A

PROJECT REPORT
ON

ADSORPTION OF METHYLENE BLUE

BY
MOSS AND ALGAE

BY

SAMAR-UR-REHMAN

UNDER THE SUPERVISION OF

Dr. (Mrs.) SEEMA UPADHYAY

HEAD of DEPARTMENT
BIOTECHNOLOGY DEPARTMENT

IMS ENGINEERING COLLEGE

NH-24, ADHYATMIC NAGAR
GHAZIABAD
MAY 2007 (BATCH 2003-2007)

ACKNOLWEDGEMENT
This is not one man’s work. We express our sincere thanks to the following
individuals: when contributing in various ways for completion of our
project. The successful culmination of our efforts reminds of our indebtness
towards our venerated guide “Dr. (Mrs.) Seema Upadhyay” HoD of
department of biotechnology IMS Engineering college Ghaziabad for the
valuable guidance and though providing encouragement through out the
semester.

We are very thankful to “Mrs. Rashmi Chandra”, “Mr. Vinay dwivedi”,

“Mr. Santosh Kumar mishra” for their valuable advice at right time. when
needed , providing the requisite facilities for completion of our project work.

We are very thankful to Dr. I.C.Aggarwal, Director IMS engineering

college, Ghaziabad for allowing us to do the project and providing us the
requisite facilities for the completion of the project work.

We are also thankful to all the faculty members of the department of

biotechnology, IMS engineering college, Ghaziabad for their valuable
suggestion and provided to us from time to time.
 INDEX

PAGE NO

1 ABSTRACT 1

2 INTRODUCTION 2-7

1.1 Introduction
1.2 History
1.3 Review of literature.

3 METHYLENE BLUE AND BIOMASS 8-12

3.1 Methylene blue

3.2 Biomass

4 MATERIAL & METHODS 13-18

4.1 Preparation of adsorbent

4.2 Pretreatment with CaCl2
4.3 Pretreatment with HCl
4.4 Pretreatment with formaldehyde
4.5 Preparation of dye solution
4.6 Dye adsorption experiment

5 RESULT & DISCUSSION 19-25

6 CONCLUSION 26

7 REFRENCES 27
 ABSTRACT

Methylne blue adsorption on moss & algae investigated using visible

Absorption spectroscopy. Different pretreatments protonation and chemical

cross linking with CaCl2 or formaldehyde have been tested in order to

improve the Stability as well as the adsorption capacity of biomass. Then

percentage of the absorption is obtained in both Moss & algae. The results

obtained have shown that this type of material especially moss with

pretreatment CaCl2 has a high adsorption capacity for the Methylene blue

dye, this feature together with the short time needed to reach the equilibrium

suggest that Moss can be used as a low cost biosorbent in waste water

treatment process.

Key word: Biosorption, Moss,algae,cross-linking,adsorption.

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1.1INTRODUCTION

Biosorption processes have attained a great importance from an

environmental point of view as they can be used to remove toxic

compound from industrial waste water. Many industries used dyes

to color their final products and their discharge into natural water

causes severe problems because they are toxic to aquatic life and

damage the aesthetic nature of the environment. Moreover, these

effluents are rather difficult to treat by conventional biological and

physical-chemical techniques due to the complex structure of the

dyes. Therefore, adsorption processes provide an attractive

alternative for the treatment of coloured waters.

Activated carbon is the most popular and widely used

adsorbent, but there are certain problems with its use since it is

expensive and its regeneration is difficult. For this reason, interest

has been recently focused on low-cost materials, ranging from

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waste products from other industries to naturally abandoned

biomass: sewage sludge and peanut shell, silk cotton hull, coconut

tree sawdust and coir pith,moss,banana pith and water hyacinth

roots, parthenium plant, bacteria and fungi. In particular, marine

algae are very promising materials to be used as biosorbents in

wastewater treatments because they represent a cheap source of

biosorbent, as they are radily available in large quantities and it has

been shown that they display a high metal binding capacity, mainly

due to carboxylic and sulphonate groups from the algal

polysaccharides.

