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RESULTS

IX. COLLECTION, ISOLATION, PURIFICATION, IDENTIFICATION AND

SCREENING OF ALGAL STRAINS

A. Collection of Samples

Algal samples were collected from the water bodies suspected to contain heavy

metals. These included Sutlej river, from the point where its water gets polluted with

Buddha Nullah, Ludhiana, Punjab (11 collections), a polluted stream at Bilaspur, H.P.

(5 collections) and Badi Nadi, Patiala, Punjab from the point where effluents of Diesel

Loco Modernisation Works (DLMW) Patiala fall (4 collections).

Algal samples from these water bodies were collected in the sterilized polythene

bags and wide mouth bottles. These samples were brought to the laboratory for isolation,

purification and identification purposes.

B. Isolation and Purification of Algal Samples

The samples were observed under the microscope for the presence of algae. The

samples contained members of green algae and cyanobacteria. Total 61 strains of algae

(green as well as blue green) were isolated and purified. Each isolate was given an

accession number which indicated site of collection and the number of isolate (Table 8).

C. Propagation and Maintenance of Purified Strains

Isolated and purified strains of algae were propagated and maintained in Chu-10

and BG-11 medium in the culture room at 28 ±2 °C under ideal growth conditions. Since

Table 8: Comparison of heavy metal removal potential of algal isolates from imidazole-HCl buffer (0.2 M) and nutrient medium.

Metal removed (µg ml-1 )

S. No.

Isolate Green/ Blue-green

algae

Cadmium Nickel Copper

Buffer Medium Buffer Medium Buffer Medium

1 SR1 Blue-green 2.28±0.06 1.64±0.05 0.89±0.02 0.49±0.01 1.49±0.09 0.64±0.13

2 SR2 Green 2.3±0.04 1.49±0.09 0.89±0.07 0.41±0.12 1.5±0.10 0.95±0.15

3 SR3 Blue-green 1.95±0.08 1.19±0.11 0.91±0.04 0.35±0.10 1.35±0.14 0.25±0.09

4 SR4 Blue-green 1.8±0.19 1.00±0.14 0.81±0.05 0.19±0.09 1.39±0.09 0.37±0.01

5 SR5 Green 1.73±0.18 0.63±0.18 0.69±0.02 0.28±0.09 1.32±0.11 0.66±0.07

6 SR6 Blue-green 3.13±0.09 2.43±0.03 1.47±0.02 0.81±0.02 2.04±0.07 1.14±0.10 7 SR7 Blue-green 1.45±0.06 0.79±0.09 0.59±0.05 0.14±0.06 1.09±0.10 0.61±0.11

8 SR8 Blue-green 1.09±0.11 0.29±0.06 0.53±0.08 0.32±0.11 1.12±0.16 0.65±0.11

9 SR9 Blue-green 1.45±0.17 0.6±0.01 0.61±0.13 0.48±0.10 1.21±0.07 0.50±0.09

10 SR10 Green 1.1±0.09 0.31±0.04 0.62±0.18 0.22±0.05 1.16±0.03 0.39±0.16

11 SR11 Blue-green 1.27±0.05 0.12±0.12 0.75±0.17 0.51±0.15 1.38±0.06 0.79±0.17

12 SR12 Blue-green 1.31±0.13 0.62±0.10 0.81±0.10 0.32±0.09 1.13±0.12 0.65±0.10

13 SR13 Blue-green 1.37±0.12 0.49±0.11 0.63±0.02 0.19±0.11 1.41±0.10 0.57±0.07

14 SR14 Blue-green 1.75±0.05 1.20±0.07 1.12±0.09 0.70±0.02 1.97±0.04 1.42±0.04 15 SR15 Green 1.33±0.05 0.99±0.13 0.59±0.15 0.11±0.01 1.01±0.10 0.71±0.07

16 SR16 Blue-green 1.41±0.14 0.17±0.17 0.31±0.11 0.16±0.10 1.17±0.15 0.19±0.01

17 SR17 Blue-green 1.09±0.08 0.55±0.10 0.65±0.16 0.21±0.14 0.99±0.09 0.22±0.11

18 SR18 Blue-green 1.17±0.14 1.01±0.12 0.44±0.10 0.19±0.17 1.09±0.04 0.34±0.14

18 SR19 Green 1.65±0.12 0.11±0.10 0.67±0.01 0.32±0.11 1.48±0.03 0.71±0.10

20 SR20 Green 1.67±0.13 0.29±0.16 0.54±0.04 0.19±0.12 1.12±0.09 0.13±0.09

21 SR21 Blue-green 1.59±0.07 0.35±0.01 0.37±0.09 0.13±0.09 1.28±0.04 1.09±0.11

22 SR22 Blue-green 2.45±0.06 1.55±0.09 0.88±0.04 0.50±0.04 1.85±0.02 1.09±0.15 23 SR23 Green 1.11±0.03 0.61±0.11 0.54±0.03 0.19±0.07 1.1±0.02 0.93±0.10

24 SR24 Blue-green 1.53±0.17 0.46±0.18 0.42±0.07 0.31±0.10 0.89±0.11 0.15±0.02

25 SR25 Blue-green 1.54±0.09 0.39±0.14 0.31±0.04 0.25±0.10 0.98±0.14 0.25±0.08

26 SR26 Blue-green 1.55±0.09 0.43±0.17 0.51±0.12 0.16±0.18 1.22±0.10 0.89±0.05

27 SR27 Green 1.49±0.04 1.10±0.08 0.70±0.16 0.58±0.13 1.27±0.18 1.02±0.11

28 SR28 Green 1.46±0.11 1.03±0.06 0.93±0.11 0.43±0.16 0.93±0.08 0.29±0.13

29 SR29 Blue-green 1.46±0.10 0.81±0.02 0.93±0.09 0.58±0.08 1.57±0.03 0.79±0.04

30 BN1 Blue-green 1.32±0.13 0.11±0.01 1.07±0.06 0.67±0.06 1.05±0.09 0.65±0.15

31 BN2 Green 1.19±0.11 0.31±0.10 1.14±0.08 0.25±0.02 1.1±0.05 0.31±0.06

31 BN3 Blue-green 1.13±0.09 0.59±0.14 1.07±0.14 0.37±0.10 1.4±0.11 1.02±0.03

33 BN4 Blue-green 1.12±0.02 0.85±0.13 0.76±0.10 0.15±0.07 1.39±0.13 0.83±0.09

34 BN5 Blue-green 1.39±0.19 1.07±0.18 0.61±0.01 0.31±0.04 1.05±0.03 0.72±0.11

Continued……..

35 BN6 Blue-green 1.07±0.16 0.57±0.20 0.48±0.11 0.29±0.10 1.38±0.05 0.55±0.15 36 BN7 Green 1.15±0.17 0.56±0.11 0.60±0.12 0.37±0.16 0.99±0.07 0.30±0.18

37 BN8 Blue-green 1.31±0.14 0.19±0.05 0.89±0.18 0.42±0.10 0.54±0.14 0.24±0.12

38 BN9 Blue-green 1.17±0.11 0.13±0.09 0.59±0.09 0.40±0.05 1.09±0.12 0.65±0.11

39 BN10 Blue-green 1.29±0.13 1.01±0.14 0.81±0.03 0.20±0.03 1.37±0.10 1.15±0.10

40 BN11 Blue-green 1.4±0.11 0.83±0.10 0.24±0.06 0.10±0.01 0.69±0.08 0.11±0.05

41 BN12 Green 1.49±0.03 0.77±0.07 0.32±0.10 0.15±0.06 0.89±0.05 0.20±0.02

42 BN13 Blue-green 1.43±0.07 1.16±0.09 0.59±0.08 0.16±0.09 0.99±0.09 0.35±0.05

43 BN14 Blue-green 3.45±0.03 2.75±0.02 1.26±0.04 0.62±0.04 2.38±0.01 1.40±0.10

44 BN15 Blue-green 1.01±0.06 0.15±0.10 0.24±0.09 0.14±0.04 1.33±0.06 1.05±0.12

45 BN16 Blue-green 0.88±0.09 0.49±0.16 0.39±0.07 0.26±0.06 0.91±0.20 0.17±0.04

46 BN17 Green 0.95±0.05 0.63±0.12 0.31±0.19 0.10±0.10 0.85±0.07 0.45±0.02

47 BN18 Green 0.87±0.01 0.24±0.17 0.19±0.12 0.10±0.01 1.12±0.09 0.82±0.17

48 BN19 Blue-green 1.09±0.08 0.21±0.08 0.89±0.14 0.33±0.11 1.13±0.11 0.73±0.16

49 PSB1 Blue-green 1.35±0.20 1.07±0.04 0.93±0.10 0.13±0.13 1.27±0.13 0.16±0.10

50 PSB2 Blue-green 0.99±0.17 0.32±0.10 0.73±0.06 0.54±0.10 1.15±0.16 1.01±0.11

51 PSB3 Blue-green 1.16±0.15 1.49±0.06 0.49±0.08 0.26±0.09 1.09±0.07 0.62±0.05

52 PSB4 Green 1.27±0.11 0.27±0.07 0.84±0.05 0.60±0.01 0.92±0.09 0.46±0.11

53 PSB5 Blue-green 1.41±0.11 1.01±0.09 0.19±0.03 0.11±0.03 0.73±0.07 0.33±0.02

54 PSB6 Green 1.14±0.19 1.01±0.12 0.39±0.11 0.21±0.11 1.11±0.11 0.79±0.18

55 PSB7 Blue-green 0.91±0.10 0.15±0.15 1.09±0.07 0.40±0.05 0.19±0.13 0.12±0.16

56 PSB8 Green 0.69±0.09 0.18±0.02 1.01±0.16 0.75±0.07 1.08±0.18 0.65±0.13

57 PSB9 Blue-green 0.52±0.08 0.16±0.07 0.67±0.12 0.27±0.03 0.81±0.12 0.41±0.09

58 PSB10 Green 1.10±0.14 0.75±0.10 0.12±0.18 0.10±0.01 0.89±0.15 0.23±0.05 59 PSB11 Blue-green 1.30±0.17 0.47±0.12 0.69±0.11 0.40±0.07 1.27±0.01 0.99±0.03

60 PSB12 Blue-green 1.09±0.11 0.32±0.16 0.45±0.05 0.20±0.09 1.15±0.09 0.26±0.10

61 PSB13 Green 1.00±0.16 0.57±0.13 0.39±0.02 0.11±0.08 1.06±0.08 0.49±0.13

(Initial metal concentration: 5 µg ml-1; biomass: 0.1 mg protein ml-1; pH of buffer: 5.0; pH of medium: 7.8; contact time: 90 min; Temp: 28±2°C)

(SR = Sutlej river, BN = Buddha Nullah, PSB = Polluted stream at Bilaspur)

all the test organisms selected for the present study were cyanobacteria, for their

cultivation and propagation only modified Chu-10 medium was used.

D. Screening of Algal Strains for Heavy Metal Bioremediation Potential

Results of these experiments revealed that isolates SR 6, SR 14, SR 22, SR 29 and BN

14 among all the isolates removed maximum amount of Cd2+, Cu2+ and Ni2+ from buffer as

compared to nutrient medium. Their metal removal efficiencies ranged from 29.2-69.0% for Cd2+,

31.4-47.6% for Cu2+ and 17.6-29.4% for Ni2+ from imidazole-HCl buffer and from nutrient

medium these ranges were 16.2-55.0% for Cd2+, 15.8-28.4% for Cu2+ and 10.0-16.2% for Ni2+

(Table 8)

These five isolates which showed maximum metal (Cd, Cu or Ni) removal efficiency were

selected for further studies.

E. Identification of Selected Algal Isolates

All the five selected isolates belonged to cyanophyceae and incidentally all, except one,

were isolates from the collections of polluted water of Sutlej river which is being polluted through

Buddha Nullah by industrial wastes and sewage of Ludhiana city in Punjab, State of India. One

isolate (BN 14), was from the collections of Badi Nadi, Patiala which is receiving effluents from

Diesel Loco Modernization Works Patiala, Punjab. These isolates were identified on the basis of

their phenotypic characters following Desikachary (1959) named as shown in Table 9.

Table 9: Identification of algal isolates selected for the present study on the basis of phenotypic characters. S. No. Isolate Organism Order

1 SR6 Synechocystis pevalekii Ercegovic Chroococcales

2 BN14 Lyngbya spiralis Geitler Oscillatoriales

3 SR22 Oscillatoria chlorina Kütz. ex Gomont Oscillatoriales

4 SR14 Phormidium molle (Kütz.) Gomont Oscillatoriales

5 SR29 Anabaena torulosa (Carm.) Lagerh. ex Born. et Flah Nostocales

The morphological identification features of the selected isolates are as follows:

a) Synechocystis pevalekii Ercegovic

Cells prokaryotic generally single or in group of two cells, spherical in shape, 2.49-3.32 µm

in diameter. The cells appear hemispherical immediately after division and blue green in colour.

(Plate 1)

Locality: Sutlej river

b) Lyngbya spiralis Geitler

Filaments of this organisms are spirally coiled, 6.64-8.3 µm broad; unlamellated,

colourless, thick, firm sheath is present; trichomes are not constricted at the cross walls. Cells of the

trichomes are 1.66-2.49 µm long, 4.98-6.64 µm broad, pale-blue in colour; end cells are rounded,

calyptra is absent. (Plate 1)

Locality: Badi Nadi, Patiala

c) Oscillatoria chlorina Kütz. ex Gomont

Thallus of the organism is very thin; yellowish green in colour; tichomes straight; slightly

constructed at the cross wall. Cells are 3.8-6.8 µm long and 3.67-4.5 µm broad. The cross walls of

the cells are ungranulated; end cells somewhat attenuated; calyptra absent. (Plate 1)


d) Phormidium molle (Kütz.) Gomont

Thallus of this organisms is mucilaginous, light blue-green in colour, filaments

are straight; sheath diffluent, colourless; trichomes constricted at the cross walls. Cells

are longer than broad, 3.02-5.42 µm long and 3.02-3.46 µm broad; end cells are rounded,

calyptra absent. (Plate 1)


e) Anabaena torulosa (Carm) Lagerh. ex Born. et Flah.

In this organism thallus is mucilaginous, blue-green in colour; trichomes are

straight, cells are barrel shaped, 4.98-5.85 µm long, 4.15-4.98 µm broad. Heterocysts

spherical in shape, 5.85-7.0 µm long, 4.15-6.64 µm broad; Akinetes are in series,

sometimes single, 5.98-9.96 µm long, 6.64-8.30 µm broad and pale brown in colour.

(Plate 1)


X. OPTIMIZATION OF PARAMETERS FOR METAL REMOVAL

Since test organisms removed more amounts of metal from buffer compared to from

nutrient medium (Table 8), all metal removal experiments were performed using buffer

only. Parameters such as contact time, pH, biomass load, initial metal concentration and

temperature were optimized for metal (Cd2+, Cu2+ and Ni2+) removal by test organisms.

a) Optimization of Contact Time

Contact time between biomass and metal solution was varied from 0-60 minutes.

With increase in contact time, Cd2+ removal efficiency of test organisms increased up to

10 min (Fig. 3). The results revealed that rate of Cd2+ removal from aqueous solution by

all the test organisms was maximum during initial 10 minutes of contact and after that

rate of metal removal slowed down. The system reached at equilibrium in 60 min.

