Mapping of Research Outcome on Remediation of Dyes, Dye Intermediates and Textile Industrial Waste a Research Compendium

Mapping of Research Outcome on Remediation of Dyes, Dye Intermediates and Textile Industrial Waste A Research Compendium

Department of Biotechnology Ministry of Science & Technology Government of India

Mapping of Research Outcome on Remediation of Dyes, Dye Intermediates and Textile Industrial Waste A Research Compendium

SARDAR PATEL UNIVERSITY VALLABH VIDYANAGAR

Department of Biotechnology Ministry of Science & Technology Government of India New Delhi

Mapping of Research Outcome on Remediation of Dyes, Dye Intermediates and Textile Industrial Waste

Compiled By: Datta Madamwar, Onkar Tiwari, Kunal Jain

Edition : First Edition June 2019

Published By : Sardar Patel University Vallabh Vidyanagar

Department of Biotechnology Ministry of Science and Technology Government of India New Delhi

Available At : Bhikaka Library Sardar Patel University Vallabh Vidyanagar

Printed By : University Press Sardar Patel University Vallabh Vidyanagar

PREFACE

It has been more than a couple of decades since Government of India through Department of Biotechnology, Ministry of Science and Technology and other agencies such as DST, MoEFCC, CSIR, UGC etc., providing huge grants to various researchers to develop treatment system for remediation of damaged environment. The trajectory growth of industrialization, urbanization and steep increases in domestic effluent load and other anthropogenic activities have caused heavy environmental stress in India. Anthropogenic activities over the past centuries have released the compounds of xenobiotic origin in various quantities in open environment and are the major cause of ground and surface water pollution.

Widespread use and production of dyes, dye intermediates have left legacy of pollution. Hence, it become inevitable to develop technologies for the treatment of industrial effluents in order to reduce the impact of pollution on various sites in the vicinities of industrial estates and biological methods continue to offer and alternative to engineering and chemical methods of treating such pollution. Restoration of damaged ecosystems is given priority in environmental sciences and technology.

This compendium intends to cover in consistent way the current methodologies to most innovative and systematic advances in treatment of dyes, dye intermediates manufacturing and textile processing industrial effluents.

Initially it provided comprehensive of the current methodologies, applications and challenges to the treatment of industrial effluents discharged by dyes and dye intermediates and textile processing industries, with plethora of new technologies available.

Datta Madamwar Onkar Tiwari Kunal Jain

Acknowledgement

In releasing the compendium on “Mapping of research outcome on remediation of dyes, dye intermediates and textile industrial waste”, authors owe a sense of gratitude to all the individuals who have generously assisted and supported during the course of preparation of compendium.

The inspiration for the need of reviewing the current status on remediation technology developed for dye and textile industry wastewater was provided by Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India, New Delhi, by providing financial support and motivation for this compendium which was well anchored by Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar.

We foremost thank all the members of Task Force on ‘Environmental Biotechnology’ of DBT, New Delhi for their appreciative remarks and guidance provided during compiling the compendium. Authors are thankful to Dr. Renu Swarup, Secretary, Department of Biotechnology, New Delhi for her persistent support. We equally acknowledge Prof. Shirish Kulkarni, Hon’ble Vice-Chancellor, Sardar Patel University, Vallabh Vidyanagar for his support. We also acknowledge Dr. Mohd. Aslam, Advisor and Group Head of DBT Environmental Biotechnology Programme for his valuable suggestions and support.

Authors are indebted to Dr. Tapan Chakrabarti, Former Chairman, Dr. C. R. Babu, current Chairman, Task Force on ‘Environmental Biotechnology’ of DBT, New Delhi for their advice and suggestions provided during the preparation of compendium. Dr. K. S. Charak, Scientist G, Former Adviser and Dr. Mohd. Aslam, Scientist G and Adviser DBT, New Delhi is greatly being acknowledged for their valuable suggestions and support.

Authors are gratified and expressed their sincere thanks to Dr. Hemant Purohit and Dr. Atya Kapley from NEERI, Nagpur; Prof. S. P. Govindwar and Prof. Jyoti Jadhav from Shivaji University, Kolhapur; Prof. Prince Sharma, Punjab University, Chandigarh; Prof. H. S. Saini, GNDU, Amritsar; Prof. S. R. Dave , Gujarat University, Ahmedabad and Dr. Venkat Mohan, IICT, Hyderabad who have gratuitously agreed and shared their research work, experience, suggestions and feedback required for the compendium.

Financial support for DBT through grant number BT/EnvBC/01/2014 dated 11-11-2015 is also gratefully acknowledgement.

Datta Madamwar Onkar Tiwari Kunal Jain

Contents

No. Item Page No. i Preface ii Forward, Secretary, DBT iii Forward Vice-Chancellor, SPU iv Forward, Mohd. Aslam v Acknowledgement 01 Introduction 01 02 Need of this compendium 04 03 Steps involved in textile processing 07 04 Management of wastes, rules and laws for environment 24 protection 05 Methods evolved for removal of dyes 30 06 History of bioremediation 36 07 Report on projects supported by DBT in the area of dye 41 bioremediation 08 Report on projects supported by other national agencies in the 55 area of dye bioremediation 09 National and international case studies 63 10 Relevant Statistics 77 11 Textile clusters of India and their waste management 83 practices 12 Textile waste management practices in other countries 98 13 Patent Analysis 103 14 Work of some Indian researchers 112 15 Suggestions and Recommendations 146 16 References 155 17 Appendices 169

Introduction  01 Introduction

1.1 Opening Remarks 02 1.2 The Problem 03 1.3 Indian Senario 03

Introduction

1.1 Opening Remarks

From the ages of the foot print of first human being, the planet earth has changed both radically and resourcefully. As it is said ‘Necessecity is the mother of invention’, they have always astonished themself since the first invention of ‘Wheel’. And the discovery of fire has provided the lethal (sic) combination for galaxies of ‘discoveries and inventions’ which have paved the way for modern earth. The nature has endowed human being with such an amazing inquisitiveness, today looking back to the time of billions of years ago even mother earth would find difficult to recognize herself due to the changes we brought within few hundred of years of time. Since the time of modern industrialization during twentieth century and coincided with the early phase of human population rise, it marks a major turning point in human history. The revolution began an era of per-capita economic growth and has completely changed the living style and raised socio-economic standard of human. This unprecedented population rise and urbanization has brought along with it an ever increasing industrial appetite that has resulted in enormous amount of waste generation for which we are unprepared. The early excitement of industrialization has now compelled us to think about developing environmental remediation strategies on priority basis to save the basic essential components of life. The deterioration of pristine environment has directly or indirectly affected human health. This has aggravated the need for understanding the impact of recalcitrant pollutants in an ecosystem. Moreover, the problem has become worse with the pollution of potable water which bears the most and sever impact of all components of habitable ecosystem. Our ancestors were amused with the shades of colour of nature. Ever since primitive people could create, they have been endeavouring to add colour to the world around them. They used natural materials to stain hides, decorate shells and feathers and paint their story on the walls of ancient caves. We can able to date the pigments (black, yellow, reddish, white) made from ochre used by primitive man in cave painting to over 15,000 BC. The historic use of dye was evidently of natural and organic origin. It was of plant extract as the Indigo from Dyer's wood (Tinctoria isatis), the red alizarin from Madder (Rubia tinctorum), also from insects (Persian scarlet), mollusks (Tyrian purple), few fungi and lichens. These natural resources were more native from the regions where they have used for dyeing processes. Until sixteen centaury dyeing procedure were not known globally, but later on techniques developed and skilled by Egyptian was remained in use until without much improvement took place in nineteen centaury. The earliest evidence of dyeing in India comes from Indus civilization (2300 – 1750 BC), when a piece of cotton at Mohen-jo-daro dyed with madder (a vegetable dye) was found. Archaeologist found various pigments, yellow, red, blue, white, green and black in paintings at Ajanta and Bagh caves. Paintings also depict men wearing colour garments which indicate the advancement made by dyeing industries in India. In 1630 Mr. Higginson of Salem from North Carolina noted for local vegetation based dye “here to be divers roots and berries wherewith the Indians dye excellent holiday colours that no rain or washing can alter”. Since the first introduction of synthetic dye in mid nineteenth centaury, colourant industry was well developed in European and other markets. However, with the environmental concern and strict legislation, in last few decades there has been a notable transition in global production of dyes, production base from Europe, USA (i.e. west) were shifted towards east and Asia has emerged as manufacturing hub of dyestuff production.

Introduction

1.2 The Problem Considering the volume and composition of the wastewater industrial effluents, dyeing (both manufacturing and application) industry is rated as one of the foremost industrial sectors that pollutes environment. It is actually difficult to estimate the actual global production and consumption of dyes, but it is estimated that worldwide production of dyes and pigments would reach 9.0 million tons by volume of US$ 30.0 billion by the 2020. However, according to one estimates there was an annual production of >7.5 x 105 metric tons of different dyes and nearly 280,000 tons (i.e. 2-50%) of textiles dyes are discharged into the effluents. To dye 1 kg of cotton, it requires 70-150 L water, 30-60 g dyestuff, 0.6-0.8 kg NaCl, which at the end of the process generates nearly 20-30% of applied unfixed dyes at a concentration of 2000 ppm along with high salt content and other auxiliary compounds. The raw materials used for the manufacturing of dyes and dye intermediates are benzene, toluene, xylene and naphthalene along with certain heavy metals. The dyeing industry uses different dyes, fastening and binding agents, soda-ash, caustic soda, organic sequestering agents, and other accessory chemicals depending of the dyeing method and fabric use for dyeing. Therefore, industrial effluent containing wastewater from the textile industry is a complex mixture of many xenobiotic substances ranging from dye and dye intermediates, organochlorine- based pesticides to heavy metals associated with dyes and the dyeing process. They are typically characterized by residual color, alkaline pH (mostly), excess TDS content, high COD and relatively low BOD values. In addition to the common chemical constituents, textile waste also contains a host of auxiliary chemicals. Thus, the wastewater, upon discharging into the water bodies without any adequate treatment, affects the multi segments of the environment leading to irreversible persistent changes.

1.3 Indian Scenario The indigenous setup of dye industries was started in the year 1940 and today India has emerged as second biggest exporter of dyestuff in the global market. India currently shares around 12.5 % of the global market in colourant industries (out of which 60 % are exported), having production capacity of more than 200,000 tonnes per annum, with an estimated value of US$ 3.4 billion (for the year 2010). More than 95 % of the domestic requirement was met by indigenous industries, out of which textile industry consumes nearly 60 % and the remaining is shared by paper, leather & other consumer industries. The major route by which dyes enters the environment is via industrial effluent. The wastewater contains different dye classes depending upon its usage and to know the relative share of each dye class in the effluent, dye consumption data should be considered together with the degree of fixation of the different dye classes. It can be observe that degree of dye fixation on for reactive dyes are lowest while basic dyes have higher affinity for its substrates. It is estimated that about 800 mg/l of hydrolysed reactive dye remains in the dye bath after dyeing process. Several modifications in dyeing process are being made to increase the rate of fixation of reactive dyes; still degree of fixation varies.

Need of this compendium  02

Need for this compendium

2.1 Need of this compendium 05 2.2 Environemntal problem 05 2.3 Indian Senario 06 2.4 Scope of the Compendium 06

Need of this compendium

2.1 Need for this Compendium

Since the first introduction of synthetic dye in mid nineteenth centaury, colourant industry was well developed in European and other markets. However, with the environmental concern and strict legislation, in last few decades there has been a notable transition in global production of dyes, production base from Europe, USA (i.e. west) were shifted towards east and Asia has emerged as manufacturing hub of dyestuff production. Dyes and pigments are primarily used in the production of consumer products (like textiles, printing inks, paints, plastics, papers, plastics, foods, etc.) for adding the colour and patterns of materials. The technological evolution during 19th centaury have lead to the gradual shift from Natural dyes (which have been used since 3500 B.C. from extracts of vegetables, fruits and flowers) to synthetic chemical dyes that provides richer colour which retain on the materials for longer period throughout wash and exposure.

2.2 Environmental Problem As a matter of fact, it is difficult to estimate the actual global production and consumption of dyes, but it is estimated that worldwide production of dyes and pigments would reach 9.0 million tons by volume of US$ 24.2 billion by the 2025 (Global Industry Analysts). However, according to one estimates there was an annual production of >7.5 x 105 metric tons of different dyes and nearly 280,000 tons (i.e. 2-50%) of textiles dyes are discharged into the effluents. Because of the infinite structural variation there exists a vast pool of synthetic dyes, each of them having its own individual production process. But, benzene/naphthalene, toluene/xylene, sulphuric acid, metallic elements (such as chromium, copper) is commonly used throughout the dye manufacturing and application industry (where dyes are synthesized in a reactor, mixed, filtered for impurities, dried and finally blended). The textile industry alone consumes more than 3,600 different types of chemical dyes. According to the Pure Earth (Blacksmith Institute), USA* and Green Cross, Switzerland* reports on of 2016, “The World’s Worst Pollution Problems 2016: The Toxics Beneath Our Feet” dye and textile industries are ranked Tenth and accounts for 220,000–430,000 Disability-Adjusted Life Years (DALYs) in low- and middle-income countries (including India). Along the way of production and application of dyes, many other accessory chemicals for fastening and binding agents, soda-ash, caustic soda, organic sequestering agents, additives, solvents are added as a catalyst for reactions. For example, in textile industry, the number and variety of chemicals used is directly correlates with the intricacy of the designs and desired patterns required in the clothing. Their manufacturing generates large amounts of vapours during dyeing, printing and curing of dye or colour pigments. Dust emission is associated with fiber processing. Besides these, the textile mills also use wood, coal or oil fired boilers and thermic fluid heaters which becomes point emission sources for pollutants. The unorganized small scale units disproportionately add to the problem of environmental pollution, besides the large organized production units. To dye 1 kg of cotton, it requires 70-150 L water, 30-60 g dyestuff, 0.6-0.8 kg NaCl, which at the end of the process generates nearly 20-30 % of applied unfixed dyes at a concentration of 2000 ppm along with high salt content and other auxiliary compounds. To an estimate nearly 9 trillion gallons of water is used for cooling, cleaning, rinsing and processing of dyes and textile, besides its use during the production of dye itself. This massive use of water is in fact a key component of pollution. 5

Need of this compendium

Considering the volume and composition of the wastewater industrial effluents, dyeing (both manufacturing and application) industry is rated as one of the foremost industrial sectors that pollutes environment. The complexity and heterogeneity of the textile effluents is because of high variation in chemical use, which is closely tied to the continuous and constantly evolving high demands for variable patterns and unique colors for clothing and other textiles. The Pure Earth’s database propose that, chromium, lead, cadmium, sulphur, nitrates, chlorine compounds, arsenic, mercury, nickel and cobalt are few of the top pollutants arise from dye industries. The textile industrial effluent are typically characterized by residual color, alkaline pH (mostly), excess TDS content, high COD and relatively low BOD values. Therefore, the wastewater effluents of dye industries pose a direct, serious and complex threat to the environment and health of the surrounding populations. 2.3 Indian Scenario The indigenous setup of dye industries was started in the year 1940 and today India has emerged as second biggest exporter of dyestuff in the global market. India currently shares around 12.5 % of the global market in colourant industries (out of which 60 % are exported), having production capacity of more than 200,000 tonnes per annum, with an estimated value of US$ 3.4 billion (for the year 2010). More than 95 % of the domestic requirement was met by indigenous industries, out of which textile industry consumes nearly 60 % and the remaining is shared by paper, leather & other consumer industries. In legacy, during earlier days of pollution awareness, it was found that wastewater from dye industry is directly dumped into surface waters or surrounding natural fresh water streams without treatment. An observation made by World Bank noted that, dye and textile and industries contribute upto 17-20 % of total industrial water pollution. There are several general underlying reasons for dye related wastewater problem (particularly in India): • The foremost and key issue is, many dyeing facilities throughout the world are small (sometimes family-run) scale and is prohibitively expensive to afford the proper effluent treatment plant for every unit. • Another significant concern is poor practices for control of effluent, coupled often with lack of evolved technology for management and treatment of textile wastewaters • Oversight of regulation and legislation by the industries as well as implementing agencies for discharge of effluents • The presence of such industries close to or within densely human populated areas

2.4 Scope of the Compendium While the problem of industrial pollution by dyes, dye intermediates and pigments remains prevalent in country like India, different cost-effective solutions to treat and prevent the problem already exist. Governments are making important progress in dealing with the problem but further efforts are required to improve the quality of life for millions of people affected by toxic pollution. Alone government and efforts from handful of agencies/organizations are not sufficient. The challenges are two fold: (1) finding enough financial resources for more interventions and (2) to scale up the already effective approaches to extend the support and assistance to the million of individuals in the communities which are struggling with pollution menace, requires immediate attention. As it stands major bottle neck in pollution control in

Need of this compendium developing (as in India) and under- develop countries is lack of proper funding mechanism that can specially address pollution. With this backdrop, there cannot be a better opportunity (when the head of the state is insisting about “Swachh Bharat) to address the short-fall on technology for the treatment methodology for industrial wastes of dyes, dye intermediates, pigments and textiles being carried out in the country.

Steps involved in textile processing  03 Steps involved in textile processing

3.1 Opening remarks 08 3.2 Steps involved in textile processing 08 3.3 Type of textile waste product 09 3.4 Impact of textile effluents 13 3.5 Textile effluent disposal standard 20 3.6 Conclusion 22

Steps involved in textile processing

3.1 Opening Remarks To devise an effective wastewater remediation technology for textile effluents, it is important to understand the processes involved in textile processing. Because the quantity and quality of the effluent produced at the end of each steps varies depending upon the raw material and fabric used for dyeing. This would help to recognize the difficulty faced during devising the technology and the inefficiency of the selected methodology for the treatment.

3.2 Steps involved in textile processing In textile industry the transformation of raw cotton to final usable form involves wet processing of several stages as follows: 3.1.1 Sizing / Slashing This is the initial process which involves sizing of yarn with either with starch or polyvinyl alcohol (PVA) or carboxy methyl cellulose (CMC) to provide necessary tensile strength and smoothness required for weaving. During this stage requirement of water varies from 0.5 to 8.2 l/kg of yarn with an average of 4.35 l/kg. 3.1.2 Desizing The sizing components which are rendered water soluble during sizing are removed from the cloth to make it suitable for dyeing and further processing. This can be done either through acid (sulphuric acid) or with enzymes. The required water at this stage varies from 2.5 to 21 l/kg with an average of 11.75. 3.1.3 Scouring / Kiering This process involves removal of natural impurities such as greases, waxes, fats and other impurities. The desized cloth is subjected to scouring. This can be done either through conventional method (kier boiling) or through modern techniques (continuous scour). Kiering liquor is an alkaline solution containing caustic soda, soda ash, sodium silicate and sodium peroxide with small amount of detergent. The water required for this process varies from 20 – 45 l/kg with an average of 32.5. 3.1.4 Bleaching Bleaching removes the natural colouring materials and renders the cloths white. More often the bleaching agent used is alkaline hydrochloride or chlorine. For bleaching the good quality fibre, normally peroxide is used. The chemicals used in peroxide bleaching are sodium peroxide, caustic soda, sulphuric acid and certain soluble oils. The water and chemical requirement and the effluent generation normally vary based on the type of operation and the material (yarn / cloth) to be processed. Bleaching the yarn both through hypo-chloride and hydrogen peroxide methods require same quantity of water and it varies between 24 to 32 l/kg. But in the cloth bleaching, the water requirement is much higher and it fluctuates between 40-48 l/kg. 3.1.5 Mercerising The process of Mercerisation provides lustre, strength, dye affinity and abrasion resistance to fabrics. It is generally carried out for cotton fabrics only for easy dyeing. Mercerisation can be carried out through cold caustic soda solution followed by washing with water several times. The water required for this process varies from 17 to 32 l/kg, with an average of 24.5. 3.1.6 Dyeing Dyeing is the most complex step in wet processing which provides attractive colour for the product. Dyeing is carried out either at the fiber stage, or as yarn or as fabrics. For dyeing process,

Steps involved in textile processing hundreds of dyes and auxiliary chemicals are used. In brief, the water requirement for dyeing purpose (include all types and shades) varies from 36 – 176 l/kg with an average of 106. The effluent generation during dyeing process fluctuates from 35 to 175 l/kg with an average of 105 l/kg.

3.3 Type of textile waste product Textile industries are the one of the most water intensive industrial segments. The industry also uses variety of chemicals for its different manufacturing steps. Water is mainly used for (i) the application of chemicals onto textiles and (ii) rinsing the manufactured textiles. The amount of water required and consumed would vary from industry to industry, depending upon the type of fabrics produced and dyeing process employed. An average amount of water consumed by different fabrics is listed in Table 3.1 and the percentage of water required during the wet processes is as shown in Table 3.2.

Table 3.1: Average consumption of water during production and processing of various fabrics

Water Consumption (m3/ ton fibre material) Processing Subcategory Minimum Median Maximum Wool 111 285 659 Woven 5 114 508 Knit 2 84 377 Carpet 8.3 47 163 Stock/yarn 3.3 100 558 Non-woven 2.5 40 83 Felted fabric finishing 33 213 933

Table 3.2: Percentage of water consumed during the wet process

Process Water Consumed (%) Bleaching 38 Dyeing 16 Printing 8 Boiler 14 Other uses 24

Within textile industry, wet processes use variety of chemicals in large amount as well as water. It is estimated that to produce 1 kg of fabrics, it requires 80-150 m3 water and nearly 1,000-3,000 m3 of wastewater is generated after processing of between 12 and 20 tonnes of textiles per day. It can be seen from the Table 3.3 and Table 3.4, industries using cotton, consumes more water as compared to synthetic textiles.

Steps involved in textile processing

Table 3.3: Amount of water required by cotton textile industry for the wet process

Process Water Consumption (L/1000 kg of products) Sizing 500-8200 Desizing 2500-21000 Scouring 20000-45000 Bleaching 2500-25000 Mercerizing 17000-32000 Dyeing 10000-300000 Printing 8000-16000

Table 3.4: Water required by synthetic textile industry for its wet process

Process Water Requirements (L/1000 kg of products) Rayon Acetate Nylon Acrylic/ Polyester Modacrylic Scouring 17000- 25000- 50000- 50000- 25000- 34000 84000 67000 67000 42000 Salt bath 4000- - - - - 12000 Bleaching - 33000- - - - 50000 Dyeing 17000- 34000- 17000- 17000- 17000- 34000 50000 34000 34000 34000 Special finishing 4000- 24000- 32000- 40000- 8000- 12000 40000 48000 56000 12000

At the end of each stage of textile dyeing, large amount of wastewaters are generated containing unused dyes (in large quantity) and other accessory chemicals. Type of chemicals and quantity used during bleaching and dyeing (i.e. in wet processes) is listed in the Table 3.5. Due to inherent limitation of textile dyeing process there always that a portion of unfixed dyes washed and released along with wastewater.

Table 3.5: Type of chemicals used and amount required during wet process

Chemical Utilization (kg/100 kg of cloth)

Steps involved in textile processing

Soft Flow Machine Winch Wetting agent 0.5 0.5 Caustic soda 2.5 4.0 Peroxide 3.0 4.0 Lubricants 0.2 0.3 Stabilizers 0.2 0.3 Peroxide killer 1.0 1.0 (oxidizing agent) Acetic acid 2.0 2.0

Table 3.6: Proportion of different dyes remains unfixed on various textiles during dyeing process

Fibre Dye Type Unfixed Dye (%) Acid dyes/ reactive 7-20

Wool and nylon dyes for wool

Pre-metallised dyes 2-7 Azoic dyes 5-10 Reactive dyes 20-50

Direct dyes 5-20 Cotton and viscose Pigment 1 Vat dyes 5-20

Sulphur dyes 30-40 Polyester Disperse 8-20 Acrylic Modified basic 2-3

Therefore, textile wastewater always found to be rich in unfixed dyes (which pollutes the environment are difficult to treat), as shown in Table 3.6. As it can be seen the based on the dye class and the fiber to which they are applied wastewater contains un-used dyes ranging from 2 – 50 %.

The solid wastes generally are not found to be hazardous, are originated from dry process, but the wet process produce only a small amount of solid wastes. Solid wastes are either fabrics or packing materials, while from wet process, dried up sludge is a major source of solid waste (Table 3.7). Besides solid waste, textile industries also emit gaseous pollutants. In textile industry, boilers, ovens and storage tanks are the three major sources of air pollution (Table 3.8). The boilers generate sulphur oxides and nitrogen, while the drying process which requires high temperature emits hydrocarbons.

Steps involved in textile processing

Table 3.7: Types and sources of solid wastes generated after each stage of textile processing and manufacturing

Source Type of Solid Waste Mechanical operations of cotton and synthetics Yarn preparation Fibres and yarns Knitting Fibres and yarns Weaving Fibres, yarns and cloth scraps Dyeing and finishing of woven fabrics Sizing, desizing, mercerizing, beaching, Cloth scraps washing and chemical finishing Mechanical finishing Flock Dyeing/printing Dye containers Dyeing/printing (applied finish) Chemical containers Dyeing and finishing of knitted fabrics Cloth scraps, dye and chemical containers Dyeing and finishing of carpets Tufting Yarns and sweepings Selvage trim Selvage Fluff and shear Flock Dyeing, printing and finishing Dye and chemical containers

Table 3.8: Type and source of gaseous pollutants emitted after different stages of textile processing Process Source Pollutants Energy Emission from boiler Particulates, nitrous production oxides(Nox), sulphur dioxide (SO2) Coating, drying Emission from high Volatile organic and curing temperature ovens components (VOCs) Cotton handling Emission from preparation, Particulates activities carding, combing and fabrics manufacturing Sizing Emission from using sizing Nitrogen oxides, sulphur compound (gums, PVA) oxide, carbon monoxide Bleaching Emission from using chlorine Chlorine, chlorine compound dioxide Dyeing Disperse dyeing using Carriers carriers sulphur dyeing H2S Aniline dyeing Aniline vapours

Steps involved in textile processing

Printing Emission Hydrocarbons, ammonia Finishing Resin finishing heat setting of Formaldehydes synthetic fabrics Carriers – low molecular weight Polymers- lubricating oils Chemical storage Emission from storage tanks Volatile organic for commodity and chemicals components (VOCs) Waste water Emission from treatment Volatile organic treatment tanks and vessels components, toxic emissions

3.4 Impact of textile effluents The characteristics of textile effluents always vary from industries to industries (or within the industries) and depend of the textiles (fibers) which are being manufactures and chemicals used during the production. The textile effluents usually contains, suspended and dissolved solids, unused chemicals and dyes (colour), biological oxygen demand (BOD), chemical oxygen demand (COD) and odour. In many cases BOD/COD ratios are found to be near 1:4, which clearly indicates the presence of bio-recalcitrant, xenobiotic and non-biodegradable compounds in the effluents. Typical characteristics of textile effluent are as described in the Table 3.9 and the possible pollutants present in the effluents at the end of each stage of wet process are listed in the Table 3.10. It is not necessarily that values of each parameter would fall in the range described in the tables. These can change depending on the type of fiber manufactured in respect to market demands and process employed, viz. pH of the textile effluent can reach between (as low as) 2 and 3. Textile effluents also contain inorganic chemicals (and salts) like, sodium hydroxide, sodium sulphide, hydrochloric acid, sodium hypochlorite along with unused dyes.

Table 3.9: A typical characteristics of untreated textile effluents

Parameter Range pH 6-10 Temperature (°C) 35-45 BOD (mg/L) 80-6,000 COD (mg/L) 150-12,000 Total Suspended solids (mg/L) 15-8,000 Total Dissolved Solids (mg/L) 8,000-12,000 Chlorine (mg/L) 1,000-6,000 Free chlorine (mg/L) <10 Sodium (mg/L) 70% Trace elements (mg/L) Fe <10

Steps involved in textile processing

Zn <10 Cu <10 As <10 Ni <10 B <10 F <10 Mn <10 V <10 Hg <10

PO4 <10 Cn <10 Cr <10 Oil & grease (mg/L) 10-30 TNK (mg/L) 10-30

NO3-N (mg/L) <5 Free ammonia (mg/L) <10

SO4 (mg/L) 600-1000 Silica (mg/L) <15 Total Kjeldahl Nitrogen (mg/L) 70-80 Color (Pt-Co) 50-2,500

Table 3.10: Type of pollutants found after various stages of textile processing

Process Possible Pollutants Nature of Effluent Desizing Starch, glucose, PVA, Very small volume, high BOD resins, fats and waxes do (30-50% of total), PVA not exert a high BOD. Kiering Caustic soda, waxes, soda Very small, strongly alkaline, dark ash, sodium silicate and colour, high BOD values (30% of fragments of cloth. total) Bleaching Hypochlorite, chlorine, Small volume, strongly alkaline, caustic soda, hydrogen low BOD (5% of total) peroxide, acids. Mercerizing Caustic soda Small volume, strongly alkaline, low BOD (Less than 1% of total) Dyeing Dye stuff, mordant and Large volume, strongly coloured, reducing agents like fairly high BOD (6% of total) sulphides, acetic acids and soap

Steps involved in textile processing

Printing Dye, starch, gum oil, china Very small volume, oily clay, mordants, acids and appearances, fairly high BOD. metallic salts Finishing Traces of starch, tallow, Very small volume, less alkaline, salts, special finishes, etc. low BOD. PVA – Poly Vinyl Alcohol; BOD – Biological Oxygen Demand; COD – Chemical Oxygen Demand

The dye concentration in textile effluents usually ranges between 0.01 g/L and 0.25 g/L, depending on the type of the dye used and the process carried out for dyeing. Indigo dyes are used at a concentration of 0.02 g/L and Vat dyes are used at a concentration in the range of 0.05 to 0.1 g/L. Textile effluents also contains a large amount of organic compounds which are difficult to degrade and are resistant to aerobic degradation. Under anaerobic conditions these compounds are found to be reduced into toxic and carcinogenic intermediates. Few of the prominent carcinogenic compounds produced due to the degradation of azo dyes are as shown in Table 3.11. The possible pollution loads after different wet processes are as listed in Table 12 shows the pollution loads from wet processing plants. The pollution loads after processing various textiles are as shown in Tables 3.13-3.16.

Table 3.11: Toxic aromatic amines derivatives from azo dyes and their level of carcinogenicity

Aromatic Amine Group Degree of Human Carcinogenicity 1-Napthylamine Slight/Mixed 2-Napthylamine Good 2,5-Diaminotoluene Slight 3,3’-Dichlorobenzidine Slight/Mixed 3,3’-Dimethoxybenzidine Slight/Mixed 3,3’-Dimethylbenzidine Slight 4-Biphenylamine Good 4-Nitrobiphenyl Slight/Mixed 4,4`-Methylenebis (2-chloroaniline) Slight Auramine Slight Benzidine Good Magenta Slight N-Phenyl-2-napthylamine Slight N,N-Bis(2-chloroethyl)-napthylamine Good

Table 3.12: Characteristics of the effluent from various wet textile processing operations [123]

Source of effluent generation Parameters pH COD BOD

Steps involved in textile processing

(mg/L) (mg/L) Process Effluent Desizing 5.83-6.50 10000-15000 1700-5200 Scouring 10-13 1200-3300 260-400 Bleaching 8.5-9.6 150-500 50-100 Mercerizing 8-10 100-200 20-50 Dyeing 7-10 1000-3000 400-1200 Wash Effluent After beaching 8-9 50-100 10-20 After acid rinsing 6.5-7.6 120-250 25-50 After dyeing (hot wash) 7.5-8.5 300-500 100-200 After dyeing (acid & soap wash) 7.5-8.64 50-100 25-50 After dyeing (final wash) 7-7.8 25-50 Printing washing 8-9 250-450 115-150 Blanket washing of rotary printer 7-8 100-150 25-50

Table 3.13: Typical pollution loads from the processing of 100 % cotton

Process pH SEV BOD COD TSS TDS Oil & (L/kg) Gas kg per 1000 kg of product Desizing Enzyme starch 6-8 2.5-9 45.5 91 89 5 5 Acid starch 6-8 45.5 91 89.5 7.5 5 Polyvinyl alcohol 6-8 2.5 5 5 48 2.5 (PVA) Carboxymethyl 6-8 4 8 5 45 9.5 cellulose (CMC) Scouring Unmercerised 12.5 2.5-43 21.5 64.5 5 50 40 greige fabric Mercerised 12.5 16.5 49.5 5 50 30 greige fabric Mercerising Greige fabric 12 231- 13 39 5 148 10 306 Scoured fabric 12 4 12 5 148 - Bleached fabric 12 2 6 5 148 - Bleaching

Steps involved in textile processing

Hydrogen 9-12 2.5- 0.5 2 4 22 peroxide 124 Sodium 9-12 1 4 4 5 - hypochlorite Dyeing Fibre reactive HE 12 6 24 - 180 dyes (woven) Basic 6-7.5 149-300 Direct 6.5-7.6 14-53 Vats 5-10 8.3-166 Sulphur 8-10 24-212 SEV - Specific Effluent Volume

Steps involved in textile processing

Table 3.14: Pollution load from the processing of 50/50 cotton-polyester blend

Source Effluent pH COD BOD TS SS TDS (l/kg) kg per 1000 kg of product Desizing Starch 12.5 6-8 38.5 97 77 20 PVA 12.5 6-8 2.5 55.4 5 50.4 CMC 12.5 6-8 3.93 59.5 5 54.5 Mixture 4.2 74 78 Scouring Unmercerised 25 12 10.8 14.8 5 9.8 Mercerised 25 12 8.34 14.7 5 9.7 Bleaching Peroxide 16.7 1.3 24 4 20 Oxidative-desize-bleach Mercerising Poly/cotton 16.7 3.2 82 5 77 Dyeing Disperse-vat 42 12 68 22.8 122 122 Vat 100 150 Disperse 80 20

Steps involved in textile processing

Direct-disperse 6-8 32 10.7 114 Sulphur-disperse 11 68 22.8 69.7 Reactive-disperse 12 41 13.8 192 Printing Pigment (woven) 6-8 5 1.26 0.13 2.5 Pigment (knit) 6-8 5 1.26 0.13 2.5 Vat (woven) 10 86 21.5 25 34 Vat (knit) 10 86 21.5 25 35 Machine wash 100 Screen wash 7 Hose vessels 30 Pigment wash 12.5 1 3 0 3 Finishing Resin finishing 6-8 22 Resin finishing flat curling 6-8 25 6.32 17.3

Steps involved in textile processing

Table 3.15: Pollution load from the processing of wool

Process pH BOD TS Scouring 9-10.4 30,000-40,000 1,129-64,448 Dyeing 4.8-8 380-3,000 2,000-10,000 Washing 7.3-10.3 4,000-11,455 4,830-19,267 Neutralising 1.9-9 28 1,241-4,830 Bleaching 6 390 908

Table 3.16: Pollution load from the processing of synthetic fibres

Fabric BOD COD SS TDS Kg per 1000 kg product Rayon 30 52 55 100 Acetate 45 78 40 100 Nylon 45 78 30 100 Acrylic 125 216 87 100 Polyester 185 320 95 150

Through varies studies and case observations, human exposure to textile dyes are reported to cause headaches, irritations in lung and skin, congenital malformations and nausea. A study reported the evidence of liver, urinary balder and kidney cancers in workers after prolonged exposure to textile dyes, besides asthma, rhinitis, nasal problems and dermatitis. Benzidine, a known carcinogen is detected in textile effluent with disperse dyes such as disperse orange 37, disperse blue 373 and disperse violet 93 dyes. In a distinct study on sever different dyes (direct violet, direct congo red, cremazoles orange 3R, direct royal blue, cremazoles blue S1, direct Bordeaux, and cremazoles brown GR), the AMES showed the evidence of mutagenic compounds.

3.5 Textile effluent disposal standard Across the globe, different countries have different limits of the disposal of treated textile effluents in the open environment. Table 17 describes the regulation imposed by the several prominent countries where textile industries are one the major contributor in national economy.

Steps involved in textile processing

Table 3.17: Discharge limit of treated textile effluent in different countries Parameter CCME China BIS Hong FEPA Mexico Thailand Philippi Indones Banglad SL Kong nes ia esh pH 6.5-8.5 6-9 5.5-9 6-10 6-9 6-8.5 5-9 6-9 6-9 6.5-9 6-8.5 Temperature 30 - 50 43 40 - - 40 - 40-45 40 (°C) Colour (Pt-Co) 100 80 None 1(Lovi 7(Lovi - - 100-200 - - 30 bond) bond) TDS mg/L 2000 - 2100 - 2000 - 2000- 1200 - 2100 2100 5000 TSS mg/L 40 150 100 800 30 - 30-150 90 60 100 500 Sulphide μg/L 200 1000 2000 1000 200 - - - - 1000 2000 Free Chlorine 1000 - 1000 - 1000 - - 1000 - - - μg/L COD mg/L 80 200 250 2000 80 < 125 120-400 200-300 250 200 600 BOD5 mg/L 50 60 30 800 50 < 30 20-60 30-200 85 150 200 Oil & Grease - - 10 20 10 - 300 5-15 5 10 30 mg/L Dissolved 6000 - - ≥ 4000 - - - 1000- - 4500- - Oxygen μg/L 2000 8000 Nitrate μg/L 13000 - 10000 - 20000 10000 - - - 10000 45000 Ammonia μg/L 0.1 - - 500 0.2 - - - - 5000 60 Phosphate μg/L <4000 1000 5000 5000 5000 - - - 2000 - 2000 Calcium μg/L - - - - 200000 - - 200000 - - 240000 Magnesium 200000 - - - 200000 - - - - - 150000 μg/L Chromium μg/L 1 - 100 100 <100 50 500 50-500 500 2000 50 Aluminium μg/L 5 - - - <1000 5000 - - - - - Copper μg/L <1000 1000 3000 1000 <1000 1000 1000 1000 2000 500 3000

Steps involved in textile processing

Manganese μg/L 5 2000 2000 500 5.0 200 5000 1000- - 5000 500 5000 Iron μg/L 300 - 3000 1500 20000 1000 - 1000- 5000 2000 1000 20000 Zinc μg/L 30 5000 5000 600 <1000 10000 - 5000- 5000 5000 10000 0 10000 Mercury μg/L 0.026 - 0.01 1 0.05 - 5 5 - 10 1

3.6 Conclusion It is well know that dyeing and textile industries are one of the biggest polluters amongst all industrial segments. The quantity of wastewater generated after textile processing is enormous (nearly 200 L of water are used to produce 1kg of textile). With three different types of fabric (cellulose fiber, protein fiber and synthetic fiber) used in the textile industries the type and level of toxic compounds found in effluents varies considerably among the industries as noted above. By knowing the types and load of toxic compounds at the end of each cycle of textile processing, it would be advantageous to develop the wastewater treatment technology more specifically.

Management of wastes, rules & laws for environnemnt protection  04 Managements of Wastes, Rules and Laws for Environment Protection

4.1 Classification of wastes 24 4.2 General classifications of wastes 24 4.3 Indian scenario for hazardous wastes 26 4.4 Laws and Regulations 27

Management of wastes, rules & laws for environnemnt protection

4.1 Classification of wastes The Basel Convention by United Nation Environment Programme (UNEP) has defined wastes as “substances or objects, which are disposed of or are intended to be disposed of or are required to be disposed of by the provisions of national law”. Waste management has been always a perennial and predicament problem across the countries. It can broadly be comprises of identification/detection, segregation, collection, transportation, recycling, treatment and reprocessing and final disposal. Depending upon the type/kind of waste management, it has varying impacts on energy consumption, carbon storage, methane emission, ecological imbalance and human health. The study by World Health Organization (WHO) suggested that nearly a quarter of the diseases prevailing today are due to prolong exposure to environmental pollution. Many of these diseases were remained undetected either acquired during childhood or manifested in alter part of adulthood. Also it was observed that there are social implications of waste due to insufficiency

4.2 General classification of wastes In a broad way on the basic of their sources (domestic as well as industrial) waste can be classified under ‘urban’, ‘industrial’, ‘biomedical’ and e-waste (Figure 4.1), These are generated during extraction, manufacturing and processing of raw materials into intermediate and final products and their consumptions.

Figure 4.1: General classification of anthropogenic wastes

Industrial waste can be further classified as Hazardous, Non-Hazardous and Waste water, which are either by-products of manufacturing processes or unused/discarded commercial products. Hazardous wastes are identified into: characteristics waste, listed wastes, universal wasters and mixed wasters (Figure 4.2).

Management of wastes, rules & laws for environnemnt protection

Figure 4.2: General classification of industrial hazardous wastes

4.2.1 Listed Wastes Wastes that are described by Environmental Protection Agency (US) can be considered as hazardous and termed as Listed Wastes. This includes the F-list (wastes from common manufacturing and industrial processes), K-list (wastes from specific industries), P-list and U-list (wastes from commercial chemical products). The F-listed wastes originate from various sectors of industial sector; generally include wastes from non-specific sources, from common manufacturing and industrial process, viz. solvents. The K-listed wastes are source specific wastes, i.e. wastes from dye and textiles industries, petroleum refining or pesticides manufacturing, including sludge and wastewater from treatment process from such industry. The P-listed and U-listed wastes are discarded specific commercial chemical products in an unused form like certain pesticides and pharmaceutical products (when discarded in open environment become hazardous).

4.2.3 Characteristic Wastes Hazardous industrial wastes which are not specifically listed where they exhibits (i) Ignitability (e.g. used solvent and waste oils, which can spontaneously combustible at < 60ºC), (ii) Corrosivity (e.g. acids or bases, pH ≤ 2 or ≥ 12.5; capable of corroding metal containers and another example is battery acid), (iii) Reactivity (e.g. wastes which are unstable under ‘normal’

Management of wastes, rules & laws for environnemnt protection conditions, that can cause explosion when heated or compressed, shoot toxic fumes/gases, like lithium-sulphur batteries), (iv) Toxicity (e.g. wastes which are harmful or fatal when ingested or absorbed, waste containing mercury or lead, etc.)

4.2.4 Universal Wastes Waste under this category includes batteries, bulbs, pesticides i.e. which are of common in use across the planet

4.2.5 Mixed Wastes Waste that contains both radioactive and hazardous waste components.

4.3 Indian Scenario for Hazardous wastes In India, inventorization of industries generating hazardous wastes and their quantification are being done by the respective State Pollution Control Boards (SPCBs). Ministry of Environment & Forests (MoEF), Government of India, has categorized the industries on the basis of the severity of pollution from a specific industry, which are Red, Orange and Green in decreasing order of severity of pollution. Seventeen industries are notified and listed under ‘RED’ category, belonging to heavily polluting units and are covered under Central Action Plan. Dyes, pigments and intermediates related industries are classified into this category. Twenty-five types of industries are classified into ‘ORANGE’ category. Small scale industries and which are not included under ‘RED’ or ‘ORANGE’ are identified in ‘GREEN’ category.

4.3.1 Industrial Waste Water The liquid industrial waste generated from different sources can broadly be classified into four categories according to nature of pollutants present in the effluent.

(A) Organic pollution Industrial effluents generated from industries like tanneries, polymer processing units, dairies, distilleries, vegetable oil and food processing units, sugar industries etc. are rich in organic contents. Domestic and municipalities wastes are also rich in organic content. These wastes have high COD and BOD.

(B) Dissolved Solids Dissolved solids are generated manufacturing and applications of Pesticides, Fertilizers, Pharmaceutical and other chemical industries.

(C) Liquid Wastes having Toxic Chemicals The industrial effluents from hazardous chemicals units, Tannery and Dye industries, Electroplating, Coke –oven, etc.

(D) Cooling Water Liquid Wastes Cooling water from Thermal Power Plants, Cable, Rolling Mills, Plastic Mounting, etc.

Management of wastes, rules & laws for environnemnt protection

4.4 Laws and Regulations By looking at the degree and level of pollution in the country, most of us would not believe that India was the first county in the world to postulate, draft and provide the constitutional safeguards for protection and prevention of (pristine nature of the) environment. In early part of 1970s, taking the lead and inspiration from Stockholm Conference (India is the only nation represented by her head of the state) various laws were notified and being implemented for waste management. The Wildlife (Protection) Act 1972 was the first statute being promulgated constitutionally. To reduce (stop) and to prevent the further pollution of rivers in the country, The Water (Prevention and Control of Pollution) Act, 1974 was came into force. The Environment (Protection) Act (EPA), 1986, directly deals to provide the constitutional framework for handling and management of hazardous materials (compounds), prior assessment of the environmental impact of major developmental projects, discharge of industrial pollutants and effluents into the environment, guidance for industrial sitting and management of chemical accidents (Ref.15). The Hazardous Wastes (Management and Handling) Rules (Manufacture, Storage and Import of Hazardous Chemical), 1989 (amended in 2000 and 2003) and Hazardous Wastes (Management, Handling and Transboundary Movement) Rules, 2008 was the first such rules to address the industrial waste issues.

Table 4.1: The Evolution of different laws for Environment Protection in India

Year Law 1974 The Water (Prevention and Control of Pollution) Act 1975 The Water (Prevention and Control of Pollution) Rules 1977 The Water (Prevention and Control of Pollution) Cess Act 1978 Water (Prevention and Control of Pollution) Cess Rules 1981 The Air (Prevention and Control of Pollution) Act 1986 The Environment (Protection) Act 1989 The Manufacture, Storage and Import of Hazardous Chemical Rules 1991 The Public Liability Insurance Act 1995 The National Environment Tribunal Act 1997 The National Environment Appellate Authority Act 1998 The Bio-Medical Waste (Management and Handling ) Rules 2001 Batteries (Management and Handling) Rules 2008 Hazardous Waste (Management, Handling & Transboundary Movement) Notified 2008 2010 National Green Tribunal Act 2011 The Public Waste (Management and Handling) Rules 2011 E-Waste (Management and Handling) Rules

Management of wastes, rules & laws for environnemnt protection

Few regulations related to dye and textile industries are discussed in brief

The Water (Prevention and Control of Pollution) Act, 1974 (As Amended in 1978 and 1988)  Specifying standards for sewage and industrial effluents discharge into water bodies  Inspection of sewage or industrial effluent  Pollution Control Board (PCB) has the Right to obtain any information regarding the construction, installation or operation of an industrial establishment or treatment and disposal system  PCB’s can issue orders restraining or prohibiting an industry from discharging any poisonous, noxious or polluting matter in case of emergencies, warranting immediate action  PCB’s power to issue direction for o Closure, prohibition or regulation of any industry, operating or Process o Stoppage or regulation of supply electricity, water or any service to industry in the prescribed manner

The Water (Prevention and Control of Pollution) Cess Act, 1977 (Amended in 1991)  The Water Cess Act provides for the levy of a cess on water consumed by specified industries given in Schedule-I of the Act and also authorities entrusted with the duty of supplying water under the laws by or under which they are constituted at the rates specified in Scheduled-II of the Act  An industry which installs and operates its effluent treatment plant is entitled to a rebate of 25 per cent of the cess payable

Hazardous Waste (Management, Handling & Transboundary Movement) Notified 2008  Gives procedure for handling hazardous wastes defining the responsibilities, grant as well as authority to cancel authorization if failed to comply. Apart from this, various standards for recycling the hazardous waste, Transboundary movement of hazardous waste in form of import & export procedure are mentioned which is to be approved by central government  Thirty six procedure (industrial operations using mineral, petroleum refining, healthcare product, electronic industry, chemical, paper industries, leather, etc.) have been identified for generating 107 hazardous waste. Waste management criteria for TSDF (Transfer, Storage & Disposal Facility) criteria have been given  Categories of hazardous waste are mentioned along with their permissible generation quantity. Industries generating any of these wastes beyond the regulatory limits are required to seek authorization form the concerned state pollution control board for its temporary storage in the premises and its disposal

The Environment (Protection) Act, 1986  Restricts areas in which any industries, operations, processes may not be carried out or shall be carried out subject to certain safeguards  Lays down safeguards for prevention of accidents and take remedial measures in case of such accidents  Lays down procedures and safeguards for handling hazardous substances  Non-compliance would lead to stoppage of supply of electricity, water or any other service for the industry  The industry, operation or process requiring consent under Water (Prevention and Control of Pollution) Act, 1974 (6 of 1974) and/or under Air (Prevention and Control of Pollution) Act, 1981 (84 of 1981) are required to submit the Environmental Statement in prescribed “Form-V”, for the Financial Year endng the 31st March to the concerned State Pollution Control Boards/Pollution Control Boards/Pollution Control Committees in the Union Territories on or before 30th September every year

Methods evovled for removal of dye  05 Methods evolved for removal of dye

5.1 Various Technology / Treatment process / Methodology used 30 for dye and textile effluent wastewaters 5.1.1 Flocculation 30 5.1.2 Sedimentation 30 5.1.3 Dissolved Air Floatation 30 5.1.4 Clarification 30 5.1.5 Neutralization 31 5.1.6 Precipitation 31 5.1.7 Activated sludge process 31 5.1.8 Biological filters 32 5.1.9 Anaerobic Treatment Systems 32 5.1.10 Granular Media Filtration 32 5.1.11 Membrane Filtration 32 5.1.12 Reverse Osmosis Systems 33 5.1.13 Ultrafiltration 33 5.1.14 Nanofiltration 33 5.1.15 Ion Exchange 33 5.1.16 Activated carbon 33 5.1.17 Ultraviolet (UV) Disinfection 34

Methods evovled for removal of dye

5.1 Various Technology / Treatment process / Methodology used for dye and textile effluent wastewaters

Depending upon the characteristic of wastewater, it can be subjected for different treatment options either from physical, chemical and/or biological processes or combination of them. These treatment technologies can be employed at preliminary, primary, secondary or tertiary and/or at more advanced stage. Few of the very commonly used methods are described below.

5.1.1 Flocculation It is a physico-chemical process that encourages the aggregation of coagulated colloidal and finely divided suspended matter by physical mixing or chemical coagulant aids. Flocculation process consists of a rapid mix tank and a flocculation tank. The process involves mixing of wastewater stream with coagulants in a rapid mix tank, which is then passed on to the flocculation basin where slow mixing of waste occurs which allows the particles to agglomerate into heavier more settleable solids. Either mechanical paddles or diffused air facilitates better mixing. The different types of chemicals used in coagulation include inorganic electrolytes, natural organic polymers and synthetic poly electrolytes. The selection of a specific chemical depends on the characteristics and chemical properties of the contaminants.

5.1.2 Sedimentation This process is aimed to remove easily settleable solids. Sedimentation chambers may also include baffles and oil skimmers to remove grease and floatable solids and may include mechanical scrapers for removal of sludge at the bottom of the chamber.

5.1.3 Dissolved Air Floatation Use of bubbles in this process is required to raise the suspended particles in wastewater up to surface level and hence make it easy for their collection and removal. Air-bubbles are introduced into the wastewater and attach themselves to the particles, thus causing them to float. This process of diffused air flotation can be used to remove suspended solids and dispersed oil and grease from oily wastewater. Wastewater is pressurised and contacted with air in a retention tank. The pressurised water that is super-saturated with air is passed through a pressure- reducing valve and introduced into at the bottom of floatation tank. As soon as pressure is released the super- saturated air begins to come out of solution in the form of fine bubbles. The bubbles get attached to suspended particles and become enmeshed in sludge flocs, floating them to surface. Float is continuously swept from the surface and sludge may be collected from the bottom. Addition of certain coagulants increases the oil removal efficiency of DAF units.

5.1.4 Clarification Clarification system uses gravity to provide continuous, low cost separation and removal of particulate, flocculated impurities and precipitates from water and generally follow the processes which generate suspended solids such as biological treatment. In a clarifier, wastewater is allowed to flow slowly and uniformly, permitting the solids to settle down. The clarified water flows from the top of the clarifier over the weir. Solids get collected at the bottom and sludge must be periodically removed, dewatered and safely disposed.

Methods evovled for removal of dye

5.1.5 Neutralization Incoming untreated wastewater has a wide range of pH, and it is difficult to treat wastewater with such a high variability of pH. Neutralization is the process used for adjusting pH to optimize treatment efficiency. Acids such as sulphuric or hydrochloric may be added to reduce pH or alkalis such as dehydrated lime or sodium hydroxide may be added to raise pH values. Neutralization may take place in a holding, rapid mix or an equalization tank. It can be carried out at the end of the treatment also to control the pH of discharge in order to meet the standards.

5.1.6 Precipitation For removal of metal compounds from the stream of wastewater, precipitation is carried out in two steps. In the first step, precipitants are mixed with wastewater allowing the formation of insoluble metal precipitants. In the second step, precipitated metals are removed from wastewater through clarification and/or filtration and the resulting sludge must be properly treated, recycled or disposed. pH is an important parameter to consider in chemical precipitation. Metal hydroxides are amphoteric in nature and their solubility increases towards higher or lower pH. Thus, there is an optimum pH for hydroxide precipitation for each metal. Wastewater generally contains more than one metal. Therefore, selecting the optimum treatment chemical and pH becomes more difficult and involves a trade-off between optimum removal of two or more metals. Various chemicals used for this process are lime, sodium hydroxide, soda ash, sodium sulphide and ferrous sulphate. Normally, hydroxide precipitation which is effective in removing metals like antimony, arsenic, chromium, copper, lead, nickel and zinc. Sulphide precipitation is used in removing mercury, lead, copper, silver, cadmium etc.

5.1.7 Activated sludge process It is a continuous flow, aerobic biological treatment process that involves suspended growth of aerobic micro-organisms to biodegrade organic contaminants. Influent is introduced in the aeration basin and is allowed to mix with the contents. A suspension of aerobic microbes is maintained in the aeration tank. A series of biochemical reactions in the basin degrade the organics and generate new bio mass. Micro-organisms oxidize the matter into carbon dioxide and water using the supplied oxygen. These organisms agglomerate colloidal and particulate solids. The mixture is passed to a settling tank or a clarifier where micro-organisms are separated from the treated water. The settled solids are recycled back to the aeration tank to maintain a desired concentration of micro-organisms in the reactor and some of the excess solids are sent to sludge handling facilities. To ensure biological stabilization of organic compounds, adequate nutrient levels of nitrogen and phosphorous must be available to the bio mass. The key variables to the effectiveness of the system include: (i) Organic loading which is described as food to micro-organism ratio (F/M) ratio or Kg of BOD applied daily to the system per Kg of biological solids in aeration tank. F/M ratio determines BOD removal, oxygen requirements and bio mass production. Systems designed and operated at lower F/M provide higher treatment efficiency. (ii) Sludge retention time (SRT) or sludge age is the measure of the average retention time of solids in the system and the SRT, similar to F/M ratio, affects the degree of treatment, oxygen requirements and the production of waste sludge. Systems designed and operated at higher SRT provide higher treatment efficiency.

Methods evovled for removal of dye

(iii) Oxygen requirements are based on the amount required for biodegradation of organic matter and the amount required for endogenous respiration of micro-organisms. Various modifications in activated sludge process are possible by changing one or more of the key parameters. Sequential batch reactor is a form of the activated sludge process where aeration, sedimentation and decantation processes are performed in a single reactor.

5.1.8 Biological filters These filters are biological reactors filled with media which provide a surface that is repeatedly exposed to wastewater and air and on which a microbial layer can grow. The two most common types of biological filters are; (i) Trickling Filters: In trickling filters treatment is provide by a fixed film of microbes that forms on the surface which adsorbs organic particles and degrades them aerobically. Wastewater is distributed over a bed made of rock or plastic and flows over the media by gravity. (ii) Rotating Biological Contractor: A rotating biological contactor (RBC) consists of a series of discs about 40% of the area is immersed in waste water and the remainder of the surface is exposed to atmosphere, provide a surface for microbial slime layer. The alternating immersion and aeration of a given portion of the disc enhances growth of the attached micro-organisms and facilitates oxidation of organic matter in a relatively short time and provides a high degree of treatment.

5.1.9 Anaerobic Treatment Systems These processes are slower than aerobic degradation and when sulphur is present, obnoxious hydrogen sulphide gas is generated. Though the capital cost is high, part of it can be compensated by recovery of bio gas. They are not so commonly used in wastewater treatment systems for CETPs except as a means for sludge stabilization.

5.1.10 Granular Media Filtration Many processes fall under this category and the common element being the use of mineral particles as the filtration medium. It removes suspended solids mainly by physical filtration. Two common types of these granular media filers are (i) Sand filters are the most common type which consists of either a fixed or moving bed of media that traps and removes suspended solids from water passing through media. (ii) Dual or multimedia filtration consists of two or more media and it operates with the finer, denser media at the bottom and coarser, less dense media at the top. Common arrangement is granite base at the bottom, sand in the middle and anthracite coal at the top. Flow pattern of multimedia filters is usually from top to bottom with gravity flow. These filters require periodic back washing to maintain their efficiency. These processes are most commonly used for supplemental removal of residual suspended solids from the effluents of chemical treatment processes.

5.1.11 Membrane Filtration This technique is used to separate particles from a liquid for the purpose of purifying it. In membrane filtration, a solvent is passed through a semi-permeable membrane. The membrane's

Methods evovled for removal of dye permeability is determined by the size of the pores in the membrane. The size of the pores has to be carefully calculated to exclude undesirable particles, and the size of the membrane has to be designed for optimal operating efficiency. The result is a cleaned and filtered fluid on one side of the membrane, with the removed solute on the other side. Microfiltration, ultrafiltration and nanofiltration are examples of membrane filtration techniques.

5.1.12 Reverse Osmosis Systems This is also a membrane separation method that is used to remove several types of large molecules and ions from solutions through application of pressure to the wastewater on one side of a selective membrane. The result is that the contaminant is retained on the pressurized side of the membrane and the treated waste water is allowed to pass to the other side.

5.1.13 Ultrafiltration Unlike reverse osmosis membranes which retain all solutes including salts, ultrafiltration membranes retain only macro molecules and suspended solids. Thus salts, solvents and low molecular weight organic solutes pass through ultrafiltration membrane with the permeate water. Since salts are not retained by the membrane, the pressure differences across ultrafiltration membrane are negligible. Flux rates through the membranes are fairly high, and hence lower pressures can be used.

5.1.14 Nanofiltration Nanofiltration can be positioned between reverse osmosis and ultrafiltration. Nanofiltration is essentially a lower pressure version membrane where the purity of permeate water is less important. The nanofiltration is capable of removing hardness elements such as calcium or magnesium together with bacteria, viruses, and colour. Nanofiltration is operated on lower pressure than reverse osmosis and as such treatment cost is lower than reverse osmosis treatment. Nanofiltration is preferred when permeate with some residual TDS but without colour, COD and hardness is acceptable. Feed water to nanofiltration should be of similar qualities as in case of reverse osmosis. Turbidity and colloids should be low. Disinfection of feed may also be necessary to remove micro-organism.

5.1.15 Ion Exchange Ion exchange is a process of exchange of ions between two electrolytes or between an electrolyte solution and a complex. Ion Exchange can be used in wastewater treatment plants to swap one ion for another for the purpose of demineralization. There are basically two types of ion exchange systems, the anion exchange resins and the cation exchange resins. It can be used for softening, purification, decontamination, recycling, removal of heavy metals from electroplating wastewaters and other industrial processes, polish wastewater before discharging , removal of ammonium ion from wastewaters, salt removal, purify acids and bases for reuse, removal of radioactive contaminants in the nuclear industry, etc.

5.1.16 Activated carbon Activated carbon is used in a large range of applications in tertiary waste water treatment. Powdered as well as granular activated carbons are used for the purpose of de-chlorination of organic compounds. Organic compounds in waste water are adsorbed on to the surface of the activated carbon. A number of factors affect the effectiveness of the activated carbon. These

Methods evovled for removal of dye include pore size, composition and concentration of the contaminant, temperature and pH of the water and the flow rate or contact time of exposure. Activated carbon can be applied on a broad spectrum of organic pollutants and is typically used to remove contaminants from water such as pesticides, aromatic compounds such as phenol, adsorbable organic halogens, non-biodegradable organic compounds, colour compounds and dyes, chlorinated/ halogenated organic compounds, toxic compounds, compounds that normally inhibits biological treatments, oil removal in process condensates, halogens, especially chlorine that oxidises downstream processes and organics that have the potential to foul ion exchange resins or reverse osmosis membranes.

5.1.17 Ultraviolet (UV) Disinfection This technique is primarily employed as a disinfection process that inactivates waterborne pathogens without use of chemicals. Additionally, UV is also effective for residual TOC removal, destruction of chloramines and ozone.

Histroty of bioremediation  06 History of Bioremediation

6.1 Brief History of Bioremediation 36 6.1.1 Courtship Period (Pre-1989 era) 37 6.1.2 Honeymoon Period (1989-1991) 37 6.1.3 Establishment Period (1992 onwards) 38 6.2 Conclusion 38

Histroty of bioremediation

6.1 Brief History of Bioremediation It is of common belief that bioremediation is a relatively modern technology. But it is not a new concept. Romans, as early as 600 B.C.E., used the complex networks of sewers for treatment of wastewaters, where microorganisms had pivotal role to play. The basic process gradually been modified and was applied to treat different contaminant. In the modern history, process of bioremediation was earnestly studied and understood in 1940s. Two decades later, in 1960s, an Assistant County Petroleum Engineer George M. Robinson, working for Santa Maria, California, spent his spare time in experimenting for cleaning up of Mucky Jar and the abandoned oil sumps of Cat Canyon Oil Filed developed the process of Bioremediation. In 1968, he experiment the process on large scale for microbial clean up of various oil spills. Besides oil spills, he also experimented, on sewage, leach fields, as well as odour and pest control and demonstrate complete clean up using bioremediation. Nearly twenty years later, the imfamous Exxon Valdez oil spill in Prince William Sound at Alaska in 1989 was the actual genesis of bioremediation globally. After successful clean up of this oil spill, bioremediation was established as technology, that is discussed, applied, researched and became an alternate option (besides physico-chemcial technology) for treatment of environmental pollutant. According to Hoff (1993), modern history of bioremediation can be divided into three developmental periods: (1) Pre-1989, (2) Between 1989 and 1991 and (3) Post 1992.

Table 6.1: A glimpses of important showcase and facts about the history of bioremediation

No. Period of the Event Description of the Event (Year) The evidence pointed that early civilization in Rome (and others) have built intricate networks of sewers during 600 B.C. The 1 600 B.C. network collects waste water which subsequently underwent to biological treatment. In Rhode Island, it was believed that disposing of waste into 2 1800 A.D. local rivers (surrounding the human habitat) would dilute the wastes. The process of bioremediation was first reported by Petroleum Engineer Mr. George M. Robinson, in California, during his 3 1960 A.D. experiments with dirty jars and abandoned oil slumps. In 1960, through U.S. Office of Naval Research supported projects oil-metabolizing (micro)-organisms were recognized. Mr. George Robinson organized first large-scale microbial 4 1968 A.D cleanup of oil spill. The U.S. Environmental Protection Agency (US-EPA) was founded

1970 A.D. 5 Dr. Anand Chakrabaty and his colleagues at General Electric, USA identified a bacterial strain (Pseudomonas), capable to degrade oil components.

Histroty of bioremediation

The bioremediation process was first commercially used to clean up a Sun Oil pipeline spill in Ambler, Pennsylvania, USA. 6 1972 A.D. In the same year fuel holding tanks of Ship RMS Queen Mary was cleaned up. The Indian Water (Prevention and Control of Pollution) Act was 7 1974 A.D implemented. The Indian Water (Prevention and Control of Pollution) Rules was formulated 8 1975 A.D. The oil-degrading superbug was developed by Dr. Ananda M. Chakrabarty at General Electric, USA. Form 1980 onwards bioremediation process was commercially 9 1980 A.D. used for the treatment of contaminated soils groundwater. Literate community has accepted the use of Bioremediation 10 1989 A.D. Following Exxon Valdez oil spill, bioremediation as a technology was established across the globe. Derek Lovely and his coworkers proposed the cleanup of 11 1990 A.D. Uranium contamination in groundwater using bioremediation in early 1900’s USEPA conducted a survey and received the information on 240 12 1992 A.D cases of bioremediation in the United States. Field level of experiments to test the efficiency of uraninite 13 2002 A.D. bioremediation were started in 2002

6.1.1 Courtship Period (Pre-1989 era) Before Exxon Valdez oil spill and its clean up, the period was seen as possible use and understanding the role of microorganisms in cleaning up of contaminated sites, i.e. dedicated to research. It is the period when bioremediation was little known outside the small community of academic, microbiologist and applied research or hazardous waste management community. A series of scientific and technical reports and articles were published after successful studies under laboratory conditions during 1970s and 1980s. This includes review papers describing mechanisms of biodegradation and results from controlled filed experiments determining degradation rates in various environments. Several studies after major oil spills (like of Amoco Cadiz spills) measured oil degradation in the environment using microorganisms, that confirms previously reported laboratory research. Bioremediation now has been established as a technology and a viable option especially for oil spill cleanup.

6.1.2 Honeymoon Period (1989-1991) In these three years bioremediation received widespread interest and attention from all the community. In this period, because of Exxon Valdez oil spill incident, a new revolutionized concept was established, what it is known today as ‘bioremediation’. Prior to 1989, there was no documented evidence of use of this technology on marine oil spills. Later on bioremediation was used (on a trial basis) in a four different oil spill in United States: (1) Seal Beach in California; (2) Prall's Island in New Jersey, (3) the Apex Barges and (4) Mega Borg spills in the Gulf of Mexico.

Histroty of bioremediation

Gradually the EPA of USA conducted series of lab scale and field studies to assess the new found concept to use on shorelines in Prince Williams Sound. The positive results, provided impetus to use bioremediation more extensively in other similar cases. In later years, two distinct type of fertilizers (Inipol EAP22™: an olepphilic fertilizer formulation and Customblen™: a granular slow-release fertilizer) were sprayed to contaminated shorelines in Prince William Sound. Followed by the initial (yet dramatic) visual changes in a test plot sprayed with Inipol EAP22™ and perhaps because bioremediation seemed like a more friendlier and east technology than other available methods for marine oil spill cleanup, bioremediation received comparatively better response from the general public. The concern during this application was use of 2-butoxy- ethanol a component in Inipol and its toxicity to cleanup workers and wildlifes. Soon the safety guidelines were framed and wildlife deterrents were used during the first 24 h of application when toxicity is of most concern. During this phase, several instances of use of bioremediation (more on experimental basis) in open-water and microbial applications with additional supplementation of nutrients on shorelines were appeared. Gradually, think-tank from National Oceanic and Atmospheric Administration (NOAA) of USA, held a cautious approach supporting the use of bioremediation, monitored its effectives to rule out potential determination impacts on marine life. It appeased to be rather controversial position during the “heyday” of bioremediation era. Soon in 1991, a wonder effect of bioremediation as potent cleanup technology started to diminish. The obvious reasons were, none of the studies conducted outside Alaska were able to provide the positive evidence of effectiveness on bioremediation in field test. Many of these approaches suffered from poor experimental designs, or conducted for a limited period of time or had limiting facilities for determining changes in oil concentrations. Several factors affected the effectiveness of bioremediation; extended studies have found the toxicity to native ecosystem, possibility of eutrophication amongst the many serious concerns were gave mixed results to the unbridled enthusiasm for bioremediation. By 1991, bioremediation has stated to pass out from limelight with initial success as ‘wonder technology’.

6.1.3 Establishment Period (1992 onwards) Though the period after 1992 can be considered as establishment phase, bioremediation as technology has achieved a certain level of acceptance, with more realist expectations than before, but the level of attention and interest was gradually decreased. At this time involvement of government in form of official guidelines, set of testing laboratory protocols as measuring product effectiveness in degradation (of standardized oil), toxicity testing aquatic organisms and a small- scale field tests. In 1993, US government issued first set of protocols for bioremediation for testing commercial products to be applied during remediation process. Bioremediation was now commonly included as an alternate option (among other remediation techniques) for cleanup of contaminated areas. Many of the proposals of bioremediation, granted for application in open environment are now being accompanied by some type of monitoring programme to access the effectiveness of technique and to prevent spreading the toxicity in applied area.

6.2 Conclusion The response that concept of bioremediation for hazardous waste received was a more typical what has been observed for any new technologies. The initial high enthusiasms with over

Histroty of bioremediation expectations were an unrealistic which was followed by disappointment and disillusionment lead to a more realistic acceptance of the technology. However, in many ways the ongoing research has helped to established and defines the limiting variables of bioremediation as technology. Often dealing with bioremediation needs patience to use the technique appropriately which may offer variable substantial benefits during long term approach.

Status report on projects supported by DBT  07 Steps involved in textile processing

7.1 Opening Remarks 41 7.2 Pre-Support/Sanction Process for the Research Proposal 41 7.3 Primary Statistics of the Sanctioned Projects 41 7.4 Detail analysis of the outcome of research projects 44 7.5 Summary and brief assessment of the results 53

Status report on projects supported by DBT

7.1 Opening Remarks The Department of Biotechnology (DBT) Ministry of Science and Technology, New Delhi, is a premier funding agency under Government of India supporting research in biotechnology and allied areas. One of the major focuses of the agency is to provide financial support for research in Environmental Biotechnology. It generously supports various research programme on developing effective and novel treatment technology for industrial effluents including liquid waste from dye, dye intermediates, pigments and textile industries.

7.2 Pre-Support/Sanction Process for the Research Proposal Through internal Task Force on Environmental Biotechnology, DBT invites research proposals (throughout the year or through special calls) on basic and applied research, innovations and development of effective technology for wastewater remediation. On receiving the research proposals, after the initial screening, they are being sent for reviewing for the suitability of the projects in terms of scope and the objectives of the work proposed, aptness of the budget requirement and general outcome of research project. After receiving the comments from the reviewers, the project investigators are invited for the brief presentation for the proposed work in Task Force Review Meeting. Post presentation if any modifications are required, the investigator are invited to submit the revised proposal for possible financial support. The research projects which scores in merit for providing the new outlook in the already existing knowledge in the proposed subject are considered for financial support. The normal tenure of the financial support ranges between 36 and 60 months.

7.3 Primary Statistics of the Sanctioned Projects Though DBT has started functioning in 1986, the first research project in the area of development of treatment technology for dye and textile effluent was sanctioned in the year 1997-1998 to Dr. T. Emilia, Department of BCP & WT Regional Research Laboratory, Thiruvananthapuram- 695019, Kerela. Since the first project, about 23 different research projects were supported by DBT till date (as on December 2017) (Figure 7.1).

Project Sanctioned Project 1

1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 Year

Figure 7.1: Distribution of the sanctioned projects from the year 1986 to 2017

Status report on projects supported by DBT

Amongst these, 03 projects were multi-institutes involving two or more institutes, while remaining 20 were supported in single institute (Figure 7.2). Ten percent of research projects were sanctioned to project investigators from Colleges, 36 % to the Institutes and 54 % to the investigators associated either with Central, State or Private Universities (Figure 7.3).

13%

87%

Single Institute Multi Institute

Figure 7.2: Distribution of sanctioned projects for Single or Multi institutes

10%

36%

54%

College University Institute

Figure 7.3: Distribution of sanctioned projects in college, university and institute

Status report on projects supported by DBT

Collectively for the 27 research projects, DBT has provided a support of Rs. 12,78,28,840/- and project investigators have spend nearly 1002 months for exploring the possibilities of developing a effective technology and treatment methodology for liquid waste for dye, dye intermediates and textile industry. Amongst the project investigator, maximum number of research projects (i.e. 05) was sanctioned to Prof. Datta Madamwar, Sardar Patel University, Vallabh Vidyanagar for the financial support of Rs. 5,10,79,000/-. Prof. S. P. Govindwar and Prof. Jyoti Jadhav from Shivaji University, Kolhapur collectively received Rs. 1,27,11,400/- for 03 research projects. Prof. Vankata Mohan, IICT, Hyderabad has worked on 02 research projects worth of Rs. 58,03,400/-. It was observed that majority of the research projects were sanctioned to the states where textile industries are in large numbers (Table 7.1). Twenty six percent of the total projects were from the investigators belonging from the state of Maharashtra (Figure 7.4). While 21 % of the projects were sanctioned to the project investigators belonging to Tamilnadu and Gujarat, followed by Telengana and Punjab (7 % each). Such observation directly suggest that, region where pollution are more due to textile industries, project investigators are more inclined towards working in developing remediation technology.

Table 7.1: State-wise distribution of textile industries

State/UT Composite Semi composite/ Total mills processing units Andhra Pradesh - 54 54 Assam 1 1 2 Bihar - 4 4 Delhi - 61 61 Gujarat 17 506 523 Haryana 1 74 75 Himachal Pradesh - 4 4 Jammu & Kashmir 1 2 3 Karnataka 8 33 41 Kerala 3 11 14 Madhya Pradesh 3 9 12 Maharashtra 27 222 249 Orissa 1 1 2 Punjab 4 378 382 Rajasthan 2 30 32 Tamil Nadu 2 739 741 Uttar Pradesh 4 76 80

Status report on projects supported by DBT

West Bengal 8 32 40 Pondicherry 1 4 5 Total 83 2241 2324

Kerela 3% 3% 3% 3% Gujarat 3% 21% Rajashtan 7% Tamilnadu

Maharashtra 7% 3% Telangana Punjab Andhra Pradesh

26% 21% Karnataka Delhi Guwahati

Figure 7.4: Distribution of the sanctioned projects amongst the state of the country

7.4 Detail analysis of the outcome of research projects

7.4.1 Objectives The heart of the any research project is its proposed objectives. A good decisive study of the objectives of the sanctioned and ongoing projects would provide the actual state of the research being carried out previously and would provide the future directions. In Table 7.2, the objectives of few of the projects (as a representative) are listed.

Table 7.2: Research objective of few projects (as representative) supported by DBT

Project I Biodegradation of textile dyes (Scarlet RR, Rubine GFL, Brown 3REL, Methyl Red, Brilliant Blue, Golden Yellow HER and Remazol Red) using Galactomyces geotrichum MTCC 1360 and consortia with Brevibacillus laterosporus Objectives 1. Standardization of media conditions and other parameters (Temperature, pH, Carbon, & nitrogen source) for maximum degradation of dyes (Scarlet RR, Rubin GFL, Brown 3 REL, Methyl Red, Brilliant Blue, Golden Yellow HER, and Remazol red) using Galactomyces geotrichum.

Status report on projects supported by DBT

2. Standardization of media conditions and other parameters (Temperature, pH, Carbon, & nitrogen source) for maximum degradation of dyes (Scarlet RR, Rubin GFL, Brown 3 REL, Methyl Red, Brilliant Blue, Golden Yellow HER, and Remazol red) using Brevibacillus laterosporus. 3. Purification of enzyme prominently responsible for degradation of these dyes and possible use for co-metabolism (most likely lignin peroxidase and laccase). 4. Study of longetivity of decolorization activity by Galactomyces geotrichum and Brevibacillus laterosporus cells alone and in consortium, and immobilized whole cells in repeated batch decolorization tests. 5. Degradation of mixture of dyes (Scarlet RR, Rubin GFL, Brown 3 REL, Methyl Red, Brilliant Blue, Golden Yellow HER, and Remazol red) using an individual strain (Galactomyces geotrichum and Brevibacillus laterosporus) and in consortium. 6. Effect of metal ions on decolorization of mixture of dyes. 7. Study of enzyme status, degradation pattern and toxicity testing. Application of this strain for textile industry waste water at laboratory bench scale Project II Development of Microbial Strains Through Biotechnological Approach for Effective Decolorisation and Degradation of Anthraquinone Dyes Objectives 1. Isolation and screening of acclimatized microorganisms for decolourization and degradation of anthraquinone dyes. 2. Few potential microorganisms have to be use during large scale production of the selected strains and to evaluate biotic and abiotic (stress) parameters affecting the degradation process. 3. The selected strains would be immobilized on appropriate material shall be fixed and to develop biotechnological and bioengineering process for dye wastewater treatment. Project III Developing Efficient Microbial Inocula for Degradation of Textile Dyes and their Amines: Genotoxicity Evaluation for Validation of their Degradation Potential Objectives 1. The primary objective of this work was to develop effective microbial inocula having a potential for mineralization of azo reactive dye including toxic amines. 2. The efficiency of the consortia was analyzed by evaluating genetoxicity potential of the untreated and biologically treated samples in mouse as a model. 3. The last objective of the study was to immobilize cells to evaluate its dye degradation efficiency in either sequential anoxic-aerobic, aerobic or plug flow system reactor for its pilot/field scale testing. Project IV Application of periodic discontinuous batch operation to enhance treatment efficiency of dye containing wastewater Objectives 1. Development of enriched bacterial mixed culture for dye and textile (Part I) industrial effluent under batch process. 2. Molecular phylogenetic detection of bacterial mixed culture by applying cultivation dependent approaches. 3. To study the syntrophic action of bacterial mixed culture during dye

Status report on projects supported by DBT

decolorization. 4. Real time Q-PCR for assessment of bacterial community structure dynamics during active bioreactor under steady static conditions and before steady static conditions with time using 16S rRNA gene approach based on species specific real time Q-PCR assay. Part II 1. To investigate the application of periodic discontinuous batch process for the treatment of dye based wastewater especially from textile industry. 2. To investigate role of various types of dyes viz., chemical class and application class on treatment efficiency 3. To elucidate the role of various metabolic functions (aerobic and anaerobic) and multiphase microenvironment/redox condition on the process performance and to investigate the influence of metabolic shift on the dye/colour removal process efficiency. 4. To study the physiological state during periodic discontinuous batch operation by monitoring the macromolecular composition (DNA, EPS, protein, etc) and microbial diversity analysis (DGGE and FISH) and measuring the robustness of the process by conducting shock load experiments. 5. To study the optimized conditions obtained from the lab scale experiments at higher scale using real field textile dye wastewater Project V Ecotechnology for treating dye waste water of textile industries: A demonstration project Objectives 1. The project investigator has (previously) demonstrated the technology for dye waste water treatment and has been validated by Sanganer Dye Manufacturers Association. 2. In this study, investigator wants to demonstrate the treatment process at a level of 30,000 liter/day and also to demonstrate the technology at 10 locations (5 in Sanganer and 5 in Punjab).

These are the few (representative) objectives of the sanctioned and completed projects. A representative four different types of projects were selected ranging from a basic laboratory scale to large scale (pilot level/small scale industry) demonstrative project. The majority of the projects sanctioned by DBT and completed can be assorted in the above four categories. The initial study of the objectives suggest that in a first category, investigators choose to work on isolation and screening of the microorganisms (with descending order of preference of Bacteria>Fungus) from environment polluted by industrial effluents either from dye and dye intermediate manufacturing industries or textile effluents (i.e. dye application). The potent strains were isolated from long term polluted soil, water sources or sediments. In recent years, because of inherent limitations of pure cultures, the dye degradation studies are more focused on developing consortia or mixed cultures of different microorganisms. Followed by isolation of strains or development of consortia/mixed-cultures, biotic and abiotic parameters were optimized at flask scale (~ 100 ml). In biotic factors, growth source (i.e. carbon and energy), potential electron donors, nitrogen sources and their concentrations was optimized in either nutritional rich synthetic media or in minimal media. In abiotic factors, pH and temperature was optimized. The effect of dissolved oxygen concentration, salt concentration, substrate (i.e. test dye) concentrations and different types of dyes were simultaneously studied. Beside a single dye, experiments were also conducted on mixture of dyes, simulated and real textile effluents.

Status report on projects supported by DBT

The optimization studies are followed by analyzing the dye decolourization and degradation pattern (of test dye compound) using different analytical techniques like UV-Vis and IR spectroscopy, HPTLC, HPLC, GS-MS, LC-MS and NMR under optimized conditions. Generally, aim of the above study is to propose the degradation pathway of test dye included in the study. Simultaneously the enzymatic studies are also performed. Measuring the activity of different enzymes (oxidoreductase and peroxidase) with study of their inductive or constitutive expression presumably indicates the involvement enzymes in dye decolourization and degradation process and suggests that degradation in biologically mediated. Enzymes like azo-reductase, laccase, lignin peroxidase, (NADH)-DICP reductase, tyrosinase, veratryl alcohol oxidase, riboflavin reductase, aminopyrine N-demethylase, etc. were studied in medium supernatant or from cell free extract. The idea behind these studies is to correlate the dye degraded intermediates (generated during dye metabolism and identified using above noted analytical techniques) with the enzymes involved in degradation of dye compounds. To demonstrate the competence of the pure cultures or consortia/mixed cultures (in complete mineralization of dye and) intermediates generated during dye metabolism is comparatively less toxic than parent compound; different toxicity assays such as phytotoxicity cytotoxicity, genotoxicity are conducted. In phytotoxicity assays, Sorghum vulgare and Phaseolus mongo are most commonly used. In second category of projects, objective is to demonstrate the treatment feasibility of developed consortia/mixed cultures (or by enriching the native microorganisms of the raw textile effluent) at laboratory scale bioreactors such as aerobic/ananerobic/sequential anaerobic (anoxic)-aerobic plugflow, upflow, downflow fixed film or other bioreactors. The treatment feasibilities are demonstrated for a volume ranging from 1 to 5 L as synthetic media or simulated industrial effluent or real textile effluents. Various parameters like optimization of packing materials, effect of HRT and OLR, measuring the BOD/COD reduction, change in DO, pH, alkalinity, TS, TDS, TSS and VSS, estimating chlorides, sulphate, phosphate, phenolics, TOC, VFA, VA, TN concentration at regular interval of time. In bioreactor based studies, another objective is to immobilize the potent pure cultures or consortia/mixed cultures in a suitable gel matrix to increase the self-life and to use the immobilized beads in treatment process. It can be noted from the Table 7.2 that, very few studies were supported for large scale on-site field applications (Project V). Under this category a laboratory (and/or pilot level) tested methods are applied for the treatment of textile effluents directly in the industries. The treatment scale can range from few hundreds to thousand liters of effluent in a day. While in a fourth category, molecular aspect of the microbial community involved in bioremediation are being studied. Understanding the microbial communities in terms of their taxonomic identification and phylogenetic identification and their functional potential at polluted sties using metagenomic library preparation and screening of clones are one of the basic objectives. Few important genes such as azoreductases were screened from metagenomic library or were amplified directly from metagenome or consortia/mixed cultures, using degenerate primers and over-expressed in surrogate host for possible use during bioremediation of coloured water. In another objective, microbial community dynamics of consortia/mixed cultures during metabolism of test dye compound were studied using qPCR. The microbial community was also studied from different stages of effluent treatment at Common Effluent Treatment Plant (CETP) on the basis of 16S rRNA gene sequences using Sanger Sequencing method or next generation massive parallel sequencing of whole metagenome.

Status report on projects supported by DBT

The objectives can be viewed in working scale of three different categories as Lab Scale, Reactor Level and Demonstrative scale Figure 7.7.

80 60

40 20 0 Lab Scale Reactor Level Demostrative

Figure 7.7: Different categories of experiments conducted in the projects

7.4.2 Results and Observations Besides analyzing the objectives, the study of results would provide more understanding about the status of the research in bioremediation of dye and textile effluents. The observed results of few of the research projects supported by DBT are mentioned below. Results of Project: Biodegradation of textile dyes (Scarlet RR, Rubine GFL, Brown 3REL, Methyl Red, Brilliant Blue, Golden Yellow HER and Remazol Red) using Galactomyces geotrichum MTCC 1360 and consortia with Brevibacillus laterosporus. The G. geotrichum showed 86% of decolorization of Golden Yellow HER within 30 h, at 30oC and pH 7.0 under microaerophilic (static) condition with significant reduction in COD (73%) and TOC (62%), respectively. This organism tolerates 500 mgl-1 of dye concentration. The degradation efficiency of this strain using carbon and nitrogen sources viz. wheat bran and glucose showed fast decolorization individually as well as glucose in combination with ammonium chloride and yeast extract responsible for faster decolorization of Golden Yellow HER. Enzymatic studies indicate the involvement of azo reductase, laccase and NADH-DCIP reductase enzymes in biotransformation. Azo dyes can be cleaved symmetrically or asymmetrically depending on the structure of substrate. The products of degradation were identified as 4(5-hydroxy, 4-amino cyclopentane) sulfobenzene and 4(5-hydroxy cyclopentane) sulfobenzene by GC-MS. In addition, when G. geotrichum was applied to decolorize textile effluent, it showed 85% of true color removal (ADMI removal) within 72 h, along with a significant reduction in TOC (42%) and COD (58%) under microaerophilic condition. Phytotoxicity study revealed less toxic nature of the formed metabolites as compared to parent dye with respect to S. vulgare and P. mungo. Malt extract medium was found to be most suitable for the degradation of Remazol Red among the other used media. The G. geotrichum showed 96% decolorization of Remazol Red (50 mgl-1) for 36 h, at 30oC and pH 11.0 under microaerophilic condition along with significant reduction in COD (62%) and TOC (41%). Peptone (5.0 g l-1), rice husk (10 g l-1 extract) and ammonium chloride (5.0 g l-1) were found to be more significant among the carbon and nitrogen sources used. The presence of tyrosinase, NADH-DCIP reductase, riboflavin reductase and induction in azo

Status report on projects supported by DBT reductase and laccase activity during decolorization indicated their role in degradation. The metabolites produced after degradation was analyzed by HPTLC, FTIR, HPLC and GC-MS. The asymmetric cleavage of azo dye Remazol Red was carried out by azo reductase into reactive intermediate and subsequently mineralized by other enzyme found in G. geotrichum. Phytotoxicity study indicated the conversion of complex dye molecules into simpler oxidizable products having less toxic nature. The degradation of disperse azo dye and its toxicological approach in the sense of Allium cepa. G. geotrichum MTCC 1360 showed 87 % decolorization of Rubine GFL (50 mg l-1) within 96 h at 30 oC and pH 7.0 under microaerophilic condition with significant reduction of COD (67%) and TOC (59%). The natural carbon and nitrogen sources like rice husk, wood shaving and bagasse was found to efficient for the decolorization of dye along with other sources used. Examination of oxidoreductive enzymes viz. laccase, tyrosinase and azo reductase confirmed their role in decolorization and degradation of Rubine GFL. Biodegradation of Rubine GFL into different metabolites was confirmed using HPTLC, HPLC, FTIR and GC-MS analysis. During toxicological studies, cell death was observed in Rubine GFL treated Allium cepa root cells. Toxicological studies before and after microbial treatment was studied with respect to cytotoxicity, genotoxicity, oxidative stress, antioxidant enzyme status, protein oxidation and lipid peroxidation analysis using root cells of Allium cepa. The antioxidant enzymes SOD and GPX were found to have the dose dependent induction, whereas CAT showed dose dependent inhibition. Similarly, lipid peroxidation and protein oxidation rates were also increased indicating the toxic nature of dye and effluent on A. cepa cells. Toxicity analysis with Allium cepa signifies that dye exerts oxidative stress and subsequently toxic effect on the root cells whereas formed metabolites relatively less toxic in nature. Phytotoxicity study reveals the less toxic nature of formed metabolite as compared to control dye Rubine GFL. The successful degradation of Brown 3 REL, Scarlet RR, Methyl Red and Brilliant Blue G was carried out by G. geotrichum. The preferential degradation of azo and non azo dye carried out in mixture. Decolorization affected by the chemical structure and surface function group near to azo bond. So we have studied time dependant degradation of textile dyes by G. geotrichum. In this study proved that the chemical structure affected the decolorization phenomenon. The G. geotrichum MTCC 1360, a yeast species showed 88% ADMI (American dye manufacturing institute) removal of mixture of structurally different dyes (Remazol red, Golden yellow HER, Rubine GFL, Scarlet RR, Methyl red, Brown 3 REL, Brilliant blue) (70 mg l-1) within 24 h at 30oC and pH 7.0 under shaking condition (120 rpm). Glucose (0.5%) as a carbon source was found to be more effective than other sources used. The medium with metal salt (CaCl2, ZnSO4, FeCl3, MgCl2, CuSO4) (0.5 mM) showed less ADMI removal as compared to control, but did not inhibit complete decolorization. The presence of tyrosinase, NADH-DCIP reductase and induction in laccase activity during decolorization indicated their role in degradation. HPTLC (High performance thin layer chromatography) analysis revealed the removal of individual dyes at different time intervals from dye mixture, indicating preferential degradation of dyes. FTIR and HPLC analysis of samples before and after decolorization confirmed the biotransformation of dye. The reduction of COD (69%), TOC (43%), and phytotoxicity study indicated the conversion of complex dye molecules into simpler oxidizable products having less toxic nature. The comparative study of decolorization of two different azo dyes Remazol red and Rubine GFL disclosed the diverse catalytic activities of B. laterosporus. It decolorized 100% of Remazol red and 95% of Rubine GFL within 30 and 48 h, respectively under static condition at 50 mg l-1 dye concentration. Significant increase was observed in azo reductase, NADH-DCIP reductase, 50

Status report on projects supported by DBT veratryl alcohol oxidase and tyrosinase in cells obtained after decolorization of Remazol red; whereas these values were much different with complete inhibition of azo reductase during decolorization of Rubine GFL. The plausible pathway of dyes obtained from Gas chromatography-Mass spectroscopy (GC-MS) data confirmed the different metabolic fate of these structurally unidentical dyes. HPLC, FTIR and HPTLC analysis of extracted metabolites confirmed the biodegradation, while phytotoxicity study assured the detoxification of both the dyes studied. The results obtained in this study suggests, i) sulpho and hydroxyl group present at ortho position to azo group stimulated reduction of azo bond by azo reductase in Remazol red, ii) the same reduction was totally hampered due to presence of ethyl-amino propanenitrile group at para position to azo group in Rubine GFL. B. laterosporus can degrade the sulphonated azo dyes having naphthol ring much faster; supporting that the sulpho and hydroxyl group present at ortho position to azo group stimulated the reduction of azo bond by azo reductase. The functional groups present in the nearby vicinity of azo bond play a central role in azo bond reduction. B. laterosporus decolorized 96% of Brown 3 REL within 48 h at pH 7, 40oC in static condition at 50 mg l-1 concentration. Presence of peptone and yeast extract showed increased decolorization efficiency of B. laterosporus, whereas glucose and starch totally inhibited the decolorization ability. Enzymatic studies indicate the involvement of Veratryl alcohol oxidase and NADH-DCIP reductase enzymes in the biotransformation at these sets of conditions. Biodegradation products of Brown 3 REL were N-carbamoyl-2-[(8-chloroquinazolin-4-yl)oxy] acetamide and N- carbamoyl-2-(quinazolin-4-yloxy)acetamide when identified by Gas Chromatography-Mass spectroscopy. These metabolites found to be much less toxic than Brown 3 REL in phytotoxicity study. In addition to the single dye decolorization, B. laterosporus strain proved its applicability into the effluent treatment plant as it decolorized textile industry effluent with effective reduction in COD and TOC. Same strain used further for biodegradation study of disperse dye Scarlet RR in which, 100% decolorization of same was obtained within 48 h with pH and temperature optima of pH 7 and 40oC, respectively. From the studied carbon and nitrogen sources, decolorization efficiency was observed better in yeast extract and peptone while carbon sources have shown reduced decolorization of Scarlet RR. At the enzyme level, induction in activities of veratryl alcohol oxidase, tyrosinase, NADH-DCIP reductase and riboflavin reductase was recorded. The Biodegradation of Scarlet RR into different metabolites was confirmed by the HPLC, FTIR and HPTLC. A possible fate of metabolism of Scarlet dye was determined using GC-MS. It revealed the proposed biodegradation pathway of Scarlet RR into final product, N-(1λ3-chlorinin-2-yl) acetamide. Phytotoxicity study revealed completely nontoxic nature of degraded metabolites to Sorghum vulgare and Phaseolus mongo plants as compared to toxic Scarlet RR. In addition, B. laterosporus can degrade the dye with high concentrations of it and can also be used repeatedly for at least five cycles. The biodegradation of a mixture containing seven commercial textile dyes with different structures and color properties has been investigated by B. laterosporus. It showed 87% decolorization in terms of ADMI removal (American dye manufacturing institute) within 24 h.

Status report on projects supported by DBT mixture. B. laterosporus can progressively degrade the dye within short span when combined with other dyes in culture; otherwise it needed long time to degrade the same dye when it concerned about single dye decolorization. B. laterosporus have broad specificity as it can degrade structurally diverse range of dyes with significant reduction in its toxicity and have preference in metabolism. The decolorization ability of a bacterial-yeast consortium was also tested with two real textile effluent having different characteristics and a simulated synthetic effluent made up of five different synthetic textile dyes. Consortium decolorized real effluent-1 and 2 with 89 and 60% decolorization efficiency respectively within 48 h. In case of simulated synthetic effluent, 69% decolorization with significant reduction in BOD and COD by consortium, 42 and 16% decolorization by G. geotrichum and B. laterosporus, respectively were detected. In all the set of experiments, decolorizing ability of consortium is much more superior to the decolorizing capability of individual microorganisms. Cumulative action of oxidoreductive enzyme in consortium was found to be responsible for faster decolorization by the same. Fourier Transform Infrared Spectroscopy (FTIR) analysis suggested effective biotransformation of dyes present in the simulated synthetic effluent by consortium as compared to individual strains. The core finding of this study is the monitoring of release of each dye present in the simulated synthetic effluent using High Performance Thin Layer Chromatography (HPTLC). As monitored by HPTLC, consortium biodegraded all the dyes except Malachite green within 1 h only. In contrast, G. geotrichum required 4 h to remove the dyes; while up to 16 h of B. laterosporus treatment, no significant change in HPTLC chromatogram was detected. This study dealt with the proficiency of consortium over individual microorganisms along with the detailed monitoring of gradual biodegradation of each dye from the simulated synthetic effluent. The decolorization efficiency of microbial treatment to real effluent 1 was in the order as, consortium > B. laterosporus > G. geotrichum; however this order was changed to consortium > G. geotrichum > B. laterosporus in case of decolorization of real effluent 2 and simulated synthetic effluent. This suggests that the decolorization efficiency of each microbial species varies with the type of pollutant to be reduced. BOD/COD ratio of real textile effluent 1 and 2 was increased to 0.35 and 0.32, respectively after consortium treatment converting the high strength effluent into easily biodegradable element which confirms the effectiveness of consortium. HPTLC analysis helped in monitoring of rapid and gradual release of each dye chromophore from the effluent. These ecofriendly methods should be used for further applications in large scale studies. But scale up of these parameters should be taken into consideration. Hence for application part of these studies, we studied the decolorization of various dyes and textile industry effluent using immobilized cells of consortium BL-GG. We extended this study be developing two different type of bioreactors i.e. Up flow fixed bed reactor (UFFBR) and Triple layered packed bed reactor (TLPBR) using immobilized cells and used for continuous decolorization of textile industry effluent along with its repeated use. Up flow fixed bed reactor (UFFBR) and Triple layered packed bed reactor (TLPBR) showed an average of 93 and 86% efficiency respectively in seven days run at flow rate of 10 and 100 ml h-1. We could use these bioreactors for repeated use after regeneration of same with nutrient feeding. These studies showed the efficiency and repeated use of immobilized cells on applied level.

Status report on projects supported by DBT

Results of Project: Decolorization and biodegradation of azo-dyes containing dye industries wastewaters

This work showed the isolation of potential microorganisms degrading naphthalene sulphonic acids (NSA) and commercial azodyes from domestic sewage treatment plant, tannery wastewater treatment plant and paper mill waste treating lagoon. Potential degraders were immobilized on insulated beads in fixed bed reactor and the columns were fed with domestic sewage and azodyes.

Results of Project: Biodegradation of textile and dyestuff industrial effluent The work describes the isolation of mixed bacterial and white rot fungal cultures capable of decolourising wide range of textile dyes such as reactive blue 28, reactive violet 5, reactive black 5, acid red 119 and ranocid. Lignolytic enzymes releaesed in the extracellular environment by white rot fungi were studied for decolourisation of dye solutions. A laboratory scale anaerobic up flow fixed film glass column bioreactor with total volume of 1.7 litres and working volume of 1 litre was constructed and decolourization parameters were studied.

Results of Project: Ecotechnology for treating dye waste water of textile industries: A demonstration project

The study has developed an efficient phytoremediation technology for degradation of reactive azodyes and the treated water can be recycled 3 times greatly reducing the requirement of water and quantity of effluent discharged. The treatment involves three steps- primary treatment including adjustment of wastewater characteristics optimal for microbial activities during secondary treatment. Secondary treatment in bioreactor will be performed with the help of a specialized microbial biofilm developed on the inert matrix. BOD of wastewater is decreased to permissible limits in this step and microbial digestion of dyes also reduces colour intensity. The partially degraded dyes are mineralized further in wetlands with the help of a different set of microbes anchored on the plant roots. This step reduces toxicity of dye waste water to minimal as per CPCB limits and also colour.

Results of Project: Development of a biocatalyst system for the treatment of dye effluents

The study describes the isolation of cultures from the textile effluent of Tirupur industrial area and developing a consortia of nine different bacteria which can degrade the mixture of 50 mg/L of Sirius yellow, Brilliant green, Direct blue, Procion Brilliant red, Crysodine, Supranol green, Proc. Navy Blue, Supranol red to 30-40% in the alkaline and salt conditions within three or four days. The work further describes adhesion of consortia into various matrices like coir, jute, brick, gramophone records, stones, laterite stones and found laterite stones to be the best. She had also experimented with packed bed reactor, trickle bed reactor and rotating disk biological contractor and could achieve upto 80% degradation of 50 mg/L. of combined dye solution at pH 9 and salinity of 3.6%. Treated dye solution was nontoxic to fish upto 96 hours.

Results of Project: Isolation, Identification and Characterization of Genes for Azo Dye degradation: An approach towards construction of efficient bioremediation strain

This multi-institute research project describes the isolation 33 distinct cultures from the polluted soil sample and development of 42 distinct consortia capable of degrading azo dyes and enriched consortium SB4 was studied in detailed, its nutritional and environmental parameters were optimized. Metagenomic DNA was extracted from a selected robust bacterial consortium by 53

Status report on projects supported by DBT employing selected robust Pseudomonas strain Azoreductase gene, screened and amplified from pUC19 vector based clones, revealed an ORF of 178 amino acids, showing 98% homology with Azoreductase gene amplified by degenerate primers was of 537bp and contained an ORF of 178 amino acids. Construction of phagemid library (cloning of large-inserts) with Stenotrophomonas acidaminiphila reported 10 clones having 75% azodye reduction potential. Azoreductase gene was cloned in pET system, which showed maximum homology with azoreductase from Bacillus cereus Q1. pET1 clone E. coli BL21(DE3) which demonstrated 90 % of dye decolourizing activity within 7 minutes only in the presence of NADPH.Na4 as co-factor.

Results of Project: Production of fungal metalloenzymes by Pleurotus ostreatus and their application in bioremediation of Azo dyes

The study demonstrated the isolation and identification of new white rot fungus Ganoderma cupreum AG-1 and its immobilization along with laccase in hydrogel of Acrylamide-Alginate (Aam - Alg) polymer showed better dye decolourization. It has been noted that the encapsulation of enzyme into Aam-Alg hoydrogel works well for immobilization of laccase enzyme with 93.96 % loading efficiency. The immobilization of Ganoderma cupreum AG 1 in hydrogel results in increased temperature and pH stability and reusability of laccase enzyme.

Results of Project: Development of microbial consortia for the biodegradation and biodecolourization of textile effluents

The work describes that the textile effluent sample was treated using microbial cells immobilized in a matrix amended with activated carbon showed good results in terms of color reduction where as the organic load reduction was found to be in a similar level as to the sample treated under aerobic condition. The effluent treated by the consortium was amended with different concentrations of carbon and nitrogen compounds as co-metabolites in order to improvise and enhance the bioremediation nature of the consortium in decreasing the lagging time required for the synthesis of required necessary enzymes. The consortium was also optimized under different physical conditions. When the same consortium was used in the treatment of effluent in an industry dealing with reactive dyes, the consortium was optimized under different conditions and studied for its bioremediation capacity.

7.5 Summary and brief assessment of the results With the type of the objectives proposed in the research projects, it was obvious that, very basic but necessary results were reported. From isolation of strains, study of requirement of carbon and other nutritional source, optimization of environmental factors, study of enzymes involved in dye decolourization and degradation, immobilization of whole cells and enzymes, reactor level demonstration of treatment efficiency of isolated strains/consortia to isolation and cloning genes involved in dye decolourization were studied and encouraging results were observed and reported. A very striking and prominent feature was that, though the results from all the projects were highly encouraging and reproducible, except couple of study none of them were carried to the next level and the lab scale results were only limited to scientific publications.

Steps involved in textile processing  08 Report the projects supported by other national agencies in the area of dye bioremediation

8.1 Opening Remarks 55 8.2 Pre-Support/Sanction Process for the Research Proposal 55 8.3 Primary Statistics of the Sanctioned Projects 55 8.4 Studying the objectives 55 8.5 Analysing the Results 57

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8.1 Opening Remarks Besides, Department of Biotechnology, many other leading national agencies (few are mention below) supports research and development in the area on developing strategies and technology for remediation of textile and dye containing effluent.

1. Department of Atomic Energy (DAE) 2. Department of Environment (DOE) 3. Department of Non-Conventional Energy Sources (DNES) 4. Department of Science and Technology (DST) 5. Council of Scientific and Industrial Research (CSIR) 6. Defense Research and Development Organization (DRDO) 7. University Grants Commission (UGC)

8.2 Pre-Support/Sanction Process for the Research Proposal Similar to DBT, the research proposals are invited through-out the year or via special call for proposal on the specific themes. The submitted proposal are bring primarily screened, sent for review and principal investigator are called for presentation in Task Force Meeting and selected meritorious research projects are supported financially. The normal tenure of the financial support ranges between 24 and 36 months.

8.3 Primary Statistics of the Sanctioned Projects DST has supported 43 research projects in this area since 1990 and collectively 1140 months were spent, while UGC has funded 87 research proposals through Major and Minor Research Projects and corresponding to 2575 months, whereas near 20 research projects (and 312 months) are being supported by CSIR and AICTE has supported 21 research projects (and 564 months) for developing novel technology for remediation of dye and textile effluents.

8.4 Studying the objectives To understand the nature of research being conducted in the projects supported by above agencies, objectives of few successfully completed projects are listed in Table 8.1.

Table 8.1: Research objective of few projects (as representative) supported by different national funding agency other than DBT

Project I Biodegradation of triphenylmethane dyes by Penicillium ochrochloron Objectives 1. To find out the potential of Penicillium ochrochloron for triphenylmethane dye degradation. 2. Standardization of media, static or shaking condition, pH, temperature for dye degradation. 3. Analysis of the product of dye metabolism and fate of metabolism. 4. Phytotoxicity and Genotoxicity study of degraded metabolites. Project II Decolourization of metal containing acid dyes Objectives 1. To isolate, enrich and characterize potential consortia degrading metal complex acid dyes (MCAD)

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2. To optimize MCAD degradation parameters for consortia 3. To adapt the consortia to extreme conditions in MCAD effluent 4. To develop process for the treatment of actual industrial effluent Project Bioremediation of dye containing wastes and their microbial diversity III Objectives 1. Development of efficient bacterial consortia for the treatment of dyes and dye containing industrial wastes. 2. Metabolic and genetic diversity profiling of all developed bacterial consortia 3. Biodegradation of selected synthetic industrial dyes in micro-aerophilic and sequential micro-aerophilic-aerobic condition 4. Elucidation of degradation pathway 5. Bio-treatment of actual industrial wastewaters of local dye manufacturing industry 6. Optimization of process parameters by conventional and advanced statistical methods 7. Scale up of developed bioprocess 8. Application of process at industrial scale Project IV Decolorization of textile dyes using Aspergillus ochraceus Objectives 1. To find out the potential of Aspergillus ochraceus NCIM-1146 for textile dye degradation. 2. Standardization of media, static or shaking condition, pH, temperature for dye degradation. 3. Analysis of the product of dye metabolism and fate of metabolism. 4. Phytotoxicity study of obtained metabolites. Project V Studies on microbial decolorization and degradation of toxic dyes from textile effluent. Objectives 1. Isolation and identification of microbial communities having potential for the decolorization and degradation of industrial textile dyes. 2. Standardization of media, growth conditions and physicochemical conditions for maximum decolorization and degradation of industrial dyes and textile effluent. 3. To study the nature of enzyme system responsible for dye degradation. 4. To study the characteristics of textile effluent, mineralization change and toxicity studies before and after microbial treatment. 5. Study of immobilized cell technology for the degradation of industrial textile dyes and effluent. 6. Development of an effective bioreactor design using immobilized microbial cells and optimization of various physicochemical and operation conditions for the treatment of industrial effluent. 7. The project is aimed for conducting systematic and detailed studies on microbial process to develop rapid, economically viable and environmental sound technology for the treatment of textile dye industry effluent and corporate technology collaboration to enhance wastewater management in India. Project VI Biodegradation of textile dyes (Golden yellow HE2R & Navy Blue 3G using Brevibacillus laterosporus

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Objectives 1. Standardization of the media conditions and other parameters (Temperature, pH, carbon and nitrogen source) for maximum degradation of textile dyes viz. Golden Yellow HE2R and Navy Blue 3G using Brevibacillus laterosporus. 2. Study of enzyme system responsible for the decolorization of Golden Yellow HE2R and Navy Blue 3G present in Brevibacillus laterosporus. 3. Study of metabolic pattern of degradation of Golden Yellow HE2R and Navy Blue 3G by Brevibacillus laterosporus using TLC, HPLC, GC. Determination of structure of liberated metabolites of Golden Yellow HE2R and Navy Blue 3G using analytical techniques viz. FTIR, GCMS. 4. Purification of enzyme prominently responsible for degradation of Golden Yellow HE2R and Navy Blue 3G and possible use for co-metabolism. 5. Study of longetivity of decolorization activity by Brevibacillus laterosporus cells, enzymes and immobilized whole cells in repeated batch decolorization tests. 6. Evaluation of toxicity of generated metabolites using plant seeds Sorghum vulgare and Phaseolus mungo (% Germination, Plumule and Radical development) and Azotobactor vinelandii and Psedomonas sp. (growth inhibition of microorganism).

The status of research supported by national funding agencies, other than DBT for developing/demonstrating any novel technology for remediation of dye containing textile effluents are no different. Isolation of potential microbial strains, development of consortia, optimization of media components and environmental factors, study of dye decolourization and degradation pattern (dye degraded intermediates), phyto-and genotoxicity analysis, enzymatic studies, laboratory scale bioreactor studies on simulated or real textile effluents, etc. are the very prominent and repetitive objectives in majority of the research projects. Very few studies were directed towards developing any novel methodology for remediation of textile effluents, but they are also limited to either lab scale or pre-pilot to pilot level. As is can be seen from the above table only Project III, have shown some application at industry level, while the academic interest are more visible than to develop any feasible technology.

8.5 Analysing the Results With the type of objectives proposed in the research projects, following results were reported which obviously shows the very basic nature of the work done in this area.

8.5.1 Results of Project I The present study dealt with the decolorization and degradation of triphenyl methane textile dye by mycelium of Penicillium ochrochloron. Spectrophotometric and visual examinations showed that the decolorization was through fungal adsorption, followed by degradation. Triphenylmethane dyes belong to the most important group of synthetic colorants and are used extensively in the textile industries for dying cotton, wool, silk, nylon, etc. They are generally considered as the xenobiotic compounds, which are very recalcitrant to biodegradation. Penicillium ochrochloron decolorizes cotton blue (50 mg l−1) within 2.5 h under static condition at pH 6.5 and temperature 25 °C. TLC, FTIR and HPLC analysis confirms biodegradation of cotton blue. FTIR spectroscopy and GC–MS analysis indicated sulphonamide and triphenylmethane as the final products of cotton blue degradation. The pH, temperature and

Steps involved in textile processing maturity of biomass affected the rate of decolorization. Presence of lignin peroxidase, tyrosinase and aminopyrine N-demethylase activities in the cell homogenate as well as increase in the extracellular activity of lignin peroxidase suggests the role of these enzymes in the decolorization process. The phytotoxicity and microbial toxicity studies of extracted metabolites suggest the less toxic nature of them. Malachite green was detoxified into p-benzyl-N,N-dimethylaniline and N,N-dimethyl-aniline hydrochloride by Penicillium ochrochloron. Degradation metabolites were analyzed by TLC, HPLC, and FTIR and identified by GCMS analysis. Phytotoxicity testing revealed the nontoxic nature of these metabolites. The percentage decolorization of malachite green (50 mg/L) was 93% in czapek dox broth after 14 h with an optimum pH of 7 at 30ºC. The induction in the activity of lignin peroxidase after degradation suggested that the degradation of malachite green was peroxidase-mediated. Fungal culture was also found to have detoxified the textile effluent. The values of TDS, TSS, COD, and BOD were reduced in the treated samples compared to the control effluent. The treated effluent was non-toxic to the plants of Triticum aestivum and Ervum lens Linn, and the amount of total chlorophyll was higher in plants with treated effluent when compared to control effluent. This study showed that fungal mycelia could effectively be used as an alternative to the traditional physico-chemical process. The main highlight of the project was Penicillium ochrochloron was found to decolorize textile dye Malachite green, Cotton blue. This study suggests that this strain could be a useful tool for textile effluent treatment and the alternative to the traditional physicochemical process.

8.5.2 Results of Project IV The study dealt with the decolorization and degradation of textile dye Reactive blue-25 (0.1 gl-1) by mycelium of Aspergillus ochraceus NCIM-1146. Spectrophotometric and visual examinations showed that the decolorization was through fungal adsorption, followed by degradation. Shaking condition was found to be better for complete and faster adsorption (7 h) and decolorization (20 d) of dye Reactive blue-25 (100 mgl-1) as compared to static condition. Presence of glucose in medium showed faster adsorption (4 h) and decolorization of dye from bound (7 d) mycelium. FTIR and GCMS analysis study revealed biodegradation of Reactive blue-25 into two metabolites phthalimide and di-isobutyl phthalate. Aspergillus ochraceus (NCIM-1146) has ability to decolorise various textile dyes viz. Purple 2R, Orange TG22, Yellow HE64, Red HERB and Golden yellow HER was determined by monitoring the decrease in absorbance of each dye in the culture supernantant. Decolorization performance of Purple 2R with various conditions such as different media, concentration of dye, agitation and static condtions were studied. The decrease in dye decolorization capability of mycelium was observed with increasing dye concentration in repeated batch mode. Spectrophotometric data revealed that the process involved in decolorization is through microbial metabolism but not biosorption. Phytotoxicity study demonstrated no toxicity of the biodegradated products for plants with respect to Phaseoulus mungo and Sorghum vulgare. Aspergillus ochraceus (NCIM-1146) has ability to decolorize various xenobiotic dyes. Biodegradation of dyes was demonstrated by their decolorisation in the culture medium. The extent of biodegradation was determined by monitoring the decrease in absorbance of each dye. Malachite green decolorisation activity is affected by various conditions such as composition of media, concentration of dye, amount of mycelia and agitation. The durability of decolorisation activity under optimum conditions was investigated in repeated batch mode. An increase in the

Steps involved in textile processing amount of mycelia positively affected the durability of decolorisation activity. The decrease in dye decolorisation capability of mycelia occurred with increasing dye concentration in repeated batch mode. This organism showed significant ability to decolorize all the dyes tested viz. malachite green (98%), cotton blue (92%), methyl violet (61%) and crystal violet (57%) in 24 h incubation. Spectrophotometric data revealed that the process involved in decolorisation is through microbial metabolism but not biosorption. This study showed that fungal mycelia (A. ochraceus) could effectively be used as an alternative to the traditional physico-chemical process. The main highlight of the work was, Aspergillus ochraceus NCIM-1146 was found to decolorize textile dye Reactive blue-25, Purple 2R, Malachite green, Cotton blue, Methyl violet and Crystal violet. This study suggests that this strain could be a useful tool for textile effluent treatment and the alternative to the traditional physicochemical process.

8.5.3 Results of Project VI Growth of B. laterosporus: The exponential phase of B. laterosporus started after 12 h of incubation and continued up to 72 h at 30oC. The status of the biotransformation enzymes was found to be optimum at 36 h of incubation which was the mid exponential phase. These results probed the use of Brevibacillus culture at its mid exponential phase for dye decolorization studies. Decolorization of dyes using B. laterosporus and the effect of physico-chemical parameters: GY-HE2R and NB-3G showed 87% and 80% decolorization respectively, within 48 h under static condition at 50 mgl-1 concentration. However, no significant change in the decolorization potential was observed under shaking conditions. Decolorization performance of all the dyes GY- HE2R and NB-3G was decreased with an increase in the initial dye concentrations from 0.1 to 1.0 g l-1. B. laterosporus steeply decreased the decolorization of Golden Yellow-HE2R and Navy Blue-3G at the concentration 1.0 g l-1 with poor performance even after extended incubation (46 and 66%, respectively within 72 h). In case of Navy Blue-3G, better performance of decolorization was observed in the quite broad range of pH 7.0 to 11.0; however decolorization of Golden Yellow-HE2R was found in the broader range (pH 5.0 to 9.0). In present study, B. laterosporus exhibited maximum decolorization at 30 °C whereas in case of Golden Yellow-HE2R and Navy Blue-3G, more than 60% of the dye was removed even at 15 °C, after an extended incubation period. Studies show that B. laterosporus has better decolorization activity of the two studied dyes during its exponential growth phase. The organism showed more efficient decolorization in 24-96 h old cultures. When 40 mg l-1 wet weight of the culture was used, the time required for complete decolorization of GY-HE2R and NB-3G was reduced to 18 h and 12 h respectively. Moreover, nitrogen sources were found to be stimulatory for the decolorization activity as compared to the studied carbon and phosphorus sources. KH2PO4 and NaH2PO4 were found to have no significant effect on the decolorization of GY-HE2R and NB-3G. Among the carbon sources studied, starch was better source whereas in presence of glucose, the cells have shown poor decolorization in all the cases. Complete decolorization of dyes occurred simply in the synthetic medium (without any supplementary components) when provided with an extended incubation. The organism retained good dye decolorization abilities even when used in repeated cycles. The growth of B. laterosporus was remarkably increased during the decolorization of GY-HER and NB-3G (45 and 56% respectively) after 24 h of incubation; whereas it decreased (18%) during the decolorization of GY-HER after the further incubation up to 72 h.

Steps involved in textile processing

Decrease in the activity of tyrosinase (31%), no significant change in the activity of laccase whereas significant increase in the activities of LiP (156%), aminopyrine N-demethylase (120%), DCIP reductase (76%) and MG reductase (40%) was found in the cell free extract obtained after 48 h of incubation during the decolorization of Golden Yellow-HER by B. laterosporus. Complete inhibition of tyrosinase, no significant change in the activities of laccase and DCIP reductase where as significant increase in the activities of LiP (54%), aminopyrine N-demethylase (122%), and MG reductase (50%) was found in the cell free extract obtained after 48 h of incubation during the decolorization of Navy Blue-3G by B. laterosporus. In addition, no significant change during the decolorization of GY-HER, significant decrease (8%) during the decolorization of NB-3G where as significant increase (~10%) during the decolorization of MG and MR was observed in the activity of LiP in to the culture supernatant (i.e. extracellular). No activities of laccase, tyrosinase as well as DCIP reductase, MG reductase and aminopyrine N- demethylase were found in to the culture supernatant.The findings of microbial and phytotoxicity studies suggest that B. laterosporus produces nontoxic metabolites with respect to the tested microorganisms and plants.

Analysis of products formed after the degradation of GY-HE2R: TLC chromatogram shown single spot (Rf = 0.98) of control GY-HER, whereas three different spots (Rf = 0.83, 0.78 and 0.71) and single spot (Rf = 0.91) of the degradation products extracted after 24 and 48 h respectively. HPLC chromatogram of control Golden Yellow-HE2R showed a single peak at the retention time 1.593, whereas three different peaks of degradation products extracted after 24 h at the retention time 2.618, 2.707 and 2.932 and two peaks of the degradation products extracted after 48 h at the retention time 2.299 and 2.453. Overall findings suggest degradation as well as further biotransformation of the dye. FTIR spectrum of control dye displayed a peak at 3431 cm-1 for N-H stretch, a peak at 2924 cm-1 -1 -1 for CH3 stretch, a peak at 1574 cm for –N=N- stretch, a peak at 1487 cm for ring vibrations, a -1 -1 peak at 671 cm for C-H bend, a peak at 1188 cm for SO2 stretch where as peaks at 1082, and 1039 cm-1 for C-N stretch as well as a peak at 615 cm-1 for C-Cl stretch suggested aromatic azo nature of the dye and confirmed its chemical structure. FTIR spectrum of degradation products displayed the peaks at 3241, 1330, and 1106 cm-1 suggested the formation of N-N-disubstituted sulfonamides from the parent dye molecules. Disappearance of a peak at 1574 cm-1 for an azo stretch clearly indicated the breaking of azo bond by B. laterosporus that would be an essential and foremost step for the color removal. A peak at 1236 cm-1 for C-N stretch of Ar–NH–R suggests the formation of aromatic amines. Biotransformation was further confirmed by HPLC chromatogram that has shown three peaks of degradation products extracted after 24 h with the retention time 2.252, 2.473 and 2.697 whereas two peaks of degradation products extracted after 48 h with the retention time 2.256 and 2.474. Disappearance of all peaks of the control dye confirmed the degradation of the Navy Blue-3G as well as its contaminants. FTIR spectrum of control Navy Blue-3G displayed a peak at 3430 cm-1 for N-H stretch, a peak at 2923 cm-1 for C-H stretch, a peak at 1738 cm-1 for –O–N=O stretch, a peak at 1698 cm-1 for >C=O stretch, a peak at 1606 cm-1 for ring vibrations, a peak at 1513 cm-1 for Ar–N=O stretch of aromatic nitro compound, a peak at 1328 cm-1 for C–N stretch of Ar–NH–R, a peak at 1174 cm-1 for asymmetric ring vibrations, a peak at 1039 cm-1 for –C–O–C stretch and a peak at 614 cm-1 for C–Br stretch confirmed the chemical structure of dye. FTIR spectrum of the degradation products extracted after 48 h retained the peaks at 3240 and 1671 cm-1 for –N=O stretch suggests 61

Steps involved in textile processing that nitroso compounds remained unvanished up to longer period. A peak at 1514 cm-1 for Ar– N=O stretch of aromatic nitro compound stretch where as various peaks in the fingerprint region for mono and di-substituted benzene rings confirmed the formation of aromatic nitrosamines (Fig. 6). A pathway has been proposed for the degradation of NB-3G based on GCMS analysis. Bromo-benzene (m/z 156, peak 1; m-2 = 154), ester substituted aniline derivative (m/z = 178, peak 2), meta-di nitro benzene (m/z 167, peak 3; m+1 = 168) and ortho benzene aniline derivative (m/z 169, peak 4; m+1 = 170) are the elected products. Decolorization of textile effluent: 100 ml batch culture of the organism decolorized 35% of textile effluent within 24 h. Approximately 5 g (wet wt) of the immobilized beads kept in 250 ml effluent under static condition (in 500 ml beaker) decolorized about 60% of the effluent within 24 h and retained 50% decolorization efficiency upto 5 cycles. Purification of intracellular lignin peroxidase: A total amount of about 280 mg of protein, corresponding to approximately 86 units of lignin peroxidase was loaded onto the column. This ion exchange column chromatography allowed the recovery of 35.55 units in 1.98 mg of protein with a yield of 40% and purification factor of approximately 57 fold. Although homogeneity was obtained and confirmed by PAGE, elution of enzyme from fraction number 39 to 69 (i.e. broad range) suggested the presence of multiple binding sites with different ionizable groups. In the present study, PAGE gel developed by Coomassie brilliant blue staining has shown single band for a protein having molecular weight about 205 kDa where the molecular weight of an enzyme was estimated using MW-standard markers. Moreover, PAGE gel developed by the activity staining using L-DOPA has shown only one band at the same run as that of developed by Coomassie brilliant blue staining. The enzyme was active throughout a broad range of temperature, its optimum temperature being 40oC while its optimum pH was found to be 4.5. Maximum activity of the enzyme was observed with 750 mM concentration of tartaric acid.

Lignin peroxidase from the B. laterosporus was found to be dependent on H2O2 since no activity exhibited in the absence H2O2. Continuous increase in lignin peroxidase activity with the increasing concentration of H2O2 from a range 10 mM to 1 M was observed. The Km value of the enzyme was found to be 1.6 mM. FTIR spectrum of the degradation products of Golden Yellow-HE2R formed by purified lignin peroxidase displayed a peak at 1720 cm-1 for carbonyl stretch, a peak at 1443 cm-1 for an -1 asymmetric bend of –CH3 of alkyl benzene derivative, a peak at 670 cm for C-H bend of benzene, a peak at 1276 cm-1 for –C–O–C–asymmetric stretch whereas the peak at 2929 cm-1 for C-H stretch of methyl group suggests the formation of aryl ethers by the transformation of dye. The major highlights of the project was to explores the dye degrading abilities of B. laterosporus, with studies of the basic underlying mechanisms behind the removal of the dyes, GY-HE2R, NB- 3G and textile effluents. The intracellular enzymes as well as whole cell systems can be used as effective bioremediating agent.

National and International case studies  09

National and International Case Studies

9.1 National Case Studies 63 9.1.1 Case study I: Environment Services Co-Operative Society Ltd., 63 Vatva (Common Effluent Treatment Plant) 9.1.2 Case study II: Sivasakthi Textile Processors, Tirupur, Tamilnadu 65 9.2 International Case Studies 69 9.2.1 Case Study III: Malaysia 69 9.2.2 Case study IV: Study of a Treatment Plant in Germany 73

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9.1 National Case Studies

9.1.1 Case Study I

(A) Environment Services Co-Operative Society Ltd., Vatva (Common Effluent Treatment Plant)

Vatva industrial estate (VIE) is one of the oldest and pioneering industrial setup in the state of Gujarat, started its operation in the year 1960, is spreaded over 572 hectares of land housing approximately 2100 small, medium and large industrial units. Dye and dye intermediates are the major units in the estate with 356 units, 90 unites are manufacturing chemicals and 12 industries are involved in textile production, while remaining industries are involved in plastics and rubbers, engineering, pharmaceuticals, foundry, metal treatment, paint and varnish, steel and furnace production . It is obvious that dye, dye intermediates and textile units are the major source of pollution in the estate. For the treatment of liquid and solid wastes, member industries in the estate have established Common Effluent Treatment Plant (CETP), which operates as Green Environment Services Co-Operative Society Ltd. (GESCSL) in the year 1992. Nearly 90 % of the effluents treated in the CETP are from dyes, intermediates, textiles and chemicals. The operating capacity of the CETP is 16 MLD, with annual running cost of Rs. 2400 lakhs. It is spread on a land area of 20,000 m3.

A.1 Treatment Technology The basic treatment facility in the CETP is conventional primary and secondary treatment with extended aeration facility. The basic design for process and operation (including technology) was provided by M/s. Advent Corporation, USA, based on the treatability study carried out by M/s. Sudarshan Chemical Industries Ltd., Pune. The detailed engineering and setup outlay was provided by M/s. Sudarshan Chemical Industries Ltd., Pune. The complete plant commissioning CETP and overall expert supervision was provided by M/s. Advent Envirocare Technology Pvt. Ltd., Ahmedabad.

A.2 Salient Features The CETP was designed and works on AIS (Advent integral system) technology. The system contains aeration basin surrounded by integrated peripheral secondary clarifier. During the upgradation after few years of the programme, integral clarifier was converted into aeration zone and two separate secondary clarifiers were additional added. The aeration is provided by medium bubble aeration grid supplemented with 12 distinct Triton type aerators supplied by M/s. Aeration Industries International Inc., Minneapolis, USA. The handling capacity (basic parameters) of the CETP is 16000 m3/day of inflow effluent, and can treat effluent having COD of 3000 mg/L, BOD of 1200 mg/L with TSS of 600 mg/L.

A. 3 Effluent Collection and Conveyance System The effluents from 674 members are conveyed to CETP from individual industrial units through underground (internal) collection system consisting of rising mains. Flow-meters and auto- samplers are installed at each inlet pipelines to monitor and measure the inlet effluent parameters. Treated wastewater from CETP is conveyed to Domestic effluent (sewage) treatment plant of 64

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Ahmedabad Municipal Corporation at Pirana through closed pipeline of ‘Naroda Pirana Pipeline Project’. The wastewater is mixed with domestic (treated) sewage to meet with the TDS parameter, before discharging into Sabarmati River.

A.4 Treatment Process As noted above CETP works with primary physico-chemical treatment and secondary biological treatment. It is mandatory for all 674 member units to provide initial primary treatment (to the effluent) in their industrial units, in order to meet the inlet norms (as mentioned above) of the CETP. A unique effluent collection network system is established by CETP. Effluents from the individual units (through overhead tank) are collected in a collection room termed as “SUMP Room”. Currently, there are 94 such SUMP rooms are constructed, where in an each SUMP room maximum of eight pipelines for as many industries are converged, having flow-meter and auto- sampler for each pipelines. From every SUMP room, again through network of pipelines, effluents are conveyed to Pumping Station which is finally channeled into CETP. A schematic diagram of the complete outlay of the system network is displayed in Figure 9.1. Another notable feature of the entire effluent collection system is a specific time slot is provided to individual units for the discharge of their primarily treated effluents; also the quantity of the flow is defined and measured regularly. After entering into the CETP, effluents are collected, sampled and pumped to an equalization tank. Heterogeneous effluents in the equalization basins are mixed through an aeration grid for homogenizing the entire content in the basin. These homogenous effluents are pumped into the flash mixer where chemical additive and supplements are added and effluent flows to flocculator. By natural gravity the flocculator overflow goes to the Dissolve Air Flotation (DAF) unit. It works on the principal of super saturation of the liquid with dissolved air, which is achieved by mixing pressurized recycled wastewater and external compressed air. The saturated recycled wastewater flow is released in DAF tank at about atmospheric pressure forming fine bubbles. The solids particles from the top and the bottom of the tank are separated by scrapper are collected in the sludge holding tank and sent to centrifuge decanter for dewatering and final disposal at Vinzol secured landfill site. Post-primary treatment, through natural gravity, influent from the DAF tank flows to aeration basin (having a holding capacity of 20600 m3). In aeration basin, organic matters of the wastewater are aerobically degraded by activated sludge in extended aeration regime. Course bubbled diffused aeration is provided to maintain the Dissolved Oxygen levels in the basin and also MLSS in the suspension from. The aeration basin plant is programmed and designed to operate at an F/M of 0.12 based on BOD/MLVSS. The treated effluents from aeration basin are pumped into Dispersion Transition and Flocuulation (DTF) channel by two air lift pump (with maximum capacity of 900 m3/h). In the channel air is lifted along with the liquid and dispersed by providing zig-zag baffles to create turbulence in the flow to allow entrap air to escape. The baffles also help in mixing mixed liquor with polyelectrolytes for enhancing the flocculation. To separate the biomass from the treated wastewater, mixed liquor is fed to the integrated secondary clarifier though a distribution network and rest of the flow is re-circulated to the aeration basin. The portion of the collected concentrated biomass is recycled from clarifier to the aeration basin through a bottom hopper, while the remaining biomass is transferred to centrifugal decanter for dewatering.

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At the end of the wastewater treatment in CETP, as noted above the treated wastewater is conveyed to Domestic treatment plant and mixed with domestic (sewage) effluent to meet the TDS norms for final discharge in to Sabarmati River. To access the treatment efficiency of CETP, Vatva, an earlier detailed study is discussed here, where physico-chemical characteristics of wastewaters from nine dyes and dye intermediates manufacturing industries and one from CETP was analyzed. The values of twenty different parameters from nine industries (I, II, III, IV, V, VI, VII, VIII, X) and CETP outlet (CO) are listed in Appendix I. The schematic outlay of the treatment process is as described in Figure 9.1.

Figure 9.1: The schematic outlay of the treatment procedures followed at CETP, Vatva

9.1.2 Case Study II B.1 Sivasakthi Textile Processors, Tirupur, Tamilnadu The river Noyyal (tributary to river Cauvery) harbors the city of Tirupur, in Coimbatore District in the state of Tamilnadu, which is well known for its cotton fabric production. It was found that quality of water of the river and the climatic conditions has been ideal for dyeing operation of fabric and yarn. The city houses about 748 dyeing and bleaching industries that generates over 90,000 m3/day of wastewater. Looking to amount of daily discharge of dyeing effluent 20 CETPs are in operation treating wastewater from 502 industries, while other industries are having local treatment plants. They were able to remove color and other organic compounds to the set stipulated standards; however treatment technology failed to arrest the inorganic contaminants.

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Both treated and untreated textile effluents are discharged in the river causing the loss of pristine ecosystem.

B.2 The Case of Sivasakthi Textile Processor The textile industrial unit at Sivasakthi has six batch flow reactors for dyeing (i.e. wetting, bleaching, neutralizing, washing, colouring, washing, etc.) of different capacities. One ton of cotton yarn requires 10 m3 of water, whereas 1 ton of polyester yarn consumes only 4 m3 in each steps. For dyeing process, it requires alkali and sodium salt. The quantity of salt (mostly sodium chloride) used is usually depends on the requirement of shade of the colour to be applied. The daily production capacity of the unit is 1500 – 2000 kg fabric and the volume of effluent generated is of the order of 100 – 200 m3/day. The effluents generated are segregated into dye bath wastewater and wash water and treated accordingly. In primary treatment plant, wash water is collected in holding tanks for flask mixing and addition of slurries of lime and ferrous sulphate with effluent and allowed for settling. Following the primary treatment, effluent is pumped in sequential manner to pressure sand filter, iron removal filter, ion exchange filter and reverse osmosis (RO) system. (The treatment facilities also possess double stage RO system, each with six spiral wound membranes. Pump pressure is maintained in the range of 21.2–28.2 kg/cm2. The filtrate of first stage RO system is sent to second RO and the final reject (~ 20 %) is carried to Multi Effect Evaporator (MEE). The condensate water from MEE process is recycled in the cleaning operations. The treated effluent from MEE having 100 g/L solid content is allowed for solar evaporation. The wastewater from dye bath water collected in a separate tank is pre-filtration (essentially required to increase the self-life of the nano-membrane) followed by nanofiltration. Nearly 30 % of the non-filtered waste water is carried for multi effect evaporation (MEE) and solar evaporation systems (SES). At the end of the process permeate is usually used for preparation of dye bath solution. A typical schematic diagram of the effluent treatment plant provided with primary, secondary and advanced treatment technologies for recycling of wastewater is presented in the Figure 9.2.

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Figure 9.2: Typical schematic diagram of advanced wastewater treatment technology for recycling of textile dyeing wastewaters The characteristics of the raw effluents, intermediate effluents and permeate are presented in the table 1&2. It can be observed that low hardness of permeate is an added advantage in the process. Reject of NF filtration contains 4.8 % salts that is mixed with more salts and used in the process. Salt recovery from the dye bath alone has 50 % returns.

Table 9.1: Average reduction of BOD, COD, TDS, Na, Cl of CETPs in Tirupur

Parameters Percentage removal BOD 88–98 COD 91–97 TDS 80–97 Sodium 96 Chloride 76–97

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Table 9.2: Characteristics of effluents of Shivasakthi Textile Processors, Tirupur

Parameter Wash water Dye bath water Inlet Outlet of RO RO Dye bath NF ETP chemical permeate rejec wastewater reject treatment t pH 9.76 9.78 7.52 8.21 10.42 8.21 Electrical 6.80 6.63 0.77 32.1 53.9 63.55 conductivity(m S/cm) Total suspended 47 26 BDL 46 76 60 solids(mg/L) Total dissolved 4280 3620 474 2167 39179 48294 solids(mg/L) 0 BOD (mg/L) 80 63 10 450 180 100 COD (mg/L) 317 204 24 1143 909 402 Total hardness 320 141 3 728 88 45 as CaCO3) (mg/L) Ca‐hardness 272 104 3 687 68 22 (mg/L) as CaCO3 Sulphate 75 116 8 328 174 362 (mg/L) Chloride 1912 1771 184 1075 19179 26432 (mg/L) 6 Sodium (mg/L) 1600 ‐ ‐ 9280 ‐ 20480 Potassium 38 ‐ ‐ 208 ‐ 62 (mg/L) % sodium 90 ‐ ‐ 95 ‐ 100 Sodium 39 ‐ ‐ 146 ‐ 1329 absorption ratio (SAR)

B.3 Cost analysis and viability of the water recycling process The cost of installing fully functional unit and it final commissioning cost in the range of Rs. 40 – 100 lakhs for small scale (< 300m3/day), Rs. 100 – 200 lakhs for medium scale (300 – 600 m3/day) and Rs. 200 – 300 lacs for large scale (> 600 m3/day) scale textile dying industries. Also the maintenance and operation cost is high as can be seen in the Table 9.3

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Table 9.3: Operating and Maintenance cost of the wastewater treatment plant

Cost Currency (INR) Chemical cost 7 – 8 /m3 Power cost 2 – 3 /m3 Sludge handling 0.75 – 1 /m3 Manpower 1 – 2 /m3 Filters/cartridges (spares) 5 – 10 /m3 RO/NF membrane maintenance 15 – 20 /m3

The total expenses for the water treatment and recovery is estimated around Rs. 80/m3 of the raw effluent. Due to excessive use to fresh water, the quality of water in Tirupur is not good for dyeing processes. Hence, it is purchased from villages located at 15 km away from the treatment facility that further adds to the cost approximately to Rs. 100/m3 (including transportation). The operating cost of MEE and maintenance of RO membrane module are very high. MEE proves to be costly system for concentration of the effluents and it requires more maintenance and also consumes more fuel at an average of Rs. 400 per m3 of premate. The recovered salt has poor purity and market value of salt is also very less (Rs. 4/kg). To reduce the chocking of RO membrane proper preliminary treatments is required. The advantage of this treatment technology is that, recovered water from the membranes is with extremely low hardness, which is always a prime requirement of textile sector for an improved finish and better quality dyeing. The treatment and maintenance cost is around Rs. 80/m3, cheaper than the input water cost (Rs. 100/m3).

9.2 International Case Studies 9.2.1 Case Study III: Malaysia Textile industries are one of the major industrial sectors in the Asian continent. India, China, Bangladesh, Malaysia, etc. are leading manufacturer and exporter of natural and synthetic textiles. Amongst them Malaysia contributes to nearly 1.3 % of synthetic textile (fibers) of the total world’s production. Malaysia’s textile industry has already crossed US$ 5 billion in terms of their export. The obvious immediate significant impact is seen in terms of environmental quality. In Malaysia, every textile industrial unit treats its own effluents and treated wastewaters are normally released to a drain. Therefore, it is mandatory for the industries to treat high polluted wastewater to meet the legislative requirement. Three different case studies are discussed below in brief to understand the textile effluent treatment facilities existing in the Malaysia and any lesson to be learned. In a state of Penang, majority of textile industries use biological methods for the treatment of their wastewater. A representative technology is as described in flow-chart (Figure 9.3). Coloured and non-coloured wastewater are separately collected from (a) fabric pre-treatment, (b) dyeing and printing and (c) fabric finishing steps into a individual equalization tank. For non-coloured wastewater, biological

National and International case studies treatment is followed by equalization step and sludge obtained after biological treatment is sent for sludge dewatering, whereas treated water is released into the drain. For coloured wastewater, post equalization, the effluent is treated in decolourization plant followed by sludge dewatering and final disposal.

Figure 9.3: A diagrammatic flow chart for treatment technology employed in Textile Industries in Malaysia While in a state of Sembilan, in many textile industries biological treatments are followed by initial physico-chemical treatment. A typical flow-chart of methodology employed for treatment of raw textile wastewater in described in Figure 9.4. Raw textile wastewater is treated in equalization tank followed by coagulation and flocculation treatment. The treated effluents are sent to clarifier and in buffer tank (for possible adjustment of pH of the effluent) followed by biological treatment. In biological treatment two stages aeration are provided. Post-aeration, portion of the treated effluent is send to sludge holding tank, while remaining fraction of the treated wastewater final disposal after holding time in clarifier. Effluent from sludge holding tank undergoes filter press (for dewatering) for final disposal in drain.

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Figure 9.4: A diagrammatic flow chart for treatment technology employed in Textile Industries in Malaysia In a third case study, a treatment technology is described from a small/medium scale textile industry, Siang Poh Knitting Sdn. Bhd., located in Kuala Lumpur. A diagrammatic representation of the technology used is described in Figure 9.5. Industry has its own Wastewater treatment facility operating since 1994, which follows Standard Activated Sludge Process (Physical, Chemical treatment followed by Biological treatment) for wastewater treatment from dyeing process.

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The facility has a capacity of handling 96 m3/day of wastewater and follows three stage treatment procedure. The annual overall chemical consumption for drying process of the industry is 4,000 kg/day.

Figure 9.5: A diagrammatic flow chart for treatment technology employed in Textile Industries in Malaysia

In a chemical (primary) treatment step, raw textile wastewater is treated in coagulation and

flocculation tank (a single time application) followed by neutralization and holing phage in primary settling tank (for separation of sludge). In a secondary (biological) stage, wastewater from primary settling stage is channeled into aeration tank for biological treatment (for possible reduction of biological oxygen demand and organic residues of the effluent). Post aeration, treated wastewater is channeled into secondary settling stage for sludge settlement. A small quantity of sludge is retained back in aeration tank for next cycle of biological treatment, while the reaming sludge is transfer into sludge drying pit. After another stage of neutralization, in a tertiary (polishing step) treatment, treated wastewater is passed through adsorption column packed with activated carbon, before final discharge of treated water. The industry claims that, after providing the above treatments to raw textile effluent, 97 % of Suspended Solids, 99 % of Biological Oxygen Demand and 99 % of Chemical Oxygen Demand (of low/medium strength influent wastewater) are removed. The operating and maintenance cost of treatment facility was projected at RM 170,000.00, which is equivalent to US$ 41600.00 and INR 26,00,000.00 (at a current currency exchange value). The water reproduced after the above treatment complies with DOC Standard A of effluent discharge standard legalized in the country. From these treatment plants, it can be concluded that conventional treatment systems such as biological treatment alone or physicochemical treatments followed by a biological treatment are commonly installed in majority textile industries. The single application of coagulation and flocculation processes to treat textile effluents would generate high amount of sludge to eventually cause further handling and disposable problems. Meanwhile, conventional biological process do not always treat the textile wastewater satisfactorily as the effluents often contain organic substances which are toxic and resistant to organism used in biological treatment. Only one of the textile effluents was further treated using 73

National and International case studies advanced treatment method (i.e., adsorption column packed with activated carbon) to comply with the standard for an effluent discharge or to prepare the treated effluent for reuse purpose. Therefore, suitable combinations of treatment processes are generally used in textile industry to achieve technically and economically feasible options.

9.2.2 Case study IV C.1 Study of a Treatment Plant in Germany This treatment plant is an activated sludge system with low food-to-microorganism ratio (F/M). It receives municipal wastewater and effluent from four large textile finishing mills. The textile wastewater is equalised and then mixed with primarily treated municipal waste water. The hydraulic percentage of textile wastewater is about 45 % and referring to COD-load about 60 %. Subsequent to primary treatment and equalisation, there is a biological treatment including nitrification / denitrification and flocculation with FeCl3 as final step. Capacity of Treatment Plant Total flow = 8377 ± 1431 m3 / day Municipal = 4562 ± 2018 m3 /day Textile waste water = 3685 ± 1431 m3 /day with F/M ratio = 0.1 Influent, effluent characteristics & Regulatory standard

Table 9.4: Influent, effluent characteristics & Regulatory standard Germany Plant No 1

Influent quality Outlet Removal European Meet effluent Efficiency National & standard Parameter Textile Municipal quality standard

mg/l mg/l mg/l mg/l mg/l

BOD5 157 ± 114 ± 50 3 ± 2 97 ± 2% < 25 mg/l √ 57 COD 791 ± 443 ± 200 59 ± 16 90 ± 4% < 125 mg/l √ 281 Ammonia 2.6 ± 30 ±14 0.1 ± 0.2 2.0 pH 9.2 ± 8 ± 0.4 6-9 √ 0.8 PVA 28-138 0.6-7.8

Nitrate, 2.9 ± 1.9 88 ± 6% NO3 Norg 19.5 ± 18 ± 7 No 88 ± 6% 7 Total 3.8 ± 6 ± 2 0.2 ± 0.2 96 ± 3 < 2.0 mg/l √ phosphorus 1.2

(Federal Environmental Agency, Germany, 2002) The municipal sewage is treated in primary treatment unit before mixing with the textile wastewater. The textile effluent is passed through bar screen to remove large waste material and then passed through a grit chamber to remove the smaller material. The water is then pumped to

National and International case studies an equalisation tank (one standby equalisation tank) and the wastewater is then mixed with the primarily treated municipal wastewater and passes to the neutralisation tank. The waste is then diluted and neutralized using H2SO4 or NaOH. Then the wastewater is treated using the activated sludge process and since it has a low F/M ratio longer retention time and aeration is required. The water is then passed to the secondary clarifiers after which FeCl3is added to destabilize the colloidal materials and cause the small particles to agglomerate into larger settleable flocs, thus aiding the coagulation and flocculation process. The treated effluent is then directly discharged to the river. The removable efficiencies are shown in Table 9.5. This system is able to meet European and Germany’s National discharge regulatory standard.

Table 9.5: Percentage removal of BOD, N and P in German Plant No 1

Parameters Percentage reduction

BOD5 97 ± 2% COD 90 ± 4 % Nitrogen 88 ± 6% Phosphorus 96 ± 3%

C.2 Sludge management Sludge from clarifier and precipitation and flocculation chamber is treated with centrifuge before sludge storage tank. Sludge from primary sediment tank used in municipal wastewater treatment is also sent to the sludge storage tank. The sludge is then mixed and it is passed through chamber filter press to separate the liquid phase from the solid phase. The solid phase is then transported to a sludge drying bed.

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Relevant statistics  10 Relevant Statistics

Relevant statistics

Any kind of successful scientific projects would yield new knowledge in the domain of research as well as it produce good publications. Therefore,

33%

1% 6% 1% 1% 5% 4%

28%

26%

CSIR UGC DST+SERB DBT MOEF BRNS DRDO Others

Figure 10.1: Scientific and technical publications (in the area of dye degradation) generated from the research projects supported by different funding agencies of India (Source: ISI Web of Knowledge)

Relevant statistics

The highest contribution in terms of scientific and technical publications in the area of remediation of effluents from dye and textile industries is by Dr. J. Wang followed by Dr. Y. Wang, Dr. Zhang of China.

BRILLAS E

SWAMINATHAN M

MADRAS G

WANG Q

OH WC

XU H

CHEN CC

KHAT AEE A

WANG L

ZHAO JC

GOVINDWAR SP

ZHANG Y

ZHANG H

LIU Y

ZHANG J

LI J

LI Y

ZHANG L

WANG Y

WANG J

0 50 100 150 200 No. of Publications

Figure 10.2: Top 20 authors (with respect to scientific and technical reports published in different international journals) in the world working in the area of dye degradation (Source: ISI Web of Knowledge)

Relevant statistics

Amongst the Indian scientists, highest numbers of scientific publications (in the area of remediation of effluents from dye and textile industries) were contributed by Dr. S. P. Govindwar, followed by Dr. G. Madras, Dr. M. Swaminathan and Dr. A. Kumar.

KUMAR P S KUMAR V

MADAMWAR D

MEHTA SK AMETA R

MURUGESAN V KUMAR R DEVI LG

S UBAS H B KANS AL SK

SINGH P

MUNEER M SHANTHI M

ANANDAN S KRISHNAKUMAR B SINGH S

GUP TA VK J ADHAV J P

UMAR A

KUMAR S KUMAR A

SWAMINATHAN M MADRAS G GOVINDWAR SP

0 10 20 30 40 50 60 70 80 90 100 No. of Publications

Figure 10.3: Top 20 Indian authors (with respect to scientific and technical reports published in different international journals) working in the area of dye degradation (Source: ISI Web of Knowledge)

Relevant statistics

In last three decades, maximum scientific publications (in the area of remediation of effluents from dye and textile industries) in the form of original articles were published in followed by review papers.

2% 2% ARTICLE

REVIEW+BOOK CHAPTER

PROCEEDINGS PAPER

RETRACTED PUBLICATION 96%

Figure 10.4: Different type of scientific and technical reports published in the area of dye degradation (Source: ISI Web of Knowledge)

With the awareness amongst the general citizens and the scientists around the globe for environmental pollution, the number of scientific publications (in the area of remediation of effluents from dye and textile industries) has been gradually increased in the last three decades as it can be observed from Figure 10.5

700

600

500

400

300

No. of Publicationsof No. 200

100

0 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016

Figure 10.5: Year-wise (from 1991) distribution of scientific and technical reports published as articles and reviews related to dye degradations (Source: ISI Web of Knowledge)

Relevant statistics

REST OF THE WORLD 9109

TURKEY 641 TAIWAN 659

JAPAN 688

SPAIN 699 BRAZIL 779

SOUTH KOREA 1068

IRAN 1543 USA 1653

INDIA 3487

PEOPLES R CHINA 6862

Figure 10.6: Top 10 countries of the world with respect to scientific and technical publications related to dye degradations (Source: ISI Web of Knowledge)

INDIAN INSTITUTE OF TECHNOLOGY IIT

SHIVAJI UNIVERSITY 4% 4% 4% 22% COUNCIL OF SCIENT IFIC INDUSTRIAL RESEARCH CSIR INDIA

5% ANNA UNIVERSIT Y

6% SARDAR PAT EL UNIVERSIT Y

ANNA UNIVERSIT Y CHENNAI 7% INDIAN INSTITUTE OF TECHNOLOGY IIT DELHI 21% 8% INDIAN INSTITUTE OF TECHNOLOGY IIT GUWAHATI NATIONAL ENVIRONMENT AL 19% ENGINEERING RESEARCH INSTITUTE INDIA NATIONAL INSTITUTE T ECHNOLOGY TIRUCHIRAPPALLI

Figure 10.7: Top 10 Indian Institutes (with respect to number of scientific and technical reports published) working in dye degradations (Source: ISI Web of Knowledge)

Textile Clusters of India and Waste Management Practices  11 Textile Clusters of India and Their Waste Management Practices

11.1 Opening Remarks 83 11.2 Structure of India Textile Industry 83 11.3 Geographical Distribution of textile industry 84 11.4 Textile Wastewater Management 86 11.5 Textile effluent treatment technologies in three states of India 86 11.5.1 Gujarat 86 11.5.2 Maharashtra 88 11.5.3 Tamil Nadu 93 11.6 Conclusion 96

Textile Clusters of India and Waste Management Practices

11.1 Opening Remarks

Textile industry is backbone of Indian Socio-Economy development, like agriculture. It is one of the oldest industrial sectors, largest and having prominent global presence. Textile industry in India is highly fragment, complex and distributed into localized clustered across all states of the country.

11.2 Structure of India Textile Industry Textile industry in our country is divided into (i) Organized (formal) sector (~ 3 %) and (ii) Unorganized (informal) sector (~ 97 %) (Figure 11.1). The organized but small sector is advanced, modernized and highly sophisticated Mill based. The large portion of Indian textile industry has been formed by large unorganized sector, segregated into handloom, powerloom and hosiery based units. A third sector also runs parallel to these both sector is ‘sub-contracting segment’. It is composed of small tailoring and fabrication units, which operates under a defined and specified contract from parent units.

Indian Textile Industry

Organized Sector Unorganized Sector Total Cloth Total Cloth Production: 3 % Production: 97 %

Mill Powerlo Handloo Hosiery Sector om m Sector

Figure 11.1: Structure of Indian Textile Industrial Sector

Textile production is broadly classified into five stages: ginning, spinning (yarn), weaving & knitting (fabrics), dying and processing and garments. Spinning: It consists of more than 1,100 small-scale independent units and > 1,500 large- scale independent units. Weaving and Knitting: India’s weaving and knitting segment is small scale and highly fragmented. It consists of handlooms, powerlooms and knitting technologies. Handloom segment is mainly rurally situated, powerlooms are traditionally prerogative of composite mills, while knit based segment are geographically concentrated centers like in Triuppur. The sector consist of

Textile Clusters of India and Waste Management Practices nearly 3.9 millions handlooms, and once used to 3,80,000 powerlooms (this segment is gradually phased out). Processing (Fabric Finishing): Processing segments includes dyeing, printing and other cloth preparation prior final production of clothing. Small-scale and independent enterprises dominate this segment. About 2300 processors are currently operating, amongst them 2100 are independent units and 200 are integrated with spinning, weaving or knitting units. Clothing: Apparel (clothing) segment consist of about 77,000 small-scale units, (as domestic and exporters manufacturers, fabricators (as (subcontractors). In organized sector nearly 65,000 garment units are estimated.

Salient Features of Indian Textile Industry  It is second largest in the world, after China.  India contributes to 60 % of total loomage of total world production, making it highest in the globe  India has 22 % of total world’s spindle capacity, making its second-highest in the globe  India is second largest producer of Cotton, Cotton Yarn, Cellulosic fibre /yarn, Silk.  India has largest producer of Jute and leading in polyester yarn production  India placed fourth in synthetic fiber/yarn production  Well supported by dye industry (export-worthy) and strong cooperation by multi-national and national companies  Dominated by small-scale domains across the value chain  Many portion of the sector is decentralized and dominated by handloom and independent processing units  Abundant availability of raw materials

11.3 Geographical Distribution of textile industry As noted above, textile industry is widespread across the country, in forms of clusters, parks, SEZs, etc. According to CPCB, New Delhi report there is nearly 140 textile clusters in India. Major and prominent clusters are situated in the state of Gujarat, Tamil Nadu, Maharashtra, Punjab, Rajasthan and Uttar Pradesh, besides many small clusters is located in other states too.

11.3.1 Punjab In the state of Punjab, three major cities, Ludhina, Amritsar, Panipat are hubs of textile and related industries. Ludhina may be called as “Manchester of Punjab”. The textile industry is in the form of small scale segments, mainly producing hosiery items. Textile units in Amritsar are based on cotton and yarn. Panipat is famous for cotton fabric production.

11.3.2 Haryana Haryana is leading state in cotton production in India, which gives added advantage to the state in textile industry. The centres like Hisar, Panipat, Sonipat, Gurugram, Faridabad are major hub for textile industry in Haryana.

Textile Clusters of India and Waste Management Practices

11.3.3 Rajasthan The Pali textile cluster in Rajasthan is one of the biggest SME textile clusters in India having over 350 industries. The units in the cluster are mainly located in two Industrial Areas namely Industrial Area Phase I & Phase II and Mandia Road Industrial Area. Some of the units hitherto functioning in residential colonies also and few industries are present in the Punayata Road Industrial area. Textile units in Pali are of two main types: (a) Hand Process Units and (b) Power Process Units, Balotra, Jodhpur and Bhilwara are other textile clusters in Rajasthan.

11.3.4 Gujarat Gujarat is well known for its Textile and Dye based industries. Large number of dye, dye intermediate manufacturing and textile industries are situated at Vatva (Ahmedabad), Ankleshwar (Bharuch), Vapi, Sachin and Surat, Umargam. Hand based textile dyeing and processing industries are dominated at Jetpur, Manavadar. The region of Kuchchh is famous for handicraft related textiles.

11.3.5 Maharashtra In Maharashtra the cotton manufacturing industries are located in Kolhapur, Sangli, Miraj, Ichalkaranji, Solapur, Pune, Kalyan, Mumbai, Thane, Chalisgaon, Jalgaon, Akola, Badnera, Phulgaon, Hinganghat and Nagpur. The yarn manufacturing industries are situated in Mumbai, Ambarnath, Amalner and Jalgaon. Silk manufacturing industries are positioned in Satara, Pune, Mumbai, Kalyan, Nashik and Pimpalgaon.

11.3.6 Tamil Nadu Besides Gujarat and Maharashtra, textile industries are also found in large numbers in the state of Tamil Nadu. Six cites: Erode, Perundurai, Coimbatore, Karur, Gobichettipalayam and Triuppur are at time referred as ‘Textile valley of India’ and they are largest garment exporters in India. Handloom based units are dominated in Madurai and Kanchuipuram. Triuppur is well known as ‘Kintware’ capital of India. It houses about 9000 knitting, dyeing, bleaching, processing and manufacturing units. Tamil Nadu, Karnataka, and Kerala are the main states for wholesale sarees Coimbatore cluster has around 919 spinning units.

Table 11.1: List of textile clusters of India

No. City/Clusters State Product 1 Ludhiana Punjab Woollen Knit Wear 2 Panipat Haryana Handloom made ups and Hand block printing, Powerloom, Woolen & Cotton Wet Processing home textiles 3 Sanganer Rajasthan Apparel Manufacturing 4 Jodhpur Rajasthan Hand Processing 5 Kanpur Uttar Pradesh Defense related textiles 6 Surat Gujarat Powerloom weaving and processing

Textile Clusters of India and Waste Management Practices

7 Kolkata West Bengal Cotton Hosiery 8 Solapur Maharashtra Powerloom towels and blankets 9 Guntur Andhrapradesh Cotton ginning 10 Kannur Kerala Home furnishing 11 Tirupur Tamilnadu Cotton knitwear 12 Coimbatore Tamilnadu Powerloom, handloom and Home furnishing 13 Doddaballapura Karnataka Textiles Handlooms 14 Bengaluru Karnataka Power loom 15 Bengaluru Karnataka Readymade Garments 16 Bellary Karnataka Jeans garments 17 Ichalkaranji Maharashtra cotton weaving Yarn manufacturing 18 Delhi NCR Delhi NCR apparel manufacturing 19 Solapur Maharashtra bed linen, towels, etc 20 Yeola Maharashtra Silk dhoties and uparnae 21 Nagpur Maharashtra Weaving 22 Nawanshahr Punjab Garmenting & knitting 23 Barnala Punjab Production of towels, melange yarn, bathrobes and training center

11.4 Textile Wastewater Management Textile wastewater management in our country is inspired from the European and Western countries. Along the line of successful practices in those countries, many technologies were adopted and translated into Indian sub-continent. These technologies revolve around physico- chemical and biological treatment methods. The treatment steps in CETPs and ETPs across the country follows primary, secondary and tertiary stages for treatment of textile and dye industrial effluents. In the state of Tamil Nadu, Zero Liquid Discharge (ZLD) practice is widely used.

11.5 Textile effluent treatment technologies in three states of India

11.5.1 Gujarat (A) Vapi Green Enviro Ltd., Vapi CEPT, Vapi The CETP at Vapi Industrial Estate, Vapi, Gujarat (one of the largest in Asia) operates on conventional biological treatment plant (Figure 11.2). The industrial effluents from individual member units were colleted at CETP through pipeline. Effluents are treated in three stage process: during initial primary process, effluents are screen for large particulate matter in screening chamber, followed by equalization treatment (by addition of lime), flax mixing and flocculation. In second stage, through primary clarifier the part of pre-processed effluent are send for aerobic treatment in aeration tank and another part of pre-processed effluent are feed to UASB reactor. 87

Textile Clusters of India and Waste Management Practices

After aerobic treatment through secondary clarifier the treated effluents are released into Daman Ganga River.

Figure 11.2: A schematic diagram and a Process Flow Chart of Vapi Green Enviro Ltd., Vapi CEPT, Vapi

(B) The Green Environment Services Co-Op. Society, Vatva CEPT, Vatva, Ahmedabad Like CETP in Vapi, CETP at Vatva Industrial Estate operates on conventional biological treatment method. Partially treated effluent from individual member units are collected at CEPT through pipe line. Post primary treatment through clarifier, equalization, flash mixer and flocculator, effluents are treated under aeration conditions in activated sludge aeration tank. In third stage, during tertiary treatment after passing through secondary clarifier, treated effluent are send to land filing sites and at terminal pumping station for final release in ocean. The schematic outlay of Vatva CETP is as describe in Figure 11.3

Textile Clusters of India and Waste Management Practices

Figure 11.3: A schematic diagram and a Process Flow Chart of CETP of The Green Environment Services Co-Op. Society, Vatva CEPT, Vatva, Ahmedabad

11.5.2 Maharashtra

(A) Greenfield CET Plant Pvt. Ltd., Solapur, Maharashtra The CETP plant at Solapur, Maharashtra works with conventional biological treatment plant (Figure 11.4). The three stages of effluent treatment technology are: initial collection and screening process followed by primary equalization process, the second stage consists of biological treatment in Upflow Anaerobic Sludge Blanket Reactor followed by aerobic process in aeration tank. In the last stage, generated sludge is sent to clarifier and sludge drying beds. The CEPT treats the industrial effluents for large and medium size textile industries, effluents from chemical and bulk drugs and oil refinery.

Textile Clusters of India and Waste Management Practices

Figure 11.4: Flow diagram of Greenfield CET Plant Pvt. Ltd., Solapur, Maharashtra

(B) Lote Parshuram Environment Protection Co-op. Society Ltd., Lote, Maharashtra Lote- Parshuram is industrialized zone, in Ratnagiri district in the state of Maharashtra, comprising more than 200 industries (small, medium and large industries units). The clusters mainly produce dyes intermediates, agro chemicals, specialty chemicals and bulk drugs. Effluents from these industries are treated by CETP which operates on conventional technology (Figure 11.5).

Textile Clusters of India and Waste Management Practices

Figure 11.5: Flow diagram of Lote Parshuram CETP, Lote, Maharashtra

The three stage process involves: initial effluent collection from member industrial units through pipeline to storage tank at CETP, followed by neutralization of effluents using Lime, which is fed to primary sedimentation tank through flash mixture. In second stage (secondary treatment), pre- treated effluents are feed to bioreactors for biological treatment followed by separation of sludge in secondary sedimentation tank. In a final stage (tertiary treatment), biologically treated effluent is feed to oxidation pond and pressure charcoal bed filters. Sludge generated during each step of effluent treatment is transferred to sludge holding tank, which after poly addition the mixture is transferred to decanter. The liquid fraction is recycled to primary equalization tank for another round of treatment and the solid fraction is send for solar drying and finally to Land Disposal site.

Textile Clusters of India and Waste Management Practices

(C) The Ichalkaranji Textile C.E.T.P. Ltd., Ichalkaranji, Dist. Kolhapur, Maharashtra Ichalkaranji is known as the ‘Manchester of Maharashtra.’ The city houses several small and medium scale textile units, majority of them are export oriented. The CETP at Ichalkaranji Textile Cluster has 62 processes and 122 sizing units, which are involved in activities such as dyeing printing and finishing of cotton, bleaching, synthetic and blended fabrics. The CETP operates on conventional three stage treatment process (Figure 11.6): primary screening, secondary biological treatment and last stage is sludge thickening or dilution from sewage treatment plant.

Figure 11.6: Flow diagram of The Ichalkaranji Textile C.E.T.P. Ltd., Ichalkaranji, Dist. Kolhapur, Maharashtra

(D) ETP of Raymond Zambaiti Ltd., Kolhapur In another example, treatment process of Effluent Treatment Plant of textile industry: Raymond Zambaiti Ltd., Kolhapur, Maharashtra, works on biological treatment biological treatment method (Fluidized Aerobic Bio-Reactor) with an average wastewater inflow of 2MLD (Figure 11.7). It consist of 1) Screen Chamber, 2) Equalization tank, 3) Flash mixer, 4) Flocculation Tank, 5) Tube Settler-I, 6) Fluidized Aerobic Bio-Reactor (FAB-I), 7) FAB-II, 8) Tube Settler-II, 9) Chlorine Contact Tank, 10) Sludge Thickener, 11) Centrifuge.

Textile Clusters of India and Waste Management Practices

Figure 11.7: A process flow diagram of ETP plant commissioned at Raymond Zambaiti Ltd., Kolhapur, Maharashtra

The textile effluent generated within the plant is collectively passed through the screen chamber to remove the floating matter present in the wastewater. The quality and quantity of the wastewater is maintained in the equilisation tank where air blower is provided for the supply of oxygen. The wastewater then comes to the flash mixer in which lime and ferrous sulphate are the coagulants added to the wastewater with detention time of 30 sec. The floc gets formed due to the slow mixing and resultant settling of floc in the first tube settler reduces total suspended solids and BOD load on the secondary treatment. The water is then allowed in the FAB-I where microorganisms are attached to the media, which inturn is suspended in the wastewater. The growth occurred on the media. The oxidation of organic matter is performed with the help of microorganisms. The sludge formed due to biological process gets settled in the tube settler II. The wastewater treated by secondary treatment is then allowed in chlorine contact tank to kill pathogens using the hypochlorite as a disinfectant. The treated wastewater is then sending to the common effluent treatment plant for further treatment. The sludge settled in the tube settlers is then sending to the sludge thickener then it is concentrated in centrifuge using poly electrolyte dosing. The concentrated sludge is send to the hazardous waste disposal site at Rajangoan, Pune.

Textile Clusters of India and Waste Management Practices

11.5.3 Tamil Nadu The state of Tamil Nadu follows the concept of ‘Zero Liquid Discharge’ (ZLD) especially for textile wastewater treatment. The city of Tirupur is one of the leading garment export centers in the country. Tirupur and its neighboring Towns houses more than 700 dyeing and bleaching industries. Several CETPs are in operation in this cluster, having total handling capacity of >120 MLD and about 85 industries have their independent ETP and recycling system. The CETPs in Tirupur process the textile effluent in three stages: (1) Pre-treatment, to prepare the raw effluent for recycling process; (2) First stage of membrane based recycling and (3) Reject management from the recycling plants. Here, four different types of pre-treatment are used: (1) Biological Process based on technology developed by Tamil Nadu Water Investment Company (TWIC); (2) Chlorination; (3) Membrane Bio Reactor Technology (MBR); (4) Ozonation

(A) Arulpuram CETP Arulpuram CETP in Tirupur is based on Biological treatment technology developed by TWIC (Figure 11.8).

Figure 11.8: General flow diagram of Arulpuram CETP, Tirupur

Textile Clusters of India and Waste Management Practices

Distinct features of Arulpuram CETP

. Pre treatment is only through biological treatment and series of specialized filters. . No chemical process is implemented during pre-treatment, which results in generation of very low amount of secondary sludge. . Characteristic of pre-treated effluent is within the prescribed limit, except TDS . Pre-treated effluent is being feed to Two Stage Reverse Osmosis plant. The R.O. treated effluent is recycled back in the industries for their utilization in various processes . The recycled treated effluents are supplied to each unit through separate pipeline. Each industrial unit is supplied 80 % of the raw effluent sent to CETP for treatment

(B) Veerapandi CETP based on Chlorination The Veerapandi CEPT were initial based on conventional methods, which along the line of ZLD practices are upgrade, where pretreatment are now chlorination based oxidation reduction reaction, which is batch process. Post pre-treatment, effluents are sent to RO plant and reject management system. The schematic diagram of CETP is as describe in Figure 11.9.

Figure 11.9: General flow diagram of Veerapandi CETP, Tirupur

(C) Angeripalayam CETP based on Membrane Bio Reactor (MBR) The conventional CETP were upgraded to follow ZLD guidelines and primary treatment is now based on Membrane Bio Reactor (MBR) process (Figure 11.10). 95

Textile Clusters of India and Waste Management Practices

Figure 11.10: General process flow diagram of Angeripalayam CETP based on Membrane Bio Reactor (MBR), Tirupur

This membrane based technology is usually a biological treatment system, where membranes are used to retain MLSS, which proves to be better than conventional activated sludge process. Post MBR treatment effluents are sent for RO plant and reject management system.

(D) Ozonation Technology at Kuppamdampalayam CETP In ozonation technology after biological treatment, effluents are treated with ozone gas, which is followed by two R.O. stages and multiphase evaporator.

Textile Clusters of India and Waste Management Practices

11.6 Conclusion Upon studying the different treatment technologies adopted, few common observations can be made. (1) In the conventional treatment technology for industrial effluent treatment process alum/ferrous sulphate with lime is used, which post-treatment generates excessive sludge. Therefore sludge handling is additional burden on treatment cost and required another round of treatment. (2) ZLD practices are limited to the state of Tamil Nadu, which needs to be adopted in other states across the country. (3) With all the practices are in force and implemented, still we rarely see decline in textile water pollution. (4) The conventional practices are needed to be upgraded on urgent basis, since at longer time they have not produce the desired results.

Textile waste management practices in other countires  12 Textile Waste Managements Practices in Other Countries

12.1 Opening Remarks 98 12.2 China 98 12.3 Malaysia 95 12.4 Europe 99 12.4.1 Germany 99 12.4.2 FOTOTEX project 100 12.4.3 PROWATER project 100 12.4.4 ADOPBIO project 100 12.4.5 BATTLE project 100 12.4.6 PURIFAST project 101

Textile waste management practices in other countires

12.1 Opening Remarks

The Indian wastewater management practices have been inspired by success stories of most established practices around the world. To understand more about how these countries manages dye, dye intermediates and textile wastewater, an overview of the current technologies prevailing are discussed here.

12.2 China China is one of the global leaders in textile industries. For many years China continued with conventional methods involving physico-chemical and biological process. But, China is now captivating multi-prolonged approach, involving public-private sectors. In recent years they have collaborated with International Atomic Energy Agency (IAEA) which led their wastewater management practices to reach advanced level. In IAEA assisted technology they are using Electron beam technology for cleaning the industrial wastewater particularly in textile industries. The technology involves generation of beams of electrons with the help of ‘electron beam accelerators’ which are irradiated into wastewaters. In the treatment process, the wastewater is passed through an armored chamber which is exposed to ionizing radiation (high energy electron beams) from the accelerators. During irradiation, the electron beams interact with complex compounds of wastewater, the reaction leads to the generation of comparatively simpler and smaller compounds. The irradiated water with less complex compounds can be further treated with either biological or in combination with physico-chemical methods, before releasing into open water bodies. The initial success of this technology suggests that, the treatment does not require additional chemicals, is energy saver and can easily be implemented at conventional wastewater plants. However, it has been claimed that, the technology that does not leave any radiation in treated water or make water radioactive, but further investigation may help to clear the doubts. The first such technology in China was initiated in the textile wastewater treatment plant at city of Jinhua, nearly 300 km south of Shanghai.

Figure 12.1: Textile wastewater treated with irradiation tehnology

Textile waste management practices in other countires

The above Figure 12.1, exemplify the success of irradiation technology in treatment of textile effluents. The first tube contains untreated textile wastewater; second tube contains irritated wastewater, while in third tube the dose of electron beams were higher. The result clearly shows that, the technology has a potential to produce much cleaner water, which can be reuse safely.

12.3 Malaysia Malaysia is one of the leading manufacturer and exporter of natural and synthetic textiles. It contributes nearly 1.3 % of synthetic textile (fibres) of the world total production. In Malaysia, every textile units treats its own effluents and treated water is normally released to a drain. One of the wastewater treatment plant in a textile units operating since 1994, follows Standard Activated Sludge Process (Physical, Chemical treatment followed by Biological treatment) for wastewater treatment from dyeing process. They follow three stage treatment procedures. In a primary treatment step, raw textile wastewater is treated in coagulation and flocculation tank followed by neutralization and holding phage in primary settling tank (for separation of sludge). In a secondary (biological) stage, pre-treated effluent is given biological treatment into aeration tank. Post biological treatment, treated wastewater is channeled into secondary settling stage for sludge settlement. Retaining the part of sludge, major portion of sludge is transfer into sludge drying pit. After another stage of neutralization, in a tertiary stage, treated wastewater is passed through adsorption column packed with activated carbon, before final discharge of treated water. In another textile unit, coloured (from dyeing and printing process) and non-coloured (fabric pre- treatment) wastewaters are treated separately. The coloured wastewater is treated in decolourization plant post equalization, followed by sludge dewatering and final disposal. While for non-coloured wastewater conventional biological treatment methods are applied as mentioned above. In one more treatment plant, wastewater from several textile industries are initially provided physico-chemcial treatment, followed by two stage aeration are provided (secondary biological treatment). The resultant sludge is treated (for filter press, i.e. dewatering) for final disposal in drain.

12.4 Europe In Europe many large scale network projects were initiated for developing treatment stratergies at larger scale. Few of them are summarized below.

12.4.1 Germany In one of the treatment plant in Germany, based on activated sludge system with low food-to- microorganism ratio (F/M). It receives municipal wastewater and effluent from four large textile finishing mills. The textile wastewater is equalised and then mixed with primarily treated municipal waste water. The hydraulic percentage of textile wastewater is about 45 % and referring to COD-load about 60 %. Subsequent to primary treatment and equalisation, there is a biological treatment including nitrification / denitrification and flocculation with FeCl3 as final step. In another example, the municipal sewage is treated in primary treatment unit before mixing with the textile wastewater. The textile effluent is passed through bar screen to remove large waste material and subsequently passed through a grit chamber to remove the smaller material. The water is then pumped to an equalisation tank (one standby equalisation tank) and the wastewater is then mixed with the primarily treated municipal wastewater and passes to the neutralisation 100

Textile waste management practices in other countires tank. The waste is then diluted and neutralized using H2SO4 or NaOH. Then the wastewater is treated using the activated sludge process and since it has a low F/M ratio longer retention time and aeration is required. The water is then passed to the secondary clarifiers after which FeCl3 is added to destabilize the colloidal materials and cause the small particles to agglomerate into larger settleable flocs, thus aiding the coagulation and flocculation process. The treated effluent is then directly discharged to the river.

12.4.2 FOTOTEX project The project demonstrated the effectiveness of photo-oxidation treatment process for removal of biodegradable and non-biodegradable organic compounds from textile effluents. Though the photo-oxidation technique removes organic compounds, it did not decrease the salinity and fails to achieve quality parameters for reuse of water in the production process of textile units. The project concludes that, single treatment system is not effective and requires combination of membrane system like microfilteration and nanofilteration.

12.4.3 PROWATER project In PROWATER project effluent recycling system was based on physico-chemical pre-treatment (coagulation + lamellar sedimentation/flotation), cross-flow ultrafiltration and ozonation. The process made claim for water reuse from technical and economic perception. Four proto-types of treatment technologies were developed during the project for four different textile wet industries and had tested various kinds of effluents. The project claims to reduce the colour of effluent by 98 % and total surfactants by 62 %. The additional claim was for the cost of purification and reuse of water, which was cost effective compared to commercial purification process.

12.4.4 ADOPBIO project The project employed Advanced Oxidation Process (UV-activated photolysis of hydrogen peroxide and thermal activated oxidation process) in conjugation with bio-flotation process for decolourization and recyclying treatment of textile finishing effluent. The process achieved complete decolourization of coloured effluent, while bio-flotation aids in degradation of residual organic compounds.

12.4.5 BATTLE project The BATTLE project essentially used and evaluated the applicability of BATs for SEMs of textile finishing sector. Before implementing the project, study was performed to collect primary information of effluents and availability of process. It based on separation of wastewater into those that have potential reuse properties and that cannot be reuse. For reusable water, membrane based technology was used followed by biological treatment. Wastewaters are segregated, collected separately and pumped into a mixing tank and send for an ultrafiltration. For reuse of treated water, membrane permeates are mixed with primary water and send to water distribution networks. Water which cannot be reuse further are mixed with membrane concentrated and were treated in existing biological treatment plant. The project concludes that developed technology is highly replicable and provides a model for other textile industries.

101

Textile waste management practices in other countires

12.4.6 PURIFAST project This project used Advanced Oxidation Process (AOPs) and ultrafiltration to demonstrate the technical as well as economic feasibilities for treatment of textile and mixed effluent. The lab scale method showed the efficiency of ultrasound in combination of ultrafilteration. While at pre- industrial scale ultrasound were replaced with AOPs. The project concludes that, treatment with ultrafilteration allows complete removal of total suspended solids and turbidity and ozonation removes colour. While combination of ozonation and ultrafilteration at pre-industrial scale achieves 90 % colour removal, 80 % COD reduction and nearly 80 % TSS removal.

102

Patent Analysis  13 Patent Analysis

13.1 Opening Remarks 103 13.2 Indian Patent Analysis 103 13.3 National Status of Patents 108 13.4 International Status of Patents 109 13.5 Conclusion 110

103

Patent Analysis

13.1 Opening Remarks

By studying the scientific literatures (in forms of research articles, reviews, comments, news, etc.) in peer and non-peer reviewed journals, would provide limited, many time academic oriented knowledge about the area of interest. Many technical and scientific research out-come are not published instantly, but they are oriented for patent grants. To get a comprehensive view of current state of knowledge about textile wastewater management practices in our country (and around the world) patent granted and patents applied were studied. 13.2 Indian Patent Analysis Though number of technologies and methodologies in the field of bioremediation have been granted patents, very few patents are been awarded for dye and textile wastewater management practices. The search for patents on textile wastewater treatment has retrieved 24 published patents awarded to Indian researcher. The detail list of all 24 patents is described in Table 13.1.

Table 13.1: List of few of the Patents awarded to Indian Scientist for developing novel technologies in dye and textile effluent waste magagment

No. Title Inventors Issue Date / Applicants Application Date 1 Biological Kumar, Rita; 20-02-2007 Council of neutralization of highly Kumar, Anil Scientific and alkaline textile Industrial industrial wastewater Research, India [CSIR- IGIB] 2 A novel process for Chandralata 02-12-2011 Council of decolorization of Raghukumar; Scientific and colored effluents Donna Trella Industrial D`Souza Ticlo Research, India [CSIR- NIO] 3 Processes for Kumar 13-09-2011 Council of decolorization of Chandralata Scientific and colored effluents Raghu; Ticlo Industrial Donna Trella Research, D'Souza India [CSIR- NIO] 4 Novel method for Annamalai, 22-01-2016 Council of removal of organic and Sivasankar; Scientific and inorganic contaminants Santhanam, Industrial caused by textile dye Manikandam; Research, effluents in the Sundaram, India [CSIR- agricultural soil by bio- Maruthamuthu; CECRI] electrokinetics Kandasamy, Subramaniyam;

104

Patent Analysis

Gopalkrishnan, Rajagopal 5 A process for the Chiya Ahmed 26-02-2010 Council of treatment of textile dye Basha Scientific and bath effluent Industrial Research, India [CSIR- CECRI] 6 Magnetic dye-adsorbent Shukla, Satyajit 28-10-2016 Council of catalyst Vishnu; Warrier, Scientific and Krishna Industrial Gopakumar; Research, Madadhin, Thazhe India [CSIR- Lajina; Narayani, IIP] Harsha; Chalappurath Pattelath Reshmi; Manoj Raama Varma 7 A Process for Shukla Satyajit 09-03-2018 Council of Decomposition of Vishnu; Warrier Scientific and Organic Synthetic Dyes Krishna Industrial Using Semiconductor- Gopakumar; Babu Research, Oxides Nanotubes Via Babitha India [CSIR- Dark Catalysis Kunnathuparambil NIIST] 8 Process for removing Raghukumar 11-10-2005 Council of dye from dye containing Chandralata; Scientific and water or soil using D'Souza; Trevor Industrial white rot-lignin- M.; Thorn; R. Research, modifying fungus Greg; Reddy; C. India [CSIR- flavadon flavus A. NIO] 9 A process for removal Chandralata 27-02-2008 Council of of dyes in dye- Raghukumar; Scientific and containing waste-waters Trevor Industrial and soil M.D'Souza; Research, R.Greg Thorn; C. India [CSIR- A. Reddy NIO] 10 An Efficient And Dr.Nupur Bahadur 19.06.2015 Amity Economical Green University Process For Primary Treatment Of Effluent As Well As Sludge Of Textile & Dyeing Industry 11 Novel Process For The Perumal, 19.11.2014 Shri Amm Treatment Of Raw Karuppan Murugappa Textile Dyeing Malleswari, Chettiar

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Effluents Ramayanam Baby Research Karuppuraj, Centre (Mcrc) Velusamy 12 Treatment Of Textile Setlur Ranganna 16.07.2010 Setlur Dyeing Effluents Ramaswamy Ranganna Ramaswamy 13 'A Process For The B.D. Thakur 27.03.2009 The Director, Treatment Of Textile Mangala Joshi Northern India Industries Effluent Textile Research Association 14 An Improved Treatment Santosh Narain 27.03.2009 Council Of Plant For Textile Kaul, Girish Scientific And Wastewaters And An Ramesh Pophali Industrial Improved Process Tapas Nandy Research Thereof 15 A Process For Treating K.P.Sharma, 28.02.2009 Department of Dyes Subhasini Sharma Biotechnology, Wastewater New Delhi 16 Photocatalytic Bhaskarwar, 21.04.2017 Indian Institute Degradation Of Dyes In Ashok, Of A Photochemical Foam- N.Prof. Technology, Bed Reactor New Delhi 17 A Process For Sarabjeet Singh 30.09.2016 General Decolorizing Dye- Ahluwalia Shivdev Singh Containing Wastewater Diwan Using Dye Degrading Gurbachan Bacteria Singh Khalsa College, Patiala 18 Algal-Go Subhashsa Nigam 09.06.2017 Amity Nanocomposites For University The Reduction Of Textile Dyes 19 Decolorization And S. Sandhya 26.08.2011 Council Of Degradation Of Textile Scientific And Industry Wastewater Industrial Using Laboratory Research Isolated Consortia Containing Pseudomonas Aeruginosa, Alcaligenes Sp And Bacillus Latrosphorous 20 Α-Fe2o3/Zncr-Ldh Kulamani Parida 21.04.2017 Institute For Composite; A Proactive Technical Photocatalyst For Education And

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Visible Light Driven Research Photocatalytic Degradation Of Textile Dyes, Phenol And A Process For The Preparation Thereof 21 Method For Preparation Sandip 12.11.2010 Amity Of Catalyst, Its Activity Chakrabarti University And Method For Treatment Of Textyle Wastewater 22 Treatment Of Dye S. Raghu 28.11.2008 S. Raghu Effluent And Simutaneous Recovery Of Sodium Hydroxide Using Electro-Chemical Ion Exchange Membrane Process 23 Satellitism : A Novel Dr. Arun Kumar 06.05.2016 Dr. Arun Approach For The Kumar Decolorization Of Azo Dyes 24 An Electrokinetic Cell Annamalai 22.01.2016 Council Of Reactorand A Method Sivasankar Scientific & For Removal Of Industrial Organic And Inorganic Research Contaminants From The Dye Contaminated Soil Using The Said Reactor

The preliminarily analysis of these patents revealed that technologies for textile wastewater treatment were based on chemical, physico-chemical, biological methods, including integrated processes (biological-physico-chemical). It further indicated that, technologies based on biological methods are more preferred (Figure 13.1), than chemical or physico-chemical methods.

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Figure 13.1: Broad classification of treatment technologies developed for dye and textile industrial effluent

The technologies based on biological methods can be further classified into following: (1) Use of single microbial cultures (viz. Bacteria, Fungus, Algae) (2) Use of microbial consortium (3) Use of novel apparatus or devise involving microorganisms (4) Use of novel composition

(1) Use of Single Microbial cultures Conventionally, the most common biological method to decolorize and degrade the dye containing and textile effluent is use of single pure microbial cultures favorably (due to number of advantages) different species of bacteria and fungi. More than half of the patents studied made of use of either Pseudomonas spp., Bacillus spp., Kocuria sp. (bacteria), Flavodon sp. (fungi) or Scenedesmus sp. (algae). The approach describes isolation, screening and use of most effective microbial strains for treatment of simulated/raw dye and/or textile effluents under laboratory/in situ conditions. To discuss couple of patents under this category, Raghu et al., (2011) in their patent “Processes for decolorization of colored effluents” describes the use of a unidentified fungus ‘NIOCC #2a’, isolated from a mangrove wood, for decolourization of effluents from textile and dye manufacturing industries, molasses spent wash from alcohol distilleries and paper and pulp industries. The decolourization process was carried at 30 to 60ºC and at pH 3 to 6, having a salinity of 25 ppt, using either free mycelia or immobilized fungus or extracellular culture fluids or extracellular polymeric substances. In another patent Sarabjeet Singh Ahluwalia (2016) have reported the use of bacterial strain Kocuria rosea capable of decolourizing and degrading simulated and raw effluent released from

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Patent Analysis dye, textile and paper-pulp industries with a efficiency of removing 53-99 % colour from wastewater.

(2) Use of microbial consortium/mixed cultures Although microbial consortia/mixed cultures are used widely in many laboratories across the country for dye and textile wastewater remediation, exceedingly few of them have approach for patenting. In one of the rare examples, patent obtained by S. Sandhya (2011) of CSIR laboratories, has reported using of bacterial consortia consisting of Pseudomonas aeruginosa, Alcaligenes sp. and Bacillus latrosphorous for bioremediation of dye and textile wastewater under microaerophili-aerobic consortium.

(3) Use of novel apparatus or devise involving microorganisms Under this category, patent granted in the year 2016 to Sivasankar et al., on “Novel method for removal of organic and inorganic contaminants caused by textile dye effluents in the agricultural soil by bio-electrokinetics” describes the use of three distinct species of Bacillus and a Pseudomonas sp. which is based on electrokinetic appraoch. This bio-electrokinetic process used starch along with bacterial strains as anolyte, which moves towards cathode via electro-osmosis process, resulting in reduction of COD in the range of 70-80 %. The inorganic contaminants (like chloride, sulphate and trace metals) from the effluent during the treatment were removed (recovered) through electro-migration process. The patent claims to improve the fertility of agricultural soils after this treatment.

(4) Use of novel composition The fourth type of technology describes the preparation of novel composition including algal stain for treatment of water containing azo dye Direct Red 31 (DR31). Subhashsa Nigam (2017) from Amity University, in the patent “Algal-Go Nanocomposites for the Reduction of Textile Dyes” mentions the preparation of nano-composite consisting of cells of Scenedesmus sp. and graphene oxide nano sheets for reduction of DR31. The lyophilized Scenedesmus sp. and graphene oxide were mixed at optimum ratio in deionized water (with constant stirring for 12 h under ambient conditions) and the resultant mixture was again lyophilized to treat dye containing wastewater by photocatalytic activity.

13.3 National Status of Patents Amongst the different research institutes and universities, Council for Scientific and Industrial Research (CSIR), New Delhi is leading in obtaining patents for developing technology for treatment of dye and textile effluents. As observed from Figure 13.2, half of the patents (51 %) came from CSIR labs. Two patents were awarded to Amity University, while one patent from University of Rajasthan, Rajasthan were supported by Department of Biotechnology (DBT) New Delhi. Three patents were filed by three different individuals that can be categorized as Private/Individual patents.

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Figure 13.2: Patent distribution according to its funding source

13.4 International Status of Patents Globally, China leads with 171 patents (i.e. 64 %) (amongst the patent considered in this study), followed by USA (13 %), India (9 %), Korea (4 %) for developing technologies for treatment of dye and textile effluents (Figure 13.3). The other countries include DE (3 %), Japan (3 %), EP (2 %).

Figure 13.3: Distribution of patents for development of novel technologies in dye and textile waste management (top 12 countires)

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As noted above, the international patents for textile wastewater management practice also can broadly classified into physical, chemical, physico-chemical, biological methods, including integrated processes (biological-physico-chemical). The partial list of patetns obtained by scientists of top ranking conutires is described in Appendix.

13.5 Conclusion From the above study, it can be suggested that, though few patents were gratned, commerlization of patents are missing in India. Also India lags behind to top ranking countires in obtaining patents in the area of bioremediation. More research activites in this direction along with more finincal investment is necessary. We need patents which can be adopted by the ETPs, CETPs or textile mills directly.

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Work of some Indian researchers  14 Work of Some Indian Researchers

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14.1 Research outcomes from Prof. Sanjay Govindwar’s Lab

14.1.1 Decolorization of textile dyes using Aspergillus ochraceus

(A) Results, summary and major outcomes The present study dealt with the decolorization and degradation of textile dye Reactive blue 25 (0.1 gL-1) by mycelium of Aspergillus ochraceus NCIM-1146. Spectrophotometric and visual examinations showed that the decolorization was through fungal adsorption, followed by degradation. Shaking condition was found to be better for complete and faster adsorption (7 h) and decolorization (20 d) of dye Reactive blue 25 (100 mgL-1) as compared to static condition. Presence of glucose in medium showed faster adsorption (4 h) and decolorization of dye from bound (7 d) mycelium. FTIR and GCMS analysis study revealed biodegradation of Reactive blue 25 into two metabolites phthalimide and di-isobutyl phthalate. Aspergillus ochraceus (NCIM-1146) has ability to decolorize various textile dyes viz. Purple 2R, Orange TG22, Yellow HE64, Red HERB and Golden yellow HER was determined by monitoring the decrease in absorbance of each dye in the culture supernantant. Decolorization performance of Purple 2R with various conditions such as different media, concentration of dye, agitation and static condtions were studied. The decrease in dye decolorization capability of mycelium was observed with increasing dye concentration in repeated batch mode. Spectrophotometric data revealed that the process involved in decolorization is through microbial metabolism but not biosorption. Phytotoxicity study demonstrated no toxicity of the biodegradated products for plants with respect to Phaseoulus mungo and Sorghum vulgare. Aspergillus ochraceus (NCIM-1146) has ability to decolorize various xenobiotic dyes. Biodegradation of dyes was demonstrated by their decolorisation in the culture medium. The extent of biodegradation was determined by monitoring the decrease in absorbance of each dye. Malachite green decolorisation activity is affected by various conditions such as composition of media, concentration of dye, amount of mycelia and agitation. The durability of decolorisation activity under optimum conditions was investigated in repeated batch mode. An increase in the amount of mycelia positively affected the durability of decolorisation activity. The decrease in dye decolorisation capability of mycelia occurred with increasing dye concentration in repeated batch mode. This organism showed significant ability to decolorize all the dyes tested viz. malachite green (98 %), cotton blue (92 %), methyl violet (61 %) and crystal violet (57 %) in 24 h incubation. Spectrophotometric data revealed that the process involved in decolorisation is through microbial metabolism but not biosorption. This study showed that fungal mycelia (A. ochraceus) could effectively be used as an alternative to the traditional physico-chemical process.

(B) Major highlights of the work done Aspergillus ochraceus NCIM-1146 was found to decolorize textile dye Reactive blue-25, Purple 2R, Malachite green, Cotton blue, Methyl violet and Crystal violet. This study suggests that this strain could be a useful tool for textile effluent treatment and the alternative to the traditional physicochemical process.

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14.1.2 Biodegradation of textile dyes (Golden yellow HE2R & Navy Blue 3G using Brevibacillus laterosporus

(A) Results, summary and major outcomes Growth of B. laterosporus: The exponential phase of B. laterosporus started after 12 h of incubation and continued up to 72 h at 30°C. The status of the biotransformation enzymes was found to be optimum at 36 h of incubation which was the mid exponential phase. These results probed the use of Brevibacillus culture at its mid exponential phase for dye decolorization studies. Decolorization of dyes using B. laterosporus and the effect of physico-chemical parameters: GY- HE2R and NB-3G showed 87 % and 80 % decolorization respectively, within 48 h under static condition at 50 mgL-1 concentration. However, no significant change in the decolorization potential was observed under shaking conditions. Decolorization performance of all the dyes GY- HE2R and NB-3G was decreased with an increase in the initial dye concentrations from 0.1 to 1.0 gL-1. B. laterosporus steeply decreased the decolorization of Golden Yellow-HE2R and Navy Blue-3G at the concentration 1.0 gL-1 with poor performance even after extended incubation (46 and 66 %, respectively within 72 h). In case of Navy Blue-3G, better performance of decolorization was observed in the quite broad range of pH 7.0 to 11.0; however decolorization of Golden Yellow-HE2R was found in the broader range (pH 5.0 to 9.0). In present study, B. laterosporus exhibited maximum decolorization at 30°C whereas in case of Golden Yellow-HE2R and Navy Blue-3G, more than 60 % of the dye was removed even at 15°C, after an extended incubation period. Studies show that B. laterosporus has better decolorization activity of the two studied dyes during its exponential growth phase. The organism showed more efficient decolorization in 24-96 h old cultures. When 40 mgL-1 wet weight of the culture was used, the time required for complete decolorization of GY-HE2R and NB-3G was reduced to 18 h and 12 h respectively. Moreover, nitrogen sources were found to be stimulatory for the decolorization activity as compared to the studied carbon and phosphorus sources. KH2PO4 and NaH2PO4 were found to have no significant effect on the decolorization of GY-HE2R and NB-3G. Among the carbon sources studied, starch was better source whereas in presence of glucose, the cells have shown poor decolorization in all the cases. Complete decolorization of dyes occurred simply in the synthetic medium (without any supplementary components) when provided with an extended incubation. The organism retained good dye decolorization abilities even when used in repeated cycles. The growth of B. laterosporus was remarkably increased during the decolorization of GY-HER and NB-3G (45 and 56 % respectively) after 24 h of incubation; whereas it decreased (18 %) during the decolorization of GY-HER after the further incubation up to 72 h. Decrease in the activity of tyrosinase (31 %), no significant change in the activity of laccase whereas significant increase in the activities of LiP (156 %), aminopyrine N-demethylase (120 %), DCIP reductase (76 %) and MG reductase (40 %) was found in the cell free extract obtained after 48 h of incubation during the decolorization of Golden Yellow-HER by B. laterosporus. Complete inhibition of tyrosinase, no significant change in the activities of laccase and DCIP reductase where as significant increase in the activities of LiP (54 %), aminopyrine N-demethylase (122 %), and MG reductase (50 %) was found in the cell free extract obtained after 48 h of incubation during the decolorization of Navy Blue-3G by B. laterosporus. In addition, no significant change during the decolorization of GY-HER, significant decrease (8 %) during the decolorization of NB-3G where as significant increase (~10 %) during the

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Work of some Indian researchers decolorization of MG and MR was observed in the activity of LiP in to the culture supernatant (i.e. extracellular). No activities of laccase, tyrosinase as well as DCIP reductase, MG reductase and aminopyrine N-demethylase were found in to the culture supernatant.The findings of microbial and phytotoxicity studies suggest that B. laterosporus produces nontoxic metabolites with respect to the tested microorganisms and plants. Decolorization of textile effluent: 100 ml batch culture of the organism decolorized 35 % of textile effluent within 24 h. Approximately 5 g (wet wt) of the immobilized beads kept in 250 ml effluent under static condition (in 500 ml beaker) decolorized about 60 % of the effluent within 24 h and retained 50 % decolorization efficiency upto 5 cycles. Purification of intracellular lignin peroxidase: A total amount of about 280 mg of protein, corresponding to approximately 86 units of lignin peroxidase was loaded onto the column. This ion exchange column chromatography allowed the recovery of 35.55 units in 1.98 mg of protein with a yield of 40 % and purification factor of approximately 57 fold. Although homogeneity was obtained and confirmed by PAGE, elution of enzyme from fraction number 39 to 69 (i.e. broad range) suggested the presence of multiple binding sites with different ionizable groups. In the present study, PAGE gel developed by Coomassie brilliant blue staining has shown single band for a protein having molecular weight about 205 kDa where the molecular weight of an enzyme was estimated using MW-standard markers. Moreover, PAGE gel developed by the activity staining using L-DOPA has shown only one band at the same run as that of developed by Coomassie brilliant blue staining. The enzyme was active throughout a broad range of temperature, its optimum temperature being 40oC while its optimum pH was found to be 4.5. Maximum activity of the enzyme was observed with 750 mM concentration of tartaric acid.

(B) Major highlights of the work done The present work explores the dye degrading abilities of B. laterosporus, with studies of the basic underlying mechanisms behind the removal of the dyes, GY-HE2R, NB-3G and textile effluents. The intracellular enzymes as well as whole cell systems can be used as effective bioremediating tools for the removal of textile dyes.

14.1.3 Biodegradation of textile dyes (Scarlet RR, Rubine GFL, Brown 3REL, Methyl Red, Brilliant Blue, Golden Yellow HER and Remazol Red) using Galactomyces geotrichum MTCC 1360 and consortia with Brevibacillus laterosporus

(A) Results, summary and major outcomes The G. geotrichum showed 86 % of decolorization of Golden Yellow HER within 30 h, at 30°C and pH 7.0 under microaerophilic (static) condition with significant reduction in COD (73 %) and TOC (62 %), respectively. This organism tolerates 500 mgL-1 of dye concentration. The degradation efficiency of this strain using carbon and nitrogen sources viz. wheat bran and glucose showed fast decolorization individually as well as glucose in combination with ammonium chloride and yeast extract responsible for faster decolorization of Golden Yellow

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HER. Enzymatic studies indicate the involvement of azo reductase, laccase and NADH-DCIP reductase enzymes in biotransformation. Azo dyes can be cleaved symmetrically or asymmetrically depending on the structure of substrate. The products of degradation were identified as 4(5-hydroxy, 4-amino cyclopentane) sulfobenzene and 4(5-hydroxy cyclopentane) sulfobenzene by GC-MS. In addition, when G. geotrichum was applied to decolorize textile effluent, it showed 85% of true color removal (ADMI removal) within 72 h, along with a significant reduction in TOC (42 %) and COD (58 %) under microaerophilic condition. Phytotoxicity study revealed less toxic nature of the formed metabolites as compared to parent dye with respect to S. vulgare and P. mungo. Malt extract medium was found to be most suitable for the degradation of Remazol Red among the other used media. The G. geotrichum showed 96 % decolorization of Remazol Red (50 mgL- 1) for 36 h, at 30°C and pH 11.0 under microaerophilic condition along with significant reduction in COD (62 %) and TOC (41 %). Peptone (5.0 gL-1), rice husk (10 gL-1 extract) and ammonium chloride (5.0 gL-1) were found to be more significant among the carbon and nitrogen sources used. The presence of tyrosinase, NADH-DCIP reductase, riboflavin reductase and induction in azo reductase and laccase activity during decolorization indicated their role in degradation. The metabolites produced after degradation was analyzed by HPTLC, FTIR, HPLC and GC-MS. The asymmetric cleavage of azo dye Remazol Red was carried out by azo reductase into reactive intermediate and subsequently mineralized by other enzyme found in G. geotrichum. Phytotoxicity study indicated the conversion of complex dye molecules into simpler oxidizable products having less toxic nature. The degradation of disperse azo dye and its toxicological approach in the sense of Allium cepa. G. geotrichum MTCC 1360 showed 87 % decolorization of Rubine GFL (50 mgL-1) within 96 h at 30°C and pH 7.0 under microaerophilic condition with significant reduction of COD (67 %) and TOC (59 %). The natural carbon and nitrogen sources like rice husk, wood shaving and bagasse was found to efficient for the decolorization of dye along with other sources used. Examination of oxidoreductive enzymes viz. laccase, tyrosinase and azo reductase confirmed their role in decolorization and degradation of Rubine GFL. Biodegradation of Rubine GFL into different metabolites was confirmed using HPTLC, HPLC, FTIR and GC-MS analysis. During toxicological studies, cell death was observed in Rubine GFL treated Allium cepa root cells. Toxicological studies before and after microbial treatment was studied with respect to cytotoxicity, genotoxicity, oxidative stress, antioxidant enzyme status, protein oxidation and lipid peroxidation analysis using root cells of Allium cepa. The antioxidant enzymes SOD and GPX were found to have the dose dependent induction, whereas CAT showed dose dependent inhibition. Similarly, lipid peroxidation and protein oxidation rates were also increased indicating the toxic nature of dye and effluent on A. cepa cells. Toxicity analysis with Allium cepa signifies that dye exerts oxidative stress and subsequently toxic effect on the root cells whereas formed metabolites relatively less toxic in nature. Phytotoxicity study reveals the less toxic nature of formed metabolite as compared to control dye Rubine GFL. The successful degradation of Brown 3 REL, Scarlet RR, Methyl Red and Brilliant Blue G was carried out by G. geotrichum. The preferential degradation of azo and non azo dye carried out in mixture. Decolorization affected by the chemical structure and surface function group near to azo bond. So we have studied time dependant degradation of textile dyes by G. geotrichum. In this study proved that the chemical structure affected the decolorization phenomenon. The G. geotrichum MTCC 1360, a yeast species showed 88 % ADMI (American dye manufacturing institute) removal of mixture of structurally different dyes (Remazol red, Golden yellow HER, Rubine GFL, Scarlet RR, Methyl 116

Work of some Indian researchers red, Brown 3 REL, Brilliant blue) (70 mgL-1) within 24 h at 30°C and pH 7.0 under shaking condition (120 rpm). Glucose (0.5 %) as a carbon source was found to be more effective than other sources used. The medium with metal salt (CaCl2, ZnSO4, FeCl3, MgCl2, CuSO4) (0.5 mM) showed less ADMI removal as compared to control, but did not inhibit complete decolorization. The presence of tyrosinase, NADH-DCIP reductase and induction in laccase activity during decolorization indicated their role in degradation. HPTLC (High performance thin layer chromatography) analysis revealed the removal of individual dyes at different time intervals from dye mixture, indicating preferential degradation of dyes. FTIR and HPLC analysis of samples before and after decolorization confirmed the biotransformation of dye. The reduction of COD (69 %), TOC (43 %), and phytotoxicity study indicated the conversion of complex dye molecules into simpler oxidizable products having less toxic nature. The comparative study of decolorization of two different azo dyes Remazol red and Rubine GFL disclosed the diverse catalytic activities of B. laterosporus. It decolorized 100 % of Remazol red and 95 % of Rubine GFL within 30 and 48 h, respectively under static condition at 50 mgL-1 dye concentration. Significant increase was observed in azo reductase, NADH-DCIP reductase, veratryl alcohol oxidase and tyrosinase in cells obtained after decolorization of Remazol red; whereas these values were much different with complete inhibition of azo reductase during decolorization of Rubine GFL. The plausible pathway of dyes obtained from Gas chromatography-Mass spectroscopy (GC-MS) data confirmed the different metabolic fate of these structurally unidentical dyes. HPLC, FTIR and HPTLC analysis of extracted metabolites confirmed the biodegradation, while phytotoxicity study assured the detoxification of both the dyes studied. The results obtained in this study suggests, i) sulpho and hydroxyl group present at ortho position to azo group stimulated reduction of azo bond by azo reductase in Remazol red, ii) the same reduction was totally hampered due to presence of ethyl-amino propanenitrile group at para position to azo group in Rubine GFL. B. laterosporus can degrade the sulphonated azo dyes having naphthol ring much faster; supporting that the sulpho and hydroxyl group present at ortho position to azo group stimulated the reduction of azo bond by azo reductase. The functional groups present in the nearby vicinity of azo bond play a central role in azo bond reduction. B. laterosporus decolorized 96 % of Brown 3 REL within 48 h at pH 7, 40°C in static condition at 50 mgL-1 concentration. Presence of peptone and yeast extract showed increased decolorization efficiency of B. laterosporus, whereas glucose and starch totally inhibited the decolorization ability. Enzymatic studies indicate the involvement of Veratryl alcohol oxidase and NADH-DCIP reductase enzymes in the biotransformation at these sets of conditions. Biodegradation products of Brown 3 REL were N-carbamoyl-2-[(8-chloroquinazolin-4-yl)oxy] acetamide and N- carbamoyl-2-(quinazolin-4-yloxy)acetamide when identified by Gas Chromatography-Mass spectroscopy. These metabolites found to be much less toxic than Brown 3 REL in phytotoxicity study. In addition to the single dye decolorization, B. laterosporus strain proved its applicability into the effluent treatment plant as it decolorized textile industry effluent with effective reduction in COD and TOC. Same strain used further for biodegradation study of disperse dye Scarlet RR in which, 100 % decolorization of same was obtained within 48 h with pH and temperature optima of pH 7 and 40°C, respectively. From the studied carbon and nitrogen sources, decolorization efficiency was observed better in yeast extract and peptone while carbon sources have shown reduced decolorization of Scarlet RR. At the enzyme level, induction in activities of veratryl alcohol oxidase, tyrosinase, NADH-DCIP reductase and riboflavin reductase was recorded. The biodegradation of Scarlet RR into different metabolites was confirmed by the HPLC, FTIR and HPTLC. A possible fate of metabolism of Scarlet dye was determined using 117

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GC-MS. It revealed the proposed biodegradation pathway of Scarlet RR into final product, N- (1λ3-chlorinin-2-yl) acetamide. Phytotoxicity study revealed completely nontoxic nature of degraded metabolites to Sorghum vulgare and Phaseolus mongo plants as compared to toxic Scarlet RR. In addition, B. laterosporus can degrade the dye with high concentrations of it and can also be used repeatedly for at least five cycles. The biodegradation of a mixture containing seven commercial textile dyes with different structures and color properties has been investigated by B. laterosporus. It showed 87 % decolorization in terms of ADMI removal (American dye manufacturing institute) within 24 h.

The effective decolorization of dye mixture was attained in the presence of metal salt-CaCl2 and nitrogen sources. The activity of oxidative enzymes in cellular organization amplifies during the stress of structurally different dyes. High performance thin layer chromatography exposed the mechanism of preferential biodegradation of dyes at different time periods. Significant change in the High pressure liquid chromatography and Fourier transform infrared spectroscopy of sample before and after treatment confirmed the biodegradation of dye mixture. Phytotoxicity study revealed the much less toxic nature of the metabolites produced after the degradation of dyes mixture. B. laterosporus can progressively degrade the dye within short span when combined with other dyes in culture; otherwise it needed long time to degrade the same dye when it concerned about single dye decolorization. B. laterosporus have broad specificity as it can degrade structurally diverse range of dyes with significant reduction in its toxicity and have preference in metabolism. The decolorization ability of a bacterial-yeast consortium was also tested with two real textile effluent having different characteristics and a simulated synthetic effluent made up of five different synthetic textile dyes. Consortium decolorized real effluent-1 and 2 with 89 and 60 % decolorization efficiency respectively within 48 h. In case of simulated synthetic effluent, 69 % decolorization with significant reduction in BOD and COD by consortium, 42 and 16 % decolorization by G. geotrichum and B. laterosporus, respectively were detected. In all the set of experiments, decolorizing ability of consortium is much more superior to the decolorizing capability of individual microorganisms. Cumulative action of oxidoreductive enzyme in consortium was found to be responsible for faster decolorization by the same. Fourier Transform Infrared Spectroscopy (FTIR) analysis suggested effective biotransformation of dyes present in the simulated synthetic effluent by consortium as compared to individual strains. The core finding of this study is the monitoring of release of each dye present in the simulated synthetic effluent using High Performance Thin Layer Chromatography (HPTLC). As monitored by HPTLC, consortium biodegraded all the dyes except Malachite green within 1 h only. In contrast, G. geotrichum required 4 h to remove the dyes; while up to 16 h of B. laterosporus treatment, no significant change in HPTLC chromatogram was detected. This study dealt with the proficiency of consortium over individual microorganisms along with the detailed monitoring of gradual biodegradation of each dye from the simulated synthetic effluent. The decolorization efficiency of microbial treatment to real effluent 1 was in the order as, consortium > B. laterosporus > G. geotrichum; however this order was changed to consortium > G. geotrichum > B. laterosporus in case of decolorization of real effluent 2 and simulated synthetic effluent. This suggests that the decolorization efficiency of each microbial species varies with the type of pollutant to be reduced. BOD/COD ratio of real textile effluent 1 and 2 was increased to 0.35 and 0.32, respectively after consortium treatment converting the high strength effluent into easily biodegradable element which confirms the effectiveness of consortium. HPTLC analysis helped in monitoring of rapid and gradual release of each dye chromophore from the effluent. 118

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(B) Major highlights of the work done These ecofriendly methods should be used for further applications in large scale studies. But scale up of these parameters should be taken into consideration. Hence for application part of these studies, we studied the decolorization of various dyes and textile industry effluent using immobilized cells of consortium BL-GG. We extended this study be developing two different type of bioreactors i.e. Up flow fixed bed reactor (UFFBR) and Triple layered packed bed reactor (TLPBR) using immobilized cells and used for continuous decolorization of textile industry effluent along with its repeated use. Up flow fixed bed reactor (UFFBR) and Triple layered packed bed reactor (TLPBR) showed an average of 93 and 86 % efficiency respectively in seven days run at flow rate of 10 and 100 mlh-1. We could use these bioreactors for repeated use after regeneration of same with nutrient feeding. These studies showed the efficiency and repeated use of immobilized cells on applied level.

14.1.4 Studies on microbial decolorization and degradation of toxic dyes from textile effluent

(A) Results, summary and major outcomes A novel bacterium was isolated from the soil of Ichalkaranji textile industrial area. Through 16S rRNA sequence matching and morphological observation it was identified as Lysinibacillus sp. RGS. This strain has ability to decolorize various industrial dyes among which, it showed complete decolorization and degradation of toxic sulfonated azo dye C.I. Remazol Red (at 30°C, pH 7.0, under static condition) with higher chemical oxygen demand (COD) reduction (92 %) within 6 h of incubation. Various parameters like agitation, pH, temperature and initial dye concentrations were optimized to develop faster decolorization process. The supplementation of cheap co-substrates (e.g., extracts of agricultural wastes) could enhance the decolorization performance of Lysinibacillus sp. RGS. Induction in oxidoreductive enzymes presumably indicates involvement of these enzymes in the decolorization/degradation process. Analytical studies of the extracted metabolites confirmed the significant degradation of Remazol Red into various metabolites. The phytotoxicity assay (with respect to plants Phaseolus mungo and Sorghum vulgare) revealed that the degradation of Remazol Red produced nontoxic metabolites. Finally Lysinibacillus sp. RGS was applied to decolorize mixture of dyes and actual industrial effluent showing 87 % and 72 % decolorization (in terms of decrease in ADMI value) with 69 % and 62 % COD reduction within 48 h and 96 h, respectively. Lysinibacillus sp. RGS degrades sulfonated azo dye Reactive Orange 16 (RO16) efficiently. Superoxide dismutase and catalase activity were tested to study the response of Lysinibacillus sp. RGS to the oxidative stress generated by RO16. The results demonstrated that oxidative stress enzymes not only protect the cell from oxidative stress but also has a probable role in decolorization along with an involvement of oxidoreductive enzymes. Formation of three different metabolites after degradation of RO16 has been confirmed by GCMS analysis. FTIR analysis verified the degradation of functional groups of RO16, and HPTLC confirmed the removal of auxochrome group from the RO16 after degradation. Toxicity studies confirmed the genotoxic, cytotoxic, and phytotoxic nature of RO16 and the formation of less toxic products after the treatment of Lysinibacillus sp. RGS. Lysinibacillus sp. RGS, an effective dye degrading bacteria immobilized on Luffa cylindrica (Loofa) was used to decolorize sulphonated azo dye Blue HERD and 50 % real textile effluent in

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Work of some Indian researchers an upflow column bioreactor. Blue HERD (200 mgL-1) and 50 % real textile effluent were degraded at volumetric flow rate 40 mLh-1 and 15 mLh-1, respectively. Induction in reductive (NADH-DCIP reductase and azoreductase) as well as oxidative (laccase, tyrosinase and veratryl alcohol oxidase) enzymes proved enzymatic decolorization of Blue HERD and 50 % textile effluent. Significant reduction in COD, BOD, TOC, hardness and alkalinity of decolorized samples of Blue HERD and 50 % effluent confirmed mineralization. HPTLC, FTIR and GCMS analysis confirmed degradation of Blue HERD into simple intermediates during decolorization in bioreactor. Less toxic nature of metabolites was confirmed using genotoxicity, cytotoxicity and phytotoxicity tests. Complete decolorization and detoxification of Reactive Orange 4 within 5 h (pH 6.6, at 30°C) by isolated Lysinibacillus sp. RGS was observed. Significant reduction in TOC (93 %) and COD (90 %) was indicative of conversion of complex dye into simple products, which were identified as naphthalene moieties by various analytical techniques (HPLC, FTIR, and GCMS). Supplementation of agricultural waste extract considered as better option to make the process cost effective. Oxido-reductive enzymes were found to be involved in the degradation mechanism. Finally, Loofa immobilized Lysinibacillus sp. cells in a fixed-bed bioreactor showed significant decolorization with reduction in TOC (51 and 64 %) and COD (54 and 66 %) for synthetic and textile effluent at 30 and 35 mLh-1 feeding rate, respectively. The degraded metabolites showed non-toxic nature revealed by phytotoxicity and photosynthetic pigments content study for Sorghum vulgare and Phaseolus mungo. In addition nitrogen fixing and phosphate solubilizing microbes were less affected in treated wastewater and thus the treated effluent can be used for the irrigation purpose.

(B) Major highlights of the work done a) Lysinibacillus sp. RGS has a better perspective of bioremediation for textile dyes viz. Remazol Red, Reactive Orange, 16 Blue HERD and Reactive Orange 4. b) Loofa immobilized Lysinibacillus sp. cells in a fixed-bed bioreactor showed significant decolorization with reduction in TOC of synthetic and textile effluent. c) These results increase the applicability of the strain for the treatment of industrial wastewaters containing dye pollutants. d) This work could be useful for the development of efficient and ecofriendly technologies to reduce dye content in the wastewater to permissible levels at affordable cost.

14.1.5 Construction of wetland- a phytoremediation treatment process for the degradation of dyes from textile industrial effluent

(A) Results, summary and major outcomes Salvinia molesta, an aquatic fern was observed to have a potential of degrading azo dye Rubine GFL up to 97 % at a concentration of 100 mgL-1 within 72 h using 6072 g of root biomass. Both root as well as stem tissues showed induction in activities of the enzymes such as lignin peroxidase, veratryl alcohol oxidase, laccase, tyrosinase, catalase, DCIP reductase and superoxide dismutase during decolorization of Rubine GFL. FTIR, GC-MS, HPLC and UV–visible spectrophotometric analysis confirmed phytotransformation of the model dye into smaller molecules. Analysis of metabolites revealed breakdown of an azo bond of Rubine GFL by the action of lignin peroxidise and laccase and formation of 2-methyl-4-nitroaniline and N- 120

Work of some Indian researchers methylbenzene-1,4-diamine. Anatomical tracing of dye in the stem of S. molesta confirmed the presence of dye in tissues and sub sequent removal after 48 h of treatment. The concentration of chlorophyll pigments like chlorophyll a, chlorophyll b and carotenoid was observed during the treatment. Toxicity analysis on seeds of Triticum aestivum and Phaseolus mungo revealed the decreased toxicity of dye metabolites. In situ treatment of a real textile effluent was further monitored in a constructed lagoon of the dimensions of 7 m x 5 m x 2 m (total surface are a 35 m2) using S. molesta for 192 h.This large scale treatment was found to significantly reduce the -1 values of COD, BOD5 and ADMI by 76, 82 and 81 % considering initial values 1185, 1440 mgL and 950 Units, respectively. Alternanthera philoxeroides Griseb. a macrophyte was found to degrade a highly sulfonated textile dye Remazol Red (RR) completely within 72 h at a concentration of 70 mgL-1. An induction in the activities of azoreductase and riboflavin reductase was observed in root and stem tissues; while the activities of lignin peroxidase, laccase and DCIP reductase were induced in leaf tissues. Some enzymes namely tyrosinase, veratryl alcohol oxidase, catalase and superoxide dismutase displayed an increase in their activity in all the tissues in response of 72 h exposure to Remazol Red. There was a marginal reduction in contents of chlorophyll a (20 %), chlorophyll b (5 %) and carotenoids (16 %) in the leaves when compared to control plants. A detailed anatomical study of the stem during uptake and treatment revealed a stepwise mechanism of dye degradation. UV-vis spectrophotometric and high performance thin layer chromatographic analyses confirmed the removal of parent dye from solution. Based on the enzymes activities and gas chromatography-mass spectroscopic analysis of degradation products, a possible pathway of phytotransformation of RR was proposed which revealed the formation of 4-(phenylamino)- 1,3,5-triazin-2-ol, naphthalene-1-ol and 3-(ethylsulfonyl)phenol. Toxicity study on Devario aequipinnatus fishes showed that the anatomy of gills of fishes exposed to A. philoxeroides treated RR was largely protected. The plants were further explored for rhizofiltration experiments in a pilot scale reactor. A. philoxeroides could decolorize textile industry effluent of varying pH within 96 h of treatment which was evident from the significant reductions in the values of American dye manufacturers' institute color, chemical oxygen demand, biological oxygen demand, total dissolved and total suspended solids. Ipomoea aquatica, a macrophyte was found to degrade a highly sulfonated and diazo textile dye Brown 5R up to 94 % within 72 h at a concentration of 200 mgL-1. Induction in the activities of enzymes such as azoreductase, lignin peroxidase, laccase, DCIP reductase, tyrosinase, veratryl alcohol oxidase, catalase and superoxide dismutase was observed in leaf and root tissue in response to Brown 5R exposure. There was significant reduction in contents of chlorophyll a (25 %), chlorophyll b (17 %) and carotenoids (30 %) in the leaves of plants. HPLC, FTIR, UV-vis spectrophotometric and HPTLC analyses confirmed the biotransformation and removal of parent dye from solution. Enzymes activities and GC-MS analysis of degradation products lead to the proposal of a possible pathway of phytotransformation of dye. The proposed pathway of dye metabolism revealed the formation of Napthalene-1,2-diamine and methylbenzene. Toxicity study on HepG2 cell lines showed a 3 fold decrease in toxicity of Brown 5R after phytoremediation by I. aquatica. Hydrophytic nature of I. aquatica leads to its exploration in a combinatorial phytoreactor with Ipomoea hederifolia soil bed system. Rhizofiltration with I. aquatica and soil bed treatment by I. hederifolia treated 510 L of effluent effectively within 72 h. I. aquatica along with I. hederifolia could decolorize textile industry effluent within 72 h of treatment as evident from the significant reductions in the values of COD, BOD, solids and ADMI. Further on field trials of treatment of textile wastewater was successfully carried out in a constructed lagoon. In vitro grown untransformed adventitious roots (AR) culture of Ipomoea hederifolia and its endophytic fungus (EF) Cladosporium cladosporioides decolorized Navy Blue HE2R (NB- HE2R) at a concentration of 20 ppm up to 83.3 and 65 %, respectively within 96 h. Whereas the

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AR-EF consortium decolorized the dye more efficiently and gave 97 % removal within 36 h. Significant inductions in the enzyme activities of lignin peroxidase, tyrosinase and laccase were observed in roots, while enzymes like tyrosinase, laccase and riboflavin reductase activities were induced in EF. Metabolites of dye were analyzed using UV-vis spectroscopy, FTIR and gas chromatography-mass spectrometry. Possible metabolic pathways of NB-HE2R were proposed with AR, EF and AR-EF systems independently. Looking at the superior efficacy of AR-EF system, a rhizoreactor was developed for the treatment of NB-HE2R at a concentration of 1000 ppm. Control reactor systems with independently grown AR and EF gave 94 and 85 % NB-HE2R removal, respectively within 36 h.The AR-EF rhizoreactor, however, gave 97 % decolorization. The endophyte co-ionization additionally increased root and shoot lengths of candidate plants through mutualism. Combined bioreactor strategies can be effectively used for future eco-friendly remediation purposes. Wild plant and tissue cultures of Ipomoea hederifolia decolorize Scarlet RR (SRR) dye at a concentration of 50 mgL-1 up to 96 % and 90 % within 60 and 96 h, respectively. Significant induction in the enzyme activities of Lignin peroxidase, laccase, 2,6-dichlorophenol indophenol reductase, superoxide dismutase, catalase and tyrosinase was found in the plant roots and shoots during decolorization. I. hederifolia was also able to treat a dye mixture and a real textile effluent efficiently with a reduction in the American Dye Manufacturers Institute (ADMI) value (color removal) up to 85 % and 88 %, BOD up to 65 % and 63 % and COD up to 62 % and 68 %, respectively. Detailed anatomical studies of the stem and root cells of I. hederifolia during uptake and degradation were carried out, showing a stepwise and mechanistic degradation of the model dye SRR. Products formed after dye degradation was analyzed by UV-Vis spectroscopy, FTIR, HPLC and HPTLC, which confirmed the phytotransformation of SRR, dye mixture and textile effluent. A possible pathway for the phytotransformation of SRR was proposed based on GCMS analysis, which confirmed the formation of different metabolites with lower molecular weights. The phytotoxicity study revealed the non-toxic nature of the formed products.

(B) Major highlights of the work done a) Wild plant and tissue cultures of Ipomoea hederifolia decolorize Scarlet RR, Navy Blue HE2R and Brown 5R, and textile effluent. b) Alternanthera philoxeroides Griseb. a macrophyte and Salvinia molesta, an aquatic fern was found to degrade a highly sulfonated textile dye Remazol Red (RR) azo dye Rubine GFL, respectively. c) All these species were found to be useful for textile effluent treatment in constructed phytoreactors and in lagoons.

14.2 Research outcomes from Dr. Venkata Mohan’s Lab

14.2.1 Introduction The dyestuffs are water-soluble dispersible organic colourants, having high potential use in various industrial applications (mostly in the textile and paper/leather industry as a colouring material). The textile sector consumes about 60% of the total dye production for colouration of various fabrics and out of it, around 10–15% of the dyes come out through the effluents. Dyes are classified into various application classes (Direct, Acid, Basic, Disperse, Reactive, Vat, etc.) and chemical classes (Azo, Anthraquinone, Phthalocyanine, Xanthene, Nitroso, Nitro, Thiazine, etc.).

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Dye containing wastewater from the textile industry is a mixture of many polluting substances, ranging from organochlorine-based pesticides to heavy metals associated with dyes and chemicals used in the dyeing process. The colored dye effluents are considered to be highly toxic to the aquatic biota and affect the symbiotic processes and are also considered to be carcinogenic to humans. Hence, it becomes imperative that dyes should be removed from the effluents before it is disposed in water bodies.

14.2.2 Physicochemical Physicochemical methods such as adsorption, precipitation, chemical oxidation, photodegradation, or membrane filtration are used for treatment of dye based wastewater. Among various treatment technologies, adsorption onto activated carbon is proved to be one of the effective and reliable physicochemical treatment processes. However, the cost of activated carbon and regeneration problems necessitated the search for other low cost adsorbents. In this context, various materials like inorganic clay materials, industrial waste (by-products) materials, etc. have been widely studied. The potential of different coal based sorbents are widely available in huge quantities and have all physical and chemical requirements of an adsorbent. Low cost sorbent charfines, lignite coal, bituminous coal are studied in the process of adsorptive colour removal of a reactive dye Trisazo direct dye and C.I.Direct Brown mixed in a proportion of 1:1 was studied and compared with activated carbon (F400). The results pertaining to the batch kinetic studies performed on the coal-dye system indicated the varied dye sorption capacity of the tested adsorbents (charfines and activated carbon). Ranking of the sorbents in terms of color removal capacity was of the following order, activated carbon > charfines > lignite coal > bituminous coal. The study concludes that the initial high uptake of dye on coal may be reasoned due to chemisorption interaction while gradual uptake by activated carbon may be indicative of physisorption interaction. Monoazo acid dye, C.I. Acid Red 88, by adsorption onto coal based sorbents like Lignite coal, Charfines and Bituminous coal and F-400 Activated carbon and by chemical coagulation employing Aluminum sulfate, Ferric chloride, Manganese sulfate, Manganese chloride and Barium chloride. Various adsorbents derived from natural materials like silica, coal, soil, minerals, chitosan, etc. and waste materials like fly ash, rice husk, biogas slurry, silica fumes, saw dust, etc. could also studied in place of activated carbon. Although the above said physical and/or chemical methods have been used to treat dyes in wastewater or effluents, they possess inherent limitations such as high cost, formation of hazardous byproducts and intensive energy requirements.

14.2.3 Biological Despite the existence of a variety of physical and chemical treatment processes, bioremediation of textile effluent is seen as an attractive and sustainable solution due to its low cost and environmental friendly nature. Biological processes such as biosorption, bioremediation, etc. have shown potential application in removal of dyes from textile wastewater.

(A) Biosorption by viable algae Biosorption process is mainly manifested by cell surface sequestration; cell wall modification could greatly alter the adsorption capability by enhancing the binding capacity of the biomass. Algae has a high surface area to volume ratio imparting significant potential for sorption and has been recognized with respect to the persistent organochlorine compounds. Algal species of

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Spirogyra found in the oxidation ponds of wastewater treatment plants was used for treatment of dye based effluents. Direct Red 28 and Reactive Red 2 were studied using Spirogyra sp. in viable form. The study concluded that the Spirogira sp. is capable of removing azo dye colour from aqueous phase and also it is dependent on initial algal inoculum, dye concentration and nature of the dye. The colour removal was more in the case of Direct Red 28 when compared to Reactive Red 2.

(B) Biosorption by non-viable algae Biosorption employing non-viable algal species (Spirogyra sp. IO2) form was evaluated as biosorbent to study its potential to adsorb azo dye from aqueous phase at different pH and temperature (Venkata Mohan et al., 2008). Highest biosorption values (80 %) were observed at acidic pH (2.0) which could be attributed to the anionic sorption and indicated that the adsorbent surface is of H+ (cationic)-type. At lower pH values, the surface of the biosorbent turned out to be positively charged and this facilitated sorption of dye, probably by the anionic exchange sorption. These bio-macromolecules on the algal cell surfaces have several functional groups (such as amino, carboxyl, thiolsulfydryl and phosphate groups) and biosorption phenomena depends on the protonation or un protonation of these functional groups on the surface of the cell wall. Relatively less dye sorption observed at basic pH range which might be attributed to the increase of hydroxyl ion leading to the formation of aqua-complexes thereby retarding the sorption. However, the dye sorption efficiency at aqueous phase pH 7.0 was found to be lower than acidic pH and more than basic pH and hence with respect to the upscaling of the technology this neutral pH could be feasible. Increasing in operating temperature from 10 to 50°C showed a marked improvement in dye sorption capacity. Rapid enhancement in the dye sorption was observed with increase in temperature from 10 to 20°C. The increase of the dye uptake with increase in temperature suggests that the dye-algal-sorption process may be attributed to endothermic nature. If adsorption is governed only by physical phenomena, an increase in temperature will be followed by a decrease in adsorption capacity. The combined effect of ion-exchange and chemical sorption phenomena might be possible during the dye-algal sorption, which can be corroborated with the observation from kinetics studies. The non-viable form of algae Spirogyra IO2 revealed the ability of test biosorbent to remove azo dye from the aqueous phase. The sorption reaction is pH dependent with higher removal observed at low pH values. Based upon the results, the dye removal mechanism may be attributed to the process of biosorption, bioconversion and biocoagulation.

(C) Enzymes Researchers have been focusing their attention to study enzymatic pretreatment as a potential and viable alternative to conventional methods, due to its highly selective nature. Further, inhibition by toxic substances is minimum in enzymatic treatment and the process can operate over a broad aromatic concentration range with low retention time. Enzymes from various sources (fungus and plant based) are used for the treatment of dye based compounds. The source of the selected enzyme and its nature along with system conditions are found to have significant influence on the overall performance for pollutant removal. The study investigate to evaluation of hydrogen peroxidase oxido-reductase extracted from horseradish (EC 1.11.1.7) also called as horseradish peroxidase (HRP) in the process of acid azo dye removal. The study revealed the effectiveness of the peroxidase catalyzed enzymatic reaction in the treatment of an acid azo dye in aqueous phase. Immobilized HRP in acrylamide matrix resulted in effective performance over the free HRP,

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Work of some Indian researchers while alginate entrapped HRP yielded inferior performance over the free one. Repeated application of enzyme was observed to be feasible with immobilized HRP beads. Hence researchers have been focusing their attention to study enzymatic pretreatment as a potential and viable alternative to conventional methods, due to its highly selective nature. The study concludes that immobilized enzymes overcome the limitations of free enzymes as the stability and catalytic ability of free enzymes decrease with the complexity of the effluents. The performance of laccase-membrane reactor configurations viz., direct enzyme contact, enzyme impregnated, immobilized enzyme and a reactor system based on laccase immobilization in chitosan membranes was studied for decolorization of azo dye (acid black 10 BX) using laccase enzyme purified from white rot fungi Pleurotus ostreatus 1804. Experimental data showed that laccase has great potential for color removal without addition of external redox mediators. Various process parameters viz. aqueous phase of pH 6.0, enzyme concentration of 1.75 Uml-1, dye concentration of 20 mgL-1, temperature of 30 °C and reaction time of 120 min were optimized to achieve maximum decolorization efficiencies. Moreover, different laccase- membrane reactor configurations were tested to determine the efficacy of repeated application of laccase on dye decolorization process. Among the different reactor configurations studied, laccase encapsulated in chitosan membrane showed advantages such as short-term contact period and reusability of enzyme for a number of cycles.

(D) Anaerobic Treatment The presence of sulfo and azo groups in the dye structure protects the dye molecule from attack of oxygenases, making them resistant to oxidative biodegradation. Therefore, these compounds are more difficult to degrade through conventional aerobic treatment processes and hence require sequential redox conditions to cleave the chromophore (anaerobic) into corresponding aromatic amines and for further degradation (aerobic) of toxic aromatics into simpler compounds. The process of anaerobic degradation supports reductive breakdown of dye molecules by cleaving the chromophore into corresponding colourless aromatic amines. This breakdown is supported by the utilization of redox powers obtained by oxidation of a co-substrate in the surrounding microenvironment. Reductive microenvironment influenced the cleavage of azo dye bond to intermediate compounds, using glucose as co-substrate. Anaerobic microenvironment with diverse bacteria degraded the dyes to the aromatic amines due to the presence of azo reductase and dehydrogenase enzyme activities however complete mineralization was not observed hence advanced biological wastewater treatments such as sequencing batch reactor and biolectrochemical treatment systems could be used.

(E) Sequencing batch reactor Dye based effluents are normally not amenable for conventional biological wastewater treatment due to their recalcitrant and inhibitory nature. However, microbial methods are highly useful and potentially advantageous for the treatment of toxic compounds due to their effectiveness, ecofriendly nature, energy saving and less usage of chemicals. Conventional biological treatment processes are seldom capable of achieving the required degree of dye degradation, while anaerobic processes alone cannot handle the complete mineralization of the dye molecule. The ability to achieve complete mineralization of azo dye depends on the redox condition of the process. Sequencing batch reactor (SBR) or Periodic Discontinuous Batch Reactor (PDBR) could be used is a batch analog process allows the integration of both anoxic and aerobic redox conditions in a single reactor, making the combination of reductive and oxidative steps possible.

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SBR/PDBR enforces controlled short-term unsteady state conditions leading to stable steady state conditions in the long run. It also imposes selective pressures that can select a defined population which is able to degrade complex compounds, contrary to the continuous process. Metabolic function plays a significant role in altering the microbial metabolism of the bacteria and has specific function towards substrate utilization and recalcitrant compounds removal. Anaerobic metabolic function facilitates reductive breakdown of azo dye molecule by cleaving the azo bond to the corresponding colorless aromatic amines. These aromatic amine residues from anaerobic decolorization resist further anaerobic degradation due to their mutagenic nature. On the contrary, aromatic amines could be mineralized in aerobic microenvironment by non-specific enzymes through hydroxylation and ring-fission of aromatic compounds. Due to the flexibility of combining multiple microenvironments during operation, periodic discontinuous batch process might of significant interest to treat recalcitrant azo dyes. The functional role of anoxic-aerobic- anoxic microenvironments during periodic discontinuous batch mode operation for the treatment of azo-dye bearing wastewater was evaluated reactor. It is a prerequisite to have anoxic/microaerophilic conditions to initially breakdown the dye molecule prior to aerobic mineralization. Azo dyes are prone to get reduced under anaerobic conditions to corresponding aromatic amines like tri-aminobenzen, aniline and 2,8,9- triamino-1-hydroxynaphthaline 3,7- disulphonic acid which resisting to further anaerobic degradation. The study concludes the effective performance of aerobic biocatalyst under anoxic-aerobic-anoxic microenvironment for the azo dye removal in periodic discontinuous batch mode operation. This was supported by the microbial community analysis which documented the presence of facultative bacteria in the. Six periodic discontinuous/sequencing batch reactors (SBR) each with a total volume of 1 liter were designed and fabricated. Separate bioreactors with aerobic, anoxic and anaerobic metabolic functions were operated in suspended growth mode. Functional behavior of anoxic-aerobic- anoxic microenvironment on azo dye (C.I.Acid black 10B) degradation was evaluated in a periodic discontinuous batch mode operation for 26 cycles. Dye removal efficiency and azoreductase activity (30.50 ± 1 Units) increased with each feeding event until 13th cycle and further stabilized. Dehydrogenase activity also increased gradually and stabilized (2.0 ± 0.2 μgml-1) indicating the stable proton shuttling between metabolic intermediates providing higher number of reducing equivalents towards dye degradation. Voltammetric profiles showed drop in redox catalytic currents during stabilized phase also supports the consumption of reducing equivalents towards dye removal. Change in Tafel slopes, polarization resistance and other bioprocess parameters correlated well with the observed dye removal and biocatalyst behavior. Microbial community analysis documented the involvement of specific organism pertaining to aerobic and facultative functions with heterotrophic and autotrophic metabolism. Integrating anoxic microenvironment with aerobic operation might have facilitated effective dye mineralization due to the possibility of combining reduction-oxidation functions. Functional role of anoxic and anaerobic metabolic functions during the periodic discontinuous batch mode operation in comparison with aerobic metabolic function during the treatment of azo dye bearing wastewater was evaluated to understand the shifts in biocatalyst metabolic activities with the function of terminal electron acceptor (TEA). Higher dye removal was observed under anoxic operation followed by the anaerobic and aerobic. The study concludes as competence of oxygen with dye as TEA might be the reason for the observed lower dye degradation under aerobic operation. Moreover, function of dye as a TEA along with the micro-aerophilic conditions, facilitating the oxidation of dye metabolites, might be the reason for the observed higher dye removal. Inducing anoxic microenvironment in multi-phase metabolic shift (MP) 126

Work of some Indian researchers strategy during periodic discontinuous batch/sequencing batch (PDBR/SBR) mode operation showed enhanced degradation of azo dye. Two reactors with aerobic and anoxic metabolic conditions were operated by varying time intervals of anoxic and aerobic microenvironments (before multiphase (BMP); after multiphase (AMPI, AMPII, AMPIII). Higher dye removal was observed after applying multiphase strategy (anoxic-AMPIII, 62 %; aerobic-AMPIII, 58.5 %) compared to BMP (anoxic, 49 %; aerobic, 42 %). Higher azo reductase (31 ± 0.13 U), dehydrogenase activities (3.18 µgml-1) and redox catalytic currents were observed in anoxic- AMPIII operation compared to corresponding aerobic-AMPIII. Extending multiple anoxic phases resulted in secreting the redox shuttles by biocatalyst for their survival against the toxic dye environment. MP strategy extending multiple anoxic phases aided in expanding the capabilities of SBR/PDBR operation for more viable treatment application. Irrespective of the operating conditions, the carbon source will undergo degradation during biocatalyst metabolic activities to form end products along with the generation of reduced electron carriers (NAD+, FAD+, FMN+, etc.). These reducing powers should be re-oxidized in the cell to re-enter the metabolic pathways or else all the redox reactions get lagged and hence their re-oxidation is considered crucial. Effect of dye shock load on the process performance of biofilm and suspended configured periodic discontinuous batch mode operation (PDBR) in response to the treatment of azo dye (C.I. Acid Black 10B) bearing wastewater was studied. Reactors were operated at varying dye/COD ratio (0.16, 0.23, 0.33 and 0.41) (Kumnar et al., 2014). During higher dye loading operation (1250 mg dyeL-1) biofilm mode resulted in higher dye removal (74.5 %) than the corresponding suspended mode operation (42 %). With each additional dye loading event azo reductase and dehydrogenase enzyme activity also increased. Azo reductase enzyme was isolated from both the cultures and its molecular weight was determined by SDS-PAGE and was found to be 94 kDa. The performance of PDBR operation on treatment of real-field wastewater was evaluated using PDBR/SBR and continuous mode operation and by varying microenvironment (aerobic, anoxic and anaerobic). Among the operation mode studies, PDBR mode documented significant higher performance in the treatment of wastewater. In the case of reactor microenvironment, anoxic and aerobic system documented treatment efficiency than the corresponding anaerobic operation.

(F) Multi-phase metabolic shift strategy PDBR/SBR operation is integration of two variable microenvironments in a single reactor system, especially beneficial for the dye-based wastewater treatment. Due to its unique flexibility to combine multiple metabolic functions (redox conditions) during operation, PDBR/SBR process attracted significant interest in treating recalcitrant compounds. The consecutive variation of anoxic and aerobic conditions facilitated cyclic sequence of anoxic and aerobic microenvironment in the multiphases operation in a single cycle. Though the biocatalyst (aerobic) was the same in all the strategies, variations in the dye degradation was observed with and without multiphase operation. Shifts in microbial metabolism with the function of aerobic, anaerobic and anoxic microenvironments were evaluated in periodic discontinuous batch mode operation in response to the treatment of azo dye (C.I.Acid Black 10B) bearing wastewater. Anoxic microenvironment depicted higher dye removal efficiency (95.18 %) followed by anaerobic (93.67 %) and aerobic (68.77 %) microenvironments. Switching between aerobic and anaerobic microenvironment during anoxic operation facilitated the reduction of dye to its intermediates (anaerobic) followed by the mineralization of these intermediates (aerobic). Function of dye as a TEA along with the limited oxygen available might be the reason for the observed higher dye removal under anoxic microenvironment. Azo reductase activity showed marginal variation with the function of anoxic

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Work of some Indian researchers and anaerobic microenvironment indicating the similar capabilities of the biocatalyst. However, dehydrogenase activity showed significant variation with the function of microenvironment indicating the differences in the delivery of reducing powers from the substrate metabolism towards dye removal. The bio-electro kinetics showed well correlation with the redox reactions, enzyme activities and dye removal. Microbial inventory analysis documented the involvement of organisms with anaerobic and facultative metabolic functions having heterotrophic and autotrophic nutrition modes. Aerobic operation documented the dominance of facultative (83 %) followed by aerobic (17 %) bacteria while anoxic and anaerobic operations facilitated the distribution of facultative (67 % and 83 % respectively) and anaerobic (33 % and 17 % respectively) bacteria. Anoxic microenvironment switches between oxidation and reduction depending on the oxygen availability. Study was evaluated to understand the metabolic shifts by altering operating microenvironment specifically inducing anoxic condition on azo dye degradation during PDBR process. The study concluded the advantage of multi phasing and incrementing anoxic phasing on the degradation of recalcitrant azo dye during PDBR/SBR operation. Varying anoxic and aerobic microenvironments more anoxic conditions initially followed by more aerobic conditions in latter stages of cycle operation enhanced the overall treatment efficiency. Moreover, these studies will help to comprehend and design effective redox integration mechanism for treatment of complex wastewater.

(G) Biofilm verses Suspension microbes influence Microbes/bacteria play important role in the biological degradation processes in which the processes under goes diverse biochemical pathways. Hence to understand the functional role of biofilm (self immobilized) and suspended growth bioreactor configurations in response to the treatment of azo-dye bearing wastewater was evaluated in periodic discontinuous batch mode operation at varying dye concentrations. Relatively, biofilm system depicted higher dye removal efficiency compared to suspended mode. Functional role of biofilm and suspended growth bioreactor configurations in response to the treatment of azo-dye (C.I. Acid Black 10B) bearing wastewater was evaluated in periodic discontinuous batch mode operation at varying dye concentrations. The biofilm system depicted higher dye removal efficiency (93.14 %) compared to suspended mode (84.29 %) at 350 mg dyeL-1 operation. Both reactor configurations didn’t show much process inhibition at the higher dye loads studied. Azo reductase and dehydrogenase enzyme activities showed significant variation indicating the different metabolic capabilities of the native-microflora, stable proton shuttling between metabolic intermediates, and differences in the delivery of reducing powers from the substrate metabolism towards dye removal. Voltammograms visualized marked variations in electron discharge properties with the function of reactor configuration, time intervals and dye load. Higher redox catalytic currents, lower Tafel slopes and polarization resistance showed good correlation with enzyme activities and dye removal. Self-immobilization of microflora as biofilm results in high biomass hold up, thus enables the process to be operated significantly at higher liquid throughputs and organic load which are well suited for the treatment of wastewater containing poorly degradable compounds. Biofilm systems are particularly useful where high hydraulic loading variations occur and where slowly growing organisms with special metabolic capacities are to be protected from washout. Moreover, attached biofilm acts as a buffer to reduce the concentration of toxic chemicals during process operation thereby providing advantage for the treatment of dye based wastewater containing recalcitrant compounds. The study concludes that effective performance of biofilm

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Work of some Indian researchers mode of operation compared to suspended growth configurations with regard to azo dye removal during PDBR operation.

14.2.4 Bioelectrochemical treatment systems (BET) Bioelectrochemical treatment systems are typically a type of microbial fuel cell (MFC), a promising technology for electricity production, which can be advantageously combined with applications in wastewater treatment. When the major attention is on treatment rather than electricity production, MFC can also be termed as bioelectrochemical treatment (BET). BET use electrodes as solid electron acceptor for bacteria respiration and exploit microbial catabolic activities to generate electrons (e_) and protons (H+) by degrading organic molecules. The microbial metabolism is linked via electron donating and accepting conditions through the presence of artificial introduced electrodes (anode and cathode), which induces the development of a potential difference that acts as a net driving force for bioelectrogenic activity and complex dye removal. The functional activity of anaerobic bacteria in the presence of an electrode as solid electron acceptor was comprehensively evaluated during the treatment of azo dye based wastewater (Sreelatha et al., 2015). The study provides a new insight into the electron acceptor dependent respiration wherein the electrode serving as a solid electron acceptor enables efficient function of anode respiring bacteria (ARB) in terms of electron flux towards dye degradation and electrogenesis. The study also documented electron acceptor dependent respiration, exemplifying the influence of conjunction between electrode and bacteria on dye degradation. A comparative study between bioelectrochemical treatment system with the presence of electrode assembly and biocatalyst, anaerobic treatment and abiotic-control comprising of an electrode assembly without biocatalyst explained the specific influence of the electrode assembly as a solid electron acceptor in the presence of biocatalyst are of crucial importance as the terminal reduction reactions for dye degradation. The BET system functioned with coupled action of the electrode assembly as a solid electron acceptor as well as bacteria as a biocatalyst, resulting in significant dye remediation with simultaneous power generation in comparison to the anaerobic treatment and abiotic-control operations. Electron flux between a microbe and solid phase are by two overlapping mechanisms such as direct and mediated transfer. Direct cell-electrode contact with bacterial outer membrane bound cytochromes serving as reductases as well as dye molecules functioning as electron shuttles. Electrode assembly induces the development of potential difference by the biocatalytic action of electrochemically active bacteria enriched around the electrodes in carrying out the simultaneous redox reactions towards the breakdown of dye molecules. Besides, the breakdown of complex dye molecule results in the generation of reduced dye intermediates that in turn acts as redox mediators/electron shuttles during the transfer of electrons to the solid electron acceptor/ for the other dye molecule reduction enabling the mediated electron acceptor. Bio-electrogenic activity enhanced when bacterial respiration was coupled with the electrode assembly in treating/utilizing the azo dye based wastewater which can be attributed to the electron acceptor-dependent respiration. The study clearly documented that absence of electrode assembly in AnT system and bacteria in abiotic-control system respectively, resulted in low performance in terms of azo dye degradation however compiling both the processes in the single system as BET procures the advantage of electron acceptor dependent respiration offering dual benefits of dye degradation and power generation. Integration of two multiple processes aids in achieving enhanced treatment efficiency, more specifically with dye based wastewaters by considering the advantage of both the processes SBR

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Work of some Indian researchers followed by BET was developed to achieve enhanced treatment efficiency (Nagendranatha and Venkata Mohan, 2016). BET was designed to evaluate complete mineralization of partially treated dye effluent obtained from anoxically operated Periodic discontinuous batch reactor (PDBR) for simultaneous bioelectricity generation and recovery of nutrients (nage. In MET bioreactor, anode and cathode chambers were fed with designed synthetic wastewater (DSW) and + PDBR dye effluents. The dye metabolite (NH4 ) will be converted to nitrates by the activity of aerobic biocatalyst present in cathode chamber to be used as biofertilizer. Dye removal of 90.2 % was observed with good electrogenic activity (voltage (OCV)/current; 395 mV/1.77 mA). The mineralization of dye and its intermediates were assessed by reduction in overall toxicity from 23 to 4 %. Chemical oxygen demand (COD) removal efficiency of 75 % (anode) and 88 % (cathode) were observed in correspondence to higher azoreductase (18.7 U; 48 h) and dehydrogenase (1.66 μgml-1 of toluene; 24 h) enzyme activities which correlated well with metabolic activities of biocatalyst. Bioelectrocatalytic behavior of mixed biocatalyst on the basis of redox catalytic currents and prevalence of redox mediators signified the specific function of electron transfer toward dye mineralization. The results obtained suggest that the use of BET can considerably degrade toxic pollutants and provides nitrate rich solution (biofertilizer). Utilization of recovered nutrients directly to farms without any energy intensive methods is reported in this communication. The possible integration of two different processes paves a way towards sustainability by addressing product recovery and bioremediation. The increment of nitrates concentration in cathode chamber correlates well with the decrease in ammonical nitrogen, dye and toxicity. The catholyte containing high nitrates and low COD may be used directly in the farms for plant growth as the nitrates are present in easy uptake form. Over all the above mentioned advanced biological processes could be an effective solution for the treatment of dye based wastewater. These technologies could remediate the areas affected by dye based wastewaters and a pave way to reopen the textile industries. Apart from dye based wastewater, these advanced biological and bioelectrochemical processes could be used to treat wastewaters originating from chemical and allied industries which are highly complex in nature. Developing sustainable wastewater treatment technologies for management of complex wastewater in a cost effective approach is in need. Valorisation of the waste achieved through cascade level integration of various biotechnology processes that utilize biogenic segment of the waste and produce high value bioproducts. Finally, waste mining deemed as one of the prominent areas with superior commercial interest considering the various possibilities of resource recovery. Integrating various bioprocesses by in a sequential unit operation and processes with simultaneous production of value added products and biofuel generation could significantly contribute to the society in terms of waste management, pollution control, climate change and global warming along with energy recovery and value added products.

14.3 Research Outcomes from the Prof. Jyoti Jadhav’s Lab

14.3.1 Biodegradation of triphenylmethane dyes by Penicillium ochrochloron

(A) Results, summary and major outcomes The present study dealt with the decolorization and degradation of triphenyl methane textile dye by mycelium of Penicillium ochrochloron. Spectrophotometric and visual examinations showed that the decolorization was through fungal adsorption, followed by degradation.

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Triphenylmethane dyes belong to the most important group of synthetic colorants and are used extensively in the textile industries for dying cotton, wool, silk, nylon, etc. They are generally considered as the xenobiotic compounds, which are very recalcitrant to biodegradation. Penicillium ochrochloron decolorizes cotton blue (50 mgL−1) within 2.5 h under static condition at pH 6.5 and temperature 25 °C. TLC, FTIR and HPLC analysis confirms biodegradation of cotton blue. FTIR spectroscopy and GC–MS analysis indicated sulphonamide and triphenylmethane as the final products of cotton blue degradation. The pH, temperature and maturity of biomass affected the rate of decolorization. Presence of lignin peroxidase, tyrosinase and aminopyrine N-demethylase activities in the cell homogenate as well as increase in the extracellular activity of lignin peroxidase suggests the role of these enzymes in the decolorization process. The phytotoxicity and microbial toxicity studies of extracted metabolites suggest the less toxic nature of them. Malachite green was detoxified into p-benzyl-N,N-dimethylaniline and N,N-dimethyl-aniline hydrochloride by Penicillium ochrochloron. Degradation metabolites were analyzed by TLC, HPLC and FTIR and identified by GCMS analysis. Phytotoxicity testing revealed the nontoxic nature of these metabolites. The percentage decolorization of malachite green (50 mgL-1) was 93 % in czapek dox broth after 14 h with an optimum pH of 7 at 30oC. The induction in the activity of lignin peroxidase after degradation suggested that the degradation of malachite green was peroxidase-mediated. Fungal culture was also found to have detoxified the textile effluent. The values of TDS, TSS, COD, and BOD were reduced in the treated samples compared to the control effluent. The treated effluent was non-toxic to the plants of Triticum aestivum and Ervum lens Linn, and the amount of total chlorophyll was higher in plants with treated effluent when compared to control effluent. This study showed that fungal mycelia could effectively be used as an alternative to the traditional physico-chemical process. (B) Major highlights of the project Penicillium ochrochloron was found to decolorize textile dye Malachite green, Cotton blue. This study suggests that this strain could be a useful tool for textile effluent treatment and the alternative to the traditional physicochemical process.

14.4 Research outcomes from Prof. S. R. Dave’s Lab

14.4.1 Bioremediation of dye containing wastes and their microbial diversity

(A) Objectives  Development of efficient bacterial consortia for the treatment of dyes and dye containing industrial wastes.  Metabolic and genetic diversity profiling of all developed bacterial consortia  Biodegradation of selected synthetic industrial dyes in micro-aerophilic and sequential microaerophilic-aerobic condition  Elucidation of degradation pathway  Bio-treatment of actual industrial wastewaters of local dye manufacturing industry  Optimization of process parameters by conventional and advanced statistical methods  Scale up of developed bioprocess  Application of process at industrial scale

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(B) Results, summary and major outcomes  Different eight bacterial consortia were developed from soil and water contaminated sources  Metabolic and genetic profiling of all developed consortia showed dominance of Bacillus sp.  Degradation pathway of Red 111, Violet 5R and Orange 3R were elucidated  Bio-treatment of industrial wastewaters showed more than 80 % COD and 85 % colour reduction of the waste along with removal of heavy metals and toxicity  Scale up of bioprocess was carried out from 2 L reactor to 10 L sequential reactor

(C) Major highlights of the work  Bioremediation of highly contaminated wastewater by bacterial consortia  Development of bio-treatment process  Scale up of bioprocess

14.4.2 Bacterial Degradation of Selected Metal Complex Acid Dyes and its Effluent

(A) Objectives

 To isolate, enrich and characterize potential consortia degrading metal complex acid dyes (MCAD)  To optimize MCAD degradation parameters for consortia  To adapt the consortia to extreme conditions in MCAD effluent  To develop process for the treatment of actual industrial effluent

(B) Results, summary and major outcomes

 Eleven indigenous consortia were developed  Five consortia and five dyes were selected after screening  Cultivable bacteria were isolated, identified and deposited to gene bank  Decolourization was optimized in terms of pH, temperature, culture condition, dye concentration, NaCl concentration and inoculum concentration  UV-Vis spectra were studied before and after decolourization

 Consortium development for MCADs degradation  Treatment of industrial effluents  Process development for effluent treatment

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14.5 Research Outcomes from the Prof. Datta Madamwar’s Lab

14.5.1 Textile dye decolorization using cyanobacteria (A) Results, summary and major outcomes Cyanobacterial cultures isolated from sites polluted by industrial textile effluents were screened for their ability to decolorize cyclic azo dyes. Gloeocapsa pleurocapsoides and Phormidium ceylanicum decolorized Acid Red 97 and FF Sky Blue dyes by more than 80 % after 26 days. Chroococcus minutus was the only culture which decolorized Amido Black 10B (55 %). Chlorophyll a synthesis in all cultures was strongly inhibited by the dyes. Visible spectroscopy and TLC confirmed that color removal was due to degradation of the dyes.

(B) Major highlights of the work The present study confirms the ability of cyanobacteria to decolorize and degrade structurally different dyes. Further studies are needed to identify the biochemical machinery involved in the degradation.

14.5.2 Microaerophilic Symmetric Reductive Cleavage of Reactive Azo Dye-Remazole Brilliant Violet 5R by Consortium VIE6: Community Synergism

(A) Results, summary and major outcomes The textile-dyeing industry is rated as one of the foremost industrial sectors that explodes large amount of pollutants to the environment. Reactive azo dye degradation, being a major constituent of these pollutants and perilous material, has been constantly receiving scientific attention. In textile industry, use of Remazole Brilliant Violet 5R (RBV5R) as reactive azo dyes is more frequent. Highly competent, RBV5R-degrading bacterial consortium VIE6 was developed from the soil of the Vatva Industrial Estate, Gujarat, India. Consortium VIE6 comprised of five bacterial strains Bacillus sp. DMB1, Staphylococcus sp. DMB2, Escherichia sp. DMB3, Enterococcus sp. DMB4, and Pseudomonas sp. DMB5. These strains convened a better decolorization efficiency between 200 and 1000 mgL-1 of dye concentration and were much stable at pH 6.5, 37 °C. Azoreductase, laccase, and lignin peroxidase activities of consortium showed significant variation throughout the degradation process indicating the different metabolic capabilities of the existing microflora. The community interactions and synergism were shown to facilitate the biotransformation of RBV5R by combination of various electron donors. Voltammograms revealed the variations in electron discharge properties which coincide with the dynamics of community derived using qPCR assays. The variation in catabolic capabilities of the individual strains was observed during active metabolism of RBV5R degradation pertaining to the aerobic and facultative functions

(B) Major highlights of the work The present bioremediation strategy demonstrates a potent consortium VIE6 that not only reductively cleave substituted sulfonated aromatic dyes but also simultaneously detoxify them along with consistent growth without much requirement of persistent nutrients. The results suggested that microaerophilic bacterial azoreduction was a respiratory mechanism and generated enough energy through an electron transport process to support consortial growth. This respiration process enhances the azoreduction process, which plays a significant and direct role in 133

Work of some Indian researchers azo dye metabolism. To decipher the specific mechanism of microaerophilic bacterial azo reduction, it requires further study; however, here we have demonstrated a synergistic mechanism for possible bioremediation strategy for sites contaminated with azo dyes.

14.5.3 Kinetic Modeling and Community Dynamics of Microaerophilic Treatment of Textile Dyes Containing Effluent by Consortium VIE6

(A) Results, summary and major outcomes A semi-synthetic designed medium (SDM), containing azo dyes, salts and other additives, was treated in a laboratory-scale upflow microaerophilic fixed-film bioreactor (UMFB) at various hydraulic retention times (HRT) in order to obtain efficient COD removal and decolorization using consortium VIE6. Grau’s second order and modified Stover-Kincannon substrate removal kinetic models were the best fitting models for the steady-state experimental data of UMFB. The efficacy of microaerophilic process to treat SDM at varying shock-loads (high dye and salt concentrations) was assessed simultaneously. The best organic matter removal efficiency, measured as Chemical Oxygen Demand (COD), was 98 % and decolorization was 99 % at 2 d HRT. UMFB endured shock-loads of dye and salts up to 500 mgL-1 and 80 gL-1, respectively, at 2 d HRT. Bio-film configured system operating in UMFB, having charcoal as supporting material; showed overall better efficiency in treating SDM, and even after reactor completion, the bio-film remained in immobilized form showed its stability to withstand the toxic shock loads. In addition, metabolites produced during microaerophilic processes were determined along with community dynamics of consortium VIE6. The role of each organism in a community was elucidated based on their dominance in the effluent at 2 d HRT. Moreover, phytotoxicity analysis of SDMand its bio-degraded products after treatment showed acute decline in the toxicity of metabolites as compared to SDM.

(B) Major highlights of the work The present bioremediation strategy demonstrated the application of the potent consortium VIE6 that not only reductively cleaved substituted sulfonated aromatic dyes but simultaneously detoxified them along with consistent growth without much requirement of persistent nutrients under UMFB process. Consortium VIE6 could withstand the large shock-loads of dye (500 mgL- 1) and salts (80 gL-1) during UMFB treatment. The increased capability of consortium to degrade dye is due to integration of two microenvironments in a single process. It is apparent from the study, that charcoal, being porous inert material, enhanced the consortial biofilm formation and propped it firmly to withstand various shocks-loads. The UMFB process here not only degraded the dye and organic matter but also a certain level of phosphate, sulfate, phenolics and nitrogenous compounds were removed during the process. However, community synergism of consortium VIE6 was commendable during treatment processes which completely detoxified the effluent.

14.5.4 Transformation of Textile Dyes by White-Rot Fungus Trametes versicolor

(A) Results, summary and major outcomes We have investigated transformation of eight industrial dyes by a white rot fungus, Trametes versicolor. The fungus was found to decolorize Reactive Golden Yellow R, Procion Red,

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Reactive Violet 5, Reactive Blue 28, and Ponceau Red 4R at an initial dye concentration of 80 ppm within 72 h of incubation, whereas it took 5 d to completely decolorize Reactive Black 5 (40 ppm). However, it did not significantly decolorize Reactive Red 152 and Novatic Blue BC S/D. During decolorization in liquid medium, laccase and manganese-independent peroxidase (MiP) activities were detected in culture filtrate of T. versicolor. Dye-decolorizing activity of the culture was found to be associated with H2O2-dependent activity of the culture filtrate. Furthermore, dye-decolorizing activity of the culture filtrate was not influenced by Mn2+ or veratryl alcohol, thus suggesting a role of extracellular MiP in decolorization of synthetic dyes by T. versicolor.

(B) Major highlights of the work Our results can be useful in understanding the fact that a relationship was not observed between MnP activity and the decolorization rate Pleurotus ostreatus produces MiP, which is involved in decolorization of Remazol Brilliant Blue R Heinfling et al. have demonstrated transformation of several industrial dyes by MiP isoenzymes purified from Bjerkandera adusta and Pleurotus eryngii. Our results further support the role of MiP enzyme obtained from T. versicolor in dye decolorization.

14.5.5 Decolorization screening of synthetic dyes by anaerobic methanogenic sludge using a batch decolorization assay

(A) Results, summary and major outcomes The nonspecific ability of anaerobic sludge bacteria obtained from cattle dung slurry was investigated for 17 different dyes in a batch assay system using sealed serum vials. Experiments using Reactive violet 5 (RV5) showed that sludge bacteria could effectively decolorize solutions having dye concentrations up to 1000 mgL-1 with a decolorization efficiency of above 75 % during 48 h of incubation. Headspace gas composition of anaerobic batch systems for varying dye concentration revealed that lower concentrations of RV5 (upto 500 mgL-1) were found to be stimulatory to the methanogenic activity of sludge bacteria. However at higher dye concentrations, the headspace gas composition was found to be similar to batch assay controls without dye, indicating that dye at higher concentrations was inhibitory to methanogenic bacteria of sludge. The optimum inoculum and incubation temperature for maximum decolorization of RV 5 was found to be 9.0 gL-1 (in terms of total solids) and 37 ºC, respectively. Of sixteen other dyes tested, nine (Reactive Black 5, Reactive Blue 31, Reactive Blue 28, Reactive Red HE8B, Reactive Yellow, Reactive Golden Yellow, Mordant Orange, Novatic Olive R S/D & Navilan Yellow GL) were decolorized with more than 88% efficiency; three (Orange II, Navy Blue HER & Novatic Blue BC S/D) were decolorized with about 50 – 65 % efficiency, whereas other three dyes (Procion Orange H2R, Procion Brilliant Blue HGR & Novatic Blue BC S/D) were decolorized with less than 40 % efficiency. Though Ranocid Fast Blue was decolorized with about 92.5 % efficiency, this was merely due to sorption, whereas the other dyes were decolorized due to biotransformation.

(B) Major highlights of the work It is noteworthy to mention here that the anaerobic sludge bacteria used in this study were not previously exposed to these kinds of xenobiotic compounds; however they were able to act on azo

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Work of some Indian researchers dyes. This observation that unadapted sludge bacteria can decolorize azo dyes is important because it opens up the prospect of developing anaerobic treatment systems which can easily decolorize azo dyes in industrial wastewaters and also possesses potential advantages over systems using defined bacterial cultures

14.5.6 Co-metabolic degradation of diazo dye-Reactive blue 160 by enriched mixed cultures BDN

(A) Results, summary and major outcomes Mixed cultures BDN (BDN) proficient in decolourizing diazo dye – reactive blue 160 (RB160) consist of eight bacterial strains, was developed through culture enrichment method from soil samples contaminated with anthropogenic activities. The synthrophic interactions of BDN have led to complete decolourization and degradation of RB160 (100 mgL-1) within 4 h along with co- metabolism of yeast extract (0.5 %) in minimal medium. BDN microaerophilicaly decolourized even 1500 mgL-1 of RB160 underhigh saline conditions (20 gL-1 NaCl) at 37ºC and pH 7.0. BDN exhibited broad substrate specificity and decolourized 27 structurally different dyes. The reductase enzymes symmetrically cleaved RB160 and oxidative enzymes further metabolised the degraded products and five different intermediates were identified using FTIR, 1HNMR and GC– MS. The phytotoxicity assay confirmed that intact RB160 was more toxic than dye degraded intermediates. The BDN was able to colonize and decolourized RB160 in soil model system in presence of indigenous miocroflora as well as in sterile soil without any amendment of additional nutrients, which signifies it useful and potential application in bioremediation.

(B) Major highlights of the work Bacterial mediated remediation of recalcitrant dyes of anthro-pogenic origin offers great opportunity for the restoration of dyecontaminated environments in an ecologically acceptable manner.As noted above bacteria in community rather than as individ-ual pure cultures are well efficient for complete mineralization ofany pollutants. Therefore, we have developed the potent bacterialmixed cultures BDN that not only cleaved substituted sulphonatedaromatic dye but simultaneously detoxify the degraded interme-diates at a faster rate under ambient conditions. The consistentgrowth of BDN at flask conditions and ability to colonize in soilsystem with minimal nutrient requirement and concurrent degra-dation of toxic dye compounds signifies its potential application forin situ bioremediation.

14.5.7 Decolorization and degradation of azo dye – Reactive Violet 5R by an acclimatized indigenous bacterial mixed cultures-SB4 isolated from anthropogenic dye contaminated soil

(A) Results, summary and major outcomes Azo dyes an important group of synthetic compounds are recalcitrant xenobiotics. Conventional technologies are unsuccessful to efficiently remove these compounds from contaminated environment. However, consorted metabolic functioning of innate microbial communities is a promising approach for bioremediation of polluted environment. Bacterial mixed cultures SB4 proficient in complete decolorization of azo dye – Reactive Violet 5R was developed through culture enrichment technique. Bacterial community composition based on 16S rRNA gene

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Work of some Indian researchers analysis revealed that mixed cultures SB4 composed of six bacterial strains namely Bacillus sp. V1DMK, Lysinibacillus sp. V3DMK, Bacillus sp. V5DMK, Bacillus sp. V7DMK, Ochrobacterium sp. V10DMK, Bacillus sp. V12DMK. SB4 grew well in minimal medium containing low amount of glucose and yeast extract (YE) (1 gL-1) and decolorized 200 mgL-1 of RV5 within 18 h under static condition. Mixed cultures SB4 decolorized wide range of azo dyes and maximum rate of decolorization was observed at 37 ºC and pH 7.0. Decolorization efficiency was found to be unaltered under high RV5 and salt concentration where 1500 mgL-1 of RV5 was decolorized in presence of 20 gL-1 NaCl. We propose the asymmetric cleavage of RV5 and Fourier transformed infrared (FTIR), NMR and gas chromatography–mass spectrometry (GC– MS) confirmed the formation of four intermediatory compounds 1-diazo-2-naphthol, 4- hydroxybenzenesulphonic acid, 2-naphthol and benzenesulphonic acid.

(B) Major highlights of the work In the present scenario of environmental pollution, its restoration is a mammoth challenge. The acquired knowledge of their hazards and the strict legislation has led for implementation of several strategies for cleaning up the environment. Inspite of having spent several decades we are still searching for an optimistic approach and in view of that present bioremediation strategy demonstrated the potency of enriched bacterial mixed cultures SB4 efficiently decolorizing and degrading high concentration of azo dye RV5 with minimal nutritional requirement. The ability to decolorize wide array of dyes under ambient conditions and complete mineralization of RV5 indicated the useful application of SB4 for ex situ bioremediation. The synergy of results obtained has forced us to study further and now our lab is working to isolate the genes responsible for dye degradation from these mixed cultures with an aim to design a strain capable to degrading several dyes.

14.5.8 Production of Ligninolytic Enzymes for Dye Decolorization by Cocultivation of White-Rot Fungi Pleurotus ostreatus and Phanerochaete chrysosporium Under Solid- State Fermentation

(A) Results, summary and major outcomes Lignocellulosic wastes such as neem hull, wheat bran, and sugarcane bagasse, available in abundance, are excellent substrates for the production of ligninolytic enzymes under solid-state fermentation by white-rot fungi. A ligninolytic enzyme system with high activity showing enhanced decomposition was obtained by cocultivation of Pleurotus ostreatus and Phanerochaete chrysosporium on combinations of lignocellulosic waste. Among the various substrate combinations examined, neem hull and wheat bran wastes gave the highest ligninolytic activity. A maximum production of laccase of 772 Ug-1 and manganese peroxidase of 982 Ug-1 was obtained on 20 d and lignin peroxidase of 656 Ug-1 on 25 d at 28 ± 1°C under solid-state fermentation. All three enzymes thus obtained were partially purified by acetone fractionation and were exploited for decolorizing different types of acid and reactive dyes.

(B) Major highlights of the work Our study demonstrates successful optimization of lignocellulosic waste decomposition and production of ligninolytic enzymes, which could be further exploited for decolorization of synthetic dyes.

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15.5.9 Decolourization of synthetic dyes by a newly isolated strain of Serratia marcescens (A) Results, summary and major outcomes A novel dye-decolourizing strain of the bacterium Serratia marcescens efficiently decolourized two chemically different dyes Ranocid Fast Blue (RFB) and Procion Brilliant Blue-H-GR (PBB- HGR) belonging respectively to the azo and anthraquinone groups. Extracellular laccase and manganese peroxidase (MnP) activity were detected during dye decolourization. The involvement of MnP activity was found in the decolourization of both dyes. More than 90 % decolourization of PBB-HGR and RFB was obtained on days 8 and 5, respectively at 26 ºC under static conditions at pH 7.0. MnP activity was increased by the addition of Mn2+. At 50 μM Mn2+, high MnP (55.3 Uml-1) but low laccase activity (8.3 Uml-1) was observed. Influence of oxalic acid on MnP activity was also observed.

(B) Major highlights of the work The present study confirms that the use of the newly isolated Serratia marcescens strain for decolourization of structurally different synthetic dyes is possible. The dye decolourization was very fast compared to white rot fungi. Nevertheless, the fungal-based treatment system is less efficient and time-consuming due to the slow growth rate of fungi as well as high adsorption to the biomass. The exploitation of S. marcescens would be help to overcome the difficulties associated with the use of mycelial organisms for dye decolourization and to develop an efficient technology for the removal of dyes from dyes-containing effluents using a bacterial species.

14.5.10 Decolorization of azo dyes using Basidiomycete strain PV 002

(A) Results, summary and major outcomes Basidiomycete PV 002, a recently isolated white-rot strain from decomposed neem waste displayed high extracellular peroxidase and rapidly decolorized azo dyes. In this study, the optimal culture conditions for efficient production of ligninolytic enzymes were determined with respect to carbon and nitrogen. An additional objective was to determine the efficiency of PV 002 to degrade the azo dyes. White-rot strain PV 002 efficiently decolorized Ranocid Fast Blue (96 %) and Acid Black 210 (70 %) on day 5 and 9 respectively under static conditions. The degradation of azo dyes under different conditions was strongly correlated with the ligninolytic activity. The optimum growth temperature of strain PV 002 was 26 ºC and pH 7.0.

(B) Major highlights of the work The Basidiomycete strain PV 002 efficiently transformed azo dyes. The dye decolorization was very fast compared with well-known selected white-rot fungi. Since nutrient limitation is not required for ligninolytic activity in Basidiomycete strain PV 002, further studies should attempt to increase degradation of the other recalcitrant compounds (e.g. PAH) by cheap organic rich supplements, which are known to stimulate peroxidase production. Such a biological process could be adopted as a cost-effective, safer and efficient approach for decolorization of effluents. Further work needs to be performed using this fungal system with the objective of reducing cost of the process significantly.

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14.5.11 Decolourization of textile dye Reactive Violet 5 by a newly isolated bacterial consortium RVM 11.1

(A) Results, summary and major outcomes Soil samples collected from contaminated sites of Vatva, Gujarat, India were studied for screening and isolation of organisms capable of decolourizing textile dyes. A bacterial consortium RVM11.1 was selected on the basis of rapid dye decolourization. Reactive Violet 5 (RV 5) was used as model dye. The consortium exhibited 94 % decolourization ability within 37 h under a wide pH range from 6.5 to 8.5 and temperature ranging from 25 to 40 ºC. The bacterial consortium was able to grow and decolourize RV5 under static conditions in the presence of glucose and yeast extract and also showed an ability to decolourize in the presence of starch in place of glucose. Maximum decolourization efficiency was observed at 200 ppm (mgL-1) concentration of RV 5. Bacterial consortium RVM11.1 had the ability to decolourize 10 different dyes tested. The transformation and degradation products after decolourization were examined by HPTLC.

(B) Major highlights of the work Hence the mixed culture RVM11.1 exhibited good decolourization ability in the range of pH from 6.5 to 8.5 and temperature from 25 to 40 ºC which are normal operational parameters for conventional wastewater treatment systems. Our culture offers obvious advantages, since oxygen can be easily depleted under stationary conditions, thus creating conditions favourable for decolourization of azo dyes. The requirement of supplementary co-substrate by our bacterial consortium is quite low as compared to other reports and suggests its suitability for treatment of dye-bearing wastewaters. RVM 11.1 could decolourize dyes much above the reported dye concentration in wastewaters, and thus could be successfully employed for treatment of dye- bearing industrial wastewaters. Moreover it exhibited efficient decolourizing ability for 10 of the dyes tested. The ability of a culture to utilize cheap co-substrate such as starch for dye decolourization gives it an advantage for treatment of textile industry wastewaters. However the potential of the culture needs to be demonstrated for its application in treatment of real dye- bearing wastewaters using appropriate bioreactors.

14.5.12 Isolation, characterization and decolorization of textile dyes by a mixed bacterial consortium JW-2

(A) Results, summary and major outcomes Soil samples collected from dye contaminated sites of Jetpur, Gujarat were exploited for isolation of dye decolorizing organism. A microbial consortium JW-2 was selected based on its efficiency, showing maximum and faster decolorization of textile dyes. The consortium consisted of three isolates. Identification of isolates by 16S rRNA technique revealed that the organisms were Paenibacillus polymyxa, Micrococcus luteus and Micrococcus sp. The concerted metabolic activity of these isolates led to complete decolorization of Reactive Violet 5R (100 ppm) within 36 h whereas individual isolates could not show decolorization even on extended incubation. The culture exhibited good decolorization ability in the pH range from 6.5 to 8.5 and temperature from 25 to 37 ºC. The consortium showed complete decolorization utilizing low amount of

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Work of some Indian researchers cosubstrates like glucose (0.1 % w/v) and yeast extract (0.05 % w/v) and could also utilize a cheaper carbon source like starch in place of glucose as an alternative co-substrate. The consortium had the ability to decolorize nine different dyes amongst 10 tested. Potential of this consortium JW-2 to decolorize textile effluent containing a mixture of textile dyes is to be carried out using appropriate bioreactors.

(B) Major highlights of the work The consortium containing bacterial cultures P. polymyxa, M. luteus and Micrococcus sp. exhibited good decolorization ability in a mixed form. These bacterial species have not been reported so far to our knowledge for dye decolorization. The culture exhibited good decolorization ability at pH from 6.5 to 8.5 and temperature from 25 to 37 ºC, which are normal operation parameters for conventional wastewater treatment systems. The bacterial consortium was able to grow and decolorize dyes under static conditions. Agitated cultures, though exhibited good growth, showed poor decolorization. The mixed culture seems to have potential application in treatment of dye bearing wastewaters as it exhibited efficient decolorizing ability for nine out of 10 dyes tested. The ability of culture to utilize cheap co-substrate such as starch for dye decolorization gives it an advantage for treatment of textile industry wastewaters. However, potential of culture needs to be demonstrated for its application in treatment of real dye bearing wastewaters using appropriate bioreactors.

14.5.13 Response surface methodology for optimization of medium for decolorization of textile dye Direct Black 22 by a novel bacterial consortium

(A) Results, summary and major outcomes Decolorization and degradation of polyazo dye Direct Black 22 was carried out by distillery spent wash degrading mixed bacterial consortium, DMC. Response surface methodology (RSM) involving a central composite design (CCD) in four factors was successfully employed for the study and optimization of decolorization process. The hyper activities and interactions between glucose concentration, yeast extract concentration, dye concentration and inoculum size on dye decolorization were investigated and modeled. Under optimized conditions the bacterial consortium was able to decolorize the dye almost completely (>91 %) within 12 h. Bacterial consortium was able to decolorize 10 different azo dyes. The optimum combination of the four variables predicted through RSM was confirmed through confirmatory experiments and hence this bacterial consortium holds potential for the treatment of industrial waste water. Dye degradation products obtained during the course of decolorization were analyzed by HPTLC.

(B) Major highlights of the work Application of the mixed bacterial consortium DMC, comprising of P. aeroginosa PAO1, S. maltophila and P. mirabilis to decolorization of textile dye Direct Black 22 seems to be a pragmatic approach. This study shows that response surface methodology was an appropriate method to optimize the best culture conditions for obtaining maximum decolorization of the dye. The experimental and the predicted values were very close which reflected the accuracy and the applicability of RSM. By applying central composite design and RSM to the optimization experiments, we could investigate the process variables completely and achieve decolorization values up to 91 %. The culture not only decolorized the dye but it also degraded the dye which is

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14.5.14 Decolorization of Ranocid Fast Blue Dye by bacterial consortium SV5

(A) Results, summary and major outcomes Synthetic dyes are not uniformly susceptible to degradation in conventional wastewater treatment processes. A number of biotechnological processes have been suggested as of potential interest in combating these pollutants in an ecofriendly manner. We determined the optimal parameters necessary for the bacterial consortium SV5 to decolorize Ranocid Fast Blue dye. The best results were obtained with a 0.1 % (w/v) concentration of both starch and yeast extract supplemented in Bushnell Hass Medium under static conditions at a temperature of 37°C in less than 24 h with an initial dye concentration of 100 ppm.

(B) Major highlights of the work Our study demonstrates that the bacterial consortium SV5 has a very efficient azo dye- decolorizing capability. Studies of the identification and characterization of the bacterial consortium SV5 and the mode of action of decolorization of RFB by these microorganisms are under way.

14.5.15 Community genomics: Isolation, characterization and expression of gene coding for azoreductase

(A) Results, summary and major outcomes Soil collected from banks of the Khari-cut canal (Vatva, Ahmedabad, Gujarat, India), contaminated by industrial wastes, was used as inoculum for developing consortium enriched with dye degrading and/or tolerating bacteria. The consortium V9 was able to decolourize 100 ppm of Reactive Violet 5 within 24 h at 37ºC. The amplified azoreductase gene had a length of 537 bp containing an ORF of 178 amino acids. BLASTn and BLASTx analyses showed 97 % and 98 % identity to sequences of azoreductase gene of Rhodobacter sphaeroides and Bacillus cereus G9241, respectively. The azoreductase gene was cloned in expression vectors and its in vitro activity was optimized. Co-factor NADPH.Na4 (1 mM) was essential to obtain 85-90 % dye degradation by cloned azoreductase gene. Escherichia coli BL21(DE3) clone PET1 was able to degrade 90 % of dye within 7 min. Conversely, only 20-30 % of total dye was degraded with cell extracts of control strains after 3 h.

(B) Major highlights of the work The present study highlights the importance of consortium in bioremediation processes and will be of immense help in developing novel recombinant strains for application in dye contaminated waste-water treatments. Moreover, the present work also demonstrates that efficacious bioremediation strategies can be designed based on innate microbial community dynamics, structure and function. Microbial communities are fundamental components of ecosystems capable of playing critical role in the metabolism and detoxification of anthropogenic/xenobiotic compounds.

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14.5.16 Decolorization of Synthetic Textile Dyes by Lignin Peroxidase of Phanerochaete chrysosporium

(A) Results, summary and major outcomes Neem hull waste (containing a high amount of lignin and other phenolic compounds) was used for lignin peroxidase production by Phanerochaete chrysosporum under solid-state fermentation conditions. Maximum decolorization achieved by partially purified lignin peroxidase was 80 % for Porocion Brilliant Blue HGR, 83 for Ranocid Fast Blue, 70 for Acid Red 119 and 61 for Navidol Fast Black MSRL. The effects of different concentrations of veratryl alcohol, hydrogen peroxide, enzyme and dye on the efficiency of decolorization have been investigated. Maximum decolorization efficiency was observed at 0.2 and 0.4 mmolL-1 hydrogen peroxide, 2.5 mmolL-1 veratryl alcohol and pH 5.0 after a l h reaction, using 50 ppm of dyes and 9.96 mkat/L of enzyme.

14.5.17 Microaerophilic degradation of sulphonated azo dye - Reactive Red 195 by bacterial consortium AR1 through co-metabolism

(A) Results, summary and major outcomes The textile and dye industries are considered as one of the foremost sectors that pollutes environment. Technologies employing biological methods showed promising approach to remediate sites polluted with dye and dye intermediates. Bacterial consortium AR1 developed through culture enrichment method was comprised of four distinct bacterial strains. The synergistic metabolic activities of AR1 led to complete decolourization of monoazo dye Reactive Red 195 within 14 h under microaerophilic environment. The co-metabolic decolourization of Reactive Red 195 by consortium, in presence of maltose and proteose peptone (0.1 %, w/v, each) in minimal medium, easily reduced Reactive Red 195 (100 mgL-1) for five consecutive cycles without any replenishment of nutrient. The maximum decolourization was observed at pH 8.0 and 40 ºC. The consortium was acclimatized to decolourize Reactive Red 195 at higher concentration (2000 mg/L) even under high salt concentration (1 M NaCl). Consortium exhibited broad substrate specificity where it decolourized 15 structurally different dyes and more than 50% decolourization was observed in a medium containing mixture of dyes. The degradation products analyzed using FTIR, HPTLC and 1H NMR revealed the formation of 2-amino-naphthalene, 1-amino-benzene and 1-nitrobenzene. Thus the ability of bacterial consortium for simultaneous decolourization and degradation of azo compounds signifies its potential application in dye remediation.

(B) Major highlights of the work The omnipresence and great adaptability of bacteria offers a simple and inexpensive green technology for remediating the environment contaminated with xenobiotic compounds of anthropogenic origin. Plenty of existing technologies are available for cleaning up the polluted environment; still promising and alternative strategies are incessantly being developed. Thus, looking at the scenario, we have developed a potent consortium AR1 that reductively cleaved azo dye with simultaneous degradation of corresponding aromatic amines under ambient conditions without requirement of persistent nutrients. Consortium exhibited broad substrate specificity and capable of decolourizing azo dyes at higher concentrations. However, the detailed study is required to know the enzymatic and biochemical pathways involved during metabolism of azo

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14.5.18 Synergistic action of flavin containing NADH dependant azoreductase and cytochrome P450 monooxygenase in azoaromatic mineralization

(A) Results, summary and major outcomes An alkaliphilic strain Bacillus lentus BI377 was isolated from contaminated soil of the textile area of Solapur, India. The strain was able to degrade almost 98% of recalcitrant azoic compounds by a mutually regulated process of azoreductase and a monooxygenase system. An enzyme activity study and a periodical carbon monoxide (CO) binding spectra study on a UV-visible spectrophotometer revealed that the intermediate amines formed by typical azoreduction (NLN cleavage), subsequently underwent hydroxylation by the cytochrome P450 monooxygenase (CYP450) system. Azoreductase was purified by chromatographic techniques and characterization by MALDI-TOF substantiated its identity as FMN containing NADH dependent azoreductase of 32 kDa in size. Surprisingly, purified azoreductase showed the highest activity at 80 uC and pH 8.0. An increase in the activity of superoxide dismutase after decolorization confirmed the signature of oxidative stress and its involvement in the dismutation of reactive metabolites. Intermediate metabolite analysis by HPLC, GC-MS and FTIR and the removal of total organic carbon (TOC) suggested the azoaromatics’ degradation leads to mineralization via a TCA cycle.

(B) Major highlights of the work The present study emphasizes the major utility of isolate B. lentus BI377 for the bioremediation of hazardous recalcitrant azoaromatic compounds. The astonishing catabolic ability of the strain certainly provides unparalleled opportunities for understanding the fundamental molecular mechanism of the degradation of organic compounds and their utilization as an energy source. The purified and characterized flavin containing alkaliphilic azoreductase provides rich opportunities to investigate the nonspecific diverse substrate specificity and mechanism of promiscuity among the family of flavoprotiens. This study opens up a dependable and efficient way to use B. lentus to accelerate the exploration of the feasibility of the bioremediation reactions over a wide range of temperature and pH. The symbiotic action of the enzyme azoreductase, cytochrome P450 monooxygenase and superoxide dismutase may provide a selective advantage to the degradation reaction under various conditions of environmental stress. Therefore, this bacterial strain provides the potential enzymatic machinery to remove a wide variety of natural and man-made aromatic compounds discharged through geochemical cycles and urban and industrial activities as well as their subsequent transformation into CO2 and H2O.

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14.6 Research outcomes from Dr. Hemant Purohit and Dr. Atya Kapley’s Lab

14.6.1 Selection of indicator bacteria based on screening of 16S rDNA metagenomic library from a two-stage anoxic–oxic bioreactor system degrading azo dyes

(A) Results, summary and major outcomes Dye degradation has gained attention of late due to indiscriminate disposal from user industries. Enhancing efficiency of biological treatment provides a cheaper alternative vis-à-vis other advanced technologies. Dye molecules are metabolized biologically via anoxic and oxic treatments. In this study, bacterial community surviving on dye effluent working in anoxic–oxic bioreactor was analyzed using 16S rDNA approach. Azo-dye decolorizing and degrading bacterial community was enriched in lab-scale two-stage anoxic–oxic bioreactor. 16S rDNA metagenomic libraries of enriched population were constructed, screened and phylogenetically analyzed separately. Removal of ~35 % COD with complete decolorization was observed in anoxic bioreactor. Process was carried out by uncultured gamma proteobacterium constituting 48 % of the total population and 12 % clones having homology to Klebsiella. Aromatic amines generated during partial treatment under anoxic bioreactor were treated by aerobic population having 72 % unculturable unidentified bacterium and rest of the population consisting of Thauera sp., Pseudoxanthomonas sp., Desulfomicrobium sp., Ottowia sp., Acidovorax sp., and Bacteriodetes sp.

(B) Major highlights of the work The study proposes the two-stage treatment of dye wastewater using anoxic and oxic bacterial consortia as biological key. Azo dyes were decolorized anoxically to produce aromatic amines which were toxic to E. coli; and this effluent was further detoxified in oxic bioreactor. Moreover, the study brings out the importance of knowledge of dye degrading microbial population. The 16S rDNA data analysis indicated that Clostridium sp., Klebsiella sp. and Bacteroides bacterium as typical indicator for anoxic reactor whereas the Ottowia sp., Pseudoxanthomonas sp. and Acidovorax sp. indicator for oxic reactor in the case of dye degradation. Further studies are required to identify the role of defined members of microbial population.

14.7 Research outcomes from Dr. Prince Sharma’s Lab 14.7.1 Genotoxicity of Degradation Products of Textile Dyes Evaluated with rec-Assay After PhotoFenton and Ligninase Treatment

Fourteen textile dyes (from azo, anthraquinone, heterocyclic, oxazine, methine/polymethine and triphenylmethane groups) were decolourized and degraded using Photo-fenton treatment (PFT) and Phanerochaete chrysosporium crude ligninase enzyme (ED) treatment approach. The genotoxicity of the dyes and their degradation products were assayed using rec-assay method. We found that the genotoxicity was depended on the dye and on the method of degradation. In general, PFT was better than ED in decreasing the genotoxicity. Basic dyes showed complete or maximum loss of genotoxicity, whereas the vat group was more resistant. The azo group showed varied results. Crystal Violet was the only dye whose genotoxicity increased after PFT. Our results suggest that PFT and ED are two effective treatment methods to reduce the genotoxicity of dyes in waste waters.

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14.7.2 Biodegradation of textile azo-dyes by Phanerochaete chrysosporium Eighteen commercially obtained textile dyes was used in the study. Amongst the 18 dyes, eight dyes were degraded in the range of 40 to 73 % by the white rot fungus, Phanerochaete chrysosporium. The study suggested that dye removal of diazo dye Reactofix Gold Yellow by Phanerochaete chrysosporium was achieved by lignin-degrading enzyme system Degradation was best achieved by adding the dye to the medium and then inoculating with pre-grown mycelium. Inoculation with fungal spores resulted was mainly resulted in dye adsorption.

14.7.3 Use of Phanerochaete chrysosporium biomass for the removal of textile dyes from a synthetic effluent

The use of Phanerochaete chrysosporium biomass for the removal of Reactofix Golden Yellow from aqueous solution and eight textile dyes (four azo and four anthraquinone) from a synthetic effluent (0.6 gL-1) at different pH, temperature and biomass concentrations was studied. Adsorption was maximum at pH 2.0 and 40ºC using 2.45 g mycelial biomass. The rate constant of adsorption was 1.95 x 10-1min-1 for Reactofix Golden Yellow and 1.64 x 10-1min-1 for synthetic effluent. In both cases, the equilibrium data fitted well in the Langmuir but not the Freundlich model of adsorption, and the adsorption was biphasic. Adsorption decreased the COD of Reactofix Golden Yellow and synthetic effluent by 54 and 57 %, respectively. Desorption (80–84 %) of dyes from P. chrysosporium mycelial surface occurred as the pH increased from 2 to 10.

14.8 Research outcomes from Dr. Harvinder Singh Saini’s Lab Dr. Saini’s work mainly involves evaluating the metabolic potential of the cultures in immobilized cell bioreactors to transform the dye to non-toxic central metabolic intermediates, which could be further mineralized. For this different configurations of the bioreactors viz, upflow immobilized cell reactors (UICR ranging from 10 ml to 100 ml working volumes), plug flow reactor (PFR, upto 100 L working volume) and continuously stirred aerobic reactor (CSAR, upto 10 L working volume) were desigened and used. The small volume UICR had been used to carry out extensive studies with different effluents, both simulated and actual effluents, to optimize the operating parameters so as to achieve desired levels of clean up. The efficiency of treatment was validated by analytical techniques and by genotoxic studies using mouse model. Their future prospect is to apply the efficient cultures either in the equalization tanks of the industries or to treat spent dye bath liquor, after appropriate dilution, to achieve desired levels of clean up. The spent dye bath liquor needs dilution as in some dyeing process the organic load (in terms of COD) is higher, resulting in toxic shock to the microbial populations.

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15.1 Opening Remarks 146 15.2 Primary Observations 146 15.3 Recommendation to Department of Biotechnology, New Delhi 148 15.3.1 Major Opinions 148 15.3.2 Minor Opinions 150 15.4 Additional suggestions and recommendations 150 15.4.1 Recommendations and suggestions to other funding agencies 150 15.4.2 Recommendations and suggestions to regulatory bodies 151 15.4.3 Recommendations and suggestions to textile industries 152 15.4.4 Recommendations to researchers with list of thrust areas of R&D aspects 152 which needs to be taken up

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Suggestions and recommendations 15.1 Opening Remarks Till the mid-late part of last centaury (1960-70s) water has not been considered as non- renewable resources and economic cost of water was not taken into consideration when cost of industrial activities was counted. Its essence as a natural resource required for saving the basic entity of life was distinctly realized in the last decade of 20th centaury. The hard earn lesson have taught us, to minimize water consumption, minimize the contamination load while returning it back to environment, because it has limited self-purification ability. Dye, dye intermediates, pigment manufacturing and textile industries form the backbone of the industrial and agricultural development in the country. They provide building blocks for downstream industries. The inevitable part of any industrial process is generation of wastewater and in the mist of development we have neglected the impact on environment, which have directly and indirectly affected the basic essence of life. Soon we have realized the negative consequence of industrial development and have started the remediation process. An ample amount of research globally as well as in sub-continent has been directed towards development of efficient (bio)remediation technology. However, spending billions of rupees and efforts of several decades, the hunt for integrative and universal technology is still on and vast gap exists between lab research and its successful on-filed application. Therefore, there is an urgent need for an integrated perspective for effective bioremediation technology i.e. integrating the knowledge of geobiologist, biochemical engineer, microbiologist, biotechnologist and statistician for predicting and analyzing the technology outcome. Thus, to assess the status of the research carried out on bioremediation (and biodegradation of dyes and dye intermediates) in the country the compendium was buildup.

Before we put any opinion or any recommendations, some peculiar observations needed to be looked upon, to reach any decisive conclusion.

15.2 Primary Observations Currently in the country, with reference to available scientific literature, on the biological front, researchers are effectively working on bio-decolurization (of dye and other intermediates) and bio-degradation using microbial sources and plant system in association with rhizospheric bacteria. Moreover, upon analyzing the projects supported by Department of Biotechnology, Ministry of Science and Technology, New Delhi, and other national funding agencies (and which were recorded as ‘Successfully Completed’) certain peculiar features in Objectives of the Projects are highlighted below. • Many of the projects were inclined towards isolation and screening of novel microorganisms capable to degrading various dyes and raw textile effluents: so as to account for a first time reported strains. • Apprehensive need for standardization of nutritional requirement and other media conditions: because dye compounds are poor source of carbon and energy. • Obligatory optimization of environmental factors (under laboratory conditions): so as to mimic the real environmental situation and to access the competence of the isolated strains.

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• Orderly description of degradation profile and identification of degraded intermediates of test dye compound using different analytical methods: so as to demonstrate the degradative potential of the isolated strains. • Supportive studies on toxicity, majority of them are through seed germination of agriculturally important crops: so as to demonstrate the superlative ability of the isolated stains. • Need for studying enzymes involved in decolourization and degradation of dye compounds: so as to prove the biological role of decolourization and degradation. • Immobilization of microbial strains or enzymes in different matrix: so as to prevent direct damage to the enzymes and to reuse the entrapped microbes/enzymes to reduce the financial burden of treatment. • Validation of flask level results at lab scale (bio)reactors and direct study on bioreactors either with simulated effluents or real textile effluents • In a very few instances investigators wanted to demonstrate or to apply the lab driven technology at large scale for real on-site field applications

Microbial sources primarily and most widely used are Bacteria followed by Fungus and Algae and very few types of Yeast. They are used either as pure cultures or in mixed form in various mutual associations of different microbes. It was observed that in certain studies immobilizing the whole microbial cells through entrapment or attachment was performed. The entrapment would provide fibrous or porous materials for easy transfusion of dye compound into the matrix. The immobilization step proposed to provide more resilient to the cells from environmental perturbation such as pH, other toxic chemicals, etc., in comparison to suspended cells in the medium. The work is also actively being conducted on dye decolourization and degradation through microbial enzymes either directly from microbes or using various preparation of commercial source. Like immobilization of microbial cells, enzymes are also immobilized to prolong their activity and half-life. Majority of the studies were performed under laboratory conditions and at small scale (very few were successfully demonstrated at pilot scale with single industrial effluent). By citing the reason of environmental conditions, bulk of resources and good amount of time are spend on optimizing the very known abiotic and biotic factors, which generally have less significance in open environment under natural conditions. Removal of colour from waste water should not be sole aim for such treatment process. Recently the study has been diverted towards complete mineralization of dye molecules besides removing the colour portion of the effluents.

. There were more than 500 Ph.Ds were produced in last three decades, majority of these studies were focused on isolation and characterization of microbial strains, optimization of abiotic and biotic parameters, studying the probable degradation pathway of single model dye compound using analytical techniques like UV-vis spectroscopy, HPLC, HPTLC, GC-MS/LC- MS, NMR, etc., few toxicity studies at Flask scale under laboratory conditions using either pure strains of mixed cultures. . Since the work in the publications is reflection of Ph.D thesis of the researcher, by studying the publications in form of research articles of Indian authors, mostly it was found to be a routine work with isolation characterization and few optimization studies along with pathway predictions and primitive toxicity studies are reported. . Few studies have been directed towards studying simulated or real textile/dye industrial effluents at reactor levels. . More applicable research (or the technologies developed) on remediation of dye and textile effluent is being carried out at ETPs and CETPs rather than at University laboratories.

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. Actual filed scale applications are very rare from the studies originated at University level. . Recently in last five years few research works, directly emphasising on demonstrating the effective methodology for large scale treatment are coming up. . It is a matter of fact and we need to become more realistic on accepting our limitations for incapable of producing more viable and universally accepted technology. The application and concept of bioremediation are wobbling since last three decades in our country. But, with the complexity of the effluents and our own restrains (and the point of views are more academic rather that on technology development) we are still searching blindly for a possible solution. . There are different standards set for regulation of release of industrial waste / by-products directly into open environment depending upon the nature of xenobiotic compounds and their effect on biosphere. For instance, for dye containing waste water effluents discharge standards are being set by taking into consideration of the fact that human eye can detect colour at the level of 1 ppm also. . There are isolated case studies showing effective technological excellence for remediation of waste effluent, but post success it was either not adopted to national level or the developed methodologies are specific for the local region. . For the treatment of textile effluent, very rare studies (viz. Prof. S. P. Govindwar’s lab, under University set up) study where recently they are focusing on phytoremediation at field level, majority of the studies in the country is very preliminary showing most efficient results under laboratory conditions. Because many of them have not experimented or have not excelled at larger scale or at filed trails, the studies had become more of mere publications oriented or to scale academic heights. . The limitations of all the available technologies and the failures to translate the lab scale results at field levels may be attributed to one or more of following: o certain technologies may not be able to function (as antiicapted) due to unpredictable and non-anticipated complexities at field level o due to chronic pollution, some of essential components of ecosystem and ecological process might have already been damaged irreversibly o bioaugmentation and acclimatization may fail as added organism/s might not be able to compete and survive with indigenous flora o the level of pollution and the complexities of the effluents is too high to be treatment with available technology

15.3 Recommendation to Department of Biotechnology, Ministry of Science and Technology, New Delhi

15.3.1 Major Opinions 1. Foremost, we need to understand and recognize that textile effluent is highly heterogeneous because the composition of the effluent is usually a subject to daily, seasonal and/or market demand depending on the current fashion trends of the country. And which makes it extremely difficult for any technology of either physical or chemical or physico-chemical or biological origin or integrated of all three types or any hybrid technology to provide absolute treatment efficiency. Therefore, textile effluent based on its characteristic composition must be segregated into similar/dissimilar origin and the emphasis should be on developing the individual technology for particular group of effluents. 2. The current practices at few CETPs across the country of combining of treated textile/dye containing effluents along with treated sewage water or high scale dilution of treated effluent may not be sufficient. With the scale of industrial effluents being treated and released daily, there could be two possible locations for treatment of dye containing effluents. 149

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(i) at the manufacturing or dye application industry itself: treated water may be partially or completely reused for indigenous consumptions (ii) at sewage treatment plant: after primary/secondary treatment, where indigenous ongoing biological/chemical treatment method should be applied as a final polishing step 3. Today in the country more than 500 ETPs, CETPs and STPs are in operating conditions. Extensive and substantial knowledge has been generated over past two decades on the problems relating to efficiency and effectiveness of these treatment plants, for treatment of waste water and its reuse. The technical and scientific information should be used for enhancing the functional efficiency of presently operating ETPs, CETPs and STPs through modifications, additions or deletions of treatment steps. Alternatively, any novel, simple and cost effective technologies that are adaptive to local conditions should be evolved at higher level. 4. The initial success of phytoremediation (which was evident from the couple of projects being supported by Department of Biotechnology) may be extrapolated by linking CETPs and STPs with constructed wetlands. The constructed wetlands should be developed in a way that they are locally adaptive. 5. It is well known that more than 70 % of the textile and dye manufacturing industries in the country are segmented into un-organized sectors comprising of Small and Medium Scale Enterprises (SMEs). To impose the individual wastewater treatment plant in these industries, it would add a financial burden on the units. However, such industries are one of the major sources of the pollution. In certain industrial estates the SEMs do have the waste water treatment facilities, but they are rudimentary. Therefore, government should provide financial assistance to such units for setting up of primary and/or secondary treatment plants or need to devise an incentive based policies to promote the treatment facilities in such industries. 6. Today we have adequate information and basic understanding about the microbial behavioural pattern and their requirements in terms of carbon sources and environmental parameters, at flask level and lab scale reactor level (including few local successes studies). Department of Biotechnology (as well as other national funding agencies) may immediately cease any future support to the research projects which are of routine nature (as described in Chapter 07, Project Type I). DBT may focus on the research projects which demonstrate the direct application and ready to take challenge to provide treatability solution at industry level for multiple remediation cycle. The project should involve scientific expertise from academic institute, technical expertise either from CETPs or similar organization and an industrial partner where actual application has to be performed. 7. Because as noted in Chapter 03, there are several stages for the processing of textile and at the end of each steps, waste water are generated. The compositions of these waste waters considerably differ at each step which required different treatment approaches. It would be more easy to develop technology which would be more target oriented. Therefore, possibilities should be explored to establish the treatment system for different stages of wastewaters (especially for steps which are generating wastewater with more contaminants) in the individual textile units. 8. Moreover, the research projects which have shown the potential success for treating the effluents at large scale (e.g. Project No. 3, Chapter No. 07) are either not adopted at large 150

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scale or in other region of the country. Department of Biotechnology should to take enough care that such kind of projects is further extrapolated at national level. 9. One of the problems of using microbial technologies in treatment of waste water for reuse is how to immobilize microbial consortium/mixed cultures used in the treatment of industrial effluents and sewage? For long-term retention of microbes in the system, it is necessary to evolve a technology that enables microbes to remain in the rhizoplane / rhizosphere of plants used in the constructed wetlands. 10. For construction of wetlands or any phyto-remediation technology requires land. Government intervention is therefore required for implementation of large scale treatment scheme. 11. Another prominent observations made was the absolute absence commercial Patents in dye and textile effluents treatment technologies in the country. The lack of quality Patents clearly describes the current state of research in this area.

15.3.2 Minor Opinions 1. Looking at the state of research at laboratory level and limited success of various CETPs across the country it is inevitable to develop an alternative industrial scale set-up excessively for dye manufacturing and/or for textile industries to achieve the set standards of discharge limits of dye containing effluents. 2. As a premier funding agency, the Department of Biotechnology, New Delhi may recommend the Central Pollution Control Board and State Pollution Control Boards that polluters must take responsibilities for cleaning of atleast streams and small tributaries and should discharge clean water by treating effluents at the source as per Polluter pay Principle. For a certain Medium Scale Enterprises and Large Scale Textile and Dye manufacturing industries as their responsibilities, they should support R&D for development and implementation of advanced cost effective technologies for wastewater treatment. 3. After having substantial knowledge and adequate experimental trials at lab scale, pilot level, ETPs and CETP plants and at individual units, Department of Biotechnology should integrate the expertise available in the country from all sphere of science and technology with a common aim to be able to demonstrate the remediation of any one selective polluted tributary / stream / rivulet / river in a defined time frame. 4. Department of Biotechnology, New Delhi may promote entrepreneurship among young scientists/postgraduates or scheme on Start-up Grants may be provided for the development and implementation of next generation integrative bioremediation technologies for treatment of textile industrial effluents and its possible reuse in industry

15.4 Additional suggestions and recommendations Besides to above suggestions and recommendations to DBT, few additional suggestions were congregated to other national funding agencies, regulatory bodies, textile industries.

15.4.1 Recommendations and suggestions to other funding agencies . Today we have adequate information and basic understanding about the microbial behavioural pattern and their requirements in terms of carbon sources and environmental parameters, at flask level and lab scale reactor level (including few local successes studies). All Scientific and Technical Research Funding Agencies (including state funding agencies) may

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immediately cease any future support to the research projects which are of routine nature (as described in Chapter 07, Project Type I).

. It has been observed that many of the research work from different funding agencies were of repetitive in nature and non-overlapping. There should be continutity in the research and overlapping with the aim and objectives. . Moreover, the scientific and technical studies for developing a common technology for treatment of dye manufacturing and textile effluents (in our country) have been highly fragmented (and usually of shorter duration). Many of them are oriented towards achieving academic milestones rather than providing effective solution to the problem. . It has to be more targeted and if needed the research projects should be extended to longer durations (7-15 years). There should be a ‘mission’ and a coordinated, synchronized timely and highly focused work needed to be funded in a more organized manner. . They may focus on the research projects which demonstrate the direct application and ready to take challenge to provide treatability solution at industry level for multiple remediation cycle. . The individual centric research projects have provided very limited solution. Focus should be involving (a) industrial partner, mainly from Small scale units and a research groups with established scientific and technical experience in the area; (b) CETPs and a research groups

15.4.2 Recommendations and suggestions to regulatory bodies

(A) State Regulatory Bodies (SRBs) . It is of no doubt that all SRBs are performing their duties in right directions. But they need to follow their mandate religiously. . Foremost they need to persue the state government to activate the closed-down STPs and any ETPs (if any). . SRBs strictly needs to prevent ‘Diffusion Pollution’ particularly for groundwater . SRBs must focus and devise a ‘Comprehensive Rain Water Conservation Policy’ for all textile units (need to implement both for new and existing units) . SRBs on regular basis need to sensitize the owners about the environmental concerns and required to be need encouraged for implementation of any type of treatment technology at the industry itself . The textile industries in particular are clustered together in specified zones (as Small/Medium Scale Industries), across all the states in our country. SRBs need to create database (has to make it mandatory) to collect the right information about the raw materials and chemicals used from each units from time to time. This will immensely help to develop the treatment/technologies. . SRBs may encourage the each unit to switch towards the ‘cleaner productions’, water less production of their products . SRBs must take initiative and request the industrial units to participate, cooperate and must coordinate the scientific and technical work leading to establishment of treatment technology suited best for their effluent.

(B) Central Regulatory Bodies (CRBs) . There exists a frame works of rules, regulations and legislation in the country for the release and disposal of industrial liquid waste in the environment. But the implementing agencies have isolated and limited success.

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Suggestions and recommendations

. Different region of the country are following different standards, viz. the state of Tamil Nadu are following the Zero Discharge Policy, while in state of Gujarat treated effluents with defined release norms are either mixed with treated wastewater from local domestic wastewater treatment plants or are released into oceans. Therefore, with a matter of further deliberation, additionally there is an immediate need to devise a uniform national policy for discharge and release treated wastewater throughout the country. . Zero Liquid Discharge (ZLD) has shown promising results in the countries like USA, Spain, China. Even in India as noted above Tamil Nadu are following ZLD practise, especially for Textile effluent. CRBs may consider in phase manner to adopt the ZLD practises across the country. . CRBs need to devise “Integrated Concepts for Water Reuse” for water intensive industries. . CRBs may meticulously adopt the policy of ‘Polluters pay Principle’ atleast for Large Scale Mills and few Medium Scale Units. They must take responsibilities for cleaning of should discharge clean water by treating effluents at the source. . CRBs must take initiative and request the industrial units to participate, cooperate and must coordinate the scientific and technical work leading to establishment of treatment technology suited best for their effluent. . CRBs need to devise “Water Reuse Policy” particularly for textile and finishing industries.

15.4.3 Recommendations and suggestions to textile industries Environmental planning is no more considered as a luxury, but a base stone for a sustainable and developmental planning. Therefore, before commencement of any new industrial setups, a study of environmental impacts must be included as a part of feasibility studies. It is the time to move towards “Water Efficient India”. Need of the hour is to recycle and reuse water. Inspite of its great potential and accumulated evidence, reuse of water in many of the textile industry is still an uncommon practise. (A) Large Scale Textile Mills . Foremost, Large Scale industrial setups which are in forms of mills, needs to establish their In-house wastewater treatment facility, since they have enough freedom at monetary front. Many of them might having the treatmeant facility, but require upgradation according to the current need. . Many of them may directly support couple of research projects to academic or research institutes. . They need to develop the strategy to for recycling and reuse of water

(B) Small and Medium Scale Textiles Units . Most importantly, they need to become more proactive and required to show their consent and offer their space to willing research groups. Because quantity of pollution caused by SMEs is far larger than large scale units, they need to coordinate and collaborate in the mission, without aggressively depending on government initiatives and CETPs. . They need to develop the strategy to for recycling and reuse of water

15.4.4 Recommendations to researchers with list of thrust areas of R&D aspects which needs to be taken up

. Currently we have ample amount of treatment methods and technology, which have shown their potential for the treatment industrial effluent at different stages of technology development.

153

Suggestions and recommendations

. After couple of decades of intense research, the merits and demerits of each treatment methods are well know. The scope of each technology is well understood. And we have recognized that we need to reset our approaches. . Research community need to shed the perception of academic oriented study, especially for industrial effluent research. . Primary focus should to develop treatment technology directly for raw effluent from dye and dye intermediates manufacturing industries and textile and finishing industries, rather than working on individual dyes and/or simulated effluents under conditions. . Large number of laboratory scale studies has shown that, the combination of two or more methods has worked better, then the implementation of single method. Thus, integrated approach based work should be given priority.

For next decade, research must be focused in Phase-wise orderly manner, without being over ambitious (and spending time and money with repetitive type of never ending research)

Thrust Areas Development of Integrated Technologies,

 Phase – I (3-5 years)  So as to observe the repetitive effect and potentiality of developed technology on different production cycles and to counter the seasonal effect on implementing technology  Technology should be targeted for a single dye and dye intermediates manufacturing and textile and finishing units  Because, once point source pollution decrease, it would be easy to treat the effluents at CETP or effluents from different industrial source simultaneously

 Phase –II (4– 6 years)  Target should be on cluster of units producing similar type of effluents

 Phase – III (5-8 years)  Target should be on devising treatment technology for effluent collected at treatment facilities (i.e. ETPs, CETPs, etc.)

154

References  16 References

155

References

List of Publications and References

Selective list of publications produced from different work on bioremediation of dye compounds and industrial effluent containing dye and dye intermediates and the references cited in the compendium

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168

Appendices  17 Appendices

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Appendices

Table 17.1: Research Projects sanctioned by Department of Biotechnology (DBT), Ministry of Science and Technology, New Delhi on development of treatment technologies for dye and textile industrial effluents

No. Title Cost Principal Investigator (in Rs.) Rs. 1 Development of a biocatalyst 16.85 Dr. T. Emilia system for treatment of dye Department of BCP & WT Regional effluents Research Laboratory Thiruvananthapuram 2 Biodegradation of textile and 24.24 Dr. Datta Madamwar dyestuff industrial effluent Department of Biosciences Sardar Patel University Vallabh Vidyanagar 3 Ecotechnology for treating dye 07.66 Dr. K. P. Sharma Associate wastewaters of textile Department of Botany Rajasthan industries: a demonstration University project Jaipur 4 Decolorization and 14.59 Dr. N. Jothi Kumar biodegration of azo dyes National Environmental Engineering containing dye industries Research Institute Environmental wastewaters Biotechnology Division Chennai 5 Development of cellulase from 25.74 Dr. Mala Rao an extremophilic actinomycete National Chemical Laboratory for application in textile Biochemical Sciences Division industry Dr. Homi Bhaba Road Pune 6 Alkali stable cellulase 06.02 Dr. M. Rao demonstration of its National Chemical Laboratory application in textile industry Biochemical Sciences Division, Homi Bhabha Road Pune 7a Isolation, identification & 61.90 Dr. Datta Madamwar characterization genes for azo Department of Biosciences dye degradation: An approach Sardar Patel University. Vallabh towards construction of Vidyanagar efficient Bioremediation strain 7b Isolation, identification & 23.48 Dr. A. Gayatri characterization genes for azo Department of Microbiology & dye degradation: An approach Biotechnology towards construction of The M.S. University of Baroda efficient Bioremediation strain Vadodara 7c Isolation, identification & 9.48 Dr. Y.S. Shouche characterization genes for azo National Centre for Cell Sciences dye degradation: An approach Pune towards construction of

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Appendices

efficient Bioremediation strain 8 Decolorization of synthetic & 9.00 Dr. T. Palvannan real textile/paper wastewater Department of Biochemistry by the use of laccase from Periyar University Pleurotus florida (white-rot Salem fungi): Production & amp: Decolorization optimization using Response Surface Methodology 9 Biodegradation of textile 51.66 Dr. S. P. Govindwar Department of dispersive dyes (Scarlet RR & Biochemistry Rubine GFL) using Shivaji University Galactomyces Geotrichum Kolhapur MTCC 1360 & consortia with Brevibacillus laterosporus 10 Development of mixed 34.82 Dr. R. Rajendran, microbial consortia for the P.G.& Research Department of bioremediation of textile Microbiology, effluents P.S.G. College of Arts & Science Coimbatore 11a Application of periodic 51.00 Dr. Datta Madamwar discontinuous batch operation Department of Biosciences to enhance treatment Sardar Patel University efficiency of dye containing Vallabh Vidyanagar wastewater 11b Application of periodic 19.06 Dr. Venkata Mohana discontinuous batch operation Bioengineering & Environmental to enhance treatment Centre efficiency of dye containing Indian Institute of Chemical wastewater Technology Hyderabad 12 Bioremediation of dyestuff 43.224 Dr. M. H. Fulekar, effluent compounds in Mumbai University sequence bioreactor & Mumbai metagenomics study of rhizosphere 13 Developing efficient microbial 28.456 Dr. H. S. Saini inocula for degradation of Guru Nanak Dev University textile dyes & their amines: Amritsar Genotoxicity evaluation for validation of their degradation potential 14 Microbial consortia for 32.216 Dr. D. V. Laxmi effective decolorisation & Yogi Vemanna University degradation of anthraquinone Kadapa dyes 15 Promoting the use of invasive 21.56 Dr. M. Kannan,

171

Appendices

plant species in Sikkim for dye Ashoka Trust for Research in making: Ecology & The Environment A strategy for enhancing the (ATREE) livelihoods of the rural Bengaluru communities 16 Design of novel laccases for 41.118 Dr. S. Mishra, degradation of complex dyes Indian Institute of Technology-Delhi New Delhi 17 Elucidation of the degradation 09.418 Dr. K. K. Chauhan, mechanism of a mixture of Ashok & Rita Patel Institute of four reactive azo dyes by Integrated Study & Research in Comamonas sp. VS-MH2: A Biotechnology & Allied Sciences unique strategy towards water Vallabh Vidyanagar pollution abatement 18 Developing bio-restoration 33.548 Dr. R. Anandham, technology using the microbial Agricultural College & Research consortium for restoration of Institute profoundly degraded Madurai Orathupalayam dam due to accumulation of xenobiotics from textile processing units in Tiruppur, Tamil Nadu 19 Construction of wetland- a 32.312 Dr. Jyoti P. Jadhav phytoremediation treatment Department of Biotechnology process for the degradation of Shivaji University dyes from textile industrial Kholapur effluent 20 Optimization of membrane 16.45 Dr. S. Kalidass immobilized nanoparticles for Karunya University textile dye colour removal Coimbatore 21 Vetiver Based Treatment 21.00 Dr. J. Hema System for Textile Industry P.S.G. College of Arts & Science Wastewater Coimbatore 22 Structural characterization of 37.296 Dr. Pravindra Kumar dye-decolorizing peroxidase Indian Institute of Technology - enzyme from Bacillus subtilis Roorkee and Pseudomonas putida with an aim for bioremediation of industrial wastewater 23 Field scale evaluation of 50.59 Dr. Harvinder Singh Saini bioreactor developed with Guru Nanak Dev University indigenous microbial inocula Amritsar for treatment of textile industrial effluents 24a Integrated eco-electrogenic 134.878 Dr. Chirayu Desai Charotar system for efficient and University of Science and sustainable treatment of textile Technology, Changa

172

Appendices

wastewater 24b Integrated eco-electrogenic Dr. Venkata Mohan system for efficient and Indian Institute of Chemical sustainable treatment of textile Technology wastewater Hyderabad 24c Integrated eco-electrogenic Prof. Jyoti P Jadhav system for efficient and Department of Biotechnology sustainable treatment of textile Shivaji University Kolhapur wastewater 25a Metagenome Analysis for 61.32 Dr. Datta Madamwar Metabolic Pathways Present in Sardar Patel University Activated Biomass at Vallabh Vidyanagar Common Effluent Treatment Plant (CETP) 25b Metagenome Analysis for 22.83 Dr. Hemant Purohit Metabolic Pathways Present in National Environmental Engineering Activated Biomass at Research Institute Common Effluent Treatment Nagpur Plant (CETP) 26 Production of fungal Dr. Akshaya Gupte metalloenzymes by Pleurotus NVPAS College ostreatus and their application Vallabh Vidyanagar in bioremediation of azo dyes 27 Molecular and ‘-omics’ 336.57 Dr. Datta Madamwar technologies to gauge Department of Biosciences microbial communities and Sardar Patel University bioremediation of xenobiotic Vallabh Vidyanagar - contaminated sites

173

Appendices

Table 2: Research Projects sanctioned by University Grants Commission (UGC), New Delhi on development of treatment technologies for dye and textile industrial effluents

No. Title MJRP / Cost Principal Investigator MNRP (in lakhs) Rs. 1 Adsorption of dyes from dye- MJRP 02.29 Dr. R. J. Shukla house effluents on to ... bed Department of systems Chemistry Kamla Nehru Institute of Physical & Social Science Sultanpur 2 Photocatalytic treatment of MJRP 03.04 Dr. R. Ameta effluents from dyeing and Department of printing industries Chemistry Government P.G. College Banswara 3 Evaluation of occupational MJRP 03.87 Dr. K. Kalaiselvi genetic risks among dyeing and P.S.L. College of Arts bleaching industrial & Science workers Coimbatore 4 Interaction of some important MJRP 03.52 Dr. K. Misra azo dyes with specific Department of sequences of DNA/RNA Chemistry Allahabad University Allahabad 5 Photochemical of destruction of MNRP 00.40 Dr. S. P. Bansal dyes in dyeir and printing Maharaja Collage industry waste water Jaipur 6 Physico-chemical studies of MNRP 00.50 Dr. (Mrs.) S. Joshi. some co- ordination Sarojini Naidu Govt. compounds of some Girls P.G. College azo-dyestuffs Bhopal 7 Aerated packed bed upflow MJRP 04.88 Dr. T. Murugesan bioractors for continuous Anna University biodegradation of organic Alagappa College of effluents Technology, Department of Chemical Engineering Chennai 8 An integrated approach for MJRP 06.37 Dr. Datta Madamwar poto-evolution of hydrogen and Department of transformation of textile dyes Biosciences Sardar present in waste water by Patel University cyanobactreria Vallabh Vidyanagar 9 Textile effluent treatment with MJRP 03.28 Dr. A. Mishra natural gums and Mucilages Department of Chemistry Chhatrapati Shahu Ji Maharaj

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University Kanpur 10 Bacterial laccase: Cloning, MJRP 05.58 Dr. N. Capalash purification characterization Department of and applications in pulp Biotechnology Punjab delignification ...... effluent University treatment Chandigarh - 160 014 11 Studies on microbial MNRP 00.52 Dr. S. G. Gupta decolourization of dyes Department of Microbiology, Government Institute of Science Aurangabad 12 Studies on new super-absorbent MJRP 05.44 Dr. R. G. Patel, materials for dye effluent Sardar Patei University treatment Department of Chemistry Vallabh Vidyanagar 13 Treatment of textile effluents MJRP 10.08 Dr. R. Sivaraj by parthenium activated carbon Department of adsorption and bioremediation Biochemistry Karpagam Arts & Science College, Coimbatore 14 High Performance ion- MNRP 00.45 Dr. B. C. Dixit exchange resins for treatment Department of of industrial effluents Chemistry V.P. & R.P.T.P. Science College Vallabh Vidyanagar 15 Biodegradation of textile dyes MNRP 00.70 Dr. K. K. Mallesham, Pune University Pune - 411 007 16 Biodegradation of textile dyes MNRP 00.25 Dr. K. Kadam Pune University Pune-411 002 17 Effluent characterization & MNRP 00.50 Dr. S. K. Katyal treatment studies in dyeing Government College synthetic textile units of Nagaur - 341 001 Jodhpur 18 Semiconductor...... effluents MJRP 04.69 Dr. L. G. Devi Department of Studies in Chemistry Bangalore University Bengalooru 19 Bioremediation ...... MJRP 03.24 Dr. K. Girish

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industries Department of Biotechnology Mahajana First Grade College Mysore

20 Peroxidase conjugate to TiO2 MJRP 10.67 Dr. M. Sardar nanoparticles for the removal Department of of phenols & dyes in waste Biosciences Jamia water Millia Islamia University New Delhi 21 Studies on decolourization an MJRP 10.93 Dr. P. R. Thorat degradation of textile azo dyes Department of Microbiology Shri Shivaji Mahavidyalaya Barshi Solapur 22 Studies on textile dye MJRP 09.22 Dr. A. M. Deshmukh decolorisation by Department of Actinomycetes Microbiology Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 23 Investigations of dye MJRP 05.19 Dr. S. Kaur adsorption from aqueous Department of Applied solutions using unconventional Chemistry Guru Nanak adsorbents Dev University Amritsar 24 Physico-chemical studies on MJRP 06.54 Dr. A. K. Panda dye- nanoparticle aggregates Department of Chemistry North Bengal University Darjeeling 25 Sonophotochemical MJRP 04.57 Dr. H. G. Prabu degradation of dyes catalyzed Department of by different polyoxometalates Chemistry Alagappa immobilized on TiO2 University nanoparticles Karaikudi 26 Treatment of dyeing industry MJRP 01.72 Dr. P. N. Palanisamy effluents using low cost Department of adsorbents Chemistry Kongu Engineering College, Perundurai Erode 27 Utilization of clays & modified MJRP 06.23 Dr. S. G. Gupta clays as adsorbents for Department of hazardous & toxic dyes in Chemistry water B. N. College Dhubri

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28 Environmental benign MNRP 00.95 Mr. R. S. Yamgar photodegradation of dyes using S.S. & L.S. Patkar nanotechnology College of Arts & Sc. & V.P. Varde Comm. & Economics Mumbai 29 Sensitive micro-determination MNRP 01.80 Mr. G. W. Belsare of toxic metal ions such as Shri Shivaji College of Lead & Mercury with Arts, Commerce & triphenylmethane dyes in Science presence of .. Akola 30 Dehydration of azeotropic MJRP 10.62 Dr. S. Kaur alcohol & removal of dyes Department of from the effluent by surfactant Chemical Sciences & application Technology Guru Nanak Dev University Amritsar 31 Treatment of dyeing industry MJRP 01.72 Dr. P. N. Palanisamy effluents using low cost Department of adsorbents Chemistry Kongu Engineering College, Erode 32 Coupling of photo-oxidation MJRP 09.19 Dr. S. R.Thorat process with biological Department of treatment in textile & tannery Environmental industrial wastewater: Catalytic Sciences North Technological Aspect Maharashtra University Jalgaon 33 Genotoxicity & cytotoxicity of MJRP 11.27 Dr. A. Kaur azo dyes to Indian Major Carps Department of Zoology Guru Nanak Dev University Amritsar 34 Microbial decolorization of MJRP 07.52 Dr. J. Kumar textile dye effluent Department of Biotechnology Hans Raj Mahila Maha Vidyalaya Jalandhar 35 Studies on textile dyes MNRP 01.65 Dr. R. M. Khobragade decolourisation by Department of Streptomyces species Biotechnology Dr. Babasaheb Ambedkar Marathwada University Aurangabad 36 A study on removal of toxic MJRP 05.15 Dr. V. Venkateswaran metals & dyes from waste Department of

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waters using nano composites Chemistry Erode Arts College 37 Biodegradation of reactive dyes MJRP 04.60 Dr. J. H. Parikh used in textile industries by Department of macrofungi Chemistry Mafatlal Gagalbhai Science Institute Ahmedabad 38 Effective minimization of MJRP 04.36 Dr. M. Sundrarajan pollution load in reactive dye Department of bath using eco-friendly salt & Chemistry Alagappa ozonation University Karaikudi 39 Optimization study of salt-free MJRP 05.60 Dr. S. Thambidurai reactive dyeing & fiixing of Department of seaweed nono particles on Chemistry Alagappa cotton fabric for permanent University antibacterial finishing Karaikudi 40 Oxidative degradation of some MJRP 07.26 Dr. A. Goel dyes using Iridium nanocluster Department of catalyst Chemistry Kanya Gurukul Mahavidyalaya Haridwar 41 Study of water pollution of MNRP 02.00 Dr. B. S. Padhi Pikhwa town - A special Department of reference to effluents of textile Environmental Science dyeing industries R.S.S. (P.G.) College Ghaziabad 42 Studies on the impact of textile MNRP 01.20 Ms. K. Poornima effluents on freshwater fish Department of Zoology Oreachromis mossambicus HOD Poornaprajna College Udupi 43 Development of eco-friendly & MJRP 06.67 Dr. S. Meenakshi cost effective biosorption & Department of photodegradation methods for Chemistry Gandhigram the reversal of textile effluents Rural University Gandhigram 44 Biodegradation of dye effluent MJRP 06.90 Dr. R. using strains of bacteria & Kumuthakalavalli fungi isolated from Spent Department of Zoology Mushroom Substrate (SMS) & Gandhigram Rural effluent discharged sites University Gandhigram 45 Microbial & molecular MJRP 05.92 Dr. R. Ravikumar investigation of decolorization Department of Botany & degradation of textile dye Jamal Mohamed effluent through field College Tiruchirappalli

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application studies for solving the problems of textile-industry belt of Tamil Nadu 46 Characterization of bacterial MNRP 01.30 Mr. N. S. Junnarkar enzymes involved in textile dye Shree Manibhai Virani decolorization & Smt. Navalben Virani Science College Rajkot 47 Degradation of textile dye by MNRP 00.70 Dr. U. S. Patil actinomycetes & effect of Vishwasrao Naik degraded ... Mahavidyalaya Shirala Sangli 48 Studies on decolorization & MNRP 01.85 Ms. A. Y. Joshi degradation of synthetic azo Sheth Motilal dyes by bacterial isolates Nyalchand Science obtained from ... College Patan 49 Effluent dye waste water MJRP 03.95 Dr. D. R. Singh treatment using novel physico- Department of chemical & biological methods Chemistry Presidency University Chennai 50 Hydrothermal synthesis of MJRP 08.44 Dr. S. Sivanesan mesoporous carbon for the Department of effective removal of textile Chemical Engineering dyes Chennai Anna University 51 Synthesis & characterization of MJRP 06.66 Dr. D. Chakrabortty modified titania for photo- B. N. College catalytic degradation of toxic Department of dyes & phenolic compounds Chemistry Dhubri 52 A combinated effect of MNRP 00.95 Mr. G. H. Sonawane ultrasound cavitation on Department of adsorption kinetics in removal Chemistry Kisan of dyes along with College of Arts bioadsorbents Commerce & Science Jalgaon 53 Studies on the photcatalytic MNRP 0.95 Mrs. S. Shanthi degradation of dyes from Department of aqueous Soluktions of their Chemistry binary mixture, using The S.F.R. College for synthesised zink Women Sivakasi 54 Application of nanosized metal MJRP 06.26 Dr. K. Yogendra oxide particles as photocatalyst Department of in the degradation of azo dyes Environmental Science Kuvempu University Shimoga

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55 Decolorization & removal of MJRP 08.64 Dr. H. S. Patel dyes from textile industries Department of effluents Chemistry Sardar Patel University Vallabh Vidyanagar 56 Man made disasters: Mapping MJRP 6.36 Dr. P. H. Anand the geochemical hazards of dye Department of pollution in Amaravathi river Geography basin using GPS & GIS Government Arts technologies College Kumbakonam 57 Development of copolymeric MJRP 07.21 Dr. R. Singhal hydrogels based adsorbents for Department of removal of toxic heavy metal Chemical Technology ions & dyes from industrial Harcourt Butler waste waters Technological Institute Kanpur 58 Studies of new super absorbent MJRP 07.01 Dr. P. M. Patel nano materials for removal of Department of Polymer atoxic metals & dyes from Science industrial wastewater Sardar Patel University Vallabh Vidyanagar 59 Integrated bioremediation MNRP 00.90 Dr. S. Goel approach for the treatment of Department of textile & heavy metal laden Biotechnology Mata industrial effluents Gujri College Fategarh Sahib 60 Screening, isolation & MNRP 01.10 Ms. R. Agrawal characterization of azo dye Softvision College degrading bacteria from soil Indore industrial sector in Madhya Pradesh 61 The enzymatic decolorisation MNRP 17.75 Mrs. S. S. Horta of textile dyes by immobilized St. Francis College for polyphenol oxidase from peel Women Hyderabad & pulp of Tomato 62 Adsorption of dye on to MNRP 02.30 Ms. B. G. Chakraborty Polyelectolyte/surfactant Dasaratha Dev complex fabricated by layer by Memorial College layer technique Tripura University Lalchera 63 Adsorption of dyes from MNRP 03.30 Dr. G. R. Jani dyeing house waste water by Shri U.P. Arts, Smt. using .. M.G. Panchal Science & Shri V.L. Shah Commerce College North Gujarat University

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Mehsana 64 Conducting polymer/inorganic MNRP 04.50 Mrs. P. Rejani oxide nanocompositites for the N.S.S. College photo catalytic degradation of Palakkad organic dye 65 Decolorization of dyes from MNRP 02.45 Mrs. G. Kavitha industrial effluents using Gobi Arts & Science activated carbon prepared from College agricultural waste Gobichettipalayam

66 Eco-friendly method to MNRP 02.25 Dr. S. P. Govindaraj detoxify dyes from textile Saiva Bhanu Kshatriya efluent using bricks kiln ash as College Virudhunagar adsorbents Aruppukottai

67 Effect sorption of textile dyes MNRP 03.75 Mr. D. R. Shrivastava, on herbal ash & natural Government Arts & polymer chitosan thin film Commerce College combination: Study of efficacy Durg of eco-friendly binary process 68 Green synthesis & MNRP 02.65 Dr. M. Murugalakshmi, characterization of coinage S.F.R. College for metal nanoparticles using Women Sivakasi Erythrina variegata & evaluation of their dye adsorption .. 69 Photocatalytic degradation of MNRP 04.05 Dr. A. M. Sargar, various dyes with nanostructure Bharti Vidyapeeth's

Dr.Patangrao Kadam Mahavidyalaya Shivaji University Sangli 70 Physico chemical analysis of MNRP 04.15 Dr. H. Gopalappa synthesized simple & Government Science composite metal oxide College Chitradurga nanoparticles & study of their application in the colour removal of textile dyes 71 Studies on the removal of dyes MNRP 02.15 Mrs. V. T. Priya, from wastewater using clay & J.K.K.Nataraja College nanocomposites of Arts & Science Salem 72 An experimental & DFT study MNRP 01.70 Ms. J. Cyriac of the UV/Visible spectra of Sacred Heart College azo dyes & nitro substituted Ernakulam aromatic Amines

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73 Azo dyes of synthetic MNRP 02.00 Ms. M. Usha, curcuminoids and their metal Sree Neelakanta chelates Government Sanskrit College Palakkad

74 Decolorization of Sulphonated MNRP 01.85 Mr. R. Masarbo, azo dye Methyl orange by H.K.E. Society's A.V. bacterial species Patil Degree College of Arts, Science & Commerce Gulbarga

74 Investigations on the absorption MNRP 01.48 Ms. C. K. Memsy, efficiency of natural & low cost Mercy College absorbents locally available in Palakkad Kerala for removing dyes & toxic metals from water 75 Surfactant impregnated MNRP 03.40 Mr. G. Ramkumar chitosan as ADS or the removal M.V.G.R. College of of dyes from industrial .. Engineering Vizianagaram 76 A novel approach for the MNRP 03.60 Dr. K. V. Selvakumar degradation of organic dye Adhiyamaan College of compounds present in .. Engineering & Research Institute Krishnagiri 77 Analysis of waste water of MNRP 04.10 Dr. K. S. Meena textile industries of Bhilwara M.LV. Government (Rajasthan) & its treatment by College the Photo-Fenton system Bhilwara 78 Synthesis of metal oxide nano MNRP 01.80 Ms. A. K. John materials an& their application Bharata Mata College in self cleaning textiles Kochi 79 Testing the competent MNRP 03.80 Dr. V. Sreeja technique of textile effluents Vellalar College for using electrochemical & Women Erode activated carbon 80 Design & development of MJRP 12.32 Dr. K. V. S. Narayana, continuous flow packed bed Department of reactor for the removal of dyes Biotechnology Gitam from the textile industry University effluents Visakhapatnam 81 Synthesis & characterisation of MNRP 02.00 Dr. H. C. Prameela metal oxide nano particles & Government Science their application for College Hassan degradation of industrial dyes

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82 A novel & eco-friendly MJRP 14.49 Dt. T. Thayumanavan approach for complete colour Dr. G.R. Damodaran removal & reduction of total College of Science dissolved solids from textile Department of dyeing industrial effluent Environmental Science Coimbatore 83 Effects of process factors on MNRP 02.00 Mr. K. P. Dandge the efficiency of mixed aerobic Department of culture for the decolorization of Environmental Science reactive red dye North Maharashtra University Jalgaon 84 Photocatalytic degradation of MNRP 00.97 Mr. A. Garg textile dye effluent using doped Department of TiO2 catalyst Environmental Science Thapar University Patiala MJRP: Major Research Project; MNRP: Minor Research Project

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Table 3: Research Projects sanctioned by Department of Science and Technology (DST), Ministry of Science and Technology, New Delhi on development of treatment technologies for dye and textile industrial effluents

No. Title Funding Cost Principal Agency (in lakhs) Investigator Rs. 1 Application of plant (peroxidases SERC 09.13 Dr. P. Wangikar & laccases) for removal of Indian Institute of recalcitrant organic chemicals and Technology (B) dyes from industrial waste water Mumbai 2 Fungal treatment for removal of SERC 17.98 Dr. S. Sumathi textile dyes Centre for Environmental and Engineering Indian Institute of Technology (B) Mumbai 3 Decolorisation and degradation of SERC FT 09.44 Dr. R. Sanghi azo dyes using white rot fungal Indian Institute of microbes Technology (K) Facility for Ecological & Analytical Testing Kanpur 4 Studies on the screening of SERC-BS 09.68 Dr. N. chromosome abnormalities of Panneerselvam blood lymphocytes of Madura College occupationally exposed textile dye Department of unit workers Botany Madurai 5 Decolorisation and bio- SERC-EP 05.66 Dr. L. Iyengar degradation of 4- ABS containing Indian Institute of azo dyes under microaerophilic Technology (K) aerobic conditions Kanpur 6 Biodegradation of SERC-FT 11.04 Dr. J. P. Jadhav triphenylmethane dyes by Shivaji University Pemcillium ohrochloron Department of Biochemistry Kolhapur 7 Bio-remediation of textile dyes & SERC-FT 06.06 Dr. K. M. Kodam dye wastewater by akaliphiies Department of Chemistry Pune University Pune 8 Standardization of conditions for SERC-SC 14.28 Dr. O. P. Ahlawat exploitation of spent substrate for Department of decolorization of colouring dyes Crop Production

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National Research Centre for Mushroom Solan 9 Community link up for natural SST 05.92 Dr. P. S. Vankar dyeing with plants found in Southern Manipur Laboratories Indian Institute of Technology (K) Kanpur 10 Flux enhancement & fouling TSD 21.40 Dr. S. D. Gupta reduction during effluent (leather Department of & dye) treatment using membrane Chemical separation Engineering Indian Institute of Technology (Kh) Kharagpur 11 Chemical & electrochemical SERC-SC 33.76 Dr. Y. A. Naik generation of ZnO, CuO, SnO2, Department of TiO2, Fe203 & MgO nanoparticles Chemistry for the degradation of textile dyes Kuvempu from industrial effluents (low-cost, University eco- friendly and renewable Shimoga method) 12 Genetic studies on azo-dye SERC-EP 31.97 Dr. S. Garg degrading immobilized bacterial Department of consortia TJ-1 & TJ-2 Chemical Engineering Indian Institute of Technology (K) Kanpur 13 Investigations on microbial SERC-EP 12.15 Dr. A. Malik formulation of a heat & alkali Centre for Rural tolerant fungal strain for Development & decolourization of dye bearing Technology effluents Indian Institute of Technology (D) New Delhi 14 Investigations in mechanistic or SERC–FT 12.00 Dr. T. Sivasankar physical features of the Department of senochemical remediation of dyes Chemical in textile Engineering Indian Institute of Technology (G) Guwahati 15 Development of a new textile IDP 12.46 Dr. A. Basu affluent technology towards zero South India Textile

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sludge Research Association Coimbatore 16 Investigations in mechanistic or SERC–FT 12.00 Dt. T. Sivasankar physical features of the Department of senochemical remediation of dyes Chemical in textile Engineering Indian Institute of Technology (G) Guwahati 17 Removal of dyes from aqueous SERC- 17.76 Dr. Uma solutions & dye house wastewaters WOS Department of by activated carbons synthesized Applied Chemistry from waste materials Institute of Technology Banaras Hindu University Varanasi 18 Source reduction of pollutants SERC-FT 19.34 Dr. M. Sundrarajan from textile effluents by greener Alagappa route University Karaikudi 19 Biodegradation of acidic azo dyes SERC- 14.76 Ms. R. H. Dave & their industrial waste WOS Department of Microbiology & Biotechnology Gujarat University Ahmedabad 20 Development & characterization of SERC-FT 19.60 Dr. G. Pugazhenthi low cost ceramic composite Indian Institute of membrane for separation of dyes Technology from aqueous solution (Guwahati) Department of Chemical Engineering Guwahati 21 Development of effluent treatment WS 16.37 Dr. V. Gupta plant for natural dyeing unit & Magan conversion of waste into Sangrahalaya commercial products Samiti Kumarappa Road Wardha 22 Biosensor for analysis of phenolic TSG 28.98 Dr. K. Rekha compounds in textile industry C.M.R. Institute of effluent Technology Department of Biotechnology,

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Bengaluru 23 Development of effluent treatment WS 16.37 Dr. V. Gupta plant for natural dyeing unit & Magan conversion of waste into Sangrahalaya commercial products Samiti Wardha 24 Biodegradation of textile dyes by SERB 17.44 Dr. M. Z. Khan sequential Anaerobic-aerobic Department of process & recovering energy Chemistry Aligarh Muslim University Aligarh 25 Removal of toxic compounds in SERB-FT 20.90 Dr. W. Jabez textile dye effluents from polluted School of sites of Tripura District: A phyto Bioscience & & rhizoremediation approach Technology Vellore Institute of Technology University Vellore 26 An integrated approach for biofuel SERB-FT 20.80 Dr. K. production by coupling with Nanthakumar microbial based textile wastewater The Energy & treatment Resource Institute Environmental & Industrial Biotechnology Division New Delhi 27 Comparative analysis of soil SERB-FT 22.50 Dr. J. Pandey microbial diversity across textile Department of wastewater effluent stream (s) of Biotechnology Sanganer region, Rajasthan Central University of Rajasthan Ajmer 28 Loofa (Luffa cylindrica) songe as SERB-FT 24.00 Mr. R. G. Saratale carrier for microbial cell Department of immobilization & its application in Biotechnology & the treatment of dye containg Bioinformatics effluent Padmashree Dr. D.Y. Patil Vidyapeeth Navi Mumbai 29 Bacterial degradation of selected SERB- 11.40 Ms. D. K. Patel metal complex acid dyes & its WOS Department of effluent Microbiology Gujarat University

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Ahmedabad 30 Biodegradation of textile dyes by SERB-FT 22.50 Mr. M. Z. Khan sequential anaerobic-aerobic Department of process & recovering energy Biochemistry Engineering & Biotechnology Indian Institute of Technology (D) New Delhi 31 Development & demonstration on SSTP 47.58 Dr. R. Rajendran bioremediation of Indigo dye P.S.G.College of containing textile effluent using Arts & Science microbial biofilm with adapted Civil Aerodrome microorganisms (PO) Coimbatore 32 Utilization of plant bioresource for WTI 12.34 Dr. A. Sarma biosorptive removal of dyes from Morigaon College water Morigaon 33 Carbon dioxide sequestration & TSDP 18.37 Dr. N. Krishnaveni dye industrial alkaline effluent Associate remediation using fly ash Professor & Head Vellalar College of Engineering & Technology Department of Chemistry Erode 34 Treatability studies for Textile TSDP 50.70 Dr. P. N. Wastewater Treatment-I Palanisamy Professor & Head Kongu Engineering College Department of Chemistry Perundurai 35 High performance multifunctional WTI 24.95 Dr. S. Pal modified guar gum for wastewater Indian School of & industrial effluent treatment Mines Dhanbad 36 Development & application of clay SERB-FT 25.98 Dr. S. Mandal based absorbents for effective Central Leather removal of dye/color from Research Institute industrial processed streams with Centre for Eco- special focus on treatment of Testing Laboratory leather i ndustry wastewater Chennai

37 Advanced oxidation process & SERB-FT 25.00 Dr. S. S. Kumar

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Appendices

microbial treatment for toxicity- National College free textile effluent disposal Department of Biotechnology Tiruchirappalli 38 Microbial consortium for removal SERB-FT 21.00 Dr. A. Bohra, of textile pollutants Department of Biotechnology Mahila (P.