Chapter-3

Materials and Methods

3.1 Introduction

In this chapter the description of the materials used during this research, including the (i) dye

used in adsorption study, (ii) adsorbent used (iii) chemicals used for Aspergillus niger growth

and bead preparation, treatment of adsorbents etc.(iii) instruments for preparation of adsorbents

(iv) measuring instruments are given. Calibration procedure for (i) concentration and

absorbance value for MB dye (ii) peristaltic pump (iii) Lowry’s method for concentration vs

absorbance for Bovine Serum Albumin (B.S.A) are discussed. The determination of fixed carbon

content of the sample by proximate analysis is also explained. The procedure for conducting

different batch experiment for A.niger, PTMF, ASD and PTMFAC is given briefly.

3.2 Materials

3.2.1 Methylene blue dye

MB was purchased from Himedia, Bangalore. The MB (>99% dye content) was chosen because

of its known strong adsorption onto solids. The properties and characteristics along with

structure are given in Table 3.1 and Figure 3.1 respectively.

Table 3.1: Properties and characteristics of Methylene blue (Hameed & Ahmed ,2009)

Generic name Methylene blue

Chemical name(IUPAC) 3,7-bis(Dimethylamino)-phenazathionium

chloride tetramethylthionine chloride

Chemical formula C16H18ClN3S

Molecular weight (g/ mol) 319.9

Molecular volume (cm3/ mol) 241.9

Molecular diameter (nm) 0.8

Maximum Wavelength, λ max (nm) 665

Color Index 52015

Fig- 3.1 Structure of Methylene Blue

3.2.2 Aspergillus niger (Baker,2006 )

Table 3.2: Scientific classification of A. niger

Fig- 3.2 A.niger culture

3.2.3 Palm tree

Palm tree belongs to Arecaceae family, Borassus genus and species B.flabellifer. Borassus

flabillifer is native to Indian subcontinent and South East Asia. This tree is robust and can live

more than 100 years and reach a height of 30 m, with a canopy of green leaves , several dozen

fronds spreading 3 m across. The very large trunk resembles that of coconut tree and ringed with

leaf scars. There are two kinds of the Palm - the male and the female. The male and female

flowers are held by two different trees, never in one tree. Both male and female trees produce

spikes of flowers but only the female plant bears fruits(Morton,1988) .

Domain Eukaryota

Kingdom Fungi

Phylum Ascomycota

Subphylum Pezizomycotina

Class Eurotiomycetes

Order Eurotiales

Family Trichocomaceae

Genus Aspergillus

Species A. niger

3.2.4 Benteak tree Sawdust

Benteak is a deciduous tree, native to the moist forests of Western Ghats, growing to 10-15 m

tall. The bark is ash colored and peels off in strips. The oppositely arranged elliptic-lance like

leaves are 6-10 cm long, with 1 cm long stalks. Flowers are borne in large compound panicles,

20-30 cm long. Flowers are small, white, 1 cm across, with 6 oblong petals 6 mm long. Capsule

is ellipsoid, 1-2 cm long. Seeds are winged and flowering season is May-June. Common name:

Ben Teak, Botanical name: Lagerstroemia microcarpa Family: Lythraceae (Crape Myrtle

family) : Synonyms: Lagerstroemia lanceolata.

3.2.5 Chemicals and Instrument used

Table 3.3: The chemical used in the study and their manufacturers.

Sl.no Chemicals used Make

1 Dextrose Finar

2 Peptone Himedia

3 Yeast extract Himedia

4 Formalin Finar

5 Sodium alginate Finar

6 CaCl2 solution Himedia

7 Hydrochloric Acid Finar

8 Sodium hydroxide Finar

9 Phosphoric acid S D Fine Chemicals

10 Methylene Blue dye Himedia

11 Sodium bicarbonate Finar

12 Sodium nitrate Finar

Table 3.4: Instruments used and their make.

Sl.No Instruments Make Function

1 Laminar flow chamber Labline During inoculation and

chemical preparation.

2 Autoclave Napro Scientific To sterilize

3 Drier Servwell To remove moisture

4 pH meter Systronic Measurement of pH

5 Rotary shaker Thermo-Fischer Stir the contents

6 Filter Assembly Borosil Filter the solution

7 Domestic mixer Phillips Size reduction

8 Weighing balance Citizon To measure weight

9 Peristaltic pump EnerTech Constant flow rate

10 Vaccum pump During filtration of samples

11 Incubator shaker Lead Stir the contents at different

temperature

12 Muffle Furnace Shital Scientific High temperature during

carbonization

13 Scanning Electron Microscopy Zeiss Surface morphology

14 Fourier Transform Infra -red Shimadzu Chemical groups present in the

adsorbent.

