M. Tech.

Final project presentation on

Bioremediation of hazardous Metanil yellow dye by
using Trichoderma and Azotobacter biofertilizers

Presented by
Manujendra Kumar Prajapati
Under The Guidance of
Dr. K. S. Kulkarni
Associate professor
Department of Chemical Engineering,
BVU ‘COE’, Pune - M.H.
 Contents
• Introduction.
• Literature review.
• Necessity for the removal of dye.
• Toxicity properties of Metanil yellow.
• Various types of health hazards.
• Why adsorption use ?
• Adsorption process.
• Adsorption mechanism.
• Objective of the experimental work
• Optimizations study of all parameters likes Temp., Conc., pH, etc.
• Feasibility of the experimental work.
• Examination study of Langmuir and Freundlich isotherms.
• Reusability of biosorbent.
• Conclusion.
• Reference.
 Introduction
• The textile industry is one of the major contributors to water, human, soil,
and our prestigious environment pollution due to the high amount of wastewater
generated during textile processing.

• Main pollution from textile wastewater comes from dyeing and finishing processes.

• Water is used as principal medium to apply dyes and various chemical finishes.

• The experimental setup involved assessing their efficiency in dye removal of

industrial wastewater, cultivation of Trichoderma and Azotobacter in contaminated
media.

• The results demonstrated an impressive 92.89% removal of Metanil Yellow dye by

Trichoderma and even higher removal rate of 99.9% by Azotobacter.
 Literature Review
Adsorbent Dye Parameter Isotherm &
K, Kulkarni Biofertilizer containing Rhodamine-B initial dye concentration, Langmuir and
2019) Trichoderma viride contact time, solution of pH, isotherm model
temperature and adsorbent
dosage

Haq NB, Saima Chemically modified acid red 1 adsorbent dosage, initial dye pseudo
ad AT, Soo-JinP Sugarcane bagasse-based concentration, reaction kinetics ande Fr
bio composites temperature and pH. the Langmui
models

nthilKumar P, fungal biomass Malachite Green adsorbent dosage, initial dye Langmuir isoth
aravanan K, concentration, pH, contact and Lagranges m
(2020) time, temperature etc

M, Velic N, Gali Waste Wood Biomass Congo Red Initial concentration and Langmuir and F
Jakovljevic T, adsorbent dosage isotherm model
icM(2021)

Haq NB, White Rot Fungus Reactive Dye Initial concentration and Langmuir, Freu
rheen A, adsorbent dosage Sips models, ps
iham AA, Iqbal order, pseudo-se
023) and Elovich mo
 Literature Review
or Adsorbent Dye Parameter Isotherm & m
N, Amina K, Haq Polypyrrole and rice husk 2,4,6-trichloro phenol amount of adsorbent, Langmuir, Freundli
uhammad Z, temperature, pH, initial Sips models,
n A, Amani N, concentrations, contact
FA, Siham AA, time and agitation speeds
M (2022)

Z, Amina K, Urooj Eucalyptus Basic Blue 41 dye initial dye concentration, Langmuir, Freundli
-Jin P, Haq N, (camaldulensis) bark- contact time, solution of Langmuir–Freundli
based pH, temperature and models
composites adsorbent dosage

edo-Delira R. Trichoderma Biomass Congo Red solution pH on dye Freundlich isotherm

-Martínez MJ, adsorption the Langmuir isoth
Kaffure R (2021)

ali S, Dariush B, CTAB-acid modified Congo red dye initial dye concentration, Langmuir, Freundli
(2019) celery contact time, solution of Langmuir–Freundli
pH, temperature and models
adsorbent dosage
 Literature Review

or Adsorbent Dye Parameter Isotherm & m

MA, Gomaa M algal biorefinery waste congo red and Fe (II) amount of adsorbent, Langmuir, Freundl
and waste office paper temperature, pH, initial Sips models,
concentrations, contact
time and agitation speeds

l NK, Sumana K banana peel Congo red initial dye concentration, Langmuir, Freundl
contact time, solution of Langmuir–Freund
pH, temperature and models
adsorbent dosage

VV, Raut P, activated teak leaf powder Congo red solution pH on dye Freundlich isother
de A, Pathak P adsorption the Langmuir isoth

A, Shahzad M, peanut shell congo red and brilliant initial dye concentration, Langmuir, Freundl
S, Muhammad M, green dyes contact time, solution of Langmuir–Freund
, Akber S (2012) pH, temperature and models
adsorbent dosage
 Literature Review
Adsorbent Dye Parameter Isotherm
hagufta Z, , Hafiz morus Alba leaves as congo red dye initial dye concentration, Langmuir
H, Zahid S (2015) biosorbent contact time, solution of pH, isotherm mo
temperature and adsorbent
dosage