Maximum amount of Cd2+ (69.0% of 5 µg ml-1) was removed by L. spiralis and

minimum (29.2% of 5 µg ml-1) by A. torulosa (Fig. 3). t-test on data revealed that the

values of pcal were >0.025 when Cd2+ removal from 10-60 min was compared indicating

insignificant differences in metal removal.

Similar experiments were performed with Cu2+ and Ni2+ by employing all the test

organisms individually. Results revealed that rates of Cu2+ and Ni2+ removal were maximum

during initial 10 min of contact between biomass and metal with system attaining equilibrium

in 60 min (Figs. 4 & 5). Maximum amount of Cu2+ (47.6% of 5 µg ml-1) was removed by

L. spiralis and minimum (31.4% of 5 µg ml-1) by A. torulosa. While the maximum

amount of Ni2+ (29.4% of 5 µg ml-1) was removed by S. pevalekii and minimum (17.6%)

by O. chlorina. t-test on Cu2+ and Ni2+ removal data gave value of pcal was >0.025 when

metal removal from 10-60 min was compared indicating insignificant differences in

metal removal, during this period of time.

Thus, the optimum contact time for Cd2+, Cu2+ and Ni2+ removal by all the test

organisms was 60 min.

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

0 10 20 30 45 60

Time (min)

Cd2

+ rem

ove

d (µ

g m

l-1)

Fig. 3: Cd2+ removal by S. pevalekii ( ), L. spiralis ( ), O. chlorina ( ), P. molle

( ), and A. torulosa ( ).

Experimental conditions: Initial Cd2+ concentration: 5 µg ml-1; biomass: 0.1 mg protein ml-1; buffer: imidazole-HCl 0.2 M; pH: 5.0; Temp: 28±2 °C pcal> 0.025 when Cd2+ removal is compared from 10-60 min indicating insignificant differences in metal uptake.

0.00

0.50

1.00

1.50

2.00

2.50

3.00

0 10 20 30 45 60

Time (min)

Cu2

+ rem

ove

d (µ

g m

l-1)

Fig. 4: Cu2+ removal by S. pevalekii ( ), L. spiralis ( ), O. chlorina ( ), P. molle


Experimental conditions: Initial Cu2+ concentration: 5 µg ml-1; biomass: 0.1 mg protein ml-1; buffer: imidazole-HCl 0.2 M; pH: 5.0; Temp: 28±2 °C pcal> 0.025 when Cu2+ removal is compared from 10-60 min indicating insignificant differences in metal uptake.

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

0 10 20 30 45 60

Time (min)

Ni2

+ rem

ove

d (µ

g m

l-1)

Fig. 5: Ni2+ removal by S. pevalekii ( ), L. spiralis ( ), O. chlorina ( ), P. molle


Experimental conditions: Initial Ni2+ concentration: 5 µg ml-1; biomass: 0.1 mg protein ml-1; buffer: imidazole-HCl 0.2 M; pH: 5.0; Temp: 28±2 °C pcal> 0.025 when Ni2+ removal is compared from 10-60 min indicating insignificant differences in metal uptake.

b) Optimization of pH

Effect of pH on metal removal efficiency of the test organisms was studied by

varying pH of the buffer from 4.0 to 7.0. pH beyond 7.0 was not selected to avoid

precipitation of metals. The results revealed that maximum amounts of metals were

removed by all the test organisms from the buffer with pH 6.0 (Figs. 6-8). Increase or

decrease in pH of the buffer from this value resulted in decrease in metal removal

efficiency. At pH 4.0, Cd2+ removal by all the test organisms ranged from 21.6 to 58.8%

of 5 µg ml-1 which increased with increase in pH up to 6.0 and then decreased at pH 7.0

(Table 10). The maximum amount of Cd2+ was removed by L. spiralis (76.4 % of 5 µg ml-1)

followed by S. pevalekii (74.0%), O. chlorina (59.8%), P. molle (50.0%) and A. torulosa

(45.0%). The results indicated strong dependence of metal adsorption on pH.

Similar experiments were performed with Cu2+ and Ni2+. Maximum amounts of

Cu2+ and Ni2+ were removed by the test organisms at pH 6.0 i.e 70.0% Cu2+, 37.8% Ni2+

removal by S. pevalekii, 73.8% Cu2+, 35.6% Ni2+ removal by L. spiralis, 47.0% Cu2+,

25.4% Ni2+ removal by O. chlorina, 48.6% Cu2+, 29.8% Ni2+ by removal P. molle and

43.0% Cu2+, 29.4% Ni2+ by removal A. torulosa (Tables 11 & 12). Data of t-test revealed

that pcal was <0.025 when metal removal from 4-7 pH was compared indicating

significant differences in metal removal.

Thus, pH 6.0 was optimum pH for Cd2+, Cu2+ and Ni2+ removal by all the test

organisms.


( ), and A. torulosa ( ) from imidazole-HCl buffer (0.2M).

Experimental conditions: Initial Cd2+ concentration: 5 µg ml -1; biomass: 0.1 mg protein ml-1; contact time: 60 min; Temp: 28±2 °C

pcal< 0.025 when Cd2+ removal is compared from pH 4-7 indicating significant differences in metal uptake.


( ), and A. torulosa ( ) from imidazole-HCl buffer (0.2M). Experimental conditions: Initial Cu2+ concentration: 5 µg ml-1; biomass: 0.1 mg

protein ml-1; contact time: 60 min; Temp: 28±2 °C

pcal< 0.025 when Cu2+ removal is compared from pH 4-7 indicating significant differences in metal uptake.


( ), and A. torulosa ( ) from imidazole-HCl buffer (0.2M).

Experimental conditions: Initial Ni2+ concentration: 5 µg ml -1; biomass: 0.1 mg protein ml-1; contact time: 60 min; Temp: 28±2 °C

pcal> 0.025 when Ni2+ removal is compared from pH 4-5 indicating insignificant differences in metal uptake. pcal< 0.025 when Ni2+ removal is compared from pH 5-7 indicating significant differences in metal uptake.

Table 10: Effect of pH on Cd2+ removal by test organisms.

Organism

Cd2+ removed (%)

pH

4.0 5.0 6.0 7.0

S. pevalekii A46.60a±2.4 A62.60b±1.0 A74.00c±2.0 A69.40b±0.9

L. spiralis B58.80a±2.2 B69.00b±2.6 A76.40c±1.8 A72.00b±3.0

O. chlorina C30.60a±1.0 C49.00b±2.8 B59.80c±1.6 B52.80b±3.3

P. molle D21.60a±2.0 D29.20b±3.0 C51.00c±2.2 C42.00d±2.8

A. torulosa D25.00a±1.8 E35.00b±2.8 D45.00c±1.0 D33.80b±1.8

Experimental conditions: Initial Cd2+ concentration: 5 µg ml-1; biomass: 0.1 mg protein ml-1; buffer: imidazole-HCl (0.2 M); contact time: 60 min; Temp: 28±2 °C; volume: 50 ml

Arithmetic means within the same column with the same uppercase letter are not

significantly different from each other at the 95% confidence level (p > 0.025). Also,

arithmetic means within the same row with the same lowercase letter are not significantly

different from each other at the 95% confidence level (p > 0.025).

Table 11: Effect of pH on Cu2+ removal by test organisms.

Organism

Cu2+ removed (%)

pH

4.0 5.0 6.0 7.0

S. pevalekii A32.80a±1.8 A40.80b±0.80 A70.00c±2.2 A56.00d±2.8

L. spiralis A29.00a±2.2 B47.60b±2.0 A73.80c±2.6 A52.80b±2.2

O. chlorina A31.00a±1.0 A37.00a±1.8 B47.00b±2.2 B33.00a±2.8

P. molle A33.20a±2.0 A39.40a±1.8 B48.60b±2.2 B35.00a±2.8

A. torulosa B19.60a±1.8 C31.40b±2.8 B43.00c±2.4 C23.40a±3.2

Experimental conditions: Initial Cu2+ concentration: 5 µg ml-1; biomass: 0.1 mg protein ml-1; buffer: imidazole-HCl (0.2 M); contact time: 60 min; Temp: 28±2 °C; volume: 50 ml





Table 12: Effect of pH on Ni2+ removal by test organisms.

Organism

Ni2+ removed (%)

pH

4.0 5.0 6.0 7.0

S. pevalekii A27.20a±2.0 A29.40a±2.8 A37.80b±1.8 A36.20b±2.6

L. spiralis A24.80a±2.2 A25.20a±2.6 A35.60b±2.8 C28.40a±3.2

O. chlorina B15.00a±1.8 B17.60a±2.0 B25.40b±1.2 C16.80a±2.4

P. molle B16.40a±2.2 C22.40b±1.8 B29.50C±2.6 B24.40b±2.8

A. torulosa B17.20a±2.4 B18.60a±2.8 B29.40b±2.4 B24.60b±2.6

Experimental conditions: Initial Ni2+ concentration: 5 µg ml-1; biomass: 0.1 mg protein ml-1; buffer: imidazole-HCl (0.2 M); contact time: 60 min; Temp: 28±2 °C; volume: 50 ml





c) Optimization of Biomass Load

Metal removal by test organisms from the solution (pH 6.0) containing 5 µg ml-1

Cd2+ was studied by varying biomass from 0.025 to 0.6 mg protein ml-1. It was observed

that with increase in biomass load, Cd2+ removal by the test organisms increased (Fig. 9).

With increase in biomass from 0.025 mg protein ml-1 to 0.6 mg protein ml-1 Cd2+ removal

by S. pevalekii increased from 24.0 to 82.6%, by L. spiralis from 32.0 to 83.8%, by

O. chlorina from 27.4 to 65.8%, by P. molle from 24.6 to 56.6% and by A. torulosa from

18.6 to 51.6% (Table 13). Appreciable increase in Cd2+ removal was observed with

increase in biomass load up to 0.15 mg protein ml-1. With further increase in biomass

load metal removal did not increase significantly. t-test of data revealed that pcal were <

0.025 when Cd2+ removal by successive biomass loads from 0.025 to 0.15 mg protein

ml-1 were compared indicating significant differences in metal removal. But pcal were

>0.025 when Cd2+ removal by biomass load from 0.15 to 0.6 mg protein ml-1 was

compared indicating insignificant differences in metal removal. Thus, 0.15 mg protein

ml-1 was optimum biomass for metal removal from solution containing 5 µg Cd2+ ml-1.

In the same way experiments were performed to study Cu2+ and Ni2+ removal by test

organisms. Results revealed that removal of both the metals increased with increase in

biomass up to 0.15 mg protein ml-1 and after that it got saturated (Figs. 10 & 11). t- test

applied on data of both the metals revealed insignificant differences in metal removal when

metal removal by successive biomass loads from 0.15 to 0.6 mg protein ml-1 was compared

(Tables 14 & 15). Thus the optimum biomass load for Cd2+, Cu2+ and Ni2+ removal from

solution containing 5 µg metal ml-1 by all the test organisms was 0.15 mg protein ml-1.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 0.025 0.05 0.75 0.1 0.15 0.3 0.45 0.6

Biomass (mg protein ml-1

)

Cd2

+ rem

ove

d (µg m

l-1)


( ), and A. torulosa ( ) at different biomass load.

Experimental conditions: Initial Cd2+ concentration: 5 µg ml-1; Buffer: imidazole-HCl (0.2 M); pH: 6.0; contact time: 60 min; Temp: 28±2 °C

pcal< 0.025 when Cd2+ removal is compared from 0.025 to 0.15 mg protein ml-1

indicating significant differences in metal uptake. pcal> 0.025 when Cd2+ removal is compared from 0.15 to 0.6 mg protein ml-1 indicating insignificant differences in metal uptake.

Table 13: Effect of biomass load on Cd2+ removal by test organisms.

Organism

Cd2+ removed (%)

Biomass (mg protein ml-1)

0.025 0.05 0.075 0.10 0.15 0.3 0.45 0.60

S. pevalekii A24.00a±2.4 A30.80a±2.6 A42.00b±3.2 A58.80c±1.2 A76.20d±2.0 A81.00d±2.6 A83.40d±2.8 A85.60d±2.2

L. spiralis B32.00a±2.2 B43.60b±2.4 B53.60c±2.0 A60.00d±1.0 A78.40e±1.8 A82.20e±2.2 A84.60e±2.4 A86.80e±2.8

O. chlorina A27.40a±1.2 A33.40b±2.2 A43.40c±1.8 B50.80d±2.4 B60.20e±2.6 B64.20e±1.4 B68.00e±2.8 B69.80e±3.0

P. molle A24.60a±1.4 A32.20b±1.8 A40.20c±1.2 B46.40d±2.4 C51.20e±2.8 C57.40e±1.5 C59.80e±2.4 C60.60e±2.6

A. torulosa C18.60a±1.8 C23.20a±3.4 C29.00b±2.8 C32.80b±1.4 D45.80c±2.2 D51.00c±2.8 D52.20c±3.0 C56.60c±2.0

Experimental conditions: Initial Cd2+ concentration: 5 µg ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; contact time: 60 min; Temp: 28±2 °C; volume: 50 ml Arithmetic means within the same column with the same uppercase letter are not significantly different from each other at the 95%

confidence level (p > 0.025). Also, arithmetic means within the same row with the same lowercase letter are not significantly different

from each other at the 95% confidence level (p > 0.025).

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 0.025 0.05 0.075 0.1 0.15 0.3 0.45 0.6Biomass (mg protein ml-1)

Cu2

+ rem

ove

d (µ

g m

l-1)



Experimental conditions: Initial Cu2+ concentration: 5 µg ml -1; Buffer: imidazole-HCl (0.2 M); pH: 6.0; contact time: 60 min; Temp: 28±2 °C

pcal< 0.025 when Cu2+ removal is compared from 0.025 to 0.15 mg protein ml-1

indicating significant differences in metal uptake. pcal> 0.025 when Cu2+ removal is compared from 0.15 to 0.6 mg protein ml-1

indicating insignificant differences in metal uptake.

0

0.5

1

1.5

2

2.5

0 0.025 0.05 0.075 0.1 0.15 0.3 0.45 0.6


Ni2

+ re

mo

ved

(µg

ml-1)

Fig, 11: Ni2+ removal by S. pevalekii ( ), L. spiralis ( ), O. chlorina ( ), P. molle


Experimental conditions: Initial Ni2+ concentration: 5 µg ml-1; Buffer: imidazole-HCl (0.2 M); pH: 6.0; contact time: 60 min; Temp: 28±2 °C

pcal< 0.025 when Ni2+ removal is compared from 0.025 to 0.15 mg protein ml-1

indicating significant differences in metal uptake. pcal> 0.025 when Ni2+ removal is compared from 0.15 to 0.6 mg protein ml -1 indicating insignificant differences in metal uptake.

Table 14: Effect of biomass load on Cu2+ removal by test organisms.

