PHYCO-REMEDIATION OF AZO DYE CONTAMINATED...
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PHYCO-REMEDIATION OF AZO DYE CONTAMINATED WASTEWATER BY Muhammad Rashid Waqas M.Sc. (Hons.) Soil Science A thesis submitted in partial fulfillment of the requirement for the degree of DOCTOR OF PHILOSOPHY IN SOIL SCIENCE Institute of Soil and Environmental Sciences University of Agriculture Faisalabad, Pakistan 2014
Transcript of PHYCO-REMEDIATION OF AZO DYE CONTAMINATED...
PHYCO-REMEDIATION OF AZO DYE CONTAMINATED
WASTEWATER
BY
Muhammad Rashid Waqas
M.Sc. (Hons.) Soil Science
A thesis submitted in partial fulfillment of the requirement for the degree of
DOCTOR OF PHILOSOPHY
IN
SOIL SCIENCE
Institute of Soil and Environmental Sciences
University of Agriculture
Faisalabad, Pakistan
2014
iii
The Controller of Examinations
University of Agriculture,
Faisalabad
“We, the supervisory committee, certify that the contents and form of this thesis
submitted by Mr. Muhammad Rashid Waqas (Reg No. 2003-ag-2368) have been
found satisfactory, and recommend that it be processed for evaluation by the External
Examiner(s) for the award of degree”.
SUPERVISORY COMMITTEE
Chairman _______________________
(Dr. Muhammad Arshad (T.I)
Member _______________________
(Dr. Hafiz Naeem Asghar)
Member _______________________
(Dr. Muhammad Asghar)
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DECLARATION
I hereby declare that the contents of the thesis “Phyco-remediation of azo dye
contaminated wastewater” are product of my own research and no part has been copied
from any published source (except the references, standard procedures and protocols etc.). I
further declare that this work has not been submitted for any diploma/degree. This university
may take action if the information provided is found inaccurate at any stage.
Muhammad Rashid Waqas
2003-ag-23688
v
To
Who taught me first word to speak and first step to take
parents
DDeeddiiccaatteedd
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ACKNOWLEDGEMENTS
All the praises and thanks to Allah Almighty, the most Merciful Who enabled me to
complete this study. I pay a great tribute to the last prophet of the Creator – Muhammad (Peace be upon him) who is forever torch of guidance and knowledge for humanity as a whole.
I feel great pleasure to express my sincere gratitude to my kind supervisor, Dr. Muhammad Arshad (T.I.), Professor, Dean, Faculty of Agriculture and Director of Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad for his guidance and enormous support concerning my research and education. He elevated my research abilities to their highest and provided enthusiastically guidance and best cooperation in thesis write up.
I am also grateful to my thesis committee, Dr. Hafiz Naeem Asghar, Assistant Professor, Institute of Soil and Environmental Sciences, and Dr. Muhammad Asghar, Professor, Chairman, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad for their valuable time and insightful comments.
I am also highly thankful to Dr. Azeem Khalid Associate Professor, Department of Environmental Sciences, PMAS Arid Agriculture University, Rawalpindi for his enormous assistance and support in my thesis write up.
I would like to give very Special thanks to Mr. Khaliq-ur-Rehman Arshad, Dr. Zia Chishti and Dr. Sarfraz Hussain, Pesticide Quality Control Lab. Institute of Soil Chemistry and Environmental Sciences AARI, Faisalabad, for their technical support and cooperation in analytical work and in improvement of this manuscript.
I would like to pay profound gratitude to my lab fellows Zulfiqar Ahmad, Dr. Muhammad Imran, Dr. Zia-ul-Hasan, Dr. Masood Saleem, Allah Ditta, Wazir Ahmad, Farhat Bashir and Uncle Sarwar (ISES, UAF) for their company and moral support during my research work. Special thanks to my special friend Muhammad Imran (University of Sargodha), for his support on each and every step.
I would like to give my deepest appreciation to my parents, brothers and sisters who supported me in all my life for all their love, prayers and encouragement – indispensable during the course of these research studies. Very special thanks to my loving, supportive and encouraging wife, Nazia Fakhar, this research work and thesis was simply impossible without her loving support and sacrifices. Special love for my son Muhammad Ayan Rashid, his cute smile always gave me freshness and eagerness to work.
Lastly, I offer my regards and blessings to all of those who supported me in any respect during the completion of the thesis.
Muhammad Rashid Waqas 2003-ag-2368
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CONTENTS
Chapter Title Page No.
Chapter 1 INTRODUCTION 1
Chapter 2 REVIEW OF LITERATURE 5
2.1 Textile industry in Pakistan 5
2.1.1 Textile wastewater flows 8
2.1.2 Characteristics of textile wastewater 8
2.2. Azo dyes and their impact on the environment 11
2.3. Treatment of dye-containing wastewaters 14
2.3.1. Physico-chemical methods 14 2.3.2. Microbial treatment 15
2.3.3. Phycology and phyco-remediation of textile wastewaters 16
Chapter 3 MATERIALS AND METHODS 20
3.1 Sampling of water for the isolation of algae 20 3.2 Analysis of collected water samples 20 3.2.1. Electrical conductivity (EC) 20 3.2.2. pH 20 3.3. Isolation of algal strains 22 3.3.1. Screening of the azo dye degrading algal strains 23 3.4. Optimization of environmental factors 24 3.4.1. Substrate (azo dye) concentration 24 3.4.2. pH 24 3.4.3. Temperature 24 3.4.4. Effect of nitrogen (N) 24 3.4.5. Effect of phosphorus (P) 25
3.4.6. Effect of salinity 25
3.4.7. Effect of light conditions 25
3.4.8. Effect of inoculum size 25 3.5 Phyco-remediation of structurally different azo dyes 25 3.6 Decolorization of mixture of azo dyes 26 3.7. Phycoremediation of real textile wastewater 28 3.7.1. Characteristics of wastewater samples 28
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3.8. Toxicity analysis 28
3.8.1. Hemolytic activity 28
3.9. Use of Phyco-remediated wastewater as irrigation 29 3.10. Biodiesel production 30 3.10.1. Oil extraction 30 3.10.2. Trans-esterification 30
3.10.3. Separation of biodiesel 30
3.11. Statistical analysis 30
Chapter 4 RESULTS 31
4.1. Analysis of water samples 31
4.1.1. pH 31
4.1.2. TSS 32
4.2. Isolation and screening of azo dye decolorizing Algae 32
4.3. Optimization of environmental factors 35
4.3.1. Substrate (azo dye) concentration 35
4.3.2. pH 37
4.3.3. Temperature 37 4.3.4. Light conditions 40 4.3.5. Inoculum Size 40
4.3.6. Salinity 43
4.3.7. Effect of nitrogen (N) 45
4.3.8. Effect of phosphorus (P) 45
4.4. Phyco-remediation of structurally different azo dyes 48 4.4.1. Phycoremediation of reactive dyes 48 4.4.2. Phycoremediation of direct group dyes 52 4.4.3. Phycoremediation of Disperse group dyes 56 4.5. Phycoremediation of mixture of azo dyes 60 4.6. Phycoremediation of real textile wastewater 62 4.6.1. Case study I (Wastewater from Shaheen Cloth Processing Mills) 62 4.6.2. Case Study II (Wastewater from Qadafi Textile) 65 4.6.3. Case study III (Wastewater from Dawood Textile Mills) 68 4.7 Toxicity analysis 71 4.7.1 Toxicity test of mixture of azo dyes after decolorization by algal
strains 71
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4.7.2 Toxicity test of real textile wastewater after decolorization by algal strains
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4.7.2.1 Toxicity analysis: Case study I 71 4.7.2.2 Toxicity analysis: Case study II 72 4.7.2.3 Toxicity analysis: Case study III 72 4.8. Use of Phyco-remediated wastewater as irrigation 77 4.9. Biodiesel production from algal biomass 79 4.9.1. Algal biomass 79 4.9.2. Biodiesel production 79 4.9.3. Biomass recovery after oil extraction 79
Chapter 5 DISCUSSION 84
SUMMARY 89
LITERATURE CITED 92
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LIST OF FIGURES S. No. Title
Page No.
4.1 Decolorization of Reactive Blue dye by different algal strains isolated from various water samples through enrichment
34
4.2. Effect of substrate concentration on decolorization of Reactive Blue dye by the selected algal strains
36
4.3. Effect of pH on decolorization of Reactive Blue by the selected algal strains. 38
4.4. Effect of temperature on decolorization of Reactive Blue by the selected algal strains.
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4.5. Effect of day light conditions on decolorization of Reactive Blue by the selected algal strains.
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4.6. Effect of inoculums size on decolorization of Reactive Blue by the selected algal strains.
42
4.7. Effect of salinity on decolorization of Reactive Blue by the selected algal strains.
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4.8. Effect of additional N source on decolorization of Reactive Blue by the selected algal strains.
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4.9. Effect of additional P source on decolorization of Reactive Blue by the selected algal strains.
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4.10 Decolorization of Orange RR Reactive azo dye by selected stains of algae. 49 4.11 Decolorization of Red S3B Reactive azo dye by selected stains of algae. 50 4.12 Decolorization of Mixture of Reactive azo dyes by selected stains of algae. 51 4.13 Decolorization of Yellow UG Direct azo dye by selected stains of algae. 53
4.14 Decolorization of Congo Red Direct azo dye by selected stains of algae. 54 4.15 Decolorization of Mixture of direct azo dyes by selected stains of algae. 55 4.16 Decolorization of Disperse Blue ZBLN azo dye by selected stains of algae. 57 4.17 Decolorization of Scarlet Disperse azo dye by selected stains of algae. 58 4.18 Decolorization of Mixture of disperse azo dyes by selected stains of algae. 59 4.19 Decolorization of Mixture of reactive, direct and disperse group azo dyes by
selected strains of algae 61
4.20 Phycoremediation of real textile wastewater sample # 1 (collected from Shaheen Cloth Processing Mills) by algal strains.
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4.21 Comparitive phycoremediation of real textile wastewater sample # 1 (collected from Shaheen Cloth Processing Mills) by living or dead cells of algae.
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4.22 Phycoremediation of real textile wastewater sample # 2 (collected from Qadafi Textiles) by two selected algal strains.
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4.23 Comparison phycoremediation of real textile wastewater sample # 2 (collected from Qadafi Textiles) by living or dead cells of algae.
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4.24 Phycoremediation of real textile wastewater sample # 3 (collected from Dawood Textiles) by two selected algal strains.
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4.25 Comparison phycoremediation of real textile wastewater sample # 3 (collected from Dawood Textiles) by living or dead cells of algae.
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4.26 Hemolytic activity of Mixture of disperse, reactive and direct azo dyes phycoremediated by selected stains of algae.
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4.27 Hemolytic activity of real textile wastewater collected from Shaheen Cloth Processing Mills after treated with selected strains of algae
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4.28 Hemolytic activity of real textile wastewater collected from Qadafi Cloth Processing Mills after treated with selected strains of algae
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4.29 Hemolytic activity of real textile wastewater collected from Dawood Cloth Processing Mills after treated with selected strains of algae
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4.30 Growth of selected algal strains on real textile wastewater (phycoremediated) and normal culture medium for period of 5 days.
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4.31 Biodiesel production of selected algal strains grown on real textile wastewater (phycoremediated) and normal culture medium.
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4.32 Biomass recovery after oil extraction in case of algae grown on real textile wastewater (phycoremediated) vs. normal culture medium.
83
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LIST OF TABLES
S. No.
Title Page No.
2.1 Region wise distribution of large, medium and small textile industries 6
2.2 List of dyes imported in Pakistan (2009 - 2010) and cumulative assessed
value in US$
7
2.3 Estimated Wastewater generated from Textile Processing 9
2.4 Characteristics of wastewater released by textile processing industry 10
3.1 Water samples collected from different sites with their identification number
21
4.1
Water analysis and isolation of algae from water samples collected from different locations
33
4.2 Impact of real and phycoremediated wastewater on wheat growth under axenic conditions
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Abstract
Synthetic dyes are widely used in textile, leather and other dye-stuff industries. A large
fraction of the dyes applied during the dyeing processes are released into wastewater.
Therefore, the wastewater from dye-related industries is very colorful, with high chemical
oxidation and biological oxidation demand. This wastewater must be treated prior to
discharge into wastewater streams to prevent pollution of surface and groundwater, and the
risk to public health. The present study was designed with the aim to isolate potential strains
of algae capable of degrading azo dyes for the treatment of textile wastewater. Eighty-eight
algae strains were isolated on agar plates using modified MA medium. About 20 algal strains
were screened by enrichment of the medium with 100 mg L-1 Reactive Blue azo dye. Out of
20 isolates of algae, two strains CKW1 (Spirogyra sp.) and PKS33 (Cladophora sp.) were
able to decolorize 88% and 83% dye, respectively, in seven days incubation time. The
substrate (dye) 100 mg L-1, pH 8, 30 °C temperature and 16 h light duration were found to be
optimum conditions for maximum decolorization of azo dyes by these strains of algae. Under
optimal conditions, both strains were able to completely decolorize the structurally different
synthetic textile dyes and real textile wastewater in 96 h to 120 h. Algal cells showed a better
efficacy in decolorizing real textile effluent than observed with dead algae biomass (dry).