15 Particle size analyzer Cilas 1064 Average particle size

16 U-V spectrophotometer-1700 Shimadzu To find absorbance/

concentration

17 B E T analyzer Smart Instruments Surface area and pore volume

of adsorbent.

3.3 Methods

3.3.1 Biomass preparation

A laboratory strain of A. niger (ATCC #11414) was routinely maintained on potato dextrose

agar. For experimental purposes, A. niger was inoculated into a liquid medium comprising (in

g/L) the following: dextrose, 20; peptone, 10; yeast extract, 3, in deionized water with pH

adjusted to 5.0 by 1.0 N HCl. The cultures were grown in an aerobic condition at room

temperature (24±2 ◦C) with 100mL of the liquid medium in 250mL conical flasks on a rotary

shaker agitated at 125 rpm. All culture work was conducted aseptically. The fungi grew as

pellicles (Fig. 3.3) and were harvested after 4 days of growth by filtering the growth media

through a 150 µm sieve. The harvested fungal pellicles were transferred to a 1% formalin

solution for 24 h and then washed with generous amounts of deionized water and autoclaved for

30 min at 121 ◦C and 103 kPa. The autoclaved biomass was allowed to cool down, hand-pressed

(Fig. 3.4 ), and dried in an oven at 60 oC for 24 h. This dried autoclaved biomass was ground to a

powder in a mortar and sieved to obtain average particle size of 150 µm. The powder was stored

in air tight container and used for biosorption experiments.(Deepa et al.,2007)

Fig-3.3 A.niger after 4 days of growth.

Fig- 3.4 Hand pressed A.niger biomass for drying.

3.3.2 Preparation A.niger immobilized biomass

4gm of A.niger powder was dissolved in 25mL distilled water. 2g of sodium alginate was

dissolved in 75 mL water in a separate beaker. Both the solutions were mixed at room

temperature. CaCl2 solution was prepared by dissolving 6 g in 100 mL of distilled water. The

beads were formed by dripping the polymer solution from a height of approximately 20 cm into

previously prepared CaCl2 solution with a syringe and a needle at room temperature. The beads

were left in CaCl2 solution to cure for about 8 h.(Murty, 2005)

3.3.3 Estimation of A.niger by Lowry’s method

1mL of Bovine Serum Albumin (B.S.A) of concentrations 10,20,40,60,70 µg/mL was added into

five labeled test tubes(S1,S2,S3,S4,S5). Blank was prepared by adding 1mL of water in another

test tube. 1mL of test solution of unknown concentration was added in two more test tubes. To

each of the above eight test tubes, 2mL of Lowry’s reagent were added and kept in an incubator

at 37oC for approximately 10 minutes. To these test tubes 0.2 mL of Folins reagent was added

and incubated for 30 minutes at 37oC (Murty,2005). The solutions from the test tubes were

analyzed using a U-V Spectrophotometer and the absorbance was recorded. The concentration of

test solution was found using the absorbance value from the calibration data of B.S.A as shown

in Table- 3.5 and Fig- 3.5.

Table 3.5 Calibration of B.S.A

Test tube

Label

Volume of

B.S.A(mL)

Volume of

water

(mL)

Volume of

Lowrys

reagent(mL)

Volume of

Folins

reagent(mL)

Absorbance

@700nm

Conc. of

Solution

µg/mL

S1 1 -- 2 0.2 0.0675 10

S2 1 -- 2 0.2 0.148 20

S3 1 -- 2 0.2 0.2825 40

S4 1 -- 2 0.2 0.425 60

S5 1 -- 2 0.2 0.505 70

Blank -- 1 2 0.2 0.0000 00

T1 1 -- 2 0.2 0.3400 47

T2 1 -- 2 0.2 0.2540 35.5

T3 1 -- 2 0.2 0.3420 47.1

T1-CaCl2 solution, T2-wash I solution , T3-wash II solution

Fig-3.5 B.S.A calibration chart

3.4 Calibration

3.4.1 Methylene blue dye solution

Concentrations of methylene blue (MB) in the filtered solutions were estimated by measuring

absorbance at maximum wavelengths of the dye (λmax=665nm) using the calibration curve shown

in Fig-3.5. The calibration curve of absorbance against MB concentration was obtained by using

standard MB solutions. The calibration curve shows that Beer’s law is obeyed in concentration

range (0.0–12 mg L-1

).

