:
Tests
S1
S2
S3
S4
Circular
Circular
Circular
Circular
Entire
Entire
Entire
Entire
Elevation
Convex
Convex
Convex
Convex
Surface
Smooth
Smooth
Smooth
Smooth
Pigment
Creamish
Creamish
Creamish
Creamish
Opacity
Transparent
Transparent
Transparent
Transparent
+ ve
+ ve
+ ve
+ ve
Cell shape
Short rods
Short rods
Short rods
Short rods
Size (m)
1-1.5
1
1.5
1
Scattered
Scattered
Scattered
Scattered
Spore(s)
- ve
- ve
- ve
- ve
Motility
+
+
+
+
Colony
morphology
Configuration
Margin
Gram’s reaction
Arrangement
Table 4.4: Physiological tests for isolated bacterial strains:
Tests
S1
S2
S3
S4
4 C
+
+
+
+
10 C
+
+
+
+
25 C
+
+
+
+
30 C
+
+
+
+
37 C
+
+
+
+
42 C
+
+
+
+
55 C
-
+
-
-
pH 5.0
+
+
+
+
pH 6.0
+
+
+
+
pH 8.0
+
+
+
+
pH 9.0
+
+
+
+
2.0
+
+
+
+
4.0
+
+
+
+
6.0
+
+
+
+
8.0
+
+
+
+
10.0
-
-
-
-
12.0
-
-
-
-
Growth under
+
+
+
+
Growth
at
temperatures
Growth at pH
Growth
on
NaCl (%)
anaerobic
condition
Table 4.5: Biochemical tests for isolated bacterial strains:
Tests
S1
S2
S3
S4
Growth on MacConkey
lf
lf
lf
lf
Indole test
-
-
-
-
Methyl red test
-
-
-
-
Voges Proskauer test
-
-
-
-
Citrate utilization
+
+
+
+
H2S production
-
-
-
-
Gas production
-
-
-
-
Starch hydrolysis
-
-
-
-
Nitrate reduction
+
+
+
+
Catalase test
+
+
+
+
Oxidase test
-
-
-
-
Urea hydrolysis
+
+
+
+
Esculin hydrolysis
+
+
+
+
Arginine dihydrolase
+
+
+
+
Tween 20 hydrolysis
-
-
-
-
Tween 40 hydrolysis
-
-
-
-
Tween 60 hydrolysis
-
-
-
-
Tween 80 hydrolysis
-
-
-
-
Galactose
(+)
-
-
-
Mannose
+
+
-
-
Maltose
+
+
+
+
Sucrose
+
+
+
+
Fructose
(+)
(+)
(+)
(+)
Lactose
+
+
+
+
from glucose
Acid Production from
+: Positive; -: Negative ;(+): Weak positive; lf: lactose fermenting;
Table 4.6: The Box-Behnken design matrix for experimental design and observed
response for Cr(VI) biotransformation using Salmonella sp. S1 and Salmonella sp. S2
Experimental
Run
pH
(A)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
4
4
6
4
6
6
6
6
8
4
8
6
6
8
8
6
6
Temp.
(OC)
(B)
30
25
25
30
25
30
30
30
30
35
30
35
35
25
35
30
30
Initial
Cr(VI) ion
conc. (C)
40
25
40
10
10
25
25
25
10
25
40
40
10
25
25
25
25
%Cr(VI)
Biotransfomation
By Salmonella sp. S1
13
17
50
20
75
75
79
60
75
14
49
43
53
53
73
75
79
%Cr(VI)
Biotransfomation
By Salmonella sp. S1
15
20
45
20
65
75
64
74
75
18
49
40
49
50
73
74
75
Table 4.7: The Box-Behnken design matrix for experimental design and observed
response for Cr(VI) biotransformation using Salmonella sp. S3 and Salmonella sp. S4
Experimental
Run
pH
(A)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
4
4
6
4
6
6
6
6
8
4
8
6
6
8
8
6
6
Temp.
(OC)
(B)
30
25
25
30
25
30
30
30
30
35
30
35
35
25
35
30
30
Initial
Cr(VI) ion
conc. (C)
40
25
40
10
10
25
25
25
10
25
40
40
10
25
25
25
25
%Cr(VI)
Biotransfomation
By Salmonella sp. S3
17
26
44
22
73
72
72
72
68
20
47
39
55
43
70
64
74
%Cr(VI)
Biotransfomation
By Salmonella sp. S4
21
23
45
20
74
72
73
73
73
20
49
40
49
56
70
64
72
Table 4.8 Analysis of variance for RSM variables fitted to quadratic model
Bacterial
strain
Source
Sum of
squares
d.f.