About 60% decolorization of the real textile wastewater was achieved by living cells in only
24 hours and 80% decolorization in 120 h. Toxicity analyses were performed in terms of
hemolytic activity. The results showed that the treated wastewater with algae living biomass
reduced the toxicity of wastewater by 70-80%, while a reduction of 30-35% of the toxicity
was observed in the case of algae dead mass. The treated textile wastewater also improved
significantly wheat growth compared to untreated real wastewater. Using the trans-
esterification method, it was found that the algal biomass produced by the use of textile
treated effluent could be used to produce biodiesel. These findings suggest that algae could
be used to treat wastewater containing textile dyes that can be used for growing crop plants.
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INTRODUCTION
Textile industry is one of the largest industrial sectors in Pakistan (Kirk and Ehow,
2008; Ahmad, 2011). The textile industry involves numerous wet processes, including
dyeing, bleaching, desizing and printing. The estimated amount of wastewater released by
the textile processing units is 114,41167 m3/day (Govt. of Punjab, 2008). Synthetic dyes have
wide application in the textile and other dye-stuff industry. According to an estimate, global
production of synthetic dyes is more than 700,000 tonnes and textile sector alone consumes
about 60% of the total production of dyes (Robinson et al., 2001; Shinde and Thorat, 2013).
Since dyeing process is not very efficient, production of highly colored wastewater is
enormous. The amount lost in wastewater is a function of the class of dyes and in general,
their loss through discharge in the wastewater can be 2% of the initial concentration of basic
dyes to as high as 50% of a reactive dye (Tan et al., 2000; Boer et al., 2004; Wins and
Murgan, 2010). Thus, the wastewater discharged from the textile industry contains huge
amount of synthetic dyes.
In Pakistan, textile wastewater is commonly used for irrigation to grow crops by
farming community, particularly in urban and peri-urban areas. The wastewater contain
heavy pollution load in terms of high biological oxygen demand (BOD), chemical oxygen
demand (COD), total dissolved solids (TDS), total soluble solids (TSS), and organic (mainly
azo dyes) and inorganic compounds (Pathak et al., 1999; Siddique et al., 2010). Therefore,
textile wastewater could be damaging to the soil health and aquatic ecosystem (Kaur et al.,
2010). Moreover, plants can uptake dye compounds which may affect human health through
food chain (Yousaf et al., 2010).
Azo dyes are a group of synthetic dyes containing one or more azo (–N=N–)
chromophores. Such dyes are considered to be electron-deficient compounds, as they possess
the azo (–N=N–) and sulfonic acid (SO3-) electron withdrawing groups, resulting in a deficit
of electrons in the molecule which renders the compound more sensitive to oxidative
catabolism by microorganisms. Hence, azo dyes tend to persist under aerobic environmental
conditions (Rieger et al., 2002). Many synthetic dyes and their metabolic intermediate
products are found to be toxic, mutagenic and carcinogenic (Poljsak et al., 2010; Dafale et
al., 2010; Sellamuthu et al. 2011; Yang et al. 2013). Because of the toxicity and recalcitrant
nature, azo dyes have been classified as hazardous to the environment (Souza et al., 2010).
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The treatment of dye-contaminated wastewater in an environmentally safe manner is
essentially required prior to its disposal.
Various physico-chemical methods are used to remove the dyes from textile
wastewater (Wang et al., 2004; Golab et al., 2005; Saxe , 2006; Alinsafi et al. 2007; Arslan-
Alaton 2007; dos Santos et al., 2007; Wang et al., 2009). These include ozonation,
electrolysis, fenton oxidation, UV-H2O2 oxidation, adsorption, membrane filteration and
coagulation-flocculation (Hao et al., 2000; Mohanty et al., 2006; Rao et al., 2006). Usually,
these methods are not cost effective and environment friendly. Production of a large amount
of sludge also reduces their application because sludge requires an additional treatment for
safe disposal (Banat et al., 1996; Verma and Madamwar, 2003; Anjaneyulu et al., 2005).
Microbes alone or in combination with other strategies can be used to remove azo dyes from
wastewater. Several studies have documented the ability of bacteria, fungi and yeasts to
degrade azo dyes (Stolz, 2001, Prasad et al., 2011). However, it has been observed that the
presence of co-substrates into the dye solution is required to accelerate the microbial growth
and decolorization process (Prasad et al., 2011). Moreover, the presence of salts in textile
effluents, and variable pH and high temperature of the effluents can also affect the rate of
biodegradation of azo dyes (Jadhav et al., 2007; Khan et al., 2009; Prasad et al., 2011).
Therefore, microbial treatment may sometimes not function effectively for the removal of
dyes from textile effluents and alternative technologies must also be evolved to treat textile
wastewater.
One of such possible strategies is phycoremediation using algae to remove pollutants
from the environment (Dresback et al., 2001). Olguin (2003) defined phycoremediation in a
much broader sense as the use of macroalgae or microalgae for the removal or
biotransformation of pollutants, including nutrients and xenobiotics from wastewater and
CO2 from waste air. Phycoremediation comprises of several applications, including
oxygenation of the atmosphere, nutrient removal from municipal wastewater and effluents
rich in organic matter, nutrient and xenobiotic compounds removal by biosorption using
algae, treatment of acidic and metal wastewater, CO2 sequestration and biosensing of toxic
compounds by algae.
Over the last few decades, efforts have been done to apply intensive microalgal
cultures to perform the biological tertiary treatment of secondary effluents (De la Noüe et al.,
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1992, Queiroz et al., 2007). Unicellular green algae such as Chlorella spp. and Scenedesmus
spp. are widely used in wastewater treatment as they often colonize the ponds naturally and
have fast growth rates and high nutrient removal capabilities. Thus, the use of microalgae for
removal of nutrients from different wastes has been described by a number of authors
(Beneman et al., 1980; De-Bashan et al., 2002; Gantar et al., 1991; Queiroz et al., 2007).
Moreover, algae offer a low-cost and effective approach to remove excess nutrients and other
contaminants in tertiary wastewater treatment, while producing potentially valuable biomass,
because of a high capacity for inorganic nutrient uptake (Bolan et al., 2001; Muñoz and
Guieyssea, 2006).
Algae are simple photosynthetic microorganisms that can efficiently use the sun
energy to convert water and carbon dioxide from the air into biomass. These cells have the
ability to convert carbon dioxide to biomass that can further be processed downstream to
produce biodiesel, fertilizer and other useful products. Photosynthetic growth of algae
requires carbon dioxide, water, sunlight and inorganic nutrients such as phosphorus and
nitrogen. Algae considered as green-cell factories that are not only considered good
scavengers of toxic chemicals, but are also involved in oxygenation of the atmosphere and
carbon dioxide sequestration, thereby making them a better candidate among bioremediation
systems. Algae are ideal candidate for the treatment of textile wastewater because not only
huge amount of wastewater is discharged during dyeing process but it is also rich source of
nutrition. In addition, phycoremediation has advantages over other conventional physico-
chemical methods, such as ion-exchange, reverse osmosis, dialysis and electro-dialysis,
membrane separation, activated carbon adsorption and chemical reduction or oxidation, due
to its better nutrient removal efficiency and the low cost of its implementation and
maintenance.
The present study was designed with the following objectives:
Isolation and screening of efficient azo dye degrading algal strains from
textile wastewater streams, saline water and fresh water.
Optimization of the selected algal strains to the environmental and utritional
factors.
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Comparison of living as well as dead (dried) algae on biodegradation of
structurally different azo dyes.
Testing bioaugmentation potential of the selected algae for phycoremediation
of real textile wastewater.
Study the impact of phycoremediated wastewater on growth of wheat under
axenic conditions.
Production of biodiesel from algal biomass through trans-esterification.
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REVIEW OF LITERATURE
Many azo dyes and their intermediates are toxic, mutagenic and carcinogenic and
affect higher organisms in both aquatic and terrestrial systems. Azo dyes and their
intermediate degradation products are common contaminants of soil and groundwater in
developing countries where textile and other dye products are produced.
2.1. Textile industry in Pakistan
After independence of Pakistan in 1947, the greatest progress in the textile sector was
observed. Increased industrialization in Pakistan began in 1950 with the textile industry in its
center (Kirk and eHow, 2008). A large number of textile processing units are located in
different parts of Pakistan. However, the main areas where the units are located include
Faisalabad, Lahore, Multan and Karachi districts. Notably, Faisalabad is called the
Manchester of Pakistan. At present, the textile industry plays a central role in Pakistan's
economy. Despite the fundamental and important role in the economy, most textile
manufacturers are small-scale or cottage industry (Table 2.1).
Both and imported and locally manufactured dyes are used in textile industry. Efforts
were made to collect data relating to the manufacture of various types of dyes in Pakistan.
Approximately, 40% of total dyes used are manufactured locally in Pakistan. Accurate
information on the extent of production of dyes is not available because the dye
manufacturers in Pakistan do not reveal their original data relating to the amount of dyes
manufactured. However, a large number of groups are involved in the manufacture of dyes in
Pakistan. Various dyes are also imported from different countries. A list of various dyes
imported in Pakistan from various countries of the world in July 2009 to June 2010 is given
in Table 2.2. Companies and corporations such as International Jans, Alibaba and Shafi
International Corporation are working as importer or wholesalers of dyes in Pakistan and
importing mainly acid, direct, disperse and reactive dyes. Textile industries release large
amounts of wastewater in wastewater streams and soil, which is of great environmental
concern.
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Table 2.1: Region-wise distribution of large, medium and small textile industries (Govt.
of Punjab, 2008).
Unit Lahore Faisalabad Sialkot Multan Gujranwala Others Total
Large 65 105 25 4 5 8 212
Medium 255 248 98 24 31 20 676
Small 195 208 56 8 33 7 507
Total 515 561 179 36 69 35 1395
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Table 2.2: List of dyes imported in Pakistan (2009 - 2010) and cumulative assessed
value in US$ (Arshad et al., 2011)
Sr. No. Products Year
1 Dispersive dyes 6,932,564.40
2 Acid dyes 1792979.92
3 Basic dyes 3,178,735.74
4 Reactive dyes 32,022,113.35
5 Dye, synthetic 1,673,106.42
6 Dye, sulphur 2819372.28
7 others 6,780,412.12
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2.1.1 Textile wastewater flows
Textile industry is a major industrial consumer of freshwater sources and
consequently produces large volumes of wastewater. With increasing demand for textile
products, textile industry and its wastewater have increased proportionally, so it is a major
source of water pollution in developing countries (Asia et al., 2006; Andre et al., 2007).
Water consumption in the textile processing industry varies depending on the types and
number of processes, machinery and production capacity, etc. Textile industry involves many
wet processes, including scouring, decizing, mercerization, bleaching, printing and dyeing.
Estimated effluent generated from knitted and woven textile processing units (based on CTP
database) is given in Table 2.3. Amount of effluent is calculated by multiplying the total
production capacity with an estimated consumption of water to process one unit of kg of
finished product (Govt. of Punjab, 2008).
2.1.2 Characteristics of textile wastewater
Composition of the wastewater generated from different textile units varies from one
industry to another and also depends on other factors such as the technology used, chemicals
and home business practices. The effluent characteristics of weaving, dyeing, printing and
processing industry of knitted garments are given in Table 2.4. The diversity in the
composition of the chemical reagents used in the textile industry contributes too much water
pollution. Wastewater generated by different stages of production of a textile factory has high
pH, temperature, detergents, oil, suspended and dissolved solids, dispersants, leveling agents,
toxic and non-biodegradable materials, color and alkalinity. Important organic pollutants are
mainly recalcitrant toxic surfactants, chlorinated compounds and dyes. These pollutants have
harmful effects on the health of humans, animals, plants, and microbial life.
Commercially, there are over 100,000 available dyes ton 7x105 over dyestuff
generated annually from different industries (Myers et al., 1996; Zollinger, 1987). Water-
soluble reactive dyes bright and acid colors are offering major problems (Willmott et al.,
1998). Dyes even in small proportion are very dangerous and hazardous to the environment
(Robinson et al., 2001).
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Table 2.3: Estimated wastewater generated from textile processing units (Govt. of
Punjab, 2008).
Industry Annual
production
(million metric ton/
year)
Unit wastewater
flow
(liters/kg)
Wastewater
generated
(m3/day)
Woven Textile Processing Sector
Small 2510 175 146,417
Medium 5145 250 428,750
Large 1940 200 129,333
Sub-total 9595 704,500
Knitwear Processing Sector
Processing Sector 2200 100 736,667
Grand-total 11795 725 1,441,167
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Table 2.4: Characteristics of wastewater released by textile processing industry (Govt.
of Punjab, 2008)
Parameters Woven Processing Industry Knitwear
Processing
Industry
NEQS
(into inland
waters)
Dyeing Dyeing &
Printing
pH 8.3-11.7 6.3-12.0 - 6-9
BOD (mg/l) 200-570 300-480 100-300 80
COD (mg/l) 640-1200 880-1130 300-800 150
TSS (mg/l) 320-940 200-450 200-440 200
TDS (mg/l) 1280-1540 1000-1900 1000-1900 3500
Chromium
(mg/l)
0.5-3.6 1.5-12.6 - 1.0
O&G (mg/l) 17-32 11-40 11-40 10
Chloride (mg/l) 400-750 90-1100 - 1000
Copper (mg/l) 0.4-0.5 0.10 0.1 1.0
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2.2. Azo dyes and their impact on the environment
There are numerous kinds of dyes with 25 structural classes. The most important of
them are azo dyes. Azo dyes are comprised of azo (–N=N–) groups, and when bound to the
molecules, become monoazo, diazo or polyazo dyes. Azo dyes comprise 70% total dyes and
are widely used in many industries to impart colors to different materials (De Souza et al.,
2006). The largest consumer of azo dyes is textile industry of all kind. However, they are
also present in small amounts in the effluents of the industries of paper, printing, food,
pharmaceutical and leather. Color in dyes is due to the azo groups in conjugation with
aromatic substituent or ionizable group that make a very complex structure and lead to the
expression of large variation of color in dyes (O’Neill et al., 2000 and Rajaguru et al., 2000).