, Yeddou AR, Aspergillus carbonarius Congo red dye adsorbent dosage, initial dye pseudo seco
Hellel D, Holtz M333 and Penicillium concentration, reaction ande Freun
u N, Chergui A, glabrum temperature and pH. Langmuir is
2017)

an X, Zhang J, Self-Immobilized Mycelial Congo red dye adsorbent dosage, initial dye Langmuir is
lah Z, Pellets concentration, pH, contact and Lagrang
Tian Y, (2020) time, temperature etc

Rajkumar S, Electrospun Congo red dye Initial concentration and Langmuir a

Saravanan P polyacrylonitrile-Moringa adsorbent dosage isotherm mo
Olifera based nanofibrous
bio-sorbent

Khurade S, Biofertilizers Azotobacter Cngo red dye Initial concentration and Langmuir, F
(2023) adsorbent dosage Sips models
order, pseud
and Elovich
 Necessity for the removal of dye

• Wastewater originated from textile dyeing environmental pollution.

• Dyes have capability to sustain in the environment for a longer period of time due
to high thermal and photo stability.

• The presence of textile dyes in water bodies obstruct their oxygenation capacity.

• It poses a challenge for the photosynthetic process of aquatic or algae.

• The long-time presence of dyes bring toxic to the aquatic environment and results
in low aerobic biodegradability.
 Necessity for the removal of dye

• Because all of them not contained in the final product, becomes waste and cause
disposal problem

• To highlight the Human, Water, Soil, health and environmental hazards associated
with the use of Metanil Yellow dye.

• The presence of very small amounts of dyes in the water affects the superiority and
transparency of water bodies
 Toxicity properties of Metanil yellow

• A prohibited Metanil yellow has been found in various foods.

• Chemical structure: Metanil Yellow has the molecular formula

C_18H_14N_3NaO_3S. It typically consists of a diazo component (aromatic amine)
and a coupling component (aromatic sulfonic acid).

• The inducing effect of Metanil yellow on cytochrome P-450 (P-450)-dependent

monooxygenases and cytosolic detoxifying enzymes, namely glutathione S-
transferase (GST) and quinone reductase (QR), was studied.

• Oral administration of Metanil yellow (430 mg/kg body weight) for seven days
significantly induced liver P-450 (48%) and its dependent aryl hydroxylase (100%)
activity, cytosolic GST (136%) and QR (92%) activity in four animals.

• The experimental studies have shown that metanil yellow is neurotoxic and
hepatotoxic thus, should be avoided at all costs.
 Toxicity properties of Metanil Yellow

• The spectrum of induced microsomes by Metanil yellow showed an increase in P-

450, moving 2.2 nm in the Soret region.

• The results suggest that Metanil yellow is a dual-function inducer of specific

isozymes of P-450 and cytosolic enzymes, and may be involved in the induction of
cytosolic aryl hydrocarbon (Ah) receptor.

• Therefore, Metanil yellow has the potential to induce toxicity in human cells.

• Compared to other monooxygenases (64-92%), extraintestina administration of

Metanil yellow (80 mg/kg body weight) for three days in another group of four
animals resulted in a higher induction rate of ethoxyresorufin-O-deethylase
(228%), as well as GST and QR (59-95%).
 Various types of health hazards
• Human Health Risks:
• Indirect Exposure: Humans can be indirectly exposed to Metanil Yellow through
consumption of contaminated food or water, leading to potential health risks such
as gastrointestinal issues, allergic reactions, and long-term health effects including
cancer.
• Example:
1. Effects on Excretory and Reproductive System.
2. Effects on Cardiovascular System.
3. Effects on Nervous System.

• Occupational Exposure: Workers in industries using Metanil Yellow dye may face
occupational exposure through inhalation or dermal contact, leading to health
hazards such as skin irritation, respiratory problems, and systemic toxicity.
 Various types of health hazards

• Animal Health:
• Toxicity: Animals can be exposed to Metanil Yellow dye through contaminated
water or food sources. Ingestion of Metanil Yellow can lead to toxicity, testicular
damage, haematopoietic system alteration causing adverse effects on the
gastrointestinal system, liver, and kidneys.