Organism

Cu2+ removed (%)


0.025 0.05 0.075 0.1 0.15 0.3 0.45 0.6

S. pevalekii A22.60a±0.8 A43.00b±1.8 A54.60c±2.2 A62.60d±3.2 A74.00e±2.8 A78.20e±1.8 A79.80e±2.2 A82.00e±1.8

L. spiralis B32.60a±2.4 A49.20b±1.0 B63.20c±2.6 A66.00c±1.8 A75.80d±3.0 A80.20d±2.6 A81.00d±3.4 A82.40d±2.0

O. chlorina C18.20a±1.2 B23.60b±2.4 C28.80b±1.8 B38.40c±1.2 B51.00d±1.0 B54.60d±2.0 B57.80d±2.2 B59.40d±1.6

P. molle C18.40a±0.8 B27.00b±2.2 C33.20b±2.6 B41.80c±1.4 B52.00d±2.4 B57.40d±2.8 B58.20d±2.0 B61.00d±2.0

A. torulosa D4.40a±2.0 C10.40b±2.4 D15.80b±2.8 C25.40c±2.0 C45.00d±2.2 B50.00d±2.4 B51.00d±2.6 B53.80d±1.8

Experimental conditions: Initial Cu2+ concentration: 5 µg ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; contact time: 60 min; Temp: 28±2 °C; volume: 50 ml Arithmetic means within the same column with the same uppercase letter are not significantly different from each other at the 95%



Table 15: Effect of biomass load on Ni2+ removal by test organisms.

Organism

Ni2+ removed (%)


0.025 0.05 0.075 0.1 0.15 0.3 0.45 0.6

S. pevalekii A13.60a±2.2 A18.80a±1.8 A26.20b±2.0 A31.40b±1.2 A38.40c±2.8 A39.80c±1.2 A41.80c±2.8 A43.80c±2.8

L. spiralis A14.80a±1.4 A21.40b±2.4 A26.00b±2.2 A29.40b±1.8 A36.00c±1.8 A38.20c±1.4 A40.20c±2.6 A42.40c±1.8

O. chlorina A12.20a±1.5 A16.40a±2.2 B19.20a±2.0 B23.40b±2.4 B27.00b±1.6 B29.80b±2.0 B30.40b±2.4 B31.20b±2.6

P. molle A11.20a±1.4 A15.60a±1.8 B19.20b±1.8 B24.20b±2.2 B30.80c±2.6 B33.80c±1.2 B34.00c±2.6 B35.20c±2.6

A. torulosa A12.40a±2.0 A14.80a±1.8 B18.40a±2.2 B24.00b±1.4 B30.20c±2.2 B34.00c±2.8 B35.40c±2.4 B37.00c±2.4

Experimental conditions: Initial Ni2+ concentration: 5 µg ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; contact time: 60 min; Temp: 28±2 °C; volume: 50 ml Arithmetic means within the same column with the same uppercase letter are not significantly different from each other at the 95%



d) Optimization of Initial Metal Concentration

Metal removal efficiency of test organisms was studied by varying initial

concentration of each metal i.e. Cd2+, Cu2+ and Ni2+ from 0.5 to 10 µg ml-1. Optimum

biomass load (0.15 mg protein ml-1) and pH (6.0) of solution were selected. Results of

experiments for Cd2+ removal revealed that with increase in initial Cd2+ concentration in

the solution up to 5 µg ml-1 amount of metal removed by the test organisms increased and

afterwards it got saturated (Fig. 12). t-test on data revealed that the pcal were > 0.025

when Cd2+ removal by the organisms from solutions containing 5 µg ml-1 Cd2+ were

compared with solution containing 10 µg ml-1 indicating insignificant differences in

metal removal. So the optimum initial concentration for Cd2+ removal under the selected

experimental condition was found to be 5 µg ml-1. Under optimum conditions of pH, biomass

load and initial metal concentration L. spiralis removed 78.4%, S. pevalekii 76.2%,

O. chlorina 60.6%, P. molle 51.2% and by A. torulosa 45.8% Cd2+ of 5 µg ml-1 Cd2+.

In the same way experiments were performed to find out optimum initial

concentrations of Cu2+ and Ni2+. Results revealed that optimum initial concentration of

Cu2+ and Ni2+ for all the test organisms was 5 µg ml-1 of each metal (Figs. 13 & 14).

e) Optimization of Temperature

Metal removal efficiency of the test organisms was compared at different

temperatures i.e. 21±2, 28±2 and 35±2 ºC (Figs. 15-17). Keeping all other conditions

optimum, when metal removal was studied at varied temperatures it was observed that

there were not appreciable differences in the amount of Cd2+ removed by the test

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 0.5 1 2 4 5 10

Cd2+

(µg ml-1

)

Cd2

+ rem

ove

d (µ

g m

l-1)


( ), and A. torulosa ( ) from solution with graded concentrations of Cd2+.

Experimental conditions: Buffer: imidazole-HCl (0.2 M); pH: 6.0; biomass: 0.15 mg protein ml-1; contact time: 60 min; Temp: 28±2 °C

pcal< 0.025 when Cd2+ removal is compared from 0.5 to 5 µg ml-1 metal concentrations indicating significant differences in metal uptake. pcal> 0.025 when Cd2+ removal is compared from 5 to 10 µg ml-1 metal concentrations indicating insignificant differences in metal uptake.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 0.5 1 2 4 5 10Cu2+ (µg ml-1)

Cu2

+ rem

ove

d (µ

g m

l-1)


( ), and A. torulosa ( ) from solution with graded concentrations of Cu2+.


pcal< 0.025 when Cu2+ removal is compared from 0.5 to 5 µg ml-1 metal concentrations indicating significant differences in metal uptake. pcal> 0.025 when Cu2+ removal is compared from 5 to 10 µg ml-1 metal concentrations indicating insignificant differences in metal uptake.

0

0.5

1

1.5

2

2.5

0 0.5 1 2 4 5 10Ni

2+ (µg ml

-1)

Ni2

+ rem

ove

d (µ

g m

l-1)


( ), and A. torulosa ( ) from solution with graded concentrations of Ni2+.


pcal< 0.025 when Ni2+ removal is compared from 0.5 to 5 µg ml-1 metal concentrations indicating significant differences in metal uptake. pcal> 0.025 when Ni2+ removal is compared from 5 to 10 µg ml-1 metal concentrations indicating insignificant differences in metal uptake.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

S. pevalekii L. spiralis O. chlorina P. molle A. torulosa

Cd2

+ rem

ove

d (µg m

l-1)

Fig. 15: Cd2+ removal by test organisms at varied temperatures.

21±2oC ( ); 28±2oC ( ); 35±2oC ( )

Experimental conditions: Initial Cd2+ concentration: 5 µg ml-1; biomass: 0.15 mg protein ml-1; buffer: imidazole-HCl 0.2; pH: 6.0; contact time: 60 min

pcal> 0.025 when Cd2+ removal is compared from 28±2 ºC to 35±2 ºC indicating insignificant differences in metal uptake.

0

0.5

1

1.5

2

2.5

3

3.5

4


Cu2

+ rem

ove

d (µg

ml-1)

Fig. 16: Cu2+ removal by test organisms at varied temperatures.

21±2oC ( ); 28±2oC ( ); 35±2oC ( )

Experimental conditions: Initial Cu2+ concentration: 5 µg ml-1; biomass: 0.15 mg protein ml-1; buffer: imidazole-HCl 0.2; pH: 6.0; contact time: 60 min

pcal> 0.025 when Cu2+ removal is compared from 28±2 ºC to 35±2 ºC indicating insignificant differences in metal uptake.

0

0.5

1

1.5

2

2.5


Ni2

+ rem

ove

d (µg

ml-1)

Fig. 17: Ni2+ removal by test organisms at varied temperatures.

21±2oC ( ); 28±2oC ( ); 35±2oC ( )

Experimental conditions: Initial Ni2+ concentration: 5 µg ml-1; biomass: 0.15 mg protein ml-1; buffer: imidazole-HCl 0.2; pH: 6.0; contact time: 60 min

pcal> 0.025 when Ni2+ removal is compared from 28±2 ºC to 35±2 ºC indicating insignificant differences in metal uptake.

Table 16: Effect of temperature on Cd2+ removal by test organisms.

Organism

Cd2+ removed (%)

21± 2 °C 28± 2 °C 35± 2 °C

S. pevalekii A73.20a± 2.2 A76.20a± 2.8 A78.00a± 1.8

L. spiralis A75.20a± 2.6 A78.40a±2.4 A79.80a± 2.4

O. chlorina B58.40a± 1.8 B60.60a±3.0 B62.20a± 2.8

P. molle C49.80a± 2.8 C51.20a± 2.2 C52.20a± 2.6

A. torulosa C44.00a± 3.2 D45.80a± 2.6 C47.20a± 1.8

Experimental conditions: Initial Cd2+ concentration: 5 µg ml-1; biomass: 0.15 mg protein ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; contact time: 60 min; volume: 50 ml Arithmetic means within the same column with the same uppercase letter are not




organisms (Table 16). t-test on data revealed that the pcal was > 0.025 when Cd2+ removal

at 28±2 ºC was compared with metal removal at 35±2 ºC indicating insignificant

differences in metal removal .

Similar experiments were performed for Cu2+ and Ni2+ removal and results

revealed that there were not significant differences in amount of Cu2+ or Ni2+ removed by

the test organisms at varied temperatures. t-test on data revealed that the pcal was > 0.025

when Cu2+ and Ni+ removal at 28±2 ºC were compared with metal(s) removal at 35±2 ºC

indicating insignificant difference in metal removal (Tables 17 & 18).

Thus, the results indicated that change in temperature from 28 to 35 ºC or 21 ºC

had no significant effect on Cd2+, Cu2+ or Ni2+ removal by the test organisms. Since the

temperature of the culture room was maintained at 28±2 ºC so all the subsequent

experiments were performed at 28±2 ºC.

Optimum conditions for Cd2+, Cu2+ and Ni2+ removal by the test organisms thus

were found to be contact time: 60 min; pH: 6.0; biomass load: 0.15 mg protein ml-1;

metal concentration: 5 µg ml-1 and temperature: 28±2 ºC (Table 19). Under optimum

conditions, maximum amounts of Cd2+, Cu2+ and Ni2+ removed by all the test organisms are

presented in the Table 20. Among all the test organisms L. spiralis removed maximum

amount of Cd2+ (78.4%) and Cu2+ (75.8%) while A. torulosa removed minimum amounts

(45.8% of Cd2+ and 45.0% of Cu2+). Maximum amount of Ni2+ (38.4%) was removed by

S. pevalekii while minimum (27.0%) was removed by O. chlorina.

Table 17: Effect of temperature on Cu2+ removal by test organisms.

Organism

Cu2+ removed (% )

21± 2 °C 28± 2 °C 35± 2 °C

S. pevalekii A72.00a± 1.4 A74.00a± 2.4 A76.20a± 1.2

L. spiralis A73.60a± 1.0 A75.80a± 1.8 A77.80a± 0.8

O. chlorina B48.00a± 2.2 B51.00a± 1.2 B52.40a± 2.8

P. molle B50.40a± 2.4 B52.00a± 2.0 B54.20a± 1.4

A. torulosa B43.80a± 2.8 C45.00a±1.8 B47.20a± 1.8

Experimental conditions: Initial Cu2+ concentration: 5 µg ml-1; biomass: 0.15 mg protein ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; contact time: 60 min; volume: 50 ml Arithmetic means within the same column with the same uppercase letter are not




Table 18: Effect of temperature on Ni2+ removal by test organisms.

Organism

Ni2+ removed (%)

21± 2 °C 28± 2 °C 35± 2 °C

S. pevalekii A36.20a± 1.8 A38.40a±2.8 A39.20a± 1.6

L. spiralis A34.80a± 2.2 A36.20a± 1.8 A37.40a± 2.4

O. chlorina B25.60a± 1.4 B27.00a± 1.4 B28.20a± 1.0

P. molle B29.40a± 2.8 B30.40a± 2.6 B31.40a± 1.8

A. torulosa B30.20a± 2.6 B30.20a± 1.0 B31.20a± 1.4

Experimental conditions: Initial Ni2+ concentration: 5 µg ml-1; biomass: 0.15 mg protein ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; contact time: 60 min; volume: 50 ml Arithmetic means within the same column with the same uppercase letter are not




Table 19: Optimum conditions for metal (Cd2+, Cu2+ or Ni2+) removal by test organisms.

Optimum conditions

Organism Contact time (min.)

pH Biomass load

(mg protein ml-1

)

Metal (µg ml-1)

Temperature (°C)

S. pevalekii 60 6.0 0.15 5 28±2

L. spiralis 60 6.0 0.15 5 28±2

O. chlorina 60 6.0 0.15 5 28±2

P. molle 60 6.0 0.15 5 28±2

A. torulosa 60 6.0 0.15 5

28±2

Table 20: Maximum amount of metal removed (µg ml-1) by test organisms under

optimum conditions. Organism Cadmium Copper Nickel

S. pevalekii A3.81a± 0.14

(76.20)

A3.70a± 0.12 (74.00)

A1.92b±0.14 (38.40)

L. spiralis A3.92a± 0.12

(78.40)

A3.79a± 0.09 (75.80)

A1.81b± 0.09

(36.20) O. chlorina B3.03a±0.15

(60.60)

B2.55b± 0.06 (51.00)

B1.35c± 0.07

(27.00) P. molle C2.56a± 0.11

(51.20)

B2.60a± 0.10 (52.00± 2.0)

B1.52b± 0.13

(30.40) A. torulosa D2.29a± 0.13

(45.80)

C2.25a±0.09 (45.00)

B1.51b± 0.05

(30.20) Experimental conditions: Initial metal concentration: 5 µg ml-1; biomass: 0.15 mg protein ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; contact time: 60 min; Temp: 28±2 °C; volume: 50 ml Arithmetic means within the same column with the same uppercase letter are not




Values in parenthesis indicate % metal removal of 5 µg ml-1

XI. ADSORPTION ISOTHERMS

Langmuir and Freundlich adsorption isotherms are generally used for metal

adsorption studies. Thus, these were obtained from equilibrium sorption data of metal

removal experiments. The calculated Langmuir and Freundlich isotherm constants and

corresponding correlation coefficient values are shown in Tables 21-23. The results

revealed that all the data of metal sorption experiments fitted better in Freundlich

isotherm than the Langmuir isotherm as evidenced by high values of correlation

coefficients (Tables 21-23).

The correlation coefficient values obtained from the Freundlich and Langmuir

isotherms (Table 21; Figs. 18-22 and Figs. 33-37) indicated that data of Cd2+ adsorption

by all the test organisms fitted better in Freundlich isotherm with higher R2 values (R2 >

0.99) as compared to Langmuir isotherm (R2 > 0.97). Freundlich model for Cd2+ removal

that the values of adsorption intensity (n) for all test organisms ranged from 1.25 to 1.65

indicating a favourable adsorption of Cd2+ from aqueous solutions (Table 21). R2 values

for Cd2+ adsorption by all the test organisms, as per Langmuir model ranged from 0.97 to

0.99 (Figs. 33-37). The highest qmax value for Cd2+ adsorption (66.67 µg Cd2+ mg-1

protein) with lowest value of b (0.021) was showed by L. spiralis (Table 21).

In the same way data of Cu2+ and Ni2+ adsorption by all the test organisms was

fitted into the adsorption isotherms. It was observed that experimental data on Cu2+ and

Ni2+ removal by all test organisms fitted well in the Freundlich model (R2 > 0.97) as

compared to Langmuir model (R2> 0.95). The R2 values for Cu2+ adsorption by all the

test organisms ranged from 0.98 to 0.99 (Figs. 23-27 and Figs. 38-47) while for Ni2+

Table 21: A comparison of Langmuir and Freundlich adsorption isotherm charactersics

for Cd2+ removal by test organisms.