Fixation rate of these dyes could be as low as 50% (Vaidya and Datye, 1982). Effluents
discharged from dyeing sections of fabric (cotton, wool and polyester) are colored due to the
presence of both soluble and insoluble dyes. The most common concentration of dyes in such
solutions is above 1 mg l-1
and the amount of dyes in textile effluent may be as high as 300
mg l-1
(Cooper, 1995 and Laing, 1991).
Due to inefficient dyeing process, a large quantity of the dye is discharged directly
into the environment. Even if very small amount of dye is discharged in water (less than 1
mg l-1 for some dyes), produce color and is highly visible, affects the aesthetic quality,
transparency and gas solubility in several water bodies (Kangwansupamonkon et al., 2010).
Industrial effluents containing synthetic dyes reduce light penetration into water bodies as a
result affecting photosynthesis of flora and seriously affect food sources of aquatic
organisms. Moreover, dyes in water bodies decrease dissolved oxygen and increase
biological oxygen demand which consequently affects the aquatic fauna (Annuar et al.,
2009). Among azo dyes, those produced from aromatic amines, such as benzidine and 4-
biphenylamine, 4-aminobiphenyl, monoacetylbenzidine, and acetylaminobiphenyl, pose a
serious threat to the environment and human health (Manning et al., 1985; Cerniglia et al.,
1986; Choudhary, 1996; Chung 2000). Previously, several researchers have reported the
toxicity of synthetic dyes on ecosystems (Osugi et al., 2009; Fraga et al., 2009; Dafale et al.,
2010). The effluents containing dyes affect the photosynthetic activity of aquatic plants and
algae by changing the light and gas penetration into water bodies. Hence, wastewater is also
12
considered very toxic to aquatic organisms, resulting in the disruption of the ecological
balance (Modi et al., 2010). These azo dyes destroy not only water quality, but also have
adverse effects on human health (Forgacs et al, 2004; Bandala et al, 2008). Azo dyes can
cause cancer, skin irritation, allergic dermatitis, mutation, heartbeat increase, vomiting,
shock, cyanosis, jaundice, quadriplegia, and tissue necrosis in humans (Hansen et al., 2003).
Many reports demonstrated that the textile dyes and effluent had toxic effects on the rates of
germination and biomass of several plant species (Wang, 1991; Kapustka and Reporter,
1993). The properties of textile dyes such as visible color at low concentration, brightness,
resistance to light and chemical structure make them fairly resistant to microbial degradation
(Guelli et al., 2008).
The negative impacts of textile effluents have also been reported in different crops.
Highly diluted effluents from textile units caused reduction in germination of Cicer arietinum
(Dayama, 1987). Very high nitrate concentration in the effluent increases the risk of
eutrophication of estuaries and lakes (Weil et al., 1990). A negative impact was induced by
different effluents in leaf width, leaf length, stem length, flower and fruit of okra (Egbeeni et
al., 2009). The growth and germination of black gram Vigna mungo (L.) is also reported to be
affected by irrigation with effluent textile industry. With increasing awareness among the
international community about the discharge of effluents and persistence of dyes, attention
has focused on the rehabilitation of these pollutants (Ali, 2010). Treated textile effluent could
be used for irrigation of vegetables. The results of Rahman et al. (2009) indicated that the
textile effluent reduced the percentage of seed germination and seedling growth of winter
vegetables. The germination of seeds of all vegetables was not affected when irrigated with
treated effluent textiles. The inhibitory effect was more prominent in higher concentration. In
relation to the seed germination and growth response, Raphanus sativus was classified more
tolerant than Brassica napus.
Textile effluents are constantly used for irrigation in Pakistan. As the effluents
contain some nutrients, so farmers voluntarily use textile effluent for irrigation, ignoring the
harmful nature of these effluents. The diluted effluent use has been reported by several
investigators that show a positive effect on the plants (Swaminathan and Vaidheeswarm,
1991; Kumar et al., 2006; Khan et al., 2011). Wins and Murgan (2010) studied the effect of
different concentrations of textile effluents. A lower concentration of textile effluent had
13
positive effect on growth and germination compared to control. However, the gradual
decrease in the percentage germination of seeds and seedling growth with some increase in
the concentration of effluent. Different concentrations of textile mill effluent had a
significant effect on growth, seed germination and pigment content of Arachis hypogea. An
inhibitory effect (28.9%) was observed with the application of pure effluent. However, 4.7%
increase in growth in the effluent concentration of 25% compared with the control was
observed. Toxicity effect was observed on plant growth at relatively high concentrations of
textile effluent (Kumar et al., 2006). In contrast, less than 50% dilution of textile effluent
increased the total sugar content, chlorophyll content starch, reducing sugars and seed
germination of peanuts (Swaminathan and Vaidheeswarm, 1991). A study was conducted by
Khan et al. (2011) on leguminous crops such as pea (Pisum sativm L.), lentil (Lens esculentus
L.) and gram (Cicer arietinum) to evaluate the impact of textile effluent (0, 10, 25, 50 and
100) on some physical parameters and seed germination. Reduction in plant growth at higher
concentrations was observed. At lower concentrations, effect of effluents was positive on
plant growth. In Bangladesh, a pot experiment was performed at agricultural farm on growth
and yield of rice varieties (BR 14 and BR 16) using industrial effluent irrigation (Mistry and
Chatterjee, 2009). The biomass was greater in the case of textile effluent than control. The
maximum productive tillers and filled grains per hill were recorded in the case of effluent
irrigation in BR 16 variety. Similarly, a study was conducted to estimate the use of textile
effluent on yield and physiology of two varieties (viz TMV-10 and JL-24) of peanut.
Application of textile effluent enhanced the seed germination, total chlorophyll contents,
chlorophyll a and b, growth parameters, yield and yield contributing parameter. Textile
effluent had the physiochemical characteristics within permissible limit and those met the
irrigation quality parameters (Saravanamoorthy and Ranjitha-Kumari, 2007).
14
2.3. Treatment of dye-containing wastewaters
2.3.1. Physico-chemical methods
A number of physico-chemical methods are in use to remove color from textile
wastewater contaminated with azo dyes (dos Santos et al., 2007; Wang et al., 2009).
Adsorption, membrane filtration and coagulation–flocculation are the physical methods
which are commonly used to treat textile effluent, while ozonation, electrolysis, Fenton
oxidation, photocatalytic-H2O2 oxidation, photochemical oxidation are the important
chemical methods (Hao et al., 2000; Rao et al., 2006). Although, active carbon (adsorbent)
has high potential to remove dye from the textile effluent but its high cost limits its use
(Robinson et al., 2001). However, some researchers use low cost adsorbents like maize stalk,
maize cobs, peat, fly ash and ion exchangers etc., but their application as a treatment has
some draw backs such as low color removal efficiency and high quantity of sludge
production (Anjaneyulu et al., 2005). In addition, adsorbents cannot be reused and also
disposal into the environment is not environmentally healthy practice. Membrane filtration
methods also have some limitations because of high initial investment and water stream
coming out of membrane filters also requires secondary treatment (dos Santos et al., 2007).
Disperse and sulfur dyes are effectively removed by coagulation-flocculation methods while
they have low efficiency for acid, direct, reactive and vat dyes because of low coagulation-
flocculation capacity. In addition sludge production also reduces its application as a good
wastewater treatment (Vandevivere et al., 1998).
Chemical oxidation methods involve addition of various oxidizing agent in
wastewater such as hydrogen per oxide, ozone and permanganate. Although, high reactivity
of ozone with azo dyes is helpful for color removal (Alaton et al., 2002), but problems are
high cost, short life time, low potential of decolorization for dyes which are not soluble in
water (such as Disperse Dyes) and low COD removal (Anjaneyulu et al., 2005). Fenton
oxidation is a good and oldest technique of dye removal from wastewater, in which hydroxyl
radical is generated by the reaction of Fenton reagent and H2O2. Hydroxyl radical has good
dye oxidation potential. According to Robinson et al., (2001), Fenton oxidation has good
color and COD removal efficiency but production of large quantity of sludge limits it
15
application. H2O2/UV is a very efficient method of dye removal from textile wastewater as
there is no sludge production and high COD removal (Alaton et al., 2002). It has been
reported that this is not suitable for disperse or vat dyes and also produces some undesirable
by products. In addition, use of UV increases cost of treatment (Pearce et al., 2003).
According to Morawski et al., (2000), electrolysis is efficient dye removal technique but high
cost of electricity is the hurdle in its use to remove dye from wastewater. Thus, physical-
chemical techniques are either expensive or they produce large amount of sludge and also are
not environmental friendly. So, there is need of such textile wastewater treatment which is
economically feasible, environmental friendly and universal in its application.
2.3.2. Microbial treatment
Microorganisms being highly versatile group contain several enzymatic systems for
mineralization of azo dyes under certain environmental conditions (Pandey et al., 2007).
Microbial degradation has several advantages over conventional treatment procedures. These
advantages include effective for persistent and recalcitrant compounds, short treatment time,
applicable over a wide range of temperature, pH and salinity, reduction in sludge production,
ease and simplicity in handling the process (Kulshrestha and Husain 2007).
Extensive research on dye degradation using microbes has shown that biological
approaches alone and/or combination with physico-chemical treatments can offer low cost
alternative treatment (Willmott et al., 1998; Pearce et al., 2003). Microbial strategy is cost
effective as well as environmentally friendly technique (Robinson et al., 2001; Chen et al.,
2006; Khalid et al., 2009) Many microorganisms are capable of decolorizing the azo dyes
including gram positive as well as gram negative bacteria (Moosvi et al., 2007), fungi (Balan
and Monteneiro, 2001; Verma and Madamwar, 2005), yeast (Ertugrul et al., 2009) and algae
(Paramesawary et al., 2010; Rawat et al. 2011). But some problems have also been widely
reported with the use of microbes as remediation entity. The existence of co-substrates in dye
solution is essential to hasten the microbial growth and ultimately the decolorization process
(Prasad et al., 2011). Moreover, the textile wastewaters are equipped with high amount of
salts, and variable pH and high temperature which can severely influence the rate of
remediation of azo dyes (Jadhav et al., 2007; Khan et al., 2009; Prasad et al., 2011).
Therefore, microbial treatment may sometimes not function effectively for the removal of
dyes from textile effluents and alternative technologies must also be evolved to treat textile
16
wastewater. One of such possible strategies is phycoremediation using algae to remove
pollutants from the environment (Dresback et al., 2001). The autotrophic nature of algae can
make it avoid the problems faced in bioremediation by microbes.
2.3.3. Phycology and phyco-remediation of textile wastewaters
Phycology can be simply be defined as the scientific study of algae. Algae are small
unicellular organisms. They form sometime colonies. Algae have the unique ability to grow
in any habitat where water is available. Such habitats could be ranged from fresh to salt water
bodies, hot springs to ice, in or on other organisms and substrate (Guschina and Harwood,
2006). Algae are generally of two types i.e., macro-algae and micro-algae. Macro-algae stand
with the lower plants, meaning that they lack roots, stems and leaves but are composed of a
thallus and sometimes even a stem or a foot (Carlsson et al., 2007). They can be red, green or
brown. Contrasts to macro-algae, the micro-algae are microscopic organisms capable of
photosynthesis. Their photosynthetic mechanism is almost identical to land based plants.
Because of their simple cellular structure and being submerged in an aqueous environment,
they are generally considered more reliable in converting solar energy into biomass (Moreno-
Garrido, 2008). Microalgae can perform a total of 40% (estimated) of global photosynthesis
(Moreno-Garrido, 2008), as a result in some environments, their biomass can compete or
even surpass the biomass of bacteria. Recently, the use of microalgae has been drastically
enhanced in the field of biotechnology and are being used in several industries including
pharmaceutical, food, aquaculture and cosmetic industries (Borowitzka, 1998)
Algae have been used by human for centuries. Blue-green algae were used for edible
purpose for thousands years ago. However, the use of microalgae is new concept and is just
few decades old (Borowitzka, 1999). In the early 1950’s, algal biomass appeared as a good
source of alternative and unconventional protein (Becker, 1994). Commercial production of
algae at large-scale was started in the early 1960’s in Japan using the culture of Chlorella
(Iwamoto, 2004). It was followed in the early 1970’s energy crisis which motivated the
interest in the use of algae for generation of renewable energy sources (Cornet, 1998). By
1980, there were almost 50 factories producing large-scale cultures of algae only in Asia.
These factories produced more than 1000 kg of Chlorella per month (Borowitzka, 1999).