The present work deals with the adsorption of Methylene blue,

a dye often used to characterize the capacity of an adsorbent, by

algae and moss. In addition, to the best of our knowledge, studies

of dye adsorption by moss are not available; therefore, it is

desirable that this promising field of biosorbent is explored.

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In order to perform the adsorption experiment different

pretreatments for the chemical modification of the biomass, among

them protonation and chemical cross linking with calcium chloride

and formaldehyde has been tested to improve its stability as well as

its adsorption capacity, making it suitable for industrial use.

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1.2History
Many industries uses various dyes as a colouring agent for their

products such as textile industry, automobile industry,etc. but the

release of such dyes in natural water is cause of many problems

and lead to the severe water pollution. These dyes such as

Methylene blue is also harmful to aquatic life. So, it is required to

remove any such dye from the wastewater before its disposal in the

natural water from the environmental point of view. Various works

already have been done in this direction to remove various dyes.

These dyes are mainly removed by physical and chemical methods

in the recent times but these methods are not feasible and also

very costly. So, attention is given to the use of biomass to remove

such dyes now are days. Activated carbon is mostly used to

remove Methylene blue dye in recent time but it it a very costly

process. So, scientists are looking for some biological alternative

which is cheap as well as easily available. In this context work is

carried out over the possibilities of various biomass for removal of

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dyes.
For this reason, intrest has been recently focused on low cost

material, ranging from waste products from other industries to

naturally abandoned biomass: sewage sludge and peanut shell, silk

cotton hull, coconut tree sawdust and coir pith, moss, banana pith

and water hyacinth roots,parthenium plant, bacteria and fungi. In

particular, marine algae are very promising materials to be used as

biosorbents in waste water treatment process because they

represent cheap source of biosorbent., as they are readily available

in large quantities, and it has been shown that they display a high

metal-binding capacity, mainly due to carboxylic and sulphonate

groups from algal polysaccharides.

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1.3 REVIEW OF LITERATURE:

Recently, work has been carried out over different biological

alternatives for the adsorption of various dyes. For example

Saragassum muticum algae has been tested for removal of

Methylene blue.

Methylene blue is also harmful to aquatic life. So, it is required to

remove any such dye from the wastewater before its disposal in the

natural water from the environmental point of view. Various works

already have been done in this direction to remove various dyes.

These dyes are mainly removed by physical and chemical methods

in the recent times but these methods are not feasible and also

very costly. So, attention is given to the use of biomass to remove

such dyes now are days. Activated carbon is mostly used to

remove Methylene blue dye in recent time but it it a very costly

process.So, scientists are looking for some biological alternative

which is cheap as well as easily available.

3.1 Methylene blue
Methylene blue is a heterocyclic aromatic chemical compound

with molecular formula: C16H18ClN3S.

It has many uses in a range of different fields, such as biology or

chemistry. At room temperature it appears as a solid, odorless,

dark green powder, that yields a blue solution when dissolved in

water. Methylene blue should not be confused with methyl blue,

another histology stain, new Methylene blue, nor with the methyl

violets often used as pH indicators.

Chemical structure of Methylene blue

3-D structure of
Methylene blue
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Physical and Chemical Properties

Appearance: Dark green crystals with bronze luster or crystalline

powder.

Odor: Odorless.

Solubility: Soluble in water.

Specific Gravity: No information found.

pH: No information found.

% Volatiles by volume @ 21C (70F): 0

Boiling Point: Decomposes.

Melting Point: 100 - 110C (212 - 230F)

Vapor Density (Air=1): 13

Vapor Pressure (mm Hg): Not applicable.

Evaporation Rate (BuAc=1): No information found.

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Hazards Identification
WARNING! HARMFUL IF SWALLOWED.
Health Rating: 2 - Moderate
Flammability Rating: 1 - Slight
Reactivity Rating: 1 - Slight
Contact Rating: 1 - Slight
Lab Protective Equip: GOGGLES; LAB COAT; VENT HOOD;
PROPER GLOVES
Storage Color Code: Green (General Storage)

Potential Health Effects:

This material is relatively nonhazardous in routine industrial

situations.