Organism

Langmuir constants Freundlich Constants

qmax b R2 K f n R2

S. pevalekii

43.47 0.023 0.987 23.55 1.25 0.990

L. spiralis

66.67 0.021 0.990 25.52 1.49 0.991

O. chlorina

33.33 0.036 0.987 13.93 1.50 0.995

P. molle

29.41 0.043 0.988 11.53 1.62 0.990

A. torulosa

23.81 0.054 0.971 9.09 1.65 0.990


for Cu2+ removal by test organisms.

Organism


qmax b R2 K f n R2

S. pevalekii 37.04 0.024 0.991 20.32 1.26 0.999 L. spiralis 43.47 0.024 0.986 22.08 1.27 0.996 O. chlorina 27.02 0.04 0.989 10.64 1.61 0.990 P. molle 30.30 0.05 0.967 11.01 1.57 0.994 A. torulosa 23.80 0.049 0.983 9.48 1.65 0.986


for Ni2+ removal by test organisms.

Organism


qmax b R2 K f n R2

S. pevalekii 27.27 0.066 0.981

8.26 2.00 0.990

L. spiralis 25.0 0.067 0.968

7.88 1.92 0.976

O. chlorina 17.24 0.090 0.970

5.66 2.04 0.980

P. molle 18.51 0.080 0.959

6.28 1.91 0.983

A. torulosa 18.51 0.084 0.964

6.12 1.96 0.983

Table 24: Based on FTIR analysis functional groups present on the surface of the test

organisms and groups which appear to be involved metal binding.

Major functional groups on the surface of

control cultures

Functional groups involved in Cd2+,

Cu2+ and Ni2+ binding

-OH, -NH, C=C, C=O, N-O, C-O, C-N

stretch Hydroxyl and Carboxyl (-OH, C=O, C-O)

y = 0.8014x + 1.3723

R2 = 0.9904

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

-1.5 -1 -0.5 0 0.5

Log Ce

Lo

g q

Fig. 18: Freundlich adsorption plot for the adsorption of Cd2+ by S. pevalekii from

aqueous solution.


y = 0.6686x + 1.4073

R2 = 0.9917

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

-1.5 -1 -0.5 0 0.5 1

Log Ce

Lo

g q

Fig. 19: Freundlich adsorption plot for the adsorption of Cd2+ by L. spiralis from

aqueous solution.


y = 0.6658x + 1.1444

R2 = 0.9956

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

-1.5 -1 -0.5 0 0.5 1Log Ce

Lo

g q

Fig. 20: Freundlich adsorption plot for the adsorption of Cd2+ by O. chlorina from

aqueous solution.


y = 0.6187x + 1.0628

R2 = 0.9902

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

-1.5 -1 -0.5 0 0.5 1Log Ce

Lo

g q

Fig. 21: Freundlich adsorption plot for the adsorption of Cd2+ by P. molle from aqueous

solution.


y = 0.6061x + 0.9595

R2 = 0.9905

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

-1.5 -1 -0.5 0 0.5 1Log Ce

Lo

g q

Fig. 22: Freundlich adsorption plot for the adsorption of Cd2+ by A. torulosa from

aqueous solution.


y = 0.7959x + 1.308R2 = 0.9993

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

-1.5 -1 -0.5 0 0.5 1Log Ce

Lo

g q

Fig. 23: Freundlich adsorption plot for the adsorption of Cu2+ by S. pevalekii from

aqueous solution.


y = 0.787x + 1.3443

R2 = 0.9987

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

-1.5 -1 -0.5 0 0.5 1Log Ce

Lo

g q

Fig. 24: Freundlich adsorption plot for the adsorption of Cu2+ by L. spiralis from aqueous

solution.


y = 0.6218x + 1.0272

R2 = 0.9903

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

-1.5 -1 -0.5 0 0.5 1Log Ce

Log

q

Fig. 25: Freundlich adsorption plot for the adsorption of Cu2+ by O. chlorina from

aqueous solution.


y = 0.6354x + 1.0426

R2 = 0.9948

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

-1.5 -1 -0.5 0 0.5 1Log Ce

Lo

g q

Fig. 26: Freundlich adsorption plot for the adsorption of Cu2+ by P. molle from aqueous

solution.


y = 0.6072x + 0.9777

R2 = 0.9867

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

-1.5 -1 -0.5 0 0.5 1Log Ce

Lo

g q

Fig. 27: Freundlich adsorption plot for the adsorption of Cu2+ by A. torulosa from

aqueous solution.


y = 0.4993x + 0.9173

R2 = 0.9901

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

-1.5 -1 -0.5 0 0.5 1Log Ce

Lo

g q

Fig. 28: Freundlich adsorption plot for the adsorption of Ni2+ by S. pevalekii from

aqueous solution.


y = 0.5199x + 0.8975

R2 = 0.9762

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

-1.5 -1 -0.5 0 0.5 1Log Ce

Lo

g q

Fig. 29: Freundlich adsorption plot for the adsorption of Ni2+ by L. spiralis from aqueous

solution.


y = 0.489x + 0.7534

R2 = 0.9805

0

0.2

0.4

0.6

0.8

1

1.2

1.4

-1 -0.5 0 0.5 1Log Ce

Lo

g q

Fig. 30: Freundlich adsorption plot for the adsorption of Ni2+ by O. chlorina from

aqueous solution.


y = 0.5216x + 0.7988

R2 = 0.9837

0

0.2

0.4

0.6

0.8

1

1.2

1.4

-1 -0.5 0 0.5 1Log Ce

Lo

g q

Fig. 31: Freundlich adsorption plot for the adsorption of Ni2+ by P. molle from aqueous

solution.


y = 0.5092x + 0.7873

R2 = 0.9837

0

0.2

0.4

0.6

0.8

1

1.2

1.4

-1 -0.5 0 0.5 1Log Ce

Lo

g q

Fig. 32: Freundlich adsorption plot for the adsorption of Ni2+ by A. torulosa from

aqueous solution.


y = 0.0234x + 0.0234

R2 = 0.9875

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 5 10 151/Ce

1/q

Fig. 33: Langmuir adsorption plot for the adsorption of Cd2+ by S. pevalekii from aqueous

solution.


adsorption these values ranged from 0.97 to 0.99 (Figs. 28-32). As per Freundlich

isotherm adsorption affinity (n) of all the test organisms ranged from 1.26 to 1.65 for

Cu2+ and from 1.91 to 2.00 for Ni2+ suggesting favourable adsorption of both metals by

test organisms (Tables 22 & 23). R2 values obtained from Langmuir isotherms for Cu2+

removal by all the test organisms ranged from 0.96-0.99 and for Ni2+ removal from 0.95-

0.98 (Figs. 38-47). Highest qmax value for Cu2+ adsorption was shown by L. spiralis

(43.47 µg Cu2+ mg-1 protein) and for Ni2+ adsorption by S. pevalekii (27.27 µg Ni2+ mg-1

protein) (Tables 22 & 23).

Thus, the correlation coefficient (R2) values indicated that the metal adsorption by

test organisms fitted better in Freundlich isotherm as compared to Langmuir isotherm.

XII. FOURIER TRASFORMED INFRARED (FTIR) ANALYSIS

Fourier transformed infrared (FTIR) spectral analysis of control and metal

adsorbed biomass was carried out to find out the nature of chemical groups present on

cell surface which might have been involved in Cd2+, Cu2+ or Ni2+ binding. Intense

characteristic peaks corresponding to various functional groups present on cell surface of

all the test organisms were observed. FTIR spectrum of S. pevalekii biomass from control

cultures displayed peaks at 3122.86 and 3018.70 cm-1 wave numbers indicating the

presence of hydroxyl (-OH) and amine (NH) group, the peak at 1618.33 cm-1 and

1400.37 cm-1 was assigned to N-H, C=C,C-O-H and -OH group. The small peaks

observed between wave numbers 1050-1320 cm-1 were attributed to the presence of C-O

and C-N bonds (Fig. 48). The transmittance spectrum of S. pevalekii biomass loaded with

Cd2+ when compared with spectrum of unloaded biomass, it was observed that peaks at

y = 0.015x + 0.021R² = 0.990

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 5 10 15 20 251/Ce

1/q

Fig. 34: Langmuir adsorption plot for the adsorption of Cd2+ by L. spiralis from aqueous

solution.


y = 0.0306x + 0.0363

R2 = 0.9871

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 2 4 6 8 10 121/Ce

1/q

Fig. 35: Langmuir adsorption plot for the adsorption of Cd2+ by O. chlorina from aqueous

solution.


y = 0.0341x + 0.0431

R2 = 0.9884

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 2 4 6 8 10 121/Ce

1/q

Fig. 36: Langmuir adsorption plot for the adsorption of Cd2+ by P. molle from aqueous

solution.


y = 0.0426x + 0.0547

R2 = 0.9716

0

0.1

0.2

0.3

0.4

0.5

0 2 4 6 8 101/Ce

1/q

Fig. 37: Langmuir adsorption plot for the adsorption of Cd2+ by A. torulosa from aqueous

solution.


y = 0.0273x + 0.0244

R2 = 0.9914

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 2 4 6 8 10 12 141/Ce

1/q

Fig. 38: Langmuir adsorption plot for the adsorption of Cu2+ by S. pevalekii from aqueous

solution.


y = 0.0233x + 0.0269

R2 = 0.9843

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 5 10 151/Ce

1/q

Fig. 39: Langmuir adsorption plot for the adsorption of Cu2+ by L. spiralis from aqueous

solution.


y = 0.0379x + 0.0448

R2 = 0.9895

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 2 4 6 8 101/Ce

1/q

Fig. 40: Langmuir adsorption plot for the adsorption of Cu2+ by O. chlorina from aqueous

solution.


y = 0.0339x + 0.0502

R2 = 0.9679

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 2 4 6 8 10 121/Ce

1/q

Fig. 41: Langmuir adsorption plot for the adsorption of Cu2+ by P. molle from aqueous

solution.


y = 0.0428x + 0.0494

R2 = 0.9831

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 2 4 6 8 101/Ce

1/q

Fig. 42: Langmuir adsorption plot for the adsorption of Cu2+ by A. torulosa from aqueous

solution.


y = 0.0361x + 0.066

R2 = 0.9813

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 2 4 6 8 101/Ce

1/q

Fig. 43: Langmuir adsorption plot for the adsorption of Ni2+ by S. pevalekii from aqueous

solution.


y = 0.0409x + 0.0673

R2 = 0.9689

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 2 4 6 8 101/Ce

1/q

Fig. 44: Langmuir adsorption plot for the adsorption of Ni2+ by L. spiralis from aqueous

solution.


y = 0.0581x + 0.0901

R2 = 0.9702

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0 2 4 6 81/Ce

1/q

Fig. 45: Langmuir adsorption plot for the adsorption of Ni2+ by O. chlorina from aqueous

solution.


y = 0.0548x + 0.0805

R2 = 0.9593

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0 2 4 6 81/Ce

1/q

Fig. 46: Langmuir adsorption plot for the adsorption of Ni2+ by P. molle from aqueous

solution.


y = 0.054x + 0.0842

R2 = 0.9643

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0 1 2 3 4 5 6 71/Ce

1/q

Fig. 47: Langmuir adsorption plot for the adsorption of Ni2+ by A. torulosa from aqueous

solution.


wave numbers 3122.86 and 3018.70 cm-1 became broad and were shifted to 3493.20 and

3402.53 cm-1, respectively. The peaks between wave number 1400.37-1050 cm-1 became

prominent and were shifted to 1535.39 and 1242.20 cm-1 indicating involvement of –OH,

C=O and C-O, N-O or C-N groups in Cd2+ binding (Fig. 49).

Similarly FTIR spectra of S. pevalekii without and biomass loaded with Cu2+ and

Ni2+ were taken to find out the nature of groups present on cell surface which might have

been involved in binding of Cu2+ and Ni2+ (Figs. 50 & 51). In case of Cu2+ loaded

biomass, the peaks were shifted to wave number 3350, 3200 and 1737 cm-1 while peaks

at wave number 1253-1017 cm-1 became broader and prominent indicating involvement

of –OH, C=O and C-O functional groups in Cu2+ binding. In Ni2+ loaded biomass, peaks

became broader and were shifted to wave number 3458.48 cm-1 (-OH), 1548.89 cm-1

(N-O). Sharpness and little broadness in peaks between wave number 1228.70-1080 cm-1

(C-Oor C-N) were observed indicating the involvement of these groups in Ni2+ binding.

FTIR spectrum of L. spiralis biomass from control cultures displayed peaks at

wave numbers 3363.97, 2393.74, 1635.69, 1392.65 and 613.38 cm-1 indicating the

presence of –OH, NH, P-H, C=C, N-H II, C-F, C=O, C-O and C-Cl functional groups on

the surface of biomass (Fig. 52). The spectrum of Cd2+ loaded biomass showed sharp

peaks at wave number 3757.46 cm-1 (-OH) while peak at 1797.72 cm-1 wave number

became more sharp indicating the involvement of C=O group in Cd2+ binding. Peaks at

1519.96-941.44 cm-1 wave numbers became little broader and prominent showing the

involvement of C-O, N-O and C-N group in Cd2+ binding (Fig. 53). The spectrum of Cu2+

loaded biomass displayed peak at 1840.15 (C=O) while other peaks were similar as

shown by Cd2+ loaded biomass indicating the involvement of same functional groups in

Cu2+ binding as well (Fig. 54). When spectrum of Ni2+ loaded biomass was compared

with spectrum of unloaded biomass, peak at wave number 3736.24 cm-1 indicating the

involvement of hydroxyl group in Ni2+ binding. Shift and reduction in size of peak at

1695.49 cm-1 indicated the involvement of C=O stretch. The peaks between 1541.18-

1329.00 cm-1 (C-N or N-O) were reduced significantly in comparison to unloaded

biomass due to metal adsorption by the biomass (Fig. 55).

FTIR spectrum of O. chlorina biomass from control cultures showed peaks at

3743.96. 2598.20, 2360.95, 1795.79, 1518.03, 1329.0-675.11 cm-1 wave numbers

indicating the presence of -OH, NH, P-H, C=O, NH II, C-F, C-O groups and C-Cl

stretch, respectively (Fig. 56). Spectrum of Cd2+, Cu2+ or Ni2+ loaded biomass revealed

reduced sharpness in peaks at 3747.81 and 3743.96 cm-1, while peaks from 1822.79-

1745.64 cm-1 and 1518.03-949.01 cm-1 became prominent. Changes observed in spectra

of metal loaded biomass indicated the possible involvement of -OH, C=O C-N and C-O

groups in metal sorption process of O. chlorina (Figs. 57-59).

FTIR spectrum of P. molle from control cultures displayed peaks at wave

numbers 3743.96, 2694.65, 2360.95, 1795.79 and 1518.03-682.82 cm-1 indicates the

presence of -OH, P-H, C=O, N-O, C=C, C-O, C-N and C-Cl groups (Fig. 60). Cd2+, Cu2+

or Ni2+ loaded biomass revealed shifting of peaks from 3743.96 cm-1 to 3747.81 cm-1.

Sharp decrease in size of peak at wave number 1797.72 and 1791.93 cm-1 was observed

in Cd2+and Cu2+ loaded biomass while in case of Ni2+ shift in peak o 1801.57 was

observed. Changes in the spectra of metal loaded biomass of P. molle indicated the

involvement of -OH, C=O, N-O, C-N and C-O groups in metal binding (Figs. 61-63).