More recently, the large-scale production of algae began in most countries of the world
17
including USA, Australia, India, China, Korea and Europe. Thus, in a short period of about
30 years, the industries using algal biotechnology have developed and expanded substantially
throughout the world. Nowadays, the algal market produces a dry matter biomass of about
5000 tons per year and generates earnings of approximately US$ 1.25×109 per year (Pulz and
Gross, 2004). However, the algal work reported in Pakistan is very negligible considering the
current issues of energy, food and water pollution.
Over the past 20 years, plants have been used to an increasing extent in various
environments for mitigating pollutant concentrations in contaminated sites (soils or waters)
and for producing biodiesel (Fouilland, 2012). However because of low plant growth rates
and biomass yields and considerable amount of water required, intensive cultivation of
microalgae has been proposed as an alternative method for phycoremediation and producing
bioenergy (Dismukes et al. 2008; Rawat et al. 2011). Phycoremediation in a broad sense is
the use of algae (macroalgae or microalgae) for the removal or biotransformation of
pollutants from wastewater and removal of CO2 from waste air (Olguin, 2003). Microalgae
usually play an important role during the tertiary treatment of domestic wastewater in
maturation ponds or the treatment of small and middle scale municipal wastewater in
facultative or aerobic ponds (Aziz and Ng, 1993; Abeliovich, 1986; Mara and Pearson, 1986;
Oswald, 1988). They enhance the removal of nutrients, heavy metals and pathogens and
furnish O2 to heterotrophic aerobic bacteria to mineralize organic pollutants, using in turn the
CO2 released from bacterial respiration (Muñoz and Guieyesse, 2006). Photosynthetic
aeration is therefore especially interesting to reduce operation costs and limit the risks for
pollutant volatilization under mechanical aeration. Recent studied have shown that
microalgae can indeed support the aerobic degradation of various hazardous contaminants
(Muñoz et al., 2004; Safonova et al., 2004). The mechanisms involved in microalgal nutrient
removal from industrial wastewaters are similar to domestic wastewater treatment.
Microalgae capability to biodegrade hazardous organic pollutants is well known and
Chlorella, Ankistrodesmus and Scenedesmus species have been already successfully used for
the treatment of olive oil mill wastewater and paper industry wastewater (Abeliovich and
Weisman, 1978; Narro, 1987; Pinto et al., 2003; Tarlan et al., 2002).
The phycoremediation is known to clean up the azo dye contaminated wastewater
with some additional benefits. Varsha et al. (2011) reported that azo dyes (Black B, Turq
18
Blue GN, Yellow HEM, Red HEFB and Navy HER), anthraquinone and phthalocyanine dyes
are commonly used in textile and paper industries. The degradation of these dyes produces
aromatic amines, which may be carcinogenic, and mutagenic. Specific algae have the ability
not only to decolorize these dyes but also to detoxify them. Jinqi and Houtian (1992)
investigated the degradation of azo dyes by Chlorella vulgaris and Chlorella pyrenoidosa.
They found that certain dyes such as Eriochrome Blue SE and Black T, could be decolorized
by algae. The degradation was found to be an inducible catabolic process. They also found
that the algae degraded aniline, a potential degradation product of the azo dye breakdown. In
another study, Ochromonas danica, a nutritionally versatile chrysophyte, grew
heterotrophically on phenol or p-cresol using them as the source of carbon up to
concentrations of 4 mM (Semple, 1997). Similarly, La Rossa (2009) found that
phycoremediation of olive mill wastewater can be done. It was also observed that selected
algal strains have the ability to decolorize the azo dye RBBR.
Sun et al. (1999) reported that Chlorella pyrenoidosa is the most potent algae in
decolorization of azo dyes. The efficiency rate of decolorization in no-nitrogen nutrient
solution was higher than that in no-carbon or normal solution. pH had a great effect on the
decolorization, with most suitable pH in neutral range. Mohan et al. (2004) reported the algal
decolorization of two azo dyes (direct and reactive dye), using a commonly available green
algae Spirogyra sp. in viable form. Batch studies revealed the potential of algae to remove
the dye color was dependant on initial inoculum size, and concentration and class of the dye.
Maximum dye uptake was noticed on the third day for both dyes. It was also concluded that
dye color removal by the algal species could be attributed to biosorption of the dye molecules
onto the surface of algal cell and subsequent diffusion and participation in metabolism
(bioconversion). The remaining dye molecules could be further removed from the aqueous
phase by adsorption and/or chelation reaction of the exopolymers released by the algae
(biocoagulation).
El-Sheekh et al. (2009) examined the ability of Chlorella vulgaris, Lyngbya lagerlerimi,
Nostoc lincki, Oscillatoria rubescens, Elkatothrix viridis and Volvox aureus to decolorize
methyl red, orange II and G-Red (FN-3G). These algae showed different efficiency for color
removal which varied from 4 to 95% depending on the algal species, growth state and the
dye molecular structure. Mohan et al. (2002) explained the potential of commonly available
19
green algae belonging to Spirogyra species as viable biomaterials for biological treatment of
simulated synthetic azo dye (Reactive Yellow 22). The results obtained from the batch
experiments revealed the ability of the algal species in removing the dye color and was
dependent both on the dye concentration and algal biomass.
The above studies clearly demonstrate that algae are indeed capable of contributing to
the degradation of environmental pollutants, either directly by transforming the pollutant in
question or by enhancing the degradation potential of the microbial community. The biomass
resulting from the treatment of wastewaters can be easily converted in added value products.
Depending on the species used for this purpose, the resulting biomass can be applied for
different aims, including the use as additives for animal feed, the extraction of added value
products like carotenoids or other bio-molecules or the production of biofuel. Moreover,
successful implementation of this strategy would allow minimizing the use of freshwater for
the production of algal biomass.
20
MATERIALS AND METHODS
A series of experiments were conducted under laboratory conditions to study
the potential of algae to decolorize various azo dyes that are widely used in textile processing
industry.
3.1. Sampling of water for the isolation of algae
Water samples were collected from three different sources such as wastewater
stream containing textile wastewater, fresh water and saline water (Table 3.1). Most of textile
industries of Faisalabad district of Punjab, Pakistan discharge untreated azo dye
contaminated wastewater directly into stream (named as Paharang drain). The collected
samples were brought into the laboratory in plastic jars and stored in the refrigerator at 4°C.
3.2. Analysis of collected water samples
These samples were analyzed for electrical conductivity (EC), total soluble salts
(TSS) and pH before isolation of algae.
3.2.1. Electrical conductivity
Electrical conductivity of the collected samples was determined in order to assess the
presence of total soluble salts in the wastewater discharged by different textile processing
units. The EC was recorded by using Jenway Model 4510 conductivity meter. The EC was
converted into total soluble salts (meq L-1) by following the method described by U.S.
Salinity Laboratory Staff (1954).
3.2.2. pH
Since different types of dyes either acidic or basic in addition to salts are used by the
textile industry, so pH was determined. The pH was measured with the help of Jenway pH
meter (Model 671 P).
21
Table: 3.1 Water samples collected from different sites with their identification code
S. No. Sample name Sample Identification code
Real textile wastewater samples
1 Dawood Textile Processing DT
2 Crescent Textile and Processing CKW
3 Arif Processing AZ
4 Qadafi Textile QT
5 Ashraf Textile AsT
Fresh water samples
6 Fish Pond, UAF NC
7 Canal (samandri road, FSD) DA
8 Chakwal C
9 Water Channel, UAF FF
Saline water samples
10 Lilah LL
11 Pir Khara Sharif PKS
12 Kalar Kahar Lake KKL
22
3.3. Isolation of algal strains
Algal cells were concentrated by passing the water samples through a filter paper.
The filter paper was then washed with distilled water, and the cell suspension was transferred
to agar plates. After 10 to 15 days of incubation at 28°C with continuous illuminated, blooms
of algae were observed on the agar plates. The material from a bloom was plated onto
separate agar plates for the isolation of photosynthetic microalgae. Eighty eight
morphologically distinct colonies were picked and transferred to another agar plate and/or a
sterile liquid medium.
Modified MA medium (Ichimura, 1979) was used with the following composition
(mg /100 mL).
• Ca(NO3)2 • 4H2O 5
• KNO3 10
• NaNO3 5
• Na2SO4 4
• MgCl2 • 6H2O 5
• β-Na2glycerophosphate • 5H2O 10
• Na2EDTA• 2H2O 0.5
• FeCl3 • 6H2O 0.05
• MnCl2 • 4H2O 0.5
• ZnCl2 0.05
• CoCl2 • 6H2O 0.5
• Na2MoO4 • 2H2O 0.08
• H3BO3 2
• Bicine 50
• Agar (used for agar plates) 200
23
3.3.1. Screening of azo dye degrading algal strains
The isolated algal strains were further screened by growing algae on the agar
plates containing azodye (Reactive Blue @ 100 mg L-1). The plates were incubated at 30°C
for 15 days under illuminated conditions. After incubation, algal colonies that appeared on
the agar medium were re-suspended into the flasks containing fresh modified MA broth
spiked with the Reactive Blue dye. After 15 days at 30°C, the cell suspensions were once
again plated onto the agar medium. About 20 actively growing colonies on the medium were
selected for further screening.
Liquid medium was prepared and pH was adjusted to 7 by using the 0.1N HCl
or 0.1N NaOH. About 100 mL of broth medium in 250 mL conical flask were inoculated
with the respective isolates. Inoculated flasks were incubated under illuminating conditions
for a period of 10 days at 30°C. After incubation, optical density of 0.6±0.01 was achieved at
600 nm, using colorimeter to maintain uniform cell densities. Further screening was done
to select the most efficient algal strains capable of decolorizing Reactive Blue azodye
using modified MA medium in triplicate. For this purpose, liquid medium was spiked with
the dye at rate of 100 mg L-1. After sterilization, medium was inoculated with the respective
inocula (20 isolates) and incubated at 30°C for 10 days. After incubation, the medium was
centrifuged at 10,000 rpm for 10 min. Then, decolorization was determined with the help
of spectrophotometer at 597 nm. Also uninoculated blanks were run to determine abiotic
decolorization. Percent decolorization was calculated. Two isolates exhibiting maximum
decolorization ability was selected for further experimentation.
Decolorization (%) was calculated based on following formula.
% Decolorization = Final absorbance - Initial absorbance x 100
Final absorbance
24
3.4. Optimization of environmental factors
Various factors were optimized to achieve the highest decolorization of Reactive
Blue azo dye by the selected isolates of algae. All the experiments were conducted in
triplicate.
3.4.1. Substrate (azo dye) concentration
Five levels (50, 100, 200, 400 and 600 mg L-1) of Reactive Blue azo dye were
used to determine the best concentration for maximum decolorization. The inoculum was
added to the medium (10 mL) at inoculum to dye solution ratio of 1:50 and incubated at 30°C
for 7 days. Decolorization was determined by taking 1.5 mL aliquot from different test tubes
and centrifuging the solutions at 10,000 rpm for 10 min to remove the algal cells.
Absorbance of the supernatant was measured at 597 nm by using spectrophotometer.
Uninoculated blank were run to check the abiotic decolorization. Each dye level was
replicated thrice.
3.4.2. pH
Effect of different pH levels ranging from 5 to 9 on the decolorization
efficiency of the selected algal isolates was examined. Modified MA medium enriched with
Reactive Blue (100 mg L-1) was used. The above mentioned procedure in section 3.4.1 was
repeated except changing the pH of the growth medium. The pH was adjusted by using 0.01
M HCl or 0.01 M NaOH solutions.
3.4.3. Temperature
Decolorization of Reactive Blue by the selected algal isolates was studied at
different temperatures, including 20, 25, 30, 35 and 40°C by using the procedure given in
section 3.4.1. Modified MA medium was enriched with 100 mg L-1 of Reactive Blue. pH of
the medium was maintained at 7.
3.4.4. Effect of nitrogen
In order to assess the effect of additional nitrogen source on the decolorization
of azo dye by algae, ammonium nitrate (1, 2, 3, 4 and 5 g L-1) was used as nitrogen source.
MA medium spiked with Reactive Blue was used. pH of the medium was maintained at 7
25
and incubated at 30°C for 7 days. Decolorization was determined by using the protocol as
described above. The experiment was conducted in three replications.
3.4.5. Effect of phosphorus
Tri-calcium phosphate was used as P source. The MA medium spiked with
Reactive Blue @ 100 mg L-1. Different levels of P i.e. 1, 2, 3, 4 and 5 g L-1 were used. pH of
the medium was maintained at 7 and incubated at 30°C for 7 days. Decolorization was
determined by using the protocol as described above.
3.4.6. Effect of salts
In order to assess the effect of salts on dye decolorization, NaCl was used at 0,
10, 20, 30, 40 and 50 g L-1 to create the saline conditions in the dye medium. MA medium
spiked with Reactive Blue @ 100 mg L-1 was used. All other previously optimized conditions
were kept the same as described above.
3.4.7. Effect of light
Dye decolorization by algae was then optimized using light 12, 14, 16, 18 and 20
hours per day. Decolorization was determined by using the already optimized conditions of
pH, temperature, substrate concentration, N and P as described above.
3.4.8. Effect of inoculum size
The inoculum was applied at the rate of 400 µL, 600 µL, 800 µL, 1000 µL and 2000
µL. The earlier optimized conditions were kept as the same including pH, temperature,
substrate concentration, light conditions, N and P.
3.5. Phyco-remediation of structurally different azo dyes
Phyco-remediation of structurally different azodyes was determined by using
modified MA medium containing 100 mg L-1 of azo dye. The following dyes were used
individually as well as in mixtures.