Mean
square
F-value
P-value
Prob>F
Salmonella Model
Residual
sp S1
8853.56
512.20
267
247
0.9451
0.8745
9
7
3
4
983.73
73.46
89.00
61.80
13.39
0.0012
Significant
1.44
0.3560
Not Significant
Salmonella Model
Residual
sp S2
8016.92
437.20
348
89.20
0.9483
0.8818
9
7
3
4
890.77
62.46
116.00
22.30
14.26
0.0010
significant
5.20
0.0725
not-significant
Salmonella Model
Residual
sp S3
6813.71
352.05
291.25
60.80
0.9509
0.8877
9
7
3
4
757.08
50.29
97.08
15.20
15.05
0.0009
Significant
6.39
0.0526
Not Significant
Salmonella Model
Residual
sp S4
6848.57
297.55
238.75
58.80
0.9584
0.9048
9
7
3
4
760.95
42.51
79.58
14.70
17.90
0.0005
Significant
5.41
0.0682
Not Significant
Lack-of-fit
Pure error
r2
r2adj
Lack-of-fit
Pure error
r2
r2adj
Lack-of-fit
Pure error
r2
r2adj
Lack-of-fit
Pure error
r2
r2adj
Table 4.9: Cr(VI) biotransformation in electroplating effluent using different
dilutions by Salmonella sp. S1
Time
(hrs)
6
12
24
48
72
96
120
25% dilution
Salmonella sp. S1
Cr(VI) Conc. in effluent (mg/L)
50% dilution
75% dilution
12.24 ± 0.01
18.11 ± 0.025
29.80 ± 0.017
35.23 ± 0.45
39.35 ± 0.200
48.33 ± 0.26
54.29 ± 0.26
8.81 ± 0.02
12.01 ± 0.63
20.89 ± 0.47
24.55 ± 0.20
28.87 ± 0.16
31.63 ± 0.09
35.71 ± 0.65
Source of variation
Interraction
Time
% Cr(VI) Concentration
-
3.20 ± 0.23
5.89 ± 0.36
12.87 ± 0.55
13.92 ± 0.24
15.19 ± 0.10
19.91 ± 0.48
20.11 ± 0.04
Undiluted
effluent
1.25 ± 0.011
1.70 ± 0.23
6.87 ± 0.15
8.92 ± 0.90
8.99 ± 0.46
9.05 ± 0.35
9.99 ± 0.30
% of total variation
P value
8.95
< 0.0001
56.28
< 0.0001
34.73
< 0.0001
*Above values are averages of three triplicates ± SD
** Concentrations units are expressed in percentage
Table 4.10: Cr(VI) biotransformation in electroplating effluent using different
dilutions by Salmonella sp. S2
Time
(hrs)
6
12
24
48
72
96
120
25% dilution
Salmonella sp. S2
Cr(VI) Conc. in effluent (mg/L)
50% dilution
75% dilution
12.81 ± 0.55
12.99 ± 0.66
26.51 ± 0.45
35.76 ± 0.50
41.00 ± 0.75
48.88 ± 0.65
55.00 ± 0.47
10.25 ± 0.76
17.73 ± 0.81
25.55 ± 0.69
33.06 ± 0.36
36.66 ± 0.55
39.16 ± 0.65
39.98 ± 0.91
Source of variation
Interraction
Time
% Cr(VI) Concentration
-
5.51 ± 0.81
9.90 ± 0.50
10.83 ± 0.65
11.89 ± 0.80
14.43 ± 0.47
23.49 ± 0.56
25.88 ± 0.65
% of total variation
12.10
55.29
32.49
*Above values are averages of three triplicates ± SD
** Concentrations units are expressed in percentage
P value
< 0.0001
< 0.0001
< 0.0001
Undiluted
effluent
2.23 ± 0.65
2.71 ± 0.80
4.41 ± 0.47
6.41 ± 0.75
6.62 ± 0.32
7.32 ± 0.70
7.99 ± 0.65
Table 4.11: Cr(VI) biotransformation in electroplating effluent using different
dilutions by Salmonella sp. S3
Tim
e
(hrs)
6
12
24
48
72
96
120
25% dilution
10.07 ± 0.12
15.77 ± 0.10
20.09 ± 0.13
28.91 ± 0.05
36.61 ± 0.65
45.06 ± 0.38
45.21 ± 0.21
Salmonella sp. S3
Cr(VI) Conc. in effluent (mg/L)
50% dilution
75% dilution
Undiluted effluent
8.82 ± 0.43
3.86 ± 0.57
1.11 ± 0.32
10.99 ± 0.23
5.98 ± 0.42
2.33 ± 0.79
15.52 ± 0.21
8.88 ± 0.76
4.88 ± 0.31
22.80 ± 0.15
15.54 ± 0.39
6.61 ± 0.35
36.93 ± 0.19
17.72 ± 0.46
6.80 ± 0.64
40.44 ± 0.25
20 ± 0.27
7.71 ± 0.97
42.26 ± 0.74
20.02 ± 0.47
7.92 ± 0.25
Source of variation
Interraction
Time
% Cr(VI) Concentration
-
% of total variation
11.99
47.55
40.39
P value
< 0.0001
< 0.0001
< 0.0001
*Above values are averages of three triplicates ± SD
** Concentrations units are expressed in percentage
Table 4.12: Cr(VI) biotransformation in electroplating effluent using different
dilutions by Salmonella sp. S4
Tim
e
(hrs)
6
12
24
48
72
96
120
25% dilution
12.04 ± 0.42
18.88 ± 0.32
27.79 ± 0.25
38.80 ± 0.71
41.64 ± 0.42
49.73 ± 0.65
58.01 ± 0.23
Salmonella sp. S4
Cr(VI) Conc. in effluent (mg/L)
50% dilution
75% dilution
Undiluted effluent
11.01 ± 0.22
5.51 ± 0.34
1.00 ± 0.67
17.74 ± 0.11
9.90 ± 0.29
1.61 ± 0.47
26.66 ± 0.21
10.83 ± 0.35
3.70 ± 0.55
34.43 ± 0.12
11.89 ± 0.29
4.12 ± 0.79
39.11 ± 0.29
14.43 ± 0.92
4.49 ± 0.82
42.90 ± 0.65
23.49 ± 0.96
4.89 ± 0.72
45.00 ± 0.52
25.88 ± 0.82
5.02 ± 0.11
Source of variation
Interraction
Time
% Cr(VI) Concentration
-
% of total variation
11.74
63.29
24.90
*Above values are averages of three triplicates ± SD
** Concentrations units are expressed in percentage
P value
< 0.0001
< 0.0001
< 0.0001