FTIR spectrum of A. torulosa from control cultures displayed peaks at wave numbers

3740.10, 3717.07, 2598.20, 2459.02, 1701.27 and 1525.74-677.04 cm-1 indicating the

presence of -OH, NH, P-H, C=O, N-O, C=C, C-O stretching and C-Cl groups (Fig. 64).

When spectra of Cd2+, Cu2+ or Ni2+ loaded biomass were compared with unloaded

biomass, peaks corresponding to all these groups were shifted indicating the involvement

of -OH, C=O, C-N and C-O groups in metal binding (Figs. 65-67).

Thus, it is concluded that the peaks between 3200 and 3600 cm-1 corresponding to

O-H stretching (H- bonded), between 3500-3700 cm-1 corresponding to O-H stretching

mode (H- free) indicated the presence of alcoholic group. Peaks between 1800-1540 cm-1

were associated with C=O stretching mode in carbonyls and carboxylic acids while peaks

between 1300-1080 cm-1 are assigned to the C-N or C-O stretching and O-H such as in

carboxylic acids, esters, ethers and alcohols etc. The results of present study indicate that

almost same functional groups were present on the surface of cells of all the test

organisms. The main groups involved in metal sorption appear to be hydroxyl and

carboxylic acids groups (Table 24). FTIR spectra revealed that S. pevalekii and L. spiralis

exhibited high intensities of hydroxyl group as compared to other three organisms i.e.

O. chlorina, P. molle and A. torulosa.

XIII. STUDIES ON EFFECT OF CERTAIN PARAMETERS ON METAL

REMOVAL

Effects of certain parameters on metal removal by test organisms were studied to

find out whether these maintain/enhance/reduce the metal removal efficiencies of the test

organisms. The parameters studied included state and age of cultures, pre-treatment of the

cultures, effect of presence of cations, effect of presence of one metal on the removal of

other metal and immobilization of cells.

A. Effect of State of Cultures on Metal Removal

Keeping all other conditions optimum, metal removal efficiencies of the test

organisms were investigated by using three states of cultures i. e. living, oven dried and

heat killed. Results revealed that when heat killed cells were used, increase in amount of

Cd2+ removal by test organisms was observed. On the oher hand, amount of Cd2+ removal

decreased compared to live cells when oven dried cells were used. Increase in Cd2+

removal was maximum (37.5%) when heat killed cells of P. molle were used followed by

A. torulosa (25.32% increase). Heat killed cells of L. spiralis exhibited only 8% increase

in Cd2+ removal (Table 25).

Similar experiments were conducted to find out the effect of state of cultures on

Cu2+ and Ni2+ removal by the test organisms. Results revealed that Cu2+ and Ni2+

removal, compared to live cells, increased when heat killed cells were used and decreased

when oven dried cells were used. Maximum increase (34.23%) in Cu2+ removal was

observed in case of P. molle and minimum (7.56%) increase was observed with

S. pevalekii biomass (Table 26). Maximum increase in Ni2+ removal was observed when

heat killed biomass of L. spiralis (39.77% increase) was used while heat killed biomass

of O. chlorina exhibited minimum (4.44%) increase in Cu2+ removal compared to live

cultures (Table 27).

The order of metal removal by all the test organisms was heat killed cells > living cells > oven dried cells. Thus, on the basis of above data it is concluded that the heat Table 25: Amount of Cd2+ removed (µg ml-11) by live and killed cultures of test organisms.

Organism Cd2+

Live cultures Oven Dried Heat Killed

S. pevalekii

A3.81a± 0.14

A3.22b±0.12

(↓ 15.49)

A4.12c±0.11

(↑ 8.14) L. spiralis

A3.92a± 0.12

B2.78b±0.06

(↓ 29.09)

A4.23c±0.09

(↑ 8.0) O. chlorina

B3.03a±0.15

C2.32b±0.10

(↓ 23.44)

B3.67c±0.11

(↑ 21.12) P. molle

C2.56a± 0.11

C2.29a±0.12

(↓ 10.55) )

B3.52b±0.09 (↑ 37.50)

A. torulosa

D2.29a± 0.13

D1.52b±0.08

(↓ 33.63)

C2.87c±0.12

(↑ 25.32) Experimental conditions: Initial Cd2+ concentration: 5 µg ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; biomass: 0.15 mg protein ml-1; contact time: 60 min; Temp: 28±2 °C; volume: 50 ml (↓) indicates % decrease compared to live cultures (↑) indicates % increase compared to live cultures Arithmetic means within the same column with the same uppercase letter are not




Table 26: Amount of Cu2+ removed (µg ml-1) by live and killed cultures of test organisms.

Organism Cu2+


S. pevalekii

A3.70a± 0.12

A3.01b± 0.07

(↓ 18.65)

A3.98c± 0.04


A3.79a± 0.09

B2.51b± 0.09

(↓ 33.78)

B4.30c± 0.08


B2.55a± 0.06

C2.18b± 0.11

(↓ 14.51)

C3.08c± 0.12

(↑ 20.78) P. molle

B2.60a± 0.10

D1.94b± 0.14

(↓ 25.39)

D3.49c± 0.03

(↑ 34.23) A. torulosa

C2.25a±0.09

D2.01a± 0.08

(↓ 10.67)

C3.00b± 0.06

(↑ 33.33) Experimental conditions: Initial Cu2+ concentration: 5 µg ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; biomass: 0.15 mg protein ml-1; contact time: 60 min; Temp: 28±2 °C; volume: 50 ml (↓) indicates % decrease compared to live cultures (↑) indicates % increase compared to live cultures Arithmetic means within the same column with the same uppercase letter are not




Table 27: Amount of Ni2+ removed (µg ml-1) by live and killed cultures of test organisms.

Organism Ni 2+


S. pevalekii

A1.92a±0.14

A1.24b±0.11

(↓ 35.42)

A2.38c±0.11


A1.81a± 0.09

A1.31b±0.05

(↓ 27.63)

A2.53c±0.09


B1.35a± 0.07

B1.06b±0.08

(↓ 21.49)

B1.41a±0.06 (↑ 4.44)

P. molle

B1.52a± 0.13

B1.09b±0.11

(↓ 28.29)

C1.98c±0.12


B1.51a± 0.05

A1.21b±0.13

(↓ 19.57)

B1.71a±0.13 (↑ 13.24)

Experimental conditions: Initial Ni2+ concentration: 5 µg ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; biomass: 0.15 mg protein ml-1; contact time: 60 min; Temp: 28±2 °C; volume: 50 ml (↓) indicates % decrease compared to live cultures (↑) indicates % increase compared to live cultures Arithmetic means within the same column with the same uppercase letter are not




killed cells of the test organisms removed more amount of metal as compared to other

states of cultures.

B. Effect of Age of Culture on Metal Removal

Cd2+, Cu2+ and Ni2+ removal experiments were performed using cultures from

different phases (Lag, Log and Stationary) of growth.

To determine the duration of different phases of growth, growth experiments with

all the test organisms were performed for 30 days (Figs. 68-72). All the test organisms

remained in lag phase of growth up to 2 day but the log phase varied from organism to

organism. In case of S. pevalekii, log phase was observed from 3 to 12 days and

stationary phase from 12 to 18 days (Fig. 68). L. spiralis and P. molle grew

logarithmically up to 21 day and then entered in to stationary phase. Log phase of growth

for O. chlorina was observed from 3 to 18 days and stationary phase from 18 to 24 days

while in case of A. torulosa, log phase was observed from 3-21 days and then the

organism entered in to stationary phase (Table 28).

Metal removal by the test organisms was studied under optimum conditions by

employing cultures from three phases of growth i.e. lag, log and stationary. The results

revealed that metal removal efficiency of test organisms increased with age of culture.

Maximum Cd2+ removal was observed when cells from stationary phase were used.

Maximum increase in amount of Cd2+ removal (35.54%), compared to log phase cultures,

was observed when cultures of P. molle from stationary phase were used (Table 29).

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

0 3 6 9 12 15 18 21 24Days

2+L

og1

0 A

72

0

Fig. 68: Growth of S. pevalekii in basal medium in terms of increase in absorbance of cultures.

pcal< 0.025 when growth values on subsequent days from zero to 12 are compared indicating significant differences. pcal> 0.025 when growth values on subsequent days from 12 to 21 are compared

indicating insignificant differences.

Fig. 69: Growth of L. spiralis in basal medium in terms of increase in absorbance of cultures.

pcal< 0.025 when growth values on subsequent days from zero to 21 are compared indicating significant differences. pcal> 0.025 when growth values on subsequent days from 21 to 30 are compared indicating insignificant differences.

Fig. 70: Growth of O. chlorina in basal medium in terms of increase in absorbance of

cultures. pcal< 0.025 when growth values on subsequent days from zero to 18 are compared indicating significant differences. pcal> 0.025 when growth values on subsequent days from 18 to 24 are compared


Fig. 71: Growth of P. molle in basal medium in terms of increase in absorbance of

cultures.

pcal< 0.025 when growth values on subsequent days from zero to 21 are compared indicating significant differences. pcal> 0.025 when growth values on subsequent days from 21 to 30 are compared


Fig. 72: Growth of A. torulosa in basal medium in terms of increase in absorbance of

cultures. pcal< 0.025 when growth values on subsequent days from zero to 21 are compared indicating significant differences. pcal> 0.025 when growth values on subsequent days from 21 to 30 are compared indicating insignificant differences.

Table 28: Duration of growth phases of test organisms.

Organism Lag Phase (d) Log Phase (d) Stationary phase (d)

S. pevalekii 2 3-12 12-18

L. spiralis 2 3- 21 21- 27

O. chlorina 2 3- 18 18 - 24

P. molle 2 3 – 21 21- 27

A. torulosa 2 3-21 21- 27

Table 29: Amount of Cd2+ removed (µg ml-1) by cultures from different phases of growth of test organisms.

Organism Cd2+

Log Lag Stationary

S. pevalekii

A3.81a± 0.14

A3.06b±0.12

(↓ 24.50) A4.19c±0.15

(↑ 10) L. spiralis

A3.92a± 0.12

A3.21b±0.09 (↓ 22.11)

A4.38c±0.13


B3.03a±0.15

B2.02b±0.08

(↓ 50.0)

B3.69c±0.09

(↑ 21.78) P. molle

C2.56a± 0.11

B2.24b±0.10 (↓ 14.28)

B3.47c±0.8


D2.29a± 0.13

C1.79b±0.11

(↓ 27.93)

C2.99c±0.11

(↑ 30.56) Experimental conditions: Initial Cd2+ concentration: 5 µg ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; biomass: 0.15 mg protein ml-1; contact time: 60 min; Temp: 28±2 °C; volume: 50 ml (↓) indicates % decrease compared to log phase cultures (↑) indicates % increase compared to log phase cultures





Similar experiments were performed to study Cu2+ and Ni2+ removal by the test

organisms. The results revealed that when cultures from stationary phase of growth were

used maximum increase in Cu2+ (29.77%) and Ni2+ removal (34.25%) was observed in

case of A. torulosa and L. spiralis, respectively (Tables 30 & 31).

The order of metal(s) removal by all the test organisms was stationary phase > log

phase > lag phase cultures. Thus, cultures from stationary phase of growth removed

maximum amounts of metal compared to lag or log phase cultures.

C. Effect of Pre-treatment of Biomass on Metal Removal

For the modification of functional groups present on cell surface, algal biomass

was treated with different acids, alkalies and organic solvents and then used for metal

removal studies. The main purpose of these pre-treatments was to find out whether metal

removal efficiency of the test organisms increased or decreased after pre-treatment.

Pre-treated cells were used to study metal removal from solution under optimized

conditions. The amount of metal removed by treated cultures was compared with the

amount of metal removed by untreated cultures and results revealed that treatment of

cultures with some reagents resulted in increase in amount of metal removed by test

organisms. Nearly 2-16% increase in Cd2+ removal was observed when cells of test

organisms were treated with 0.1 mM each of HCl, CH3COOH, HNO3 or NaOH.

Maximum increase in Cd2+ removal by test organisms was observed when these were

treated with 90% CH3OH (8-41% increase) or 0.1 mM KOH (11-51% increase).

Reduction in Cd2+ removal (4-22%) by the test organisms was observed when cells were

Table 30: Amount of Cu2+ removed (µg ml-1) by cultures from different phases of growth

of test organisms.

Organism Cu2+

Log Lag Stationary

S. pevalekii

A3.70a± 0.12

A2.89b±0.10

(↓ 28.02)

A4.01c±0.05


A3.79a± 0.09

A2.98b±0.07

(↓ 27.18)

A4.09c±0.10


B2.55a± 0.06

B1.91b±0.11 (↓ 33.50)

B3.21c±0.09

(↑ 25.88) P. molle

B2.60a± 0.10

B2.01b±0.13 (↓ 29.35)

B3.17c±0.11


C2.25a±0.09

B2.07a±0.06 (↓ 8.69)

C2.92b±0.13

(↑ 29.77) Experimental conditions: Initial Cu2+ concentration: 5 µg ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; biomass: 0.15 mg protein ml-1; contact time: 60 min; Temp: 28±2 °C; volume: 50 ml (↓) indicates % decrease compared to log phase cultures (↑) indicates % increase compared to log phase cultures Arithmetic means within the same column with the same uppercase letter are not




Table 31: Amount of Ni2+ removed (µg ml-1) by cultures from different phases of growth

of test organisms.

Organism Ni2+

Log Lag Stationary

S. pevalekii

A1.92a±0.14

A1.38b±0.11

(↓ 39.13)

A2.41c±0.07


A1.81a± 0.09

A1.55b±0.12

(↓ 16.77)

A2.43c±0.08


B1.35a± 0.07

B0.91b±0.14

(↓ 48.35)

B1.51c±0.11

(↑ 11.85) P. molle

B1.52a± 0.13

C1.12b±0.13

(↓ 35.71)

C2.01c±0.05


B1.51a± 0.05

C1.08b±0.11

(↓ 39.81)

B1.72a±0.12

(↑13.90) Experimental conditions: Initial Ni2+ concentration: 5 µg ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; biomass: 0.15 mg protein ml-1; contact time: 60 min; Temp: 28±2 °C; volume: 50 ml (↓) indicates % decrease compared to log phase cultures (↑) indicates % increase compared to log phase cultures Arithmetic means within the same column with the same uppercase letter are not




0

0.5

1

1.5

2

2.5

3

3.5

4

4.5


Cd2

+ rem

ove

d (µg m

l-1)

Fig. 73: Cd2+ removal by test organisms after different pre-treatments.

Control ( ); HCl ( ); CH3COOH ( ); HNO3 ( ); NaOH ( ); CH3OH ( );

KOH ( ); CaCl2 ( )

Experimental conditions: Initial Cd2+ concentration: 5 µg ml-1; buffer:

imidazole-HCl (0.2 M); biomass: 0.15 mg protein ml-1; pH: 6.0; contact time: 60

min; temp: 28±2 °C; time of pre-treatment: 60 min

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5


Cu2

+ rem

ove

d (µ

g m

l-1)

Fig. 74: Cu2+ removal by test organisms after different pre-treatments.