• Bioaccumulation: Metanil Yellow has the potential to bio accumulate in the tissues
of animals, particularly those higher up in the food chain. This bioaccumulation can
lead to higher concentrations of the dye in predators, resulting in toxic effects and
potential reproductive issues.
 Various types of health hazards

• Soil Contamination:
• Persistence: Metanil Yellow dye, when disposed of improperly, can persist in the
soil for extended periods. Its slow degradation can lead to long-term soil
contamination, affecting soil health and fertility.

• Impact on Microorganisms: Soil microorganisms play a crucial role in nutrient

cycling and soil health. Metanil Yellow contamination can disrupt the microbial
community, leading to decreased soil fertility and productivity.
 Various types of health hazards

• Environmental Pollution:
• Water Pollution: Industrial effluents containing Metanil Yellow dye are often
discharged untreated into water bodies. This leads to water pollution, affecting
aquatic ecosystems and biodiversity. The dye can hinder the process of
photosynthesis in aquatic plants and disrupt the balance of the ecosystem.

• Eutrophication: Metanil Yellow dye runoff into water bodies can contribute to
eutrophication, a process in which excessive nutrients lead to algal blooms and
oxygen depletion, causing harm to aquatic life.

• Habitat Destruction: Contamination of natural habitats due to Metanil Yellow dye

pollution can lead to habitat destruction and loss of biodiversity, impacting various
species dependent on these habitats for survival.
 Why adsorptions use?

• Simplest method.

• Low capital and operating cost.

• Versatility.

• Can have good physical properties.

• Adsorbents are easily available.

• High efficiency.

• Regenerability.
 Adsorption
• A process where a material is concentrated at a solid surface from its liquid or gaseous
surrounding.

• Adsorption is considered as best wastewater treatment technique because all inclusive

its nature, modesty and simplicity of operation.

• Basically adsorption is the accumulation of substance at surface or interface.

• This process occurs due to attractive forces between the adsorbate (the substance being
adsorbed) and the adsorbent (the surface of the solid material).

• Instead, it forms a layer of adsorbate on the surface, leading to various practical

applications such as purification, separation, and catalysis.

• That’s why we are called adsorption is a surface phenomenon where molecules or

particles from a fluid (liquid or gas) adhere to the surface of a solid material.
 Pictorial presentation of adsorption process

Adsorbate-The
gas, vapour or
the solute,
which is held
to surface of
the solid. e.g.
poisonous
Adsorption process gases

Adsorbent-The solid
that takes up a gas,
vapour or a solute from
a solution, e.g. silica
gel, charcoal, clay, etc.
 Adsorption mechanism

• Adsorption is surface phenomenon.

• It arises because of unbalanced forces on the surface of solids and liquids.

• The surface is under tension due to unbalanced forces.

• The surface of the solid or liquid tends to satisfy their residual forces by attracting
and retaining the molecules of other species when brought in contact with them.
 Adsorption mechanism

• Adsorption is a surface phenomenon where molecules or atoms from a gas or liquid

(adsorbate) adhere to the surface of a solid or liquid (adsorbent). The mechanism of
adsorption involves several steps:

1. Transport of adsorbate molecules: Adsorbate molecules move from the bulk phase
(gas or liquid) to the surface of the adsorbent through diffusion or convection.

2. Adsorbate-adsorbent interaction: Once at the surface, adsorbate molecules interact

with the adsorbent surface. This interaction can occur through various forces,
including van der Waals forces, electrostatic interactions, hydrogen bonding, and
chemical bonds.

3. Adsorption equilibrium: As adsorbate molecules accumulate on the surface, a

dynamic equilibrium is established between adsorption (molecules adhering to the
surface) and desorption (molecules detaching from the surface). This equilibrium
depends on factors such as temperature, pressure, and the nature of the adsorbate-
adsorbent interaction.
 Adsorption mechanism
4. Surface coverage: The extent of adsorption is characterized by the surface coverage,
which represents the fraction of available surface sites occupied by adsorbate molecules.
At low surface coverage, adsorption may occur primarily at specific sites or defects on
the surface, while at high surface coverage, multiple layers of adsorbate molecules may
form.

5. Adsorption isotherms: The relationship between the amount of adsorbate adsorbed per
unit mass of adsorbent (adsorption capacity) and the concentration or pressure of the
adsorbate in the bulk phase is described by adsorption isotherms. Common adsorption
isotherms include the Langmuir, Freundlich, and BET (Brunauer-Emmett-Teller)
isotherms, which provide insights into the adsorption mechanism and surface properties
of the adsorbent.