Table -3.6 Calibration value for methylene blue dye

Sample No Concentration

(mg/ L)

absorbance

1 0 0.000

2 2 0.150

3 4 0.300

4 6 0.450

5 8 0.60

6 10 0.75

7 12 0.90

Fig-3.6 Absorbance Vs Concentration curve for methylene blue.

3.4.2 Peristaltic pump

Table- 3.7 Calibration for peristaltic pump

Sl.no Speed

RPM

Flow rate

(ml/min)

1 10 16.3

2 20 33.9

3 30 51.5

4 40 69.0

5 50 86.7

6 60 104.3

7 70 121.9

8 80 139.5

9 90 157.1

10 100 174.7

11 110 192.3

12 120 209.9

13 130 227.5

14 140 245

15 150 262.7

16 160 280.3

0

0.2

0.4

0.6

0.8

1

0 5 10 15

Ab

sorb

an

ce

Concentration (mg/L)

Fig-3.7 Speed Vs Volumetric flow rate of peristaltic pump.

3.5 Proximate analysis.

3.5.1 Determination of moisture content

W g of sample was transferred to a previously weighed silica crucible and heated to 105oC and

maintained at the same temperature for 1h in a muffle furnace. The sample was cooled in a

desiccator to ensure that the moisture doesn’t re-enter. The difference in the weights before and

after heating (W1 g) were noted down. The following equation was used.

% Moisture = 100*W1/ W (3.1)

3.5.2 Determination of volatile matter

The W1 g sample obtained after finding the moisture content was heated to 950oC with a lid

covered on the silica crucible and maintained at the same temperature for 7 min in a muffle

furnace. The sample was cooled in a desiccator to ensure moisture doesn’t re-enter. The

0 20 40 60 80 100 120 140 1600

50

100

150

200

250

300

speed(rpm)

volu

metr

ic f

low

rate

(ml/m

in)

difference in the weights before and after heating (W2 g) were noted down and used

indetermination of volatile matter. The following equation was used.

% volatile matter = 100*W2/ W1 (3.2)

3.5.3 Determination of ash content

W3 g of the sample was taken in silica crucible and heated to 750oC and maintained at the same

temperature for 8 h. The sample was cooled in a desiccator to ensure moisture doesn’t re-enter.

The difference in the weights before and after heating (W4 g) were noted down and used in

determination of volatile matter. The following equation was used.

% Ash content = 100*W4/ W3 (3.3)

3.5.4 Determination of fixed carbon content

% Fixed carbon = (100- ash content - volatile matter - moisture content ) (3.4)

3.6 Batch study for MB removal using powdered Aspergillus niger

3.6.1 Methylene blue solution and analysis

Stock methylene blue solution of 1000 mg/L was prepared by dissolving 1g of in 1 L of distilled

water. The working methylene blue solution was prepared by diluting the stock methylene blue

solution. The total methylene blue concentration in the solution was determined by U-V

spectrophotometer.

3.6.2 Biosorption experiments

3.6.2.1 Effect of pH

The effect of pH on the amount of color removal was analyzed over a pH range from 3-11 with

increment of 2. The pH was adjusted using 0.1 N NaOH and 0.1 N HCl solutions. In this study

100mL of dye solution of 200 mg/L was agitated with 0.25 g of A.niger using mechanical

platform shakers at room temperature( 30±1 oC). Agitation was made for 30 h which is more

than sufficient to reach equilibrium at constant agitation speed of 200 rpm. The samples were

then vacuum filtered using 0.45 µm filter paper after biosorption experiments. The clear filtrate

was analyzed using U-V spectrophotometer by monitoring the absorbance changes at a

wavelength of maximum absorbance (665nm).

3.6.2.2 Effect of biosorbent concentration

The effect of A.niger on the amount of color adsorbed was obtained by contacting 100mL of dye

solution of initial dye concentration of 200mg/L with different weighed amounts(0.05, 0.10,

0.15, 0.20, 0.25 g) of A.niger using platform shaker at room temperature ( 30±1 oC) for 30 h

which is more than sufficient to reach equilibrium at constant agitation speed of 200rpm. The

samples were then vacuum filtered using 0.45µm filter paper after biosorption experiments.

The clear filtrate was analyzed as before. All the experiment was carried out at an optimum pH

of 7.