KOH ( ); CaCl2 ( )

Experimental conditions: Initial Cu2+ concentration: 5 µg ml-1; buffer:


min; temp: 28±2 °C; time of pre-treatment: 60 min

0

0.5

1

1.5

2

2.5

3


Ni2

+ rem

ove

d (µ

g m

l-1)

Fig. 75: Ni2+ removal by test organisms after different pre-treatments.


KOH ( ); CaCl2 ( )

Experimental conditions: Initial Ni2+ concentration: 5 µg ml-1; buffer: imidazole-

HCl (0.2 M); biomass: 0.15 mg protein ml-1; pH: 6.0; contact time: 60 min;

temp: 28±2 °C; time of pre-treatment: 60 min

treated with 0.2 M CaCl2 (Table 32). Maximum decrease in Cd2+ removal (22.27%) was

observed when CaCl2 treated cultures of P. molle were used.

Increase in Cu2+ removal varied not only among organisms with same treatment

but with different treatments as well. Treatment with acids i.e. HCl, CH3COOH and

HNO3, 0.1mM each, caused increase in Cu2+ removal by O. chlorina only by 23-26%,

while in other organisms there was not significant increase/decrease in Cu2+ removal

(Table 33).

Treatment of cultures with KOH caused more increase in Cu2+ removal compared

to NaOH treatment. Treatment with KOH results in Cu2+ removal from 7% (L. spiralis)

to 46% (A. torulosa) while increase in Cu2+ removal after NaOH treatment was 0.89 to

28% (Table 33). Treatment of cultures with CH3OH resulted in increase of Cu2+ removal

from 12% (L. spiralis) to 30% (A. torulosa) while treatment with CaCl2 resulted in

decrease in Cu2+ removal by all the test organisms. Maximum increase in Cu2+ removal

was observed in O. chlorina, P. molle and A. torulosa when treated with KOH while in S.

pevalekii and L. spiralis maximum increase in Cu2+ removal was observed when treated

with CH3OH.

In the same way when Ni2+ removal by treated cultures of test organisms was

studied it was observed that increase/decrease in Ni2+ removal varied with the treatment

but all organisms exhibited maximum Ni2+ removal when KOH treated cultures were

used compared to control cultures (41-50% increase) or other treatments (Table 34).

Table 32: Effect of pre-treatments on Cd2+ removal by test organisms.

Cd2+ removed (%)

Organism

Pre-treatment with

Control

HCl (0.1 mM)

CH3COOH (0.1 mM)

HNO3

(0.1 mM) NaOH

(0.1 mM) CH3OH (90%)

KOH (0.1 mM)

CaCl2

(0.2 M) S. pevalekii

A76.20a±2.8

A80.60b±2.4 (↑ 5.77)

A77.80a±1.4 (↑ 2.10)

A82.00b±2.0 (↑ 7.61)

A78.80a±2.2 (↑ 3.41)

A82.40b±2.8 (↑ 8.14)

A86.00b±1.8 (↑ 12.86)

A73.00a±2.8 (↓ 4.20)

L. spiralis

A78.40a±2.4

A81.60a±1.8 (↑ 4.08)

A80.00a±0.8 (↑ 2.04)

A83.00b±2.8 (↑ 5.87)

A79.60a±2.0 (↑ 1.53)

A84.80b±2.6 (↑ 8.16)

A87.40b±3.0 (↑ 11.48)

A75.00a±1.0 (↓ 4.34)

O. chlorina

B60.60a±3.0

B68.00b±3.0 (↑ 12.21)

B66.20b±2.6 (↑ 9.24)

B64.20a±1.0 (↑ 5.94)

B67.20b±1.2 (↑ 10.89)

B71.20c±2.0 (↑ 17.49)

B73.00c±1.2 (↑ 20.46)

B57.00a±1.6 (↓ 5.94)

P. molle

C51.20a±2.2

C56.60a±2.2 (↑ 10.55)

C54.60a±1.8 (↑ 6.64)

B59.40b±1.8 (↑ 16.02)

C48.60a±0.8 (↓ 2.73)

C65.80c±1.0 (↑ 28.52)

B70.40c±2.8 (↑ 37.50)

C39.80d±2.4 (↓ 22.27)

A. torulosa

D45.80a±2.6

C51.00b±1.0 (↑ 11.35)

C48.40a±2.8 (↑ 5.68)

C48.00a±2.4 (↑ 4.80)

C47.80a±1.8 (↑ 4.37)

C64.80c±0.4 (↑ 41.48)

B69.20c±2.4 (↑ 51.09)

C43.40a±2.0 (↓ 5.24)

Experimental conditions: Initial Cd2+ concentration: 5 µg ml-1; biomass: 0.15 mg protein ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; contact time: 60 min; Temp: 28±2 °C; volume: 50 ml; time of pre-treatment: 60 min (↓) indicates % decrease compared to control cultures (↑) indicates % increase compared to control cultures Arithmetic means within the same column with the same uppercase letter are not significantly different from each other at the 95%



Table 33: Effect of pre-treatments on Cu2+ removal by test organisms.

Cu2+ removed (%)

Organism

Pre-treatment with

Control

HCl (0.1 mM)

CH3COOH (0.1 mM)

HNO3

(0.1 mM) NaOH

(0.1 mM) CH3OH (90%)

KOH (0.1 mM)

CaCl2


A74.00a±2.4

A79.00a±1.8 (↑ 6.76)

A76.40a±2.2 (↑ 3.24)

A78.20a±1.4 (↑5.68)

A77.00a±2.0 (↑ 4.05)

A84.20b±2.4 (↑ 13.78)

A82.20b±3.2 (↑ 11.08)

A66.20c±2.0 (↓ 10.54)

L. spiralis

A75.80a±1.8

A78.40a±1.0 (↑ 3.43)

A76.80a±1.0 (↑ 1.32)

A77.20a±1.6 (↑1.85)

A78.00a±2.6 (↑ 2.90)

A85.00b±3.0 (↑ 12.14)

A81.20b±2.2 (↑ 7.12)

A65.00c±2.4 (↓ 14.25)

O. chlorina

B51.00a±1.2

B63.20b±2.4 (↑ 23.92)

B64.40b±0.6 (↑ 26.27)

B63.80b±2.0 (↑25.10)

B64.00b±1.2 (↑ 25.49)

B65.60b±1.0 (↑ 28.63)

B68.60b±2.8 (↑ 34.51)

B45.20a±0.4 (↓ 11.37)

P. molle

B52.00a±2.0

C48.60a±2.0 (↓ 6.54)

C45.80a±3.0 (↓ 11.92)

C47.20a±2.8 (↓9.23)

C45.40a±2.4 (↓ 12.69)

B64.60b±1.8 (↑ 24.23)

B69.20b±1.8 (↑ 33.08)

B41.40c±1.4 (↓ 20.38)

A. torulosa

C45.00a±1.8

C45.80ª±1.4 (↑ 1.78)

C48.20a±2.6 (↑ 7.11)

C41.20ª±2.2 (↓8.44)

C45.40a±1.8 (↑ 0.89)

C58.80b±2.0 (↑ 30.67)

B66.00c±1.0 (↑ 46.67)

B40.20a±2.2 (↓ 10.67)

Experimental conditions: Initial Cu2+ concentration: 5 µg ml-1; biomass: 0.15 mg protein ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; contact time: 60 min; Temp: 28±2 °C; volume: 50 ml; time of pre-treatment: 60 min (↓) indicates % decrease compared to control cultures (↑) indicates % increase compared to control cultures Arithmetic means within the same column with the same uppercase letter are not significantly different from each other at the 95%



Table 34: Effect of pre-treatments on Ni2+ removal by test organisms.

Ni2+ removed (%)

Organism

Pre-treatment with

Control

HCl (0.1 mM)

CH3COOH (0.1 mM)

HNO3

(0.1 mM) NaOH

(0.1 mM) CH3OH (90%)

KOH (0.1 mM)

CaCl2


A38.40a±2.8

A43.40a±1.8 (↑ 13.02)

A39.80a±2.0 (↑ 3.65))

A44.20a±0.6 (↑ 15.10)

A45.40a±2.6 (↑ 18.23)

A49.00b±2.2 (↑ 27.60)

A55.60c±2.2 (↑ 44.79)

A27.60d±1.2 (↓ 28.13)

L. spiralis

A36.20a±1.8

B37.40a±2.6 (↑ 3.31)

A39.00a±1.0 (↑ 7.73)

B37.00a±1.6 (↑ 2.21)

A41.20a±2.4 (↑ 13.81)

A44.00b±1.8 (↑ 21.55)

A51.40c±1.0 (↑ 41.99)

B15.00d±2.0 (↓ 58.56)

O. chlorina

B27.00a±1.4

C30.40a±0.8 (↑ 12.59)

B23.00a±1.4 (↓ 14.81)

C27.00a±2.2 (↑ 0.00)

B26.00a±1.2 (↓ 3.70)

B36.80b±1.0 (↑ 36.30)

B40.20b±0.4 (↑ 48.89)

B14.20c±2.4 (↓ 47.41)

P. molle

B30.40a±2.6

C32.20a±1.4 (↑ 5.92)

C31.60a±2.8 (↓ 3.95)

C29.20a±1.2 (↓ 3.95)

C32.80a±1.8 (7.89)

B39.80b±1.6 (↑ 30.92)

B45.60c±2.4 (↑ 50.00)

A23.60d±2.2 (↓ 22.37)

A. torulosa

B30.20a±1.0

C32.60ª±2.2 (↑ 7.95)

C30.80a±2.4 (↑ 1.99)

C29.80ª±2.8 (↓ 1.32)

B27.80a±2.4 (↓ 7.95)

B39.00b±2.0 (↑ 29.14)

B44.40c±2.8 (↑ 47.02)

A28.20a±1.8 (↓ 6.62)

Experimental conditions: Initial Ni2+ concentration: 5 µg ml-1; biomass: 0.15 mg protein ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; contact time: 60 min; Temp: 28±2 °C; volume: 50 ml; time of pre-treatment: 60 min (↓) indicates % decrease compared to control cultures (↑) indicates % increase compared to control cultures

Arithmetic means within the same column with the same uppercase letter are not significantly different from each other at the

95% confidence level (p > 0.025). Also, arithmetic means within the same row with the same lowercase letter are not significantly

different from each other at the 95% confidence level (p > 0.025)

Hence, KOH and CH3OH treated cultures exhibited increase in the Cd2+, Cu2+ and

Ni2+ removal efficiencies while decrease in metal removal was observed when CaCl2

treated cultures were used.

Thus, on the basis of data presented above it is concluded that the amount of

metal removal by the test organisms was strongly affected by treatment of biomass with

alkalies and organic solvents.

D. Effect of Cations on Metal Removal

Effect of presence of cations such as K+, Na+, Mg2+ and Ca2+ in metal solution on

metal removal efficiency of the test organisms was studied. Results revealed that metal

removal efficiency of the test organisms decreased in the presence of cations. Effects of

presence of these ions in metal solution on Cd2+ removal by all test organisms, except S.

pevalekii were comparable but in case of S. pevalekii Ca2+ affected Cd2+ removal more

severely than other ions (Fig. 76). Presence of these ions caused maximum decrease in

Cd2+ removal by O. chlorina and minimum decrease was observed (11-20%) in case of L.

spiralis and A. torulosa (Table 35).

Similar experiments were performed to find out the effect of presence of cations

on Cu2+ and Ni2+ removal by the test organisms and results revealed that Cu2+ and Ni2+

removal by all the test organisms was also inhibited in the presence of cations. Maximum

reduction in Cu2+ removal by P. molle (24-34%) was observed in presence of Ca2+ while

minimum reduction in Cu2+ removal by Ca2+ ions was observed in L. spiralis and

A. torulosa (Fig. 77, Table 36). Ni2+ removal by test organisms was severely inhibited in

the presence of these ions. Maximum reduction in Ni2+ removal was exhibited by S.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5


Cd2

+ rem

ove

d ( µ

g m

l-1)

Fig. 76: Cd2+ removal by test organisms in the presence of cations.

Control ( ); K+ ( ); Na+( ); Mg2+( ); Ca2+ ( )

Experimental conditions: Initial Cd2+ concentration: 5 µg ml-1; buffer:


min; temp: 28±2 °C; cation concentration: 100 µM each

Table 35: Effect of cations on Cd2+ removal by test organisms.

Organism

Cd2+ removed (%)

Control K+ Na+ Mg2+ Ca2+

S. pevalekii

A76.20a±2.8

A64.20b±1.8 (↓ 15.75)

A64.80b±2.4 (↓ 14.96)

A56.20c±3.2 (↓ 26.25)

A44.00d±2.2 (↓42.26)

L. spiralis

A78.40a±2.4

A67.60b±2.4 (↓ 13.78)

A69.00b±1.8 (↓ 11.99)

B66.20b±2.8

(↓ 15.56)

B64.60c±2.8 (↓ 17.60)

O. chlorina

B60.60a±3.0

B37.60b±1.8 (↓ 37.95)

B38.80b±1.2

(↓ 35.97)

C35.40b±2.2 (↓ 41.58)

C33.60b±1.8 (↓ 44.55)

P. molle

C51.20a±2.2

B40.20b±2.0 (↓ 21.48)

B41.60b±1.8 (↓ 18.75)

C39.00b±1.8 (↓ 23.83)

C38.20b±1.2 (↓ 25.39)

A. torulosa

D45.80a±2.6

B40.40b±1.2 (↓ 11.79)

B37.80b±2.6 (↓ 17.47)

C39.20b±1.2 (↓ 14.41)

C36.20b±2.2

(↓ 20.96) Experimental conditions: Initial Cd2+ concentration: 5 µg ml-1; biomass: 0.15 mg protein ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; contact time: 60 min; Temp: 28±2 °C; cation concentration: 100 µM each; volume: 50 ml (↓) indicates % decrease compared to control cultures Arithmetic means within the same column with the same uppercase letter are not




0

0.5

1

1.5

2

2.5

3

3.5

4


Cu2

+ rem

ove

d ( µ

g m

l-1)

Fig. 77: Cu2+ removal by test organisms in the presence of cations.

Control ( ); K+ ( ); Na+( ); Mg2+( ); Ca2+ ( )

Experimental conditions: Initial Cu2+ concentration: 5 µg ml-1; buffer:


min; temp: 28±2 °C; cation concentration: 100 µM each

Table 36: Effect of cations on Cu2+ removal by test organisms.

Organism

Cu2+ removed (%)

Control

K+ Na+ Mg2+ Ca2+

S. pevalekii

A74.00a±2.4

A61.60b±1.2 (↓ 16.76)

A56.80c±1.8 (↓ 23.24)

A60.60b±1.6 (↓ 18.11)

A59.00b±2.0 (↓ 20.27)

L. spiralis

A75.80a±1.8

A66.80b±1.8 (↓ 11.87)

B69.00b±2.8 (↓ 8.97)

B69.80b±2.4 (↓ 7.92)

B68.20b±2.8 (↓ 10.03)

O. chlorina

B51.00a±1.2

B41.60b±2.4 (↓ 18.43)

C42.60b±1.0 (↓ 16.47)

C41.20b±2.6 (↓ 19.22)

C37.60b±1.8 (↓ 26.27)

P. molle

B52.00a±2.0

C36.20b±2.0 (↓ 30.38)

C39.40b±1.4 (↓ 24.23)

C38.00b±1.8 (↓ 26.92)

C34.00b±1.6 (↓ 34.62)

A. torulosa

C45.00a±1.8

B43.40a±2.4 (↓ 3.56)

C42.00a±1.2 (↓ 6.67)

C38.60b±1.0 (↓ 14.22)

C36.00b±1.8 (↓ 20.00)

Experimental conditions: Initial Cu2+ concentration: 5 µg ml-1; biomass: 0.15 mg protein ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; contact time: 60 min; Temp: 28±2 °C; cation concentration: 100 µM each; volume: 50 ml (↓) indicates % decrease compared to control cultures Arithmetic means within the same column with the same uppercase letter are not




pevalekii in the presence of Ca2+ ions and by A. torulosa in the presence of Mg2+ ions

(Fig. 78, Table 37)

Thus, it is concluded that presence of cations in the metal solution decreased the

metal removal efficiency of the test organisms.