• Overall, the adsorption mechanism involves the transport of adsorbate molecules to the
adsorbent surface, interaction between adsorbate and adsorbent, establishment of
adsorption equilibrium, determination of surface coverage, and characterization of
adsorption behavior using adsorption isotherms. Understanding these mechanisms is
essential for designing adsorption processes in various applications such as gas
separation, purification, catalysis, and environmental remediation.
 Objective

• The objective is to investigate the removal of Metanil Yellow dye through

adsorption processes, utilizing biofertilizers such as Trichoderma and Azotobacter
as potential adsorbents.

• The study aims to understand the adsorption mechanism by fitting experimental

data to adsorption isotherm models.

• Parameters including time, initial dye concentration, temperature, adsorbent dose,

and agitation speed will be varied to optimize the adsorption process.

• Langmuir isotherm model and Freundlich, Langmiuer and isotherm model is used
to fit experimental data.
 Optimizations study of Optimum condition of
biosorbent dose, (Trichoderma & Azotobacter)
% Removal Efficency VS Adsorbent Dose Loading % removal Efficiency VS Adsorbent Loading in gm
(Azotobactor)
100 100
99.9
98
99.8
96 05-ppm 99.7 05-ppm
% Removal Efficiency

% Removal Efficiency
94 10-ppm 99.6 10-ppm
92 15-ppm 99.5 15-ppm
20-ppm 99.4 20-ppm
90 99.3
25-ppm 25-ppm
88 99.2
86 99.1
99
84
0 0.5 1 1.5 2 2.5 3 3.5
0 0.5 1 1.5 2 2.5 3 3.5
Adsorbent Dose loading in gm Adsorbent Loading in gm

Optimization study of optimum condition of Optimization study of optimum codition of

biosorbent dose of Trichoderma biosorbent dose of Azotobacter
 Optimizations study of initial biosorbent concentration
(Trichoderma & Azotobacter)

% Removal efficiency VS Concentration % Removal efficiency VS Concentration

100 100
98 99.9
99.8
% Removal efficiency

96 05 ppm 99.7 05 ppm

% Removal efficiency
94 10 ppm 99.6 10 ppm
92 15 ppm 99.5 15 ppm
20 ppm 99.4 20 ppm
90
25 ppm 99.3 25 ppm
88 99.2
86 99.1
84 99
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7

Concentration Concentration

Initial biosorbent concentartion of Initial biosorbent concentration of

Trichoderma Azotobacter
 Optimization study of Contact time,
(Trichoderma & Azotobacter)

% Removal Efficiency VS Contact time in

minute % Removal Efficiency VS Contact time in minute
100
100
99
% Removal Efficiency

98 99.8

% Removal Efficiency
97 05-ppm 99.6 05-ppm
10-ppm 10-ppm
96 99.4
15-ppm 15-ppm
95 20-ppm 99.2 20-ppm
94 25-ppm 25-ppm
99
93
92 98.8
10 30 50 70 90 110 130 10 30 50 70 90 110 130

Contact time in minute Contact time in minute

Contact time of Trichoderma Contact time of Azotobacter

 Optimization study of pH,
(Trichoderma & Azotobacter)
% Removal efficiency VS different pH values
% Removal efficiency VS different pH Values
100.1
98.2
98 100
97.8
% Removal efficiency

05-ppm 99.9 05-ppm

% Removal efficiency
97.6
10-ppm 10-ppm
97.4 99.8
15-ppm 15-ppm
97.2 20-ppm
20-ppm 99.7
97 25-ppm 25-ppm
96.8 99.6
96.6 99.5
96.4
0 2 4 6 8 10 12 14 99.4
0 2 4 6 8 10 12 14
pH Values (2 -to- 12)
pH Values (2 -to- 12)

Optimization study of pH, Trichoderma Optimization study of pH, Azotobacter

 Optimization study of Temperature,
(Trichoderma & Azotobacter)
% Removal VS Temperature % Removal VS Temperature
98.9 100
98.5 99.96
98.1 99.92 05-
05-
% Removal Efficiency

ppm 99.88 ppm

% Removal Efficiency
97.7
10- 99.84 10-
97.3
ppm 99.8 ppm
96.9 99.76
15- 15-
96.5 ppm 99.72 ppm
96.1 20- 99.68
95.7 ppm 99.64
95.3 99.6
20 30 40 50 60 70 80 90 20 30 40 50 60 70 80 90
Temperature in °C Temperature in °C

Optimizations study on Temperature Optimizations study on Temperature

effect of Trichoderma effect of Azotobacter
 Optimization study of agitation speed varying initial
dye concentration, (Trichoderma & Azotobacter)
% Removal Efficiency VS Agitation speed in rpm % Removal Efficiency VS Agitation speed in rpm