Reactive dyes
Reactive Blue BRS λmax 600 nm
Orange RR Reactive λmax 490 nm
Red S3B Reactive λmax 531 nm
Disperse dyes
26
Disperse Blue ZBLN λmax 480 nm
Scarlet Disperse λmax 498 nm
Direct dyes
Yellow UG Direct λmax 402 nm
Congo Red Direct λmax 510 nm
The medium was inoculated with living cells of algae (cell density OD 0.6) with 1:10
inoculum to medium ratio. Dye concentration was 100 mg L-1. Percent degradation was
examined after 12, 24, 48, 72, 96 and 120 h. pH of the medium was maintained at 7.0. The
decolorization of each dye was determined at maximum absorption wave length as given
above. Each experiment was performed in three replications.
3.6. Decolorization of mixture of azo dyes
Different combinations of different dyes were also used for testing the
potential of selected algae to decolorize the mixture of dyes in liquid medium. Total
concentration of the mixture was 100 mg L-1.
i) Mixture of reactive dyes (Reactive Blue, Orange RR Reactive and Red S3B
Reactive)
ii) Mixture of disperse dyes (Disperse Blue ZBLN, Scarlet Disperse)
iii) Mixture of Direct dyes (Yellow UG Direct, Congo Red Direct)
iv) Mixture of three groups of dyes (Yellow UG Direct, Congo Red Direct,
Disperse Blue ZBLN, Scarlet Disperse, Reactive Blue BRS, Orange RR Reactive and Red
S3B Reactive)
Disperse dyes were first dissolved in small volume of ethanol (95%) and then diluted
into sterilized distilled water. Nine mL of the sterilized MA medium were taken and spiked
with each dye. Final concentration of the mixture of dyes in the medium was 100 mg L-1. All
the dyes in mixture were added in equal proportion. The medium was inoculated with the
respective algal isolates by adding inocula (1:10 inocula to media) of uniform cell density
(OD 0.6). In the same experiment, the dead algal biomass (dried biomass) of both isolates
were also used (1:10 inocula to media) to check the impact of phyco-remediation between
living and dead algal mass. The algae were first sun dried then oven dried at 80°C for the
purpose to gain dead algal mass. The glass test tubes were incubated statically at 30°C under
luminous conditions. Uninoculated test tubes with MA medium containing azo dye were also
27
incubated under similar conditions to check abiotic decolorization of dye. Three replicate test
tubes were used for each strain per dye mixture. Absorbance of the supernatant was
measured at 478 nm by spectrophotometer as described above.
Reactive Blue
Reactive Orange
Reactive Red
Disperse Blue
Disperse Scarlet
Direct Yellow
Direct Red
28
3.7. Phycoremediation of real textile wastewater
Wastewater samples with high dye color were collected from the outlets of
textile industries (Shaheen Cloth Processing, Qadafi Textile and Dawood Textile Mills
located in Faisalabad, Pakistan). Two algal strains (CKW1 and PKS33) were assessed for
their ability to remove color of dyes from textile wastewater. The λmax was determined for
each type of sample by running these samples on spectrophotometer. The λmax for Shaheen
Cloth Processing, Qadafi Textile and Dawood Textile Mills was 560, 485 and 430 nm,
respectively.
Twenty mL colored wastewater were taken in glass test tubes. One mL of
algal culture (0.6 OD) was applied. Dead algal biomass (dried biomass) of both strains was
also used to compare phyco-remediation between living and dead algal mass. Dead algal
mass (dried) was added at the rate of 1:10 algae: wastewater. The amount of decolorization
was studied after 12, 24, 48, 72, 96 and 120 h by using spectrophotometer. The experiment
was performed with three replications for each treatment. Results are presented as the
average of three replications at each interval.
3.7.1. Characteristics of wastewater samples
Wastewater sample # 1 was collected from direct outlet of Shaheen Processing Mills
Faisalabad, Pakistan. Wastewater sample were analyzed for pH (8.6), ECe (2.45 dS m-1),
available P (6.25 mg kg-1), extractable K (164 mg kg-1), total N (1.66%), BOD (320 mg L-1)
and COD (860 mg L-1). Wastewater sample # 2 (Qadafi Textile) contained was analyzed for
pH (8.2), ECe (3.5dS m-1), available P (7.25 mg kg-1), extractable K (195 mg kg-1), total N
(1.75%), BOD (310 mg L-1) and COD (830 mg L-1). Wastewater sample # 3 (Dawood Textile
Mill) contained pH 9.1, ECe, 3.15dS m-1; available P, 6.75 mg kg-1; extractable K, 198 mg
kg-1; total N, 1.8%; BOD, 340 mg L-1 and COD 880 mg L-1.
3.8. Toxicity analysis
Toxicity of wastewater, treated wastewater and mixture of azodyes was
determined by measuring hemolytic activity.
29
3.8.1. Hemolytic activity
Hemolytic activity of the compound was studied by the method as described by
Sharma and Sharma (2001) and Powell et al. (2000). Three mL freshly obtained heparinized
human blood was collected from volunteers after consent and counseling and bovine from
the Department of Clinical Medicine and Surgery, University of Agriculture Faisalabad.
Blood was centrifuged for 5 min at 1000xg, plasma was discarded and cells were washed
with three times with 5 mL of chilled (4oC) sterile isotonic Phosphate-buffered saline (PBS)
pH 7.4. Erythrocytes were maintained 108 cells per mL for each assay. Hundred μL of each
compound was mixed with human (108cells/mL) separately. Samples were incubated for 35
min at 37oC and agitated after 10 min. Immediately after incubation the samples were
placed on ice for 5 min then centrifuged for 5 min at 1000xg. A hundred μL supernatant were
taken from each tube and diluted 10 time with chilled (4oC) PBS. Triton X-100 (0.1% v/v)
was taken as positive control and phosphate buffer saline (PBS) was taken as negative
control and pass through the same process. The absorbance was noted at 576 nm using
μQuant (Bioteck, USA). The % RBCs lysis for each sample was calculated.
3.9. Use of phyco-remediated wastewater for irrigation purpose
An experiment was conducted in jars to evaluate the effect of real textile wastewater
and phyco-remediated wastewater on the growth of wheat under axenic condition. Glass jars
containing sand were sterilized by autoclaving at 121°C for 20 min. Seeds of wheat (Fsd-
2008) were placed in sand with sterile forcep. Real textile wastewater and phyco-remediated
wastewater were applied for irrigation of wheat seedlings. Glass jars were kept in a growth
chamber for 2 weeks. Experiment was performed in a completely randomized design (CRD)
with the following treatments.
T1 Wastewater application
T2 Phyco-remediated wastewater (by Dead PKS33)
T3 Phyco-remediated wastewater (by Dead CKW1)
T4 Phyco-remediated wastewater (by living PKS33)
T5 Phyco-remediated wastewater (by living CKW1)
Wheat plants after 2 weeks were harvested and data on plant biomass, root/shoot
length and root/shoot weight were recorded.
30
3.10. Biodiesel production
The algal biomass obtained after phyco-remediation of wastewater was then
used to extract biodiesel. Algal biomass produced by giving modified MA medium was also
run. The methods used for oil extraction from algal biomass and conversion of extracted oil
into biodiesel are given as under:
3.10.1. Oil extraction
For extraction of oil, algal biomass was ground and then dried for 20 min at 80°C in
an incubator for removing water. It was then soaked in hexane and ether solution (40 mL and
40 mL respectively for 50 g dried algae) to extract oil. The mixture was kept for 24 h to settle
down. Biomass was collected after filtration and then weighed. The extracted oil was
evaporated in rotary evaporator to remove hexane and ether.
3.10.2. Trans-esterification
Catalyst and methanol mixture was used to make biodiesel from extracted oil. About
0.25 g NaOH was mixed with 24 mL methanol and stirred properly for 20 min. The mixture
was poured on the extracted oil in a conical flask. The reaction process is called trans-
esterification. Then, the flask containing solution was kept under shaking conditions for 3 h
in orbital shaker at 300 rpm. After shaking, the solution was kept overnight for settling.
3.10.3. Separation of biodiesel
Biodiesel was separated from sediment layers carefully. Biodiesel was washed with
5% water. Biodiesel was dried in air for 12 h. Biodiesel production was measured using
graduated cylinder. Performing a 5% water prewash stops the trans-esterification reaction
dead in its tracks by drawing much of the water-soluble methanol, lye, and glycerol out of the
biodiesel and into the glycerol layer. This can help reduce soap, inhibit emulsion formation,
and make further washing easier.
3.11. Statistical analysis
Data were entered in Microsoft® Excel 2007 spreadsheet, and means and
standard deviations were calculated. ANOVA was performed and LSD values at P< 0.05
were calculated using the MSTATC software (Michigan State University, USA).
31
RESULTS
Several water samples from dye-contaminated textile wastewater, fresh water and
saline water were collected and analyzed for pH and TSS. Algae capable of decolorizing
textile dyes were isolated from these water samples, using Reactive Blue BRS dye as a
source of carbon and nitrogen in the modified MA medium. The most efficient dye
decolorizing algae were screened in the liquid medium containing 100 mg L-1 of Reactive
Blue BRS azo dye. Various factors were optimized to accelerate the process of dye removal
by the selected strains of algae. Phyco-remediation of structurally different azo dyes was
done by using selected algal strains. The phyco-remediation efficiency of algal strains was
also investigated in the real textile wastewater containing a mixture of dyes. The phyco-
remediated wastewater was then used for irrigation purpose to grow wheat plants under
controlled conditions. Toxicity analysis was also done by measuring the hemolytic activity.
Finally, the biodiesel was extracted from algal biomass produced by using phyco-remediated
water. The results are discussed in the following sections.
4.1. Analysis of water samples
pH and TSS of textile wastewater were analyzed to determine the acidic or basic nature of
the wastewater and amount of salts present in the wastewater.
4.1.1. pH
A wide variation in pH of different water samples was recorded (Table 4.1). A pH of 12.4
was found in case of wastewater sample collected from the outlets of Qadafi Textile, and it
was highly alkaline in nature. Similarly, pH was >10 in the case of Arif and Dawood textile
processing units. A pH range of 9.0 to 9.90 was recorded in case of saline water samples
collected from Lilah area located in the Kalarkahar Salt Range of Pakistan. The pH was more
than 8.0 but less than 9.0 in case of effluents of Crescent Textile and Processing unit and
water collected from the areas including Pir Khara Sharif, Chakwal and Kalarkahar.
However, pH was between 7.0 and 8.0 in case of all fresh water samples. These results
showed that wastewater from textile processing units had a variable pH, which may affect the
efficiency of algae for removal of dye contaminants from the wastewater.
32
4.1.2. TSS
Table 4.1 represents total soluble salts (TSS) present in the different water samples.
Maximum TSS (100-150 meq L-1) was found in saline water samples (Table 4.1). In textile
wastewater, TSS observed ranged from 80-90 meq L-1, however, TSS of the fresh water was
found up to 53 meq L-1.
4.2. Isolation and screening of azo dye decolorizing algae
From water samples collected from different sources, 88 different algal strains were
isolated through enrichment technique. Based on their efficiency to remove color of the dye
from agar medium, 20 algal isolates were selected. These algal isolates were further tested
under the liquid broth medium containing 100 mg L-1 of Reactive Blue BRS dye. The results
(Fig. 4.1) clearly indicated the variable potential of algal isolates to the dye in broth medium.
Two isolates (CKW1 and PKS33) were able to decolorize more than 80% dye within 7 days
compared to abiotic control. The majority of these isolate showed a medium decolorization
potential, which ranged from 50 to 75% compared to control. Five isolates showed
decolorization below 50%.
Maximum dye decolorization was 88% by strain CKW1, followed by decolorization
(83%) by PKS33. Next most effective isolated were LL1 and PKS1 and exhibited 70%
decolorization in 7 days. Next effective algal strains in descending order were AZ5, DA4,
AZ3 and NC1. Strain DA1 was the least efficient in decolorizing the dye. Based on these
results, the most efficient isolates (CKW1 and PKS33) exhibiting the highest dye
decolorization were selected for further experiments.
33
Table 4.1. Water analysis and isolation of algae from water samples collected from different locations
Sample type Sample Location pH TSS (me L-1)
Algae isolated from water samples
1 Fresh water Faisalabad 7.32 45 DA1
2 Fresh water Faisalabad 7.32 45 DA3
3 Fresh water Faisalabad 7.32 45 DA4
4 Fish pond Faisalabad 7.48 53 NC1
5 waste canal Faisalabad 8.03 80.4 CKW1
6 waste canal Faisalabad 8.03 80.4 CKW2
7 waste canal Faisalabad 8.03 80.4 CKW5
8 waste canal Faisalabad 8.03 80.4 CKW13
9 saline water Lilah 9.3 108 LL1
10 saline water Lilah 9.3 108 LL2
11 saline water Lilah 9.3 108 LL4
12 Fresh water Chakwal 7.23 59 FF2
13 Fresh water Chakwal 7.23 59 FF4
14 saline water Salt range 8.8 150 PKS1
15 saline water Salt range 8.8 150 PKS12
16 saline water Salt range 8.8 150 PKS16
17 saline water Salt range 8.8 150 PKS21
18 saline water Salt range 8.8 150 PKS33
19 waste canal Faisalabad 10.7 88.7 AZ3
20 waste canal Faisalabad 10.7 88.7 AZ5
34
Fig. 4.1: Decolorization of Reactive Blue dye by different algal strains isolated from various water samples through enrichment.