XIV. DESORPTION OF Cd2+, Cu2+ and Ni2+

It is desired that the metal ions adsorbed on the biomass are desorbed so that the

biomass can be reused. Metal desorption efficacy of two eluents EDTA and H2SO4 (0.1M

each) was investigated. Treatment of Cd2+ loaded biomass with each of these solutions

for 60 min desorbed more than 87% of adsorbed Cd2+. Maximum desorption (>90%)

from S. pevalekii and L. spiralis was caused by treatment with EDTA (Table 38).

Similar experiments were performed to find out the Cu2+ and Ni2+ desorption

efficiency of these solutions. The results revealed that more than 84% of Cu2+ and Ni2+

were desorbed from the surface of all the test organisms when treated with these

solutions. More than 89% metal desorption was observed from the surface of S. pevalekii

and L. spiralis when treated with EDTA (Table 38).

XV. METAL REMOVAL FROM BINARY METAL SOLUTIONS

Efficiency of the test organisms to remove metal(s) from binary metal solutions

was investigated. Experiments for metal removal from binary metal solution were

performed in imidazole-HCl buffer (pH: 6.0) having biomass: 0.3 mg protein ml-1 and

concentration of each metal: 5 µg ml-1 to investigate the effect of one metal on the metal

removal efficiency of the organism for the second metal. Binary metals solutions with

0

0.5

1

1.5

2

2.5


Ni2

+ re

move

d ( µ

g m

l-1)

Fig. 78: Ni2+ removal by test organisms in the presence of cations.

Control ( ); K+ ( ); Na+( ); Mg2+( ); Ca2+ ( )

Experimental conditions: Initial Ni2+ concentration: 5 µg ml-1; buffer: imidazole-

HCl (0.2 M); biomass: 0.15 mg protein ml-1; pH: 6.0; contact time: 60 min;

temp: 28±2 °C; cation concentration: 100 µM each

Table 37: Effect of cations on Ni2+ removal by test organisms.

Organism

Ni2+ removed (%)

Control K + Na+ Mg2+ Ca2+

S. pevalekii A38.40a±2.8 A23.80b±1.6

(↓ 38.02)

A25.20b±1.8 (↓ 34.38)

A22.60b±1.2 (↓ 41.15)

A19.00c±1.0 (↓ 50.52)

L. spiralis A36.20a±1.8 B32.80a±2.0

(↓ 9.39)

B34.20a±1.6 (↓ 5.52)

B28.20b±1.8 (↓ 22.10)

B27.40b±1.8 (↓ 24.31)

O. chlorina B27.00a± 1.4 C23.20a±1.8

(↓ 14.07)

C21.00b±1.8 (↓ 22.22)

A20.40b±0.8 (↓ 24.44)

A19.80b±0.8 (↓ 26.67)

P. molle B30.40a± 2.6 C21.80b±1.6

(↓ 28.29)

C25.20b±1.0 (↓ 17.11)

A22.80b±1.2

(↓ 25.00) B25.80b±1.2

(↓ 15.13)

A. torulosa B30.20a±1.0 C21.60b±1.06

(↓ 28.48)

C23.00b±1.6 (↓ 23.84)

C14.00c±0.4 (53.64)

11A16.60c±0.8 (↓45.03)

Experimental conditions: Initial Ni2+ concentration: 5 µg ml-1; biomass: 0.15 mg protein ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; contact time: 60 min; Temp: 28±2 °C; cation concentration: 100 µM each; volume: 50 ml (↓) indicates % decrease compared to control cultures Arithmetic means within the same column with the same uppercase letter are not




Table 38: Desorption of metal ions from metal loaded biomass of the test organisms.

Metal desorbed (%)

Cd2+ Cu2+ Ni2+

Biomass EDTA H2SO4 EDTA H 2SO4 EDTA H 2SO4

S. pevalekii 92.90±2.6 85.82±1.8 93.00±1.8 90.54±2.6 94.50±2.8 90.43±2.8

L. spiralis 94.10±2.2 87.75±2.4 95.30±2.0 89.18±1.2 95.40±3.0 91.23±2.4

O. chlorina 88.81±2.8 84.44±2.0 87.28±2.4 84.79±1.8 87.94±1.8 84.59±2.2

P. molle 87.42±1.2 83.89±2.2 89.3±2.2 87.61±2.0 91.84±2.6 86.44±1.0

A. torulosa 89.08±2.4 87.88±2.6 88.44±1.8 87.11±2.4 88.11±2.0 84.74±2.0

Experimental conditions: Biomass: 0.15 mg protein ml -1; contact time: 60 min; Temp.:

28±2 °C; time of desorption: 60 min; solution used: EDTA, H2SO4

Metal was adsorbed on the biomass under conditions: Initial metal concentration: 5 µg ml-1; biomass: 0.15 mg protein ml-1; buffer: imidazole-HCl (0.2 M); pH: 6.0; contact time: 60 min; Temp: 28±2 °C; volume: 50 ml After adsorption experiments, biomass was separated, washed with water and then treated with EDTA (0.1 M)/H2SO4 (0.1 M) and amount of metal desorbed was determined

three combinations i.e. Cu+Cd, Cd+Ni and Cu+Ni were made containing equal

concentrations of either two metals from Cd2+, Cu2+ or Ni2+ i.e. 5 µg ml-1 of each metal in

the bimetallic solution. Since total metal concentration in bimetallic solution was double

(10 µg ml-1) than single metal solution (5 µg ml-1), amount of biomass in the system was

also doubled from 0.15 mg protein ml-1 to 0.3 mg protein ml-1. Results revealed that

overall metal removal efficiencies of the test organisms to remove metal(s) from solution

were not affected when compared with metal removal efficiency from single metal

solution. S. pevalekii removed 7.32 µg ml-1 of metals (sum of Cu and Cd removed from

solution containing either Cu or Cd) from single metal solution while it removed 7.05 µg

ml-1 of metals (sum of Cu and Cd removed) from bimetallic solution. Same organism

removed 5.29 µg ml-1 and 5.03 µg ml-1 in total (sum of individual metal removed), from

the solutions containing Cd+Ni and Cu+Ni respectively, while 5.41 µg ml-1 and 5.28 µg

ml-1, (sum of individual metals), were removed from single metal solution (Table 39).

Only 2-5% decrease in metal removal efficiencies was observed when bimetallic

solutions were used as compared to single metal solution. Similar experiments were

performed with other test organisms and it was observed that there was no appreciable

change in total amount of metals removal by of the test organisms from bimetallic solutions

compared to total amount of metals removed from single metal solution (Table 39).

Data presented in Table 39 show that although presence of one metal in the

solution affected the adsorption of the other metal and vice versa, but the overall total

metal removal efficiency of the test organisms did not decrease significantly. It is clear

from FTIR analyses that almost same functional groups are involved in adsorption of

Table 39: Amount of metal removed (µg ml-1) by the test organisms from single and bimetallic solutions.

Metals

Organism

aCd2+

aCu2+

aNi2+

bCd+Cu bCd+Ni bCu+Ni

Cd2+ Cu2+ Cd2+ Ni2+ Cu2+ Ni2+

S. pevalekii A7.44a±0.11 A7.19a±0.19 A3.38b±0.1 A3.6b±0.09 A3.45b±0.13 A3.53b±0.19 A1.76c±0.19 A3.34b±0.15 A1.69c±0.06

L. spiralis A7.22a±0.18 A7.35a±0.23 A3.46b±0.08 A3.61b±0.12 A3.52b±0.07 A3.51b±0.15 A1.62c±0.14 A3.59b±0.11 A1.67c±0.08

O. chlorina B5.68a±0.22 B4.92b±0.1 B2.56c±0.13 B2.79c±0.05 B2.32c±0.04 B2.75c±0.06 B1.22d±0.09 B2.37c±0.09 B1.24d±0.16

P. molle C4.9a±0.25 B5.0a±0.16 B2.8b±0.19 C2.36b±0.16 B2.33b±0.15 C2.43b±0.07 B1.3c±0.07 B2.49b±0.17 B1.24c±0.13

A. torulosa D4.28a±0.20 C4.38a±0.22 B2.84b±0.16 C2.1c±0.12 B2.0c±0.11 C2.15c±0.18 B1.25d±0.15 B2.1c±0.05 B1.3d±0.10

Experimental conditions: Initial metal concentration: 10 µg ml-1; biomass: 0.30 mg protein ml-1; buffer: imidazole-HCl (0.2M); pH: 6.0; contact time: 60 min; Temp: 28±2 °C; volume: 50 ml a = Single metal solution and concentration of each metal is 10 µg ml-1

b = Bimetal metal solution and concentration of each metal is 5 µg ml-1

Arithmetic means within the same column with the same uppercase letter are not significantly different from each other at the 95%



metal ions on the surface of cell walls of test organisms and the three metals tested do not

have any specificity for a particular functional group.

Thus, it is concluded that the overall metal removal efficiencies of the test

organisms did not vary significantly when metal removal from bimetallic solutions was

compared to metal removal efficiencies from single metal solutions.

XVI. METAL REMOVAL BY IMMOBILIZED CELLS IN BATCH CULTURE S

A. Alginate Immobilized Cells

Metal removal by alginate immobilized cells was followed from imidazole-HCl

buffer (pH: 6.0) containing biomass: 0.3 mg protein ml-1 and 10 µg ml-1 metal

concentration to find out whether immobilization affected metal removal efficiencies of

the test organisms. The results revealed that the blank alginate beads removed 2.3, 2.53

and 2.42 µg ml-1 of Cd2+, Cu2+ or Ni2+, respectively, from 10 µg ml-1 metal solution

(Table 40). Immobilization of cells in alginate beads did not affect the rate of Cd2+

removal by all the test organisms as the amount of Cd2+ removed by immobilized cells

was comparable to free cells (Table 41).

Similar experiments were performed to study Cu2+ and Ni2+ removal by alginate

immobilized biomass and results revealed that no significant differences in metal removal

efficiencies of immobilized and free cells of all the test organisms were observed (Tables

42 & 43).

Table 40: Amount of metal removed (µg ml-1) by blank agar-agar and alginate beads from

imidazole-HCl buffer.

Metal Metal removed by blank beads

Agar beads Alginate beads

Cadmium A1.85a±0.12 A2.3b±0.15

Copper B2.1a±0.09 A2.53b±0.12

Nickel A1.99a±0.14 A2.42b±0.10

Experimental conditions: Initial metal concentration: 10 µg ml-1; pH: 6.0; volume: 100

ml; time: 6 h)





Table 41: Amount of Cd2+ removed (µg ml-1) by free cells, alginate and agar immobilized

cells of test organisms from imidazole-HCl buffer.

Cd2+

Organism

Free Cells

Agar immobilized Cells

Alginate immobilized Cells

S. pevalekii A7.44a±0.11 A7.34a±0.21 A7.41a±0.17

L. spiralis A7.22a±0.18 A7.14a±0.24 A7.34a±0.20

O. chlorina B5.68a±0.22 B5.74a±0.19 B5.71a±0.25

P. molle C4.90a±0.25 C4.86a±0.23 C4.85a±0.21

A. torulosa D4.28a±0.2 C4.36a±0.15 C4.31a±0.27

Experimental conditions: Initial Cd2+ concentration: 10 µg ml-1; biomass: 0.3 mg protein

ml-1; pH: 6.0; volume: 100 ml; contact time: 6 h

Amount of Cd2+ removed by immobilized cells was obtained by subtracting the amount

of metal removed by blank beads from amount of metal removed by biomass loaded

beads. Contact time was increased to 6 h to allow metal ions to penetrate in the beads.





Thus, on the basis of data presented above it is concluded that immobilization of

cells in alginate beads did not affect significantly Cd2+, Cu2+ or Ni2+ removal efficiencies

of the test organisms.

B. Agar-Agar Immobilized Cells

Cells of all the test organisms were also immobilized in 1.5% agar-agar beads and

experiments were performed to study metal removal in the same way as were conducted

using alginate immobilized cells. The blank beads of agar removed 1.85, 2.1 and 1.99 µg

ml-1 of Cd2+, Cu2+ and Ni2+, respectively, from 10 µg ml-1 metal solution indicating that

blank beads contributed to metal removal (Table 40). When amount of Cd2+, Cu2+ or Ni2+

removed by agar immobilized cells was compared with amount removed by alginate

immobilized cells it was observed that no appreciable differences in metal removal by

agar immobilized and alginate immobilized cells of all test organisms were observed

(Tables 41-43). Data of t-test revealed that p>0.025 when removal by agar immobilized

and alginate immobilized cells was compared indicating insignificant differences in metal

removal.

Since the rates of metal removal by agar immobilized and alginate immobilized

cells were almost same, agar as immobilization matrix was chosen for further

experiments.

C. Metal Removal by Immobilized Cells in Continuous Flow Bioreactor

Two organisms, S. pevalekii and L. spiralis which exhibited highest metal

removal efficiency among all the test organisms, were selected to study metal removal by

Table 42: Amount of Cu2+ removed (µg ml-1) by free cells, alginate and agar immobilized


Cu2+

Organism

Free Cells

Agar immobilized Cells





P. molle B5.0a±0.16 B4.91a±0.17 B4.93a±0.26

A. torulosa C4.38a±0.22 C4.24a±0.14 C4.21a±0.29

Experimental conditions: Initial Cu2+ concentration: 10 µg ml-1; biomass: 0.3 mg protein


Amount of Cu2+ removed by immobilized cells was obtained by subtracting the amount

of metal removed by blank beads from amount of metal removed by biomass loaded

beads. Contact time was increased to 6 h to allow metal ions to penetrate in the beads.





Table 43: Amount of Ni2+ removed (µg ml-1) by free cells, alginate and agar immobilized


Ni2+

Organism Free Cells Agar immobilized Cells





P. molle B2.80a±0.19 B2.77a±0.18 C2.75a±0.09

A. torulosa B2.84a±0.16 B2.79a±0.09 C2.80a±0.14

Experimental conditions: Initial Ni2+ concentration: 10 µg ml-1; biomass: 0.3 mg protein


Amount of Ni2+ removed by immobilized cells was obtained by subtracting the amount of

metal removed by blank beads from amount of metal removed by biomass loaded beads.