98.2
100
98
% Removal Efficiency

99.95

% Removal Efficiency
97.8 05-ppm 05-ppm
10-ppm 99.9 10-ppm
97.6 15-ppm 15-ppm
20-ppm 99.85
20-ppm
97.4 25-ppm 25-ppm
99.8
97.2
99.75
97
80 100 120 140 160 180 200 220 99.7
80 100 120 140 160 180 200 220
Agitation Speed in rpm Agitation Speed in rpm

Optimization study of agitation Optimization study of agitation

speed of Trichoderma speed of Azotobacter
 Examination of Langmuir isotherms

Langmuir isotherm - plot of 1/Qe VS 1/Ce

2.5

2 f(x) = 0.402597598264419 x + 0.331573324287137

R² = 0.961975761843764

1.5
1/Qe

0.5

0
2 2.5 3 3.5 4 4.5 5
1/Ce
 Examination of Langmuir isotherms

Langmuir Isotherm - Plot of 1/Qe VS 1/Ce

7

6
f(x) = 0.0372561285398608 x + 0.187664753306294
5 R² = 0.96750179471189

4
1/Qe

Series1
3 Linear (Series1)

0
0 20 40 60 80 100 120 140 160

1/Ce

The Langmuir isotherm provided the best fit and shows linearity
to the equilibrium data for both (Trichoderma and Azotobacter)
biosorbents, assuming complete monolayer adsorption.
Examination of Freunlich isotherms

Freundlich isotherm - Plot of ln Ce Vs ln Qe

0
-1.6 -1.5 -1.4 -1.3 -1.2 -1.1 -1 -0.9 -0.8 -0.7
-0.1

f(x) = 0.784528270194594 x + 0.426174971929542 -0.2

R² = 0.944477552588175
-0.3
lnQe
ln Qe

-0.4 Linear (lnQe)

-0.5
-0.6
-0.7
-0.8

ln Ce
Examination of Freundlich isotherms
Freundlich Isotherm - plot of ln Ce VS ln Qe
0
-5.5 -5 -4.5 -4 -3.5 -3
f(x) = 0.882505368475651 x + 2.71022546146776
R² = 0.956646537774331
1/Qe

-1 lnQe
Linear (lnQe)

-2
1/Ce

The Freundlich isotherm provided the best fit and shows linearity
to the equilibrium data for both (Trichoderma and Azotobacter)
biosorbents, assuming complete monolayer adsorption.
 Feasibility of the experimental work

• The feasibility of using Trichoderma and Azotobacter as biofertilizers for the

adsorption process in the removal of hazardous Metanil yellow dye lies in their
ability to serve as effective biosorbents due to their high surface area and functional
groups, such as cellulose and chitin, which can interact with dye molecules.

• Additionally, Trichoderma and Azotobacter possess enzymatic activity that can

degrade or transform dye molecules, enhancing the efficiency of the adsorption
process.

• Moreover, these biofertilizers offer the advantage of promoting soil fertility and
plant growth while simultaneously reducing dye contamination, making them a
sustainable and environmentally friendly solution for wastewater treatment.
 Reusability of biosorbent

Plot of Trichoderma reusability, % removal Efficiency VS Use Times

100
90
80
70
%Removal efficiency

60
50 Series1
40
30
20
10
0
1st time use 2nd time use 3rd time use 4th time use 5th time use 6th time use
Optimised Adsorbent Used time
 Reusability of biosorbent

Plot of Azotobacter reusability, % removal efficiency VS Used time

100
90
80
% Removal Efficiency

70
60
50 Series1
40
30
20
10
0
1st time use 2nd time 3rd time 4th time 5th time 6th time 7th time 8th time
use use use use use use use

Optimised Adsorbent Used Time

The ecological constraints necessitate the importance of adsorbent reusability.

Consequently, a reusability study was conducted for both biosorbents using a 25
ppm concentration of Metanil yellow dye, as illustrated in figure . Azotobacter
exhibited diminished efficiency by the 7th cycle, while Trichoderma displayed
reduced removal efficiency in the 5th cycle.
 Conclusion

• The bacteria present on the biosorbent likely metabolize dye molecules as part of
their nutritional intake, effectively depleting the dye content from the remaining
biosorbent.

• Both biosorbents are eco-friendly and aid in enhancing agricultural output and soil
health.

• Incorporating these biosorbents offers a twofold advantage for textile industries, as

they not only cleanse wastewater of harmful substances but also promote plant
growth while maintaining environmental integrity.

• No adverse environmental impacts were detected from the utilization of these

biofertilizers.
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