Inhalation:

No adverse health effects expected from inhalation. May cause a

short period of rapid or difficult breathing.

Ingestion:

A burning sensation of the mouth may be noted following

ingestion of methylene blue. May cause nausea, vomiting,
diarrhea, and gastritis. Large doses may cause abdominal and chest
pain, headache, profuse sweating, mental confusion, painful
micturation, and methemoglobinemia.

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Skin Contact:

Not expected to be a health hazard from skin exposure. Methylene

blue may color the skin a bluish color. May cause
photosensitization.

Eye Contact:
No adverse effects expected. May cause mechanical irritation.

Chronic Exposure:

No information found.

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3.2 BIOMASS
1. Moss: Moss grass taken from nursery which is generally used
for the absorption of water.
Appearance: Greenish brown.
Density: Low.
Odour: Odourless.

2.Algae: Algae taken from local pond of Adhyatmik nagar,

Ghaziabad
Appearance: Green.
Density: higher than moss grass
Odour: foul smell.

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 MATERIAL & METHODS

4.1 Preparation of adsorbent

Preparation of the adsorbent’the adsorbent used were algae and

moss grass collected in Dasna village near GHAZIABAD

Specimens of algae and moss grass were washed with generous

amounts of distilled water and dreid in an oven at 60 C overnight.

Then, they were ground in a mixer grinder, and sieved in the size

pore range from0.5 to 1mm.Finally the biomass was chemically

modified by means of different treatment, which were carried out

as follows.

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4.2 Pretreatment with CaCl2

A sample of 2.5g of dried biomass was treated with 100cm3 of 0.2

mol dm-3 CaCl2 solution, keeping the solution pH constant at a

value of 5.0 because it is the optimum pH value for calcium

activation of biomass. The mixture was shaken for 24 hr on a

rotary

shaker at 175rpm and room temperature. The biomass was then

filtered off, followed by washing with deionised water to remove

of excess of calcium and it was then dried in an oven at 60 C for

24 hr.

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4.3 Pretreatment with HCl

A constant mass of algae and moss grass (2.5g) was treated with
100

cm3 of 0.1 mol dm-3 HCl solution for 3 hr at room temperature.

The biomass was then filtered off, followed by washing with

deionised water, and it was dried overnight at 60 C. Then, 400 cm3

of 0.1 mol dm-3 HCl solution were added to the dried biomass and

the previous procedure was repeated.

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4.4 Formaldehyde cross linking

Dried biomass (2.5gm) was added to a mixture of 17 cm3 36%

formaldehyde and 3 cm 3 of 0.1 mol dm-3 HCl solution. The

mixture was left room temperature for 1 hr under gentle mixing.

The biomass was then filtered off followed by sequential washes

with distilled water 0.2 moldm-3 Na2CO3 and finally distilled

water. It was dried overnight at 60C and finally for 2 hr at 110C.

Pre-treated biomass was kept in plastic container refrigerated at 4

C for further use

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4.5 Preparation of Dye solution-

Dye used in this study was Methylene blue

Stock solution of methylene blue, without further purification, we

prepared
by dissolving accurately weighed samples of dye in distilled water
to give a

concentration of 500mgdm-3 (0.5g/l)

4.6 Dye Adsorption Experiments-

In order to carry out adsorption experiments, a constant mass of

algal and moss grass separately (0.1 gm each) was weighed into a
conical flask and 40 cm3 of dye solution were added to it . The
mixture was shaken at 175rpm for 2 hrs , when equilibrium was
reached , then biomass were separated by decantation and the dye
concentration analyzed using a spectrophotometer UV/VS at max
665nm. The solution were diluted to 1/96 to give absorbance in
range .1 to 1 before making measurements.