Contact time was increased to 6 h to allow metal ions to penetrate in the beads.





these organisms in a continuous flow bioreactor. First of all, bioreactor was packed with

blank agar beads and Cd2+, Cu2+ or Ni2+ solution containing 40 µg ml-1 of the respective

metal was pumped into the bottom of reactor at a flow rate of 0.5 ml min-1. At regular

intervals, amount of respective metal in the effluent was determined. Sufficient biomass

of S. pevalekii or L. spiralis (containing 10 mg protein) was immobilized in agar beads

and used for metal removal studies. In 30 min time, 600 µg of respective metal passed

through the bioreactor, and results revealed that blank beads removed 49.73% of Cd2+,

51.60% of Cu2+ or 36.66% of Ni2+ during that time (Table 44). Firstly, S. pevalekii

biomass loaded agar beads (keeping the number of beads equivalent to blank beads) were

packed in the bioreactor and 40 µg ml-1 Cd2+ solution was pumped in to the bioreactor at

0.5 ml min-1 flow rate and efficiency of the metal removal by the bioreactor at different

times was determined. In the same way experiments were performed with Cu2+ or Ni2+.

Results revealed that during 30 min time, biomass loaded beads removed 66.40, 61.80,

52.20% of Cd2+, Cu2+, Ni2+, respectively, giving net removal by biomass Cd2+: 16.67%,

Cu2+: 10.20% and Ni2+: 15.54% (Table 44).

Results of similar experiments with L. spiralis are shown in Table 44 and it was

observed that net removal of Cd2+, Cu2+, Ni2+ by L. spiralis biomass was 18.87, 9.20,

12.93%, respectively (Table 44).

When the operative time of the bioreactor was extended up to 24 h and its metal

removal efficiency studied at different times, it was observed that Cd2+ (Fig. 79), Cu2+

(Fig. 80) and Ni2+ (Fig. 81) removal efficiency of S. pevalekii loaded bioreactor was

maximum during first 2 h, decreased with time up to 14 h and then remained almost

same. When amount of Cd2+, Cu2+ or Ni2+ removed by the bioreactor was calculated it

Table 44: Amount of metal removed by blank beads, biomass loaded beads and

immobilized biomass of S. pevalekii and L. spiralis in continuous flow

bioreactor.

% Metal removed

Metal Blank beads (a)

Biomass loaded beads

(b)

Immobilized biomass only

(b-a) S. pevalekii

Cd2+ A49.73a±2.48 A66.40b±3.32 A16.67c±1.00 Cu2+ A51.60a±2.06 A61.80b±2.47 B10.20c±0.75 Ni2+ B36.66a±2.19 B52.20b±1.57 A15.54c±0.93

L. spiralis Cd2+ A49.73a±2.48 A68.60b±3.5 A18.87c±1.1 Cu2+ A51.60a±2.06 B60.80b±2.2 B9.20c±0.65 Ni2+ B36.66a±2.19 C49.60b±1.7 B12.93c±1.13

Experimental conditions: Metal concentration in the influent: 40 µg ml-1; buffer:

imidazole-HCl (0.2 M, pH: 6.0); biomass load; 10 mg protein; flow rate: 0.5 ml min-1;

operative time: 30 min; Temp: 28±2 °C

Total amount of metal passed through the bioreactor during this time: 600 µg





Fig. 79: Cd2+ removal by blank ( ) and biomass loaded beads () of S. pevalekii from

imidazole-HCl buffer (0.2 M) (pH: 6.0; metal concentration: 40 µg ml-1) in

continuous flow bioreactor during different time periods of bioreactor operative

times at flow rate 0.5 ml min-1.

During 2 h period, 2400 µg of Cd2+ passed through the bioreactor and the values

of Cd2+ removed represent the removal from 2400 µg Cd2+ which passed

through the bioreactor during this period.

Fig. 80: Cu2+ removal by blank ( ) and biomass loaded beads () of S. pevalekii

from imidazole-HCl buffer (0.2 M) (pH: 6.0; metal concentration: 40 µg ml-1)

in continuous flow bioreactor at flow rate 0.5 ml min-1.

During 2 h period, 2400 µg of Cu2+ passed through the bioreactor and the

values of Cu2+ removed represent the removal from 2400 µg Cu2+ which

passed through the bioreactor during this period.

Fig. 81: Ni2+ removal by blank ( ) and biomass loaded beads () of S. pevalekii from

imidazole-HCl buffer (0.2 M) (pH: 6.0; metal concentration: 40 µg ml-1) in

continuous flow bioreactor at flow rate 0.5 ml min-1.

During 2 h period, 2400 µg of Ni2+ passed through the bioreactor and the values

of Ni2+ removed represent the removal from 2400 µg Ni2+ which passed through

the bioreactor during this period.

was observed that amount of respective metal removed increased with time but the rate of

metal removal decreased. Thus, the efficiency of the bioreactor decreased with time

(Table 45). The data given in Figs. 79-81 are data on amount of metal removed during

different time periods while data given in Table 45 are average of 2, 6 and 24 h. With

increase in operative time from 2 h to 24 h net amount of Cd2+ removed by S. pevalekii

increased from 692 to 7471 µg but efficiency in term of per cent Cd2+ removal decreased

from 28 to 25%. After 24 h Cd2+, Cu2+ and Ni2+ removal efficiency of S. pevalekii was

25.9, 11.69 and 10.95 %, respectively (Table 45).

In the same way experiments were performed employing L. spiralis and it was

observed that maximum removal of Cd2+, Cu2+ or Ni2+ in the bioreactor was during first 2

h and the rate of metal removal decreased with time (Fig. 82). Net Cd2+, Cu2+ and Ni2+

removal efficiency of L. spiralis biomass decreased from 36.2 to 30.9, 20.2 to 11.8 and

10.3 to 5.81%, respectively, from 2 to 24 h operative time of the bioreactor (Table 46).

Of the three metals, maximum amounts of Cd2+ were removed through bioreactor and the

amounts of Cu2+ and Ni2+ removed were comparable (Fig. 82). Comparative data on

metal removal by both the organisms are shown in Table 47. It was observed that L.

spiralis removed 30.97% Cd2+ on an average during 24 h while average Cd2+ removal

efficiency of S. pevalekii was 25.94%. Of Cu2+, Ni2+ and Cu2+ removal efficiency of both

organisms was almost same (~11%) while Ni2+ was removed more efficiently by S.

pevalekii (10.95%) than L. spiralis (5.81%).

Table 45: Amount of metal removed by blank beads, biomass loaded beads and immobilized biomass of S. pevalekii in continuous flow bioreactor.

S. pevalekii

Metal Flow rate (ml min -1)

Time (h)

Amount of metal passed through

bioreactor (µg)

Metal removed (µg)

Blank beads Biomass loaded beads Immobilized biomass only

Cd2+ 0.5

2 2400 A555.20a±22.20

(23.13)

A1248.00b±37.44 (52.00)

A692.80c±20.78 (28.86)

6 7200 B1254.40a±25.09

(17.42)

B3393.60b±67.87 (47.13)

B2139.20c±64.17 (29.71)

24 28800 C3286.40a±65.72

(11.41)

C10757.00b±215.14 (37.35)

C7471.20c±149.42 (25.94)

Cu2+ 0.5

2 2400 A661.60a±13.23

(27.56)

D1032.00b± 30.96 (43.00)

D370.40c±11.11 (15.43)

6 7200 B1556.40a±31.12

(21.61)

E2430.00b±72.9 (33.75)

E873.60c±26.20 (12.13)

24 28800 C3195.60a± 63.91

(11.09)

F6564.40b±65.64 (22.79)

F3368.80a±67.37 (11.69)

Ni2+ 0.5

2 2400 A501.60a±15.05

(20.90)

D960.00b±19.2 (40.00)

D458.40a±13.75

(19.1)

6 7200 B1088.00a±21.76

(15.11)

E2376.00b±47.52 (33.00)

G1288.00a±25.76 (17.88)

24 28800 C3226.80a±64.54

(11.20)

F6383.20b±63.83 (22.16)

F3156.40a±63.12 (10.95)

Experimental conditions: Metal concentration in the influent: 40 µg ml-1; biomass load: 10 mg protein; buffer: imidazole-HCl (0.2 M); pH: 6.0; flow

rate: 0.5 ml min-1; operative time: 24h; Temp: 28±2 °C

Values in the parenthesis indicate the % of metal removed of the total amount of metal passed through the bioreactor during the time indicate in the

same rows.

Arithmetic means within the same column with the same uppercase letter are not significantly different from each other at the 95% confidence level (p > 0.025). Also, arithmetic means within the same row with the same lowercase letter are not significantly different from each other at the 95% confidence level (p > 0.025).

Table 46: Amount of metal removed by blank beads, biomass loaded beads and immobilized biomass of L. spiralis in continuous flow bioreactor.

L. spiralis

Metal Flow rate (ml min -1)

Time (h)

Amount of metal passed through

bioreactor (µg)

Metal removed (µg)

Blank beads Biomass loaded beads Immobilized biomass only

Cd2+ 0.5

2 2400 A555.20a±22.20

(23.13)

A1425.60b±28.51 (59.4)

A870.40c±26.11 (36.26)

6 7200 B1254.40a±25.09

(17.42) B3936.00b±78.32 (54.66)

B2681.60c±53.63 (37.24)

24 28800 C3286.40a±65.72

(11.41) C12206.00b±244.12 (42.38)

C8920.00c±89.20 (30.97)

Cu2+ 0.5

2 2400 A661.60a±13.23

(27.56)

D1147.20b±22.94 (47.8)

D485.60c±14.56 (20.23)

6 7200 B1556.40a±31.12

(21.61)

E2630.40b±52.60 (36.53)

E1074.00c±21.48 (14.91)

24 28800 \C3195.60a± 63.91

(11.09)

F6617.60b± 66.17 (22.97)

F3422.00a±68.44 (11.88)

Ni2+ 0.5

2 2400 A501.60a±15.05

(20.90)

G748.80b±22.46 (31.20)

G247.20c±7.41 (10.30)

6 7200 B1088.00a±21.76

(15.11)

H1742.40b±34.84 (24.20)

H654.40c±19.63 (9.08)

24 28800 C3226.80a±64.54

(11.20)

F4900.80b± 98.01 (17.01)

I1674.00c±33.48 (5.81)

Experimental conditions: Metal concentration in the influent: 40 µg ml-1; biomass load: 10 mg protein; buffer: imidazole-HCl (0.2 M); pH: 6.0; flow

rate: 0.5 ml min-1; operative time: 24h; Temp: 28±2 °C

Values in the parenthesis indicate the % of metal removed of the total amount of metal passed through the bioreactor during the time indicate in the

same rows.

Arithmetic means within the same column with the same uppercase letter are not significantly different from each other at the 95% confidence level (p

> 0.025). Also, arithmetic means within the same row with the same lowercase letter are not significantly different from each other at the 95%

confidence level (p > 0.025).

Fig. 82: Cd2+ ( ); Cu2+ ( ) and Ni2+ ( ) removal by L. spiralis immobilized cells

from imidazole-HCl buffer (0.2 M) (pH: 6.0; metal concentration: 40 µg ml-1)

in continuous flow bioreactor at flow rate 0.5 ml min-1.

During 2 h period, 2400 µg of metal passed through the bioreactor and the

values of metal removed represent the removal from 2400 µg metal which

passed through the bioreactor during this period.

Table 47: Net amount of metal removed (µg) by biomass of S. pevalekii and L. spiralis in continuous flow bioreactor.

Organism Flow rate (ml min -1)

Time (h)

Cd2+/ Cu2+/Ni2+passed through bioreactor

Cd2+

Cu2+

Ni2+

S. pevalekii

0.5

2 2400 A692.80±20.78

(28.86)

A370.40±11.11 (15.43)

A458.40±13.75

(19.1)

6 7200 B2139.20±64.17

(29.71)

B873.60±26.20 (12.13)

B1288.00±25.76 (17.88)

24 28800 C7471.20±149.42

(25.94)

C3368.80±67.37 (11.69)

C3156.40±63.12 (10.95)

L. spiralis

0.5

2 2400 D870.40±26.11

(36.26)

D485.60±14.56 (20.23)

D247.20±7.41 (10.30)

6 7200 E2681.60±53.63

(37.24)

E1074.00±21.48 (14.91)

E654.40±19.63 (9.08)

24 28800 F8920.00±89.20

(30.97)

C3422.00±68.44 (11.88)

F1674.00±33.48 (5.81)

Experimental conditions: Metal concentration in the influent: 40 µg ml-1; biomass load: 10 mg protein; buffer: imidazole-HCl (0.2 M);

pH: 6.0; flow rate: 0.5 ml min-1; operative time: 24h; Temp: 28±2 °C

Values in the parenthesis indicate the % of metal removed of the total amount of metal passed through the bioreactor during the time

indicate in the same rows

Arithmetic means within the same column with the same uppercase letter are not significantly different from each other at the 95%

confidence level (p > 0.025).

D. Reuse of Biomass in the Continuous Flow Bioreactor

Biomass in the bioreactor saturated with Cd2+, Cu2+ or Ni2+ (after 24 h) was

regenerated by treating with EDTA (0.1M) for 6 h and again reused in continuous flow

bioreactor for the next sorption cycle. When treated with EDTA, more than 95% of the

metal adsorbed on the surface was desorbed (Table 48). When regenerated biomass was

used for second time in the bioreactor the results obtained revealed that no significant

change in Cd2+, Cu2+ or Ni2+ removal efficiency of S. pevalekii and L. spiralis, in the

second cycle of continuous flow bioreactor was observed (Table 49). S. pevalekii and

L. spiralis removed 28.86 and 36.26% Cd2+, respectively, in the first cycle and 25.57 and

31.67%, respectively, in the second cycle. Thus, metal removal capacity of the organisms

remained almost same with only 3-5% decrease in metal adsorption capacity of both the

organisms in the second cycle (Table 49). Similarly there was only 3-5% decrease in

Cu2+ and Ni2+ removal by both organisms during second cycle in the continuous flow

bioreactor. Thus, the immobilized biomass of the test organisms can be regenerated and

reused for metal removal purposes.

Table 48: Desorption of metals with EDTA from metal loaded biomass entrapped in agar

beads.

Metal

Desorption (%)

S. pevalekii L. spiralis

Cd2+ 94.90±4.74 95.10±2.85

Cu2+ 95.00±5.7 96.30±4.81

Ni2+ 95.50±3.82 96.40±4.10

The biomass loaded beads were recovered from the continuous flow bioreactor after

running it for 24 h and then these beads were treated with EDTA (0.1 M) for 6 h and

amount of metal in the solution was determined

Table 49: Comparison of amount of metal removed (µg) by immobilized biomass of

S. pevalekii and L. spiralis in continuous flow bioreactor during 1st and 2nd

cycle.

Organism Cycle Cd2+ Cu2+ Ni2+

S. pevalekii

1 692.80±20.78

(28.86) 370.40±11.11

(15.43) 458.40±13.75

(19.1)

2 613.70±23.37

(25.57)

297.85±28.78 (12.40)

368.40±26.10 (15.35)

L. spiralis 1

870.40±26.11 (36.26)

485.60±14.56 (20.23)

247.20±7.41 (10.30)

2

760.20±26.31 (31.67)

369.20±20.61 (15.38)

197.90±20.98 (8.24)

Experimental conditions: Metal concentration in the influent: 40 µg ml-1; biomass load:

10 mg protein; buffer: imidazole-HCl (0.2 M); pH: 6.0; flow rate: 0.5 ml min-1; operative

time: 24h; Temp: 28±2 °C

During this time 2400 µg of respective metal passed through the bioreactor and the value

in the parenthesis indicates % of metal removed of the total metal (2400 µg) passed

through the bioreactor.

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