3.6.2.3 Biosorption kinetics

Sorption Kinetics experiments were carried out at different initial dye concentration using by

agitating 200ml of dye solution of known initial concentration of 100, 150, 200mg/L with 0.5 g

of A.niger at constant agitation speed of 200 rpm. Definite volume of 4mL samples were

pipetted out at time interval of 5,10,15,20,30,40,60,90,120 min. The collected samples were then

filtered and analyzed as before. The amount of MB adsorbed per gram A.niger (qt) was obtained

using the following expression;

( )

(3.5)

where qt (mg/g) is the uptake value at any time t , Ct (mg/L) is the liquid –phase concentration at

any time t and Co is initial concentration of dye (mg/L).

3.6.2.4 Sorption equilibrium

Equilibrium experiments were carried out by contacting 0.25 g of A.niger particles with 100mL

of dye solution of different initial dye amounts, 50, 100,150,200 mg/L. in a 250 mL conical flask

at constant agitation speed of 200 rpm till equilibrium is reached. The amount of MB adsorbed

per gram A.niger (qe) was obtained using the following expression;

( )

(3.6)

where qe is the equilibrium uptake value (mg/g) , V is the sample volume (L), Ce (mg/L) is the

equilibrium MB concentration and M (g) is the dry weight of the A.niger powder. The

percentage removal of the dye is given by;

( )

(3.7)

3.7 Batch study for MB removal using powdered PTMF

3.7.1 Adsorbate

Methylene blue (C.I. name: Basic blue 9, class: Thiazine, C.I. number: 52015 ) is a cationic dye

with a molecular formula C16H18N3SCl and molecular weight of 319.9 g/mol, was purchased

from Himedia, Bangalore. The MB (>99% dye content) was chosen because of its known strong

adsorption onto solids. The maximum absorption wavelength of this dye is 665 nm.

3.7.2 Preparation of biosorbent

Palm Tree male flower (PTMF) collected from a farmhouse in Udupi, India was washed

thoroughly with distilled water to remove surface adhered particles and water soluble materials.

The PTMF was dried at 70 oC for 2 days in a hot air oven and then cut into pieces, ground in a

ball mill and sieved to obtain average particles of 150 µm. The PTMF powder was stored in an

air tight container for further use. No other chemical treatments were used prior to adsorption

experiments.

3.7.3 Characteristics of PTMF

The surface area and total pore volume of PTMF were determined using BET apparatus

(Smart Instruments, Mumbai) . The surface functional groups of PTMF were detected by Fourier

Transform Infrared (FT-IR) spectroscope (Shimadzu, Japan). About 150 mg KBr disks

containing 2 % of PTMF sample was prepared before recording the FT-IR spectra in the range

450-4000 cm-1

with 16 cm-1

resolution.

The zero point charge was determined by suspending 1.0 g of the PTMF in 1 millimol/ L

NaNO3 solution for 24 h. Sixty milliliters of the suspension was measured into each of the

eight conical flasks and the initial pH was adjusted to: 2.14, 3.06, 4.1, 5.18, 6.14, 7.6, 8.48, 9.22.

The suspension in each of the flask was divided into 4 equal parts and 0.3 g of NaNO3 was

added to two sets while the other pair contained no added nitrate. They were left for 6 h and

after this the pH of the reference and test suspensions were taken as initial and final pH’s

respectively. The pairs containing no added nitrate were taken as the reference while those with

added nitrate were taken as the test samples (Ofomaja, 2007). The results were plotted as dpH

against initial pH (pHf).

3.7.4Biosorption experiment

3.7.4.1 Effect of pH

The effect of pH on amount of color removal was analyzed over a pH range of 2-9. The pH

was adjusted using 0.1 N HCl and 0.1 N NaOH solutions. In this study 100 mL of dye solution of

200 mg/L was agitated with 0.3 g of PTMF powder at room temperature of 30 oC. Agitation was

carried out for 6 h which is more than sufficient to reach equilibrium at constant agitation of 150

rpm. The samples were then filtered and analyzed using double beam U-V spectrophotometer

(UV-1700, Shimadzu, Japan) by monitoring the absorbance at a wavelength of maximum

absorbance of 665 nm.

3.7.4.2 Biosorbent dosage

The effect of PTMF concentration on the amount of color adsorbed was studied by dissolving

various quantities (0.05, 0.1, 0.15, 0.2, 0.25, 0.3 g ) of biosorbent in 100 mL of dye solution of

initial dye concentration of 200 mg/L . All the experiment were carried out at 30 oC, pH of 6

and 150 rpm for 6 h.

3.7.4.3 Biosorption Equilibrium

The equilibrium experiments were carried out at 150 rpm by adding 0.3 g of PTMF powder to

100 mL dye solution. The dye concentration was varied from 50-300 mg/ L. The amount of

MB adsorbed per gram PTMF (qe) and % removal was obtained using the Eq.(3.6) and Eq.(3.7

)

3.7.4.4 Biosorption Kinetics

Kinetic experiments were carried out by adding 0.3 g of PTMF powder to 100 mL of the dye

solution at 30oC at an optimum pH of 6 and at 150 rpm. The dye concentration was varied from

50-200 mg/L. The amount of MB adsorbed per gram PTMF (qt) was obtained using the Eq.(3.5)

3.7.4.5 Effect of initial MB concentration

Equilibrium experiments were carried out by mixing 0.3 g of PTMF powder with 100 mL of

MB solution of different initial dye concentration ranging from 50- 300 mg/L. The studies were

carried out at three temperatures ( 303 K, 313 K, 323 K ) using shaking incubator. After 6 h the

samples were filtered and analyzed for residual MB. All experiments were carried out in

duplicate under identical conditions and mean values are presented. The error obtained was 2.0-

4.0%.

3.8 Batch study for MB removal using powdered ASD

3.8.1 Preparation of the adsorbent

Sawdust (Lagerstroemia microcarpa) which is a raw waste and usually thrown away was

collected from carpentry work shop, Manipal, Karnataka, India and dried in a dryer at 110 0C for

one day; Then sawdust was ground to fine powder and sieved to 150 μm size. Sawdust was

chemically treated with hydrochloric acid (HCl) and was termed as acid activated

sawdust(ASD). A suspension containing 20 gm sawdust, 400 ml of distilled water and 10 ml of

1.33M HCl was prepared in a beaker. The contents in the beaker was stirred for 1 hr at 80 ± 2 0C

in a magnetic stirrer with hot plate. After cooling and filtering, the sawdust was washed several

times with distilled water until the filtrate pH became neutral and then dried in air at room

temperature. The material was stored in an airtight container for further experiments.

3.8.2 Preparation of MB solutions

The analytical grade methylene blue with 99.8% purity, was supplied by Hi-Media, Bangalore,

India. A stock solution of 1000 mg/L of MB were prepared and then used for different

experiments by diluting with distilled water.

3.8.3 Characteristics of ASD

The surface area of ASD were determined using BET apparatus (Smart Instruments, Mumbai) .

The surface texture of ASD was found out by Scanning Electron microscopy (SEM) analysis.

The structural information on the functional groups of ASD samples were detected by Fourier

Transform Infrared (FTIR) spectroscope (Shimadzu, Japan). The FTIR analysis was done by

using 150 mg KBr disks containing 2 % of ASD sample in the range 450-4000 cm-1

with 16

cm-1

resolution.

3.8.4 Adsorption experiments

3.8.4.1 Influence of pH

The amount of color removal were analyzed over a pH range of 2-10 by adjusting the pH using

0.1 N HCl and 0.1 N NaOH solutions. The experiments were conducted by agitating 100 mL of

dye solution of 50 mg/L with 0.1 g ASD powder at temperature of 30 oC. The agitation was

carried out for an equilibrium time of 6 hr and speed of 150 rpm. The samples collected were

then filtered and analyzed using double beam U-V spectrophotometer (UV-1700, Shimadzu,

Japan) by measuring the absorbance at 665 nm.

3.8.4.2 Influence of ASD concentration

The effect of ASD concentration on the amount of color adsorbed was studied by adding 0.1 to

0.6 g of ASD in 100 mL of MB solution of initial concentration of 200 mg/L . All the

experiment were carried out at 30oC, pH of 7 and 150 rpm for 6 hr.

3.8.4.3 Adsorption equilibrium

The equilibrium experiments were conducted by agitating 100 mL MB solution with 0.1 g

ASD at 150 rpm. The MB concentrations was varied from 50-300 mg/ L. The amount of MB

adsorbed per gram of ASD (qe) at equilibrium and the % removal was obtained by the Eq(3.6 )

and (3.7)

3.8.4.4 Adsorption Kinetics

Kinetic experiments were performed by adding 1 g ASD powder to 1000 mL of the MB

solution at 30oC at pH 7 and at constant agitation speed of 150 rpm. The MB concentrations

were varied from 50 to 200 mg/L. The amount of MB adsorbed per unit gram ASD at any time

(qt) were obtained using the Eq.(3.5).

3.8.4.5 Effect of initial MB concentration

The experiments were conducted by adding 0.1 g ASD powder with 100 mL of MB solution of

dye concentration ranging from 50- 200 mg/ , at four different temperatures (293K, 303 K, 313

K, 323 K ) using shaking incubator for 6 hr.

3.9 Batch studies with PTMFAC

3.9.1 Preparation of PTMFAC

Palm Tree male flower (PTMF) collected from a farmhouse in Udupi, India was washed

thoroughly with distilled water to remove surface adhered particles and water soluble materials.

The PTMF was dried at 70 oC for 2 days in a hot air oven and then cut into pieces, PTMF cut

into small pieces of 2–3 cm and dried in sunlight. The dried biomass was mixed with 85% Ortho

phosphoric acid (1:1.5, w/v) and heated in a muffle furnace at 350oC for 5 h under N2

atmosphere. The carbonized material so obtained was washed with distilled water several times

to remove the free acid and soaked in 1% sodium bicarbonate solution overnight to remove any

residual acid. This material called as PTMFAC was then washed with distilled water and dried at

105oC in a hot air oven for 24 h. It was ground and sieved in the average size of 150 µm. The

PTMF powder was stored in an air tight container for further use. No other chemical treatments

were used prior to adsorption experiments.

3.9.2 Characteristics of PTMFAC

The surface area and total pore volume of PTMFAC were determined using BET apparatus

(Smart Instruments, Mumbai) .

3.9.3 Adsorption experiment

3.9.3.1 Adsorbent dosage

The effect of PTMFAC concentration on the amount of color adsorbed was studied by

dissolving various quantities (0.02, 0.04, 0.06, 0.08, 1.0 ,1.2 g ) of adsorbent in 100 mL of dye

solution of initial dye concentration of 200 mg/L . All the experiment were carried out at 30 oC,

pH of 6 and 150 rpm for 6 h.

3.9.3.2 Adsorption Equilibrium

The equilibrium experiments were carried out at 150 rpm by adding 0.1 g of PTMFAC powder

to 100 mL dye solution. The dye concentration was varied from 50-300 mg/ L. The amount of

MB adsorbed per gram PTMFAC (qe) and % removal of the dye were obtained using the Eq.

(3.6) and ( 3.7).

3.9.3.3 Adsorption Kinetics

Kinetic experiments were carried out by adding 0.1 g of PTMFAC powder to 100 mL of the dye

solution at 30oC at an optimum pH of 6 and at 150 rpm. The dye concentration was varied from

50-200 mg/L. The amount of MB adsorbed per gram PTMFAC (qt) was obtained using the Eq.

(3.5);

3.9.3.4 Effect of initial MB concentration

Equilibrium experiments were carried out by mixing 0.1 g of PTMFAC powder with 100 mL of

MB solution of different initial dye concentration ranging from 50- 300 mg/L. The studies were

carried out at three temperatures ( 303 K, 313 K, 323 K ) using shaking incubator. After 6 h the

samples were filtered and analyzed for residual MB. All experiments were carried out in

duplicate under identical conditions and mean values are presented. The error obtained was 2.0-

4.0%.

3.10 Fixed bed studies using Aspergillus niger beads

Continuous flow sorption experiments were conducted in a glass column of 2.1 cm internal

diameter and 39 cm height ( as shown in Fig.3.8). At the top and bottom of the column a rubber

cork of 1.5cm was inserted. An air gap of 1cm was left followed by glass wool of 5 cms was

packed At the bottom of the column, after the glass wool a 3 cm high layer of glass beads (1.5

mm in diameter) was placed in order to provide a uniform inlet flow of the solution into the

column.

A known quantity of immobilized A. niger beads(see Appendix-III ) was packed in the column

to yield the desired bed height of the biosorbent. Methylene blue dye solution of known

concentration at pH 6 was pumped upward through the column at a desired flow rate by a

peristaltic pump. The aliquots of dye at the outlet of the column were collected at regular time

intervals. The concentration of MB in the effluent was analyzed using a U-V spectrophotometer

by monitoring the absorbance changes at a wavelength of maximum absorbance of 665 nm.

Calibration curves were plotted between absorbance and concentration of the dye solution given

in section 3.4.1.The experimental set up is shown in Fig-3.8.

Fig-3.8 Experimental set up for Fixed bed studies