35
4.3. Optimization of environmental factors
Two algal strains, CKW1 (isolated from azo dye contaminated wastewater) and PKS33
(isolated from saline water) were further investigated for the optimization of various
incubation/environmental conditions in order to achieve the maximum decolorization of
Reactive Blue azo dye.
4.3.1. Substrate (azo dye) concentration
Five levels (50, 100, 200, 400 and 600 mg L-1) of Reactive Blue dye were used to find
the optimum substrate concentration for maximum decolorization. The substrate (azo dye)
concentration significantly affected the decolorization potential of algae (Fig. 4.2).
Decolorization potential of both strains to decolorize Reactive Blue azo dye by was almost
similar at substrate concentration of 50 mg L-1 and 100 mg L-1. Thereafter, amount of
decolorization decreased with an increase in substrate concentration. Strain CKW1 showed
the highest decolorization at 50 and 100 mg L-1 dye concentration and almost 90% color
removal of 50 and 100 mg dye L-1 was observed after 7 days. The amount of decolorization
by strain PKS33 was comparable with that of CKW1 at all tested concentration of dye.
However, when dye concentration was more than 100 mg L-1, strain PKS33 showed more
decolorization of the dye than strain CKW1. Based on these results, 100 mg L-1 dye
concentration was used for subsequent studies.
36
Fig: 4.2. Effect of substrate concentration on decolorization of Reactive Blue dye by the
selected algal strains.
37
4.3.2. pH
Effect of different pH ranging from 5 to 9 was examined to find out optimal pH for the
decolorization of dye by the selected algal isolates (Fig. 4.3). It is evident from the results
that the selected strains of algae were able to decolorize the dye over a wide range of pH.
Maximum decolorization by the both tested strains was recorded at pH between 7 and 8. In
case of PKS33 (strain isolated from salt range), a rapid increase in decolorization was
observed as the pH increased from 6 to 7. However, a relative decrease in decolorization was
found when pH was increased from 8 to 9. Overall, dye decolorization was 64% at pH 5,
67% at pH 6, 83% at pH 7, 80% at pH 8 and 77% at pH 9. Decolorization of the dye by
CKW1 (strain isolated from textile wastewater) was 74% at pH 5, 83% at pH 6, 86% at pH 7,
88% at pH 8 and 78% at pH 9. In general, pH 7-8 favored both tested strains to decolorize
the dye and pH 8 was found to be optimal and used for subsequent studies.
4.3.3. Temperature
Decolorization of Reactive Blue by the selected algal strains was studied at different
temperatures such as 20, 25, 30, 35 and 40°C. It was observed that an increase in the
temperature from 25 to 30oC had a positive impact on the decolorization of Reactive Blue
(Fig. 4.4). Decolorization of the dye Reactive Blue was optimal at temperature 30oC as
CKW1 and PKS33 were able to decolorize the dye by 85 and 80 % respectively. However,
decolorization rate in both strains dropped gradually as the temperature increased to 35 or
40oC. Overall, a similar trend was observed in case of temperature regarding the
decolorization potential of both selected algal strains.
38
Fig: 4.3. Effect of pH on decolorization of Reactive Blue dye by the selected algal
strains.
39
Fig: 4.4. Effect of temperature on decolorization of Reactive Blue by the selected algal
strains.
40
4.3.4. Light conditions
Light of 12, 14, 16, 18 and 20 hours per day was maintained to examine the decolorization of
the dye by selected algal strains. There was an increase in the decolorization of azo dye by
both strains with an increase in day light conditions (Fig. 4.5). Optimal day light period to
decolorize Reactive Blue azo dye for both tested strains was 16 h. However, decolorization
rate in both strains remained constant as the day light period increased from 16 h. In general,
a similar trend was observed in case of day light period regarding the decolorization potential
of both algal strains. However, strain CKW1 performed marginally better at all tested levels
in terms of decolorization of azo dye. A day light conditions of 16 h was selected for
subsequent studies.
4.3.5. Inoculum size
The inocula were applied at the rate of 400 µL, 600 µL, 800 µL, 1 ml and 2 ml. Fig. 4.6
illustrates an increase in the decolorization of azo dye by both the strains with the increase in
inocula size from 400 µL to 1 ml. Optimal inoculum size to decolorize Reactive Blue azo dye
for both tested strains was 1 ml/10 ml substrate. Decolorization rate in both strains was
almost same when inoculum size increased from 1 ml/10ml substrate.
41
Fig: 4.5. Effect of day light conditions on decolorization of Reactive Blue dye by the
selected algal strains.
42
Fig: 4.6. Effect of inoculums size on decolorization of Reactive Blue dye by the selected
algal strains.
43
4.3.6. Salinity
Data summarized in Fig. 4.7 clearly reflects that the selected strains of algae were
able to decolorize the dye over a wide range of salt concentration, but a decrease in
decolorization rate was observed in case of both the algal strains as the salt concentration
increased. Maximum decolorization was recorded at 0 and 10 g L-1 NaCl. In case of PKS33
(strain isolated from salt range), a decrease in decolorization was found when salt
concentration increased from 10 to 50 g L-1. Overall, decolorization ranged from 90% at 0 g
NaCl L-1, 89% 10 g NaCl L-1, 82% at 20 g NaCl L-1, 68% at 30 g NaCl L-1, 58% at 40 g NaCl
L-1 and 49% at 50 g NaCl L-1. Decolorization of the dye by CKW1 (strain isolated from
textile wastewater) was almost similar to PKS33 at 0 and 10 g NaCl L-1. As the salt
concentration increased, decolorization of Reactive Blue was about 10% less compared to
PKS33 at all salt levels after 10 g L-1 NaCl.
44
Fig: 4.7. Effect of salinity on decolorization of Reactive Blue dye by the selected algal
strains.
45
4.3.7. Effect of nitrogen
In order to assess the effects of additional nitrogen source on algal decolorization,
ammonium nitrate was used as nitrogen source. It was observed that there was a gradual
increase in the decolorization of azo dye by both the strains with the increase in additional N
source (Fig. 4.8). Maximum decolorization of 95 and 93% was observed with CKW1 and
PKS33 respectively at N concentration 5 g L-1. In general, both strain showed similar
decolorization percentage when N concentration was increased from 5 to 6 g L-1. The strain
CKW1 performed marginally better than PKS33 at all tested levels.
4.3.8. Effect of phosphorus
The effect of additional phosphorus (P) source on algal decolorization was observed
using tri-calcium phosphate as P source. The data summarized in Fig. 4.9 showed that
addition of P affected the decolorization of Reactive Blue azo dye by selected algal isolates.
The results were similar to that obtained in case of N. A gradual increase in the
decolorization of dye by both the strains with the increase in additional P source was found.
Maximum decolorization of up to 90 and 88% was observed with CKW1 and PKS33
respectively at P concentration 5 g L-1. Both strain showed a slight decrease in decolorization
of dye when N concentration was increased from 5 to 6 g L-1. Overall, both strain performed
in similar trend in terms of azo dye decolorization at all the applied levels of additional P
source.
46
Fig: 4.8. Effect of additional N source on decolorization of Reactive Blue dye by the
selected algal strains.
47
Fig: 4.9. Effect of additional P source on decolorization of Reactive Blue dye by the
selected algal strains.
48
4.4. Phyco-remediation of structurally different azo dyes
Two efficient strains of algae were tested for their potential to degrade different
groups of dyes (reactive, disperse and direct dyes) and their mixture. The results are
described as under:
4.4.1. Phycoremediation of reactive dyes
Both algal strains efficiently decolorized the dye Orange RR Reactive compared to
control (figure 4.10.). Initially, strain CKW1 decolorized Orange RR Reactive azo dye up to
19% in 12 h, and achieved 54 % discolorzation after 48 h. Whereas PKS33 strain showed up
to 17% decolorization within 12 h and 53% after 48 h. The CKW1 strain was more effective
as 77% dye decolorization was achieved in 72 h, whereas PKS33 showed 66% decolorization
in 72 h. After 96 h, both strains caused decolorization up to 87% and 75% respectively. The
maximum decolorization (100%) was shown by CKW1 while 88% of decolorization was
observed in case of strain PKS33 after 120 h. In control, the decolorization of the azo dye
was negligible that is recorded only up to 7 % after 120 h.
Strain CKW1 showed maximum decolorization of Red S3B Reactive dye up 80% in 72 h
(Figure 4.11). Strain PKS33 had maximum decolorization of 67% in 72 h. In case of control,
no decolorization of the dye was observed initially. After 96 h, up to 90% and 78%
decolorization was achieved by CKW1 and PKS33, respectively. Complete decolorization of
the dye was achieved with strain CKW1 after 120 h, while maximum decolorization was
88% in the case of PKS33.
Data regarding the effect of decolorization of mixture of reactive dyes (Reactive Blue,
Orange RR Reactive and Red S3B Reactive) using algal strain is illustrated in Figure 4.12.
Decolorization of mixture of dyes was lower than individual dyes. In case of strain CKW1,
decolorization was 7% in 12 h, 22% in 24 h, 36% in 48 h, 65% in 72 h and 82% in 96 h. In
the case of strain PKS33, similar trend was observed. The highest degradation was achieved
up to 90% by CKW1 after 120 h while 85% of decolorization was achieved with strain
PKS33. Overall, both algal strains were able to decolorize individual azo dyes in less time as
compared to mixture of reactive azo dyes.
49
Fig. 4.10: Decolorization of of Orange RR Reactive azo dye by selected strains of algae.
50
Fig. 4.11: Decolorization of Red S3B Reactive azo dye by selected strains of algae.
51
Fig. 4.12: Decolorization of mixture of Reactive azo dyes by selected strains of algae.
52
4.4.2. Phycoremediation of direct group dyes
Strain CKW1 decolorized Yellow UG Direct azo dye up to 30% in 12 h, 61% in 24 and 80%
in 48 h (Figure 4.13). Strain PKS33 caused decolorization up to 27% in 12 h, 51% in 24 h
and 70% in 48 h. Strain CKW1 was more effective than PKS33. Decolorization was 90%
after 72 h in the case of strain CKW1 whereas 81% decolorization was observed with strain
PKS33 within 72 h. The complete decolorization was observed within 96 h by strain CKW1
while 120 h were taken by strain PKS33 to achieve complete decolorization.
In case of Congo Red Direct dye, maximum dye decolorization of 64% was recorded within
48 h by strain CKW1 (Figure 4.14). Decolorization of Red Direct azo dye was 52% after 48
h in the case of strain PKS33. Further, up to 85% decolorization was attained by strain
CKW1 and 70% by PKS33 after 72 h. The complete decolorization of the dye was achieved
with strain CKW1 after 96 h. On the other hand, strain PKS33 attained complete
decolorization of azo dye after 120 h. In case of control, only up to 8% decolorization was
observed in the case of control.
Like individual dyes, algal strains CKW1 and PKS33 were also very effective in
decolorization of a mixture of direct dyes,(Yellow UG Direct, Congo Red Direct) but the
decolorization rate was slower in case of mixture than the individual dyes Fig. 4.15. Strain
CKW1 decolorized the mixture up to 16% in 12 h, 34% in 24 h and 54% in 48 h. Almost
similar trend was observed in the case of strain PKS33 where decolorization of 15%, 28%
and 47% was observed in 12 h, 24 h and 48 h, respectively. After 90 h, decolorization up to
90% and 77% was achieved by strain CKW1 and PKS33, respectively. Strain CKW1 showed
complete decolorization after 120 h while PKS33 showed maximum 87% decolorization
after 120 h. Algal strain CKW1 was more effective in decolorizing individual as well as
mixture of direct dyes.
53
Fig. 4.13: Decolorization of Yellow UG Direct azo dye by selected strains of algae.
54
Fig. 4.14: Decolorization of Congo Red Direct azo dye by selected strains of algae.
55
Fig. 4.15: Decolorization of mixture of direct azo dyes by selected strains of algae
56
4.4.3. Phycoremediation of Disperse group dyes
It was observed that strain CKW1 decolorized the dye Disperse Blue ZBLN up to 65% in 48
h (Figure 4.16). Strain PKS33 exhibited lower decolorization of the dye than CKW1.
Decolorization was 15%, 32%, and 49% in 12 h, 24 h and 48 h, respectively. After 72 h,
strain CKW1 exhibited 86% decolorization whereas PKS33 showed 70% dye decolorization.
The decolorization efficiency in the case of strain CKW1 was 100% after 120 h, while strain
PKS33 showed maximum decolorization of 91% in the same period. In case of
decolorization of Scarlet Disperse type of dyes, both algal strains showed high potential to
decolorize the dye as compared to control (Figure 4.17). Initially, the algal strains showed a
slow decolorization where strain CKW1 decolorized Scarlet Disperse dye by 15% in 12 h,
39% in 24 h and 55% in 48 h. Similarly, decolorization was 11% after 12 h, 25% after 24 h,
35% after 48 h in case of strain PKS33. Complete dye decolorization was achieved by
CKW1 after 120 h, while PKS33 showed 82% decolorization.
Data on decolorization of mixture of disperse group of dyes (disperse blue ZBLN and Scarlet
Disperse dye) is given in Figure 4.18. The strain CKW1 showed a decolorization of 21%,
38% and 53% in 12, 24 and 48 h, respectively. In case of strain PKS33, the dye
decolorization was 11%, 22% and 37% within 12, 24 and 48 h, respectively. The
decolorization of mixture of disperses azo dyes by CKW1 and PKS33 after 72 h was 71 %
and 56% respectively and 84% and 68% after 96 h respectively. The CKW1 strain showed a
complete decolorization after 140 h and PKS33 stains showed up to 95% of decolorization
after 140 h.
57
Fig. 4.16: Decolorization of Disperse Blue ZBLN azo dye by selected strains of algae.
58
Fig. 4.17: Decolorization of Scarlet Disperse azo dye by selected strains of algae.
59
Fig. 4.18: Decolorization of mixture of disperse azo dyes (disperse blue ZBLN and Scarlet Disperse dye) by selected strains of algae.
60
4.5. Phycoremediation of mixture of azo dyes
The results regarding decolorization of mixture of various groups of azo dyes by
living and dead cells of algae are described as under.
Decolorization of a mixture of azo dyes, including Yellow UG Direct, Congo Red Direct,
Disperse Blue ZBLN, Scarlet Disperse, Reactive Blue BRS, Orange RR Reactive and Red
S3B Reactive by algal strains is summarized in Figure 4.19. Like individual dyes, algal
strains CKW1 and PKS33 were also very effective in decolorization of mixture of dyes.
Algal strain CKW1 showed almost complete decolorization in 120 h while PKS33
completely decolorized the mixture of dyes in 144 h. Initially, strain CKW1 decolorized the
mixture of azo dyes up to 10% after 12 h, 25% after 24 h and 50% after 48 h. The almost
similar trend was observed in the case of strain PKS33 where maximum decolorization of
mixture of dyes was 47% within 48 h. After 72 h of incubation, 72% and 63 % decolorization
of the dyes by strain CKW1 and PKS33 was observed whereas decolorization was 87 and
73% after 96 h respectively. In case of application of dead mass of PKS33 and CKW1
strains, the decolorization of mixture of azo dyes by was by 58% and 55% in 12 h and 65%
and 63% in 24 h respectively. Interestingly, no further decolorization was observed after 24 h
by dead mass of both strains. Overall, it was found that algal strains had high potential for
decolorizing individual azo dyes as compared to the mixture of azo dyes. Moreover, strain
CKW1 was more effective in decolorizing both individual dyes as well as mixture of reactive
dyes.
61
Fig. 4.19: Decolorization of mixture of reactive, direct and disperse group azo dyes by selected strains of algae
62
4.6. Phycoremediation of real textile wastewater
Real wastewater was collected from three industrial units of Faisalabad city (Shaheen
Cloth Processing, Qadafi textile and Dawood textile mills Faisalabad) and color removal was
examined using strains CKW1 and PKS33.
4.6.1. Case study I (Treatment of wastewater from Shaheen Cloth Processing Mills)
The efficiency of selected algal strains (CKW1 and PKS33) for treatment of textile
wastewater collected from direct outlet of Shaheen Cloth Processing Mills is presented in
figure 4.20. Both algal strains showed a considerable decolorization of textile effluent,
however, very little decolorization was observed in case of non-augmented (control)
wastewater. In case of control, maximum color removal of 3.8% was observed after 120 h.
Strain CKW1 and PKS33 initially caused 21% decolorization in 12 h, which was further
increased up to 50% in 24 h. Further, CKW1 strain showed up to 74%, 86% and 94% color
removal of wastewater after 48, 72 and 96 h, respectively. Similarly, algal strain PKS33
decolorized the wastewater up to 69%, 81% and 88% after 48, 72 and 96 h respectively. The
complete color removal of textile wastewater occurred in 120 h by algal strain CKW1
whereas the strain PKS33 exhibited 94% decolorization.
In case of dead algal mass, both strains showed a higher efficiency than living mass and
about 66% decolorization of textile wastewater was observed in 12 h. Further, CKW1 (dead)
was able to remove color in wastewater by 74%, 77%, 79% and 82% after 24, 48, 72 and 96
h, respectively. While strain PKS33 (dead) caused decolorization up to 83% after 96 h.
Maximum decolorization (85%) was recorded with CKW1 (dead) after 120 h.
Optical density (biomass) of algal strains is illustrated in figure 4.21 which indicated that the
dead algal strain had a lower optical density than living algal biomass. In general, the living
algal strains showed a relatively slow decolorization during first 24 h and then decolorization
rate increased till 120 h. Overall, a direct relationship was found between biomass of algal
strain and decolorization of wastewater. The increase in decolorization was observed with the
increase in algal biomass.
63
Fig. 4.20. Phycoremediation of real textile wastewater sample # 1 (collected from Shaheen Cloth Processing Mills) by algal strains.
64
Fig. 4.21. Comparative phycoremediation of real textile wastewater sample # 1 by living and dead cells of algae.
65
4.6.2. Case Study II (Treatment of wastewater from Qadafi Textile)
Both algal strains were able to decolorize the real textile wastewater collected Qadafi Textile
dyeing unit. About 26% color removal was observed within 12 h by strain CKW1 after
augmentation, which was almost double (51%) after 24 h (Fig. 4.22). Decolorization was
73% and 88% with strain CKW1 after 48 and 72 h, respectively. In the case of strain PKS33,
decolorization up to 21% and 43% in 12 and 24 h, respectively, was recorded. Strain PKS33
was able to remove color up to 71% and 82% after 72 and 96 h, respectively. Strain CKW1
showed a complete decolorization of wastewater after 96 h, whereas PKS33 showed
maximum decolorization up to 90% after 120 h. Maximum color removal was 4.7% after 120
h in the case of control.
Application of dead algal mass of CKW1 and PKS33 resulted in 57% and 60% color
removal, respectively, after 12 h (Fig. 4.23). Maximum decolorization was 80 and 82% by
dead mass of strain CKW1 and PKS33 after 120 h. Strain CKW1 (dead) showed a
decolorization up to 67, 71, 75 and 78% after 24, 48, 72 and 96 h, respectively while
decolorization by strain PKS33 (dead) was 70, 73, 77 and 79% after 24, 48, 72 and 96 h,
respectively. Overall, the results revealed that dead algal mass of both strains (CKW1 and
PKS33) was able to decolorize most of the wastewater during first 24 h. It is also evident
from figure 4.23 that the optical density (OD) of algal strains was lower initially within 24 h,
but kept on increasing till 120 h. However, the OD of dead mass of algae was lower than
living algae.
66
Fig. 4.22. Phycoremediation of real textile wastewater sample # 2 (collected from Qadafi Textiles) by algal strains.
67
Fig. 4.23. Comparison phycoremediation of real textile wastewater sample # 2 (collected from Qadafi Textiles) by algal strains with their optical density at 600nm.
68
4.6.3. Case study III (Treatment of wastewater from Dawood Textile Mills)
The algal strains CKW1 and PKS33 exhibited promising results for phycoremediation of real
textile wastewater (Figure 4.24). Decolorization was only 3% after 120 h in the case of non-
augmented wastewater. Strain CKW1 was able to decolorize the wastewater completely in
120 h. Algal strain PKS33 decolorized wastewater up to 90% in 120 h.
Maximum decolorization by dead algal mass of CKW1 and PKS33 was 86 and 87%,
respectively, after 120 h (Fig. 4.25). A very low optical density was observed with the dead
algal mass. Overall, a direct relation was found between OD and decolorization of
wastewater. The increase in OD of algal strain also increased decolorization of wastewater.
69
Fig. 4.24. Phycoremediation of real textile wastewater sample # 3 (collected from Dawood Textiles) by algal strain.
70
Fig. 4.25. Comparison phycoremediation of real textile wastewater sample # 3 (collected from Dawood Textiles) by algal strains with their optical density at 600nm.
71
4.7. Toxicity Analysis
4.7.1. Toxicity test of mixture of azo dyes after decolorization by algal strains
Toxicity of mixture of azo dyes was examined by measuring the hemolytic activity and data
is presented in figure 4.26. Mixture of azo dyes (control) caused negative impact on human
red blood cells where 58 cells (out of 100) were lysed. The toxicity of azo dyes after
decolorization with dead algal mass (CKW1) was 30% lower (only 41 lysed) than that
observed in living cells. Similarly, the phycoremediation of azo dyes mixture with dead algal
mass (PKS33) showed 36% decrease in toxicity where 37 cells were lysed out of 100 cells.
Phycoremediation of mixture of azo dyes with living algal strain CKW1 reduced the toxicity
by 86% and only 8 cells were lysed. In the same way, phycoremediation of mixture of azo
dyes with living algal strain PKS33 reduced toxicity by 81%. Generally, it was observed that
phycoremediation of mixture of azo dyes using living mass had a positive effect on reducing
the toxicity.
4.7.2. Toxicity test of real textile wastewater after decolorization by algal strains
4.7.2.1. Toxicity analysis: Case study I
Toxicity analysis of wastewater sample (collected from Shaheen Cloth Processing Mill
Faisalabad) after treatment was performed by measuring the hemolytic activity (Figure 4.27).
The untreated wastewater (control) caused a negative impact on human red blood cells
(RBC) as it lysed 72 RBCs out of 100. The phycoremediation with dead algal mass (CKW1)
reduced the toxicity and 39 cells were lysed. Similarly, with the phycoremediation of
wastewater with dead algal mass of strain PKS33 caused lysis of 34 cells. Phycoremediation
of wastewater with living algal mass (strain CKW1) reduced 74% toxicity as only 12 cells
were lysed. Similarly, phycoremediation of wastewater with living algal strain PKS33 caused
58% reduction in toxicity. Generally, the phycoremediation of wastewater reduced the
toxicity.
72
Fig. 4.26: Hemolytic activity of mixture of disperse, reactive and direct azo dyes after treated with selected strains of algae.
73
Fig. 4.27. Hemolytic activity of real textile wastewater collected from Shaheen Cloth Processing Mills after treated with selected strains of algae
74
4.7.2.2. Toxicity analysis: Case study II
Toxicity analysis (hemolytic activity) of wastewater (collected from Qadafi textile) treated
with selected algal strain was carried out. Results indicated that the application of real
wastewater (control) caused lysis of 57 RBCs (out of 100) (Fig 4.28), whereas 26 and 28
cells were lysed with the application of treated water with strain CKW1 and PKS33 (dead
mass), respectively. Similarly, the wastewater treated with living biomass of CKW1 and
PKS33 lysed 12 and 8 cells, respectively.
4.7.2.3. Toxicity analysis: Case study III
The data regarding toxicity analysis of treated real wastewater collected from Dawood textile
mills Faisalabad is summarized in Figure 4.29. Sixty eight RBCs (out of 100) were lysed by
the application of real wastewater. Algal strains reduced the toxicity of wastewater. The
lowest toxicity was observed in wastewater treated with strain CWK1 and only 12 RBCs
were lysed. The wastewater treated with dead mass of CKW1 caused lysis of 34 RBCs,
whereas dead mass of strain PKS33 caused lysis of 28 RBCs.
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Fig. 4.28. Hemolytic activity of real textile wastewater collected from Qadafi Textiles after treated with selected strains of algae.
76
Fig. 4.29. Hemolytic activity of real textile wastewater collected from Dawood Textiles after treated with selected strains of algae.
77
4.8. Use of phyco-remediated wastewater for irrigation purpose
A study was conducted under axenic conditions to use phyco-remediated wastewater
as irrigation for the growth of wheat plants. Data presented in Table 4.2 clearly revealed that
irrigation with real textile effluent negatively affected the root and shoot length of wheat
while the phycoremediated wastewater enhanced the root and shoots length. The plants
receiving treated wastewater (PKS33 dead mass) increased root and shoot length up to 60
and 70% respectively as compared to untreated real textile wastewater. Similarly, CKW1
(dead mass) treated wastewater increased root and shoot length by 62 and 56% as compared
to untreated real textile wastewater. The irrigation with wastewater treated with CKW1
(living biomass) improved root and shoot length by 83 and 95% respectively as compared to
real textile effluent. Similarly, the wastewater treated with PKS33 (living biomass) improved
the root and shoot length by 80 and 90% respectively as compared to untreated real textile
effluent.
Application of real textile wastewater suppressed root/shoot weight and plant biomass
compared with phyco-remediated wastewater (Table 4.2). Plant biomass was 30 to 34% more
in case of irrigation with wastewater treated with CKW1 and PKS33 (dead mass) compared
to real textile wastewater irrigation. However, the application of wastewater treated with
living biomass of CKW1 resulted in 89% greater biomass than that observed with untreated
real textile wastewater. Similarly, the wastewater treated with PKS33 (living biomass)
improved plant biomass by 82% compared to real textile wastewater irrigation.
Overall, it was observed that the application of real textile wastewater influenced
negatively on the wheat growth parameters. The application of algae treated wastewater
reduced the negative effect. The living cells had more pronounced effect than dead biomass
of algae.
78
Table 4.2. Impact of real and phycoremediated wastewater on wheat growth under axenic conditions
Root Length (cm) Shoot Length (cm) Root Weight (g) Shoot Weight (g) Biomass (g)
Wastewater 8.0 c 13.0 c 0.09 c 0.19 c 0.28 c
Treated PKS33 (Dead) 12.8 b 22.0 b 0.16 b 0.22 b 0.38 b
Treated CKW1 (Dead) 13.0 ab 20.3 b 0.15 b 0.21 b 0.36 b
Treated PKS33 14.1 ab 24.7 a 0.20 a 0.31 a 0.51 a
Treated CKW1 14.8 a 25.4 a 0.22 a 0.32 a 0.54 a
4.9. Biodiesel production from algal biomass
79
The algal biomass obtained after phyco-remediation of wastewater was then used to extract
biodiesel. Algal biomass produced by growing cells on modified MA medium was also run
for comparison. The extracted oil was converted into biodiesel by trans-esterification. The
results are discussed in the following sections.
4.9.1. Algal biomass
The growth of algae was faster on modified MA medium than the algae grown on real textile
wastewater (Fig. 4.30). The algal biomass (strain CKW1) on modified MA medium was
18%, 44%, 76% and 106% more after 2, 3, 4 and 5 days, respectively, than the algal biomass
on real textile wastewater. Similarly, strain PKS33 grown on modified MA medium showed
24% more biomass after 2 days, 56% more after 3 days, 94% more after 4 days and 146%
more after 5 days. When these strains were grown on real textile wastewater, strain CKW1
produced 82% more biomass after 5 days, while PKS33 produced 100% more biomass after
5 days compared to real textile wastewater. Overall, both the algal strains grown on real
textile wastewater had less growth than that observed on modified MA medium.
4.9.2. Biodiesel production
It was found that algal strain CKW1 grown on real textile wastewater (phycoremediated
algae) produced almost 34% less biodiesel compared to CKW1 grown on normal culture
medium. Similarly, algal strain PKS33 (phycoremediated water) produced 37% less biodiesel
ompared to CKW1 grown on normal culture medium. In general, it was observed that CKW1
had the ability to produce 10% more biodiesel than PKS33 from the same amount of biomass
under both normal and phycoremediated conditions.
4.9.3. Biomass recovery after oil extraction
The data presented in Fig. 4.32 clearly illustrated the biomass recovery of algal strains after
extraction of oil for biodiesel production. Algal strain CKW1 grown on real textile
wastewater (phycoremediated algae) recovered 60% more biomass (after oil extraction) than
CKW1 grown on normal culture medium from t he same amount of algal biomass. Similarly,
biomass recovery was 50% more in case of algal strain PKS33 (phycoremediated water) than
80
strain CKW1 grown on normal culture medium. In general, biomass recovery was 20% more
in the case of PKS33 than CKW1 in normal culture medium and 11% more under
phycoremediated conditions.
81
Fig. 4.30. Growth of selected algal strains on phycoremediated real textile wastewater and normal culture medium over a period of 5 days.
82
Fig. 4.31. Biodiesel production from alglal biomass produced by using real textile wastewater (phycoremediated) and normal culture medium.
83
Fig. 4.32. Comparative biomass recovery (after oil extraction) in case of algae grown on phycoremediated real textile wastewater vs. normal culture medium.
84
DISCUSSION
The results of this study demonstrated that some algal strains isolated through
enrichment of liquid media with Reactive Blue azo dye were able to decolorize synthetic
dyes that are commonly used by textile industry. Different algal strains showed a variable
potential for decolorization of dyes. In this study, selected strains CKW1 (Spirogyra sp.) and
PKS33 (Cladophora sp.) were capable of decolorizing azo dyes efficiently in liquid medium.
These strains were isolated from textile wastewater and saline water. The results also imply
that a saline dye-contaminated wastewater also contain algae that are able to decolorize azo
dyes in the presence of salts, most likely due to direct exposure and acclimatization to grow
under such conditions. Consequently, these algal strains could perform decolorization
activities efficiently in liquid medium containing dyes and high salt concentration. Various
salts are added in dye baths to improve the dying efficiency. About 40-100 g L-1 NaCl or
NaNO3 is used in fabric dying process (Carliell et al., 1998). Previously, a very few studies
indicated that algae had a potential to decolorize azo dyes (Jinqi and Houtian, 1992; Ogugbue
and Oranusi, 2005).
In this study, it was also observed that the decolorization of Reactive Blue dye was
concentration-dependent. Maximum decolorization of the dye by both algal strains was
observed at 100 mg dye L-1 liquid medium. Decolorization of both strains reduced when the
concentration of dyes increased above 100 mg L-1 liquid medium. High substrate (dye)
concentrations are probably toxic to algae, inhibiting degradation of the dye. Previous
investigations show that the dye concentration can affect the rate of biodegradation of dyes
and the optimum dye level could also vary from species to species. In general, high color
removal efficiencies have been observed at medium dye concentrations (Rajaguru et al.,
85
2000; Kapdan and Oztekin, 2003; Sponza and Isik, 2005; Khalid et a l., 2008 a, b).
Furthermore, some azo dyes contain one or more sulphonic-acid groups on aromatic rings,
which can act as a deterrent to inhibit the growth of microorganisms (Chen et al., 2003).
Another reason of the toxicity at higher concentration could be the presence of heavy metals
(metal-complex dyes) and/or the presence of non-hydrolyzed reactive groups in case of
reactive dyes (Sponza and Isik, 2005). The addition of nitrogen and phosphorus in the liquid
medium supported the biodegradation reaction. It is very likely that both nutrients improve
biomass production of algae (Kassim, 2002; Aslan and Kapdan, 2006), resulting in greater
removal of dyes by algae.
An increase in pH from 5 to 7 caused significant increase in the amount of dye
decolorization by algae, however decolorization rate was highest at pH ranging from 7 to 8.
The pH 7-8 was proved to be the best pH for all selected isolates and maximum
decolorization was observed in case of CKW1 and PKS33 at this pH. Most likely pH affects
the enzymatic activity involved in decolorization of dye in addition to cellular growth of
algae. Prasad et al. (2011) reported that biological treatment can effectively decolorize azo
dyes over a wide range of pH (6-9). However, optimum pH for growth and decolorization
was found to be 8 because maximum decolorization (90%) was recorded at this pH.
Maximum dye decolorization by the selected algal strains was observed at 35 °C and further
increase in temperature beyond 35°C inhibited decolorization of Reactive Blue. High
temperature probably caused thermal deactivation of algal enzyme(s) responsible for
decolorization of azo dyes. Previously, Guo et al. (2008) reported that 28 to 35°C may be an
optimal temperature for the decolorization of dyes.
86
Both algal strains (CKW1 and PKS33) demonstrated phycoremediation potential for
removal of structurally different azo dyes individually as well as in mixture from dye
medium. Dye decolorization rate was greater in the case of individual dyes than that
observed with the dye mixture. The amount of dye decolorization increased with the increase
in incubation time and complete color removal was observed after 96-120 h in the case of
most of dyes having different structures. Algae grow gradually over a period of time to
achieve high biomass content which results in more phycoremdiation certain time. Our
findings also confirm the previous findings that an increase in algal biomass also increases
phycoremediation (Jinqi and Houtian, 1992; La Rossa, 2009). The acclimation of microbes to
a variety of substrates has been widely reported by various researchers (Mohan et al., 2002;
Mohan et al., 2004).
The selected algal strains were also effective for phycoremediation after
bioaugmentation into real textile wastewater. In some cases, complete color removal was
observed in response to bioaugmentation by CKW1 and 90% decolorization by strain PKS33
within 120 h. The ability of algae to utilize wastewater efficiently as nutrient source provides
a favorable environment to ensure long-term survival of the algae in wastewater streams that
contain adequate levels of azo dyes to support the growth. Some researchers have reported
phycoremediation of leather industry (Rao et al., 2011), carpet mills effluent (Chinnasamy et
al., 2010), pulp and paper industry (Tarlan et al., 2002), dairy manure effluent (Mulbry et al.,
2008) and municipal effluent (Arora and Sexena, 2005).
Dead algal mass was able to decolorize most of the wastewater in its first 12-24 h,
and thereafter color removal was very slow. In contrast, the living strains have shown a
gradual increase in the dye decolorization and complete color of the wastewater was
87
observed after 96-120 h. Initially, only substrate is sorbed, resulting in negligible
decolorization of dyes. Sorption by dead mass was reported by Rosu et al. (2008).
The results of present study also reveal positive effect of phycoremediation of
wastewater in reducing the dyes toxicity. Increase in dye decolorization by algal strains
decreased the toxicity of wastewater, probably due to reduction in COD and BOD in addition
to color removal. This premise is well supported by Paramesawary et al. (2010) who reported
algae had the ability to reduce COD and BOD of wastewater. The phycoremediated
wastewater was applied to irrigate wheat plant grown under axenic conditions. Application of
real textile wastewater suppressed the wheat growth. The reduction in growth was possibly
due to high BOD and COD of textile effluent and the presence of toxic substances including
dyes (Robinson et al., 2001; Zalawadia and Raman, 1994; Pathak et al., 1999). Jadhav and
Savant (1975) reported negative growth responses because of increasing soil salinity by using
distillery and textile effluent. Inhibitory effects were prominent at higher concentration of
effluent in winter vegetables (Rehman et al., 2009; Kumar et al., 2006). The growth of wheat
was also improved by the application of wastewater treated with dead algal mass compared
to real textile wastewater, however, the results were more promising in the case of living
cells than dead biomass.
In this study, algal biomass produced after phycoremediation of dye containing textile
wastewater was used for biodiesel production. It was observed that both algal strains grown
on real textile wastewater had a lower growth compared to the growth of algae grown in
modified MA medium. In fact, the presence of adequate nutrition in the modified MA
medium supported the algal growth better than real wastewater. Usually, algae growth is
slower in wastewater than that observed under normal conditions (Ozkan et al., 2012). The
88
biodiesel production was also 30% less in the case of algal biomass produced after
phycoremediation than the algal strains biomass produced on normal culture medium. This
might be due to metabolic changes caused by wastewater in algae, affecting lipid contents
and biodiesel production (Woertz, 2007; Baxter, 2012). This premise is also supported by our
findings that the biomass recovery after oil extraction was 20% greater in case of strain
PKS33 than the same amount of biomass produced by strain CKW1 under normal culture
medium and 11% more biomass recovered under phycoremediated conditions.
89
SUMMARY
Textile effluents contain a large proportion of the azo dyes which are used in large
quantities in the textile industry because of the ease in synthesis and low-cost as compared
with natural dyes. Azo dye contaminated water represents a serious environmental problem.
Azo dyes also pose a potential danger of bioaccumulation that may eventually affect human
health by transport through the food chain. In Pakistan, the problem is getting worse due to
the direct release (without treatment) of textile effluent in wastewater streams. There is an
urgent need for effective technology for the treatment of wastewater containing azo dyes for
environmental protection.
In the present study, several water samples were collected from the outlets of various
textile industries of Faisalabad, and from sources of freshwater and saltwater. These samples
were analyzed for pH and TSS. A wide variation in the pH of different water samples was
recorded. The pH in the case of textile wastewater was as high as 12.4. Maximum amount of
TSS in textile wastewater, fresh and saline water was 90, 53 and 150 meq L-1, respectively.
To isolate efficient azo dye-degrading strains of algae, 20 isolates (out of 88) were
obtained using the azo dye Reactive Blue as the sole source of C and N in a modified MA
medium. Based on the dye decolorization capacity of algae, two strains (CKW1 and PKS33)
capable of degrading azo dyes efficiently in liquid medium were selected for further studies.
These strains were able to degrade synthetic textile dyes effectively in liquid medium at a
dye concentration of 100 mg L-1, pH 8, and 16 h of day light at 30 oC. These strains could
completely decolorize different groups (reactive, direct and disperse) of azo dyes, both
individually as well as in mixture within 120 h of incubation time. However, the
decolorization rate was slow in case of dyes mixture. The living algae cells showed more
90
efficient decolorization of both single dye and mixture of dyes in comparison with algae dead
biomass. Both strains of algae were also able to completely decolorize the real textile effluent
within 120 h while dead biomass decolorized up to 80% dye in 120 h.
Toxicity analysis of real textile wastewater revealed 70-80% reduction in toxicity
after treatment with live algae while 30% reduction after treatment with dead biomass. Wheat
growth was also better in the case of irrigation with treated wastewater than untreated water.
Moreover, the wastewater treated with living algae strains increased wheat growth by almost
100% as compared to the untreated wastewater irrigation. Similarly, up to 50 increases in
wheat growth was observed in the case of wastewater treated with dead algal biomass. Algal
biomass obtained after phycoremediation produced a biodiesel 30-35% less compared to the
same strain of algae grown in modified MA medium.
CONCLUDING REMARKS AND FUTURE PROSPECTIVE
Algae could be used to develop a biological treatment system to address the problem
of azo dyes in wastewater. In this thesis, for the first time a variety of azo dyes individually
and in various combinations were subjected to decolorization by selected strains of algae.
Two strains (PKS33 and CKW1) showed the ability to decolorize a broad variety of azo dyes
and real textile wastewater. This implies that the use of such algae isolates in biological
treatment systems could be helpful in reducing the threat posed by these pollutants in
wastewater from the textile industry. Furthermore, the application of phycoremediated
wastewater to plants will not only help reduce the threat of hazardous materials entering the
food chain, but also increase the reuse of textile wastewater for obtaining better crop
production. Algal biomass production using textile wastewater could possibly help tackle
91
fuel shortage problem which recently severely affected Pakistan. In general, the algae could
be potential candidates for the treatment of textile wastewater, so it is useful for the
production of biomass that could be used for the production of biofuel.
92
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