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ABSORBANCE TABLE FOR MOSS

.5/96 C MOSS MOSS MOSS

METHYLENE P1 P2 P3
BLUE DYE
.789 .026 .449 .314
.754 .026 .453 .307
.784 .025 .456 .314

ABSORBANCE TABLE FOR ALGAE

.5/96 C ALGAE ALGAE ALGAE

METHYLENE P1 P2 P3
BLUE DYE
.789 .596 .782 .395
.754 .608 .787 .403
.784 .614 .791 .405

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5 RESULT:
The original reading of absorbance of methylene blue at .5/96
concentration is found to be:
S.No. Mehylene blue
1. .789
2. .754
3. .784
-------------------------------------------------------
Average .78

Absorbance table for moss at 665 nm with different

pretreatments

S.No Pretreatment Pretreatment Pretreatment

1 2 3
1 .026 .449 .314
2 .026 .453 .307
3 .026 .458 .314
AVERAGE .026 .453 .312

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So percentage adsorption by moss with different pretreatment are :

Pretreatment No 1 = 97%

Pretreatment No 2 = 42%
Pretreatment No 3 = 60%

So, according to the above observation maximum adsorption is

obtained when moss is treated with the CaCl2 due to some cross
linking

100
90
80
70 pretreatment
60 no1
pretreatment
50
no2
40 pretreatment
30 no3

20
10
0

Above graph showing percentage adsorption of methylene blue by moss in

different pretreatments
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Figure shows concentration variation of methylene blue after 2 hr by moss for
pretreatment 1

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Absorbance table for algae at 665 nm with different
pretreatments

S.no Pretreatment Pretreatment Pretreatment

no1 no2 no3
1 .596 .702 .395
2 .608 .712 .401
3 .620 .722 .405
AVERAGE .61 .712 .40

So, percentage adsorption by algae with different pretreatment are

Pretreatment no1 = 22%

Pretreatment no2 = 9%

Pretreatment no 3 = 49%

So, according to the above observation maximum adsorption is obtained

when algae is treated with the formaldehyde & HCl

100

80
pretreatment
no1
60
pretreatment
no2
40
pretreatment
no3
20

Above grabh showing percentage adsorption of methylene blue by algae in

different pretreatments
 (22)

DISCUSSION ABOUT RESULT

Comparison of moss and algae in their adsorption capacity

• According to the observations, moss is found more suitable for the

adsorption for the methylene blue, especially when pretreated with
CaCl2 gives a very good result and 97% adsorption is obtained in that
case.

• In algae maximum adsorption occurred with pretreatment 3(i.e.49%)

but in moss with pretreatment 3, 60% adsorption observed which
leads to the good result for moss again.

• In pretreatment 1, just 22% adsorption occurred in case of algae while

remarkable 97%adsorption occurred in case of moss which give
success our project

• In pretreatment 2, least adsorption is obtained in both moss and algae.

the moss just 42% and in algae only 9% adsorption is observed

• Following figure are indicating comparison between the moss & algae
for pretreatment 2 & pretreatment 3

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 Moss Algae

Figure shows the comparison of methylene blue concentration

between the algae & moss grass for pretreatment no 3

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 Algae Moss
Algae

Figure shows the comparison of methylene blue concentration

between the algae & moss grass for pretreatment no 2.

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 Conclusion

According to our observation we found that adsorption is occurred

in both moss and algae but obtained when moss is prepared with

calcium chloride. In algae maximum adsorption of methylene

adsorption of methylene blue is obtained when algae is pretreated

with HCl and formaldehyde.

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 REFERENCES

1. Eugenia Rubin, Pilar Rodriguez, Roberto Herrero. J chem. Technol

Biotechnol 80:291-298(2005)

2. Lee CK, Low KS & Chow SW, Chrome sludge, as an adsorbent for
colour removal, Environ technol 17:1023-1028(1996).

3. Fu Y and Viraraghvan T, fungal decolorization of dye waste waters: A

review. Bioresour technol 85:205-206(2002).

4. Ho YS and Mc Kay G, a comparison of chemisorption kinetic models

applied to pollutant removal on various sorbents.Transactions of the
institution of chemical engineers 76:332-340(1998).

5. Davis TA, Volesky B and Vieira RHASF, saragassum seaweed has

biosorbent for heavy metals. Water Res 34:4270-4278(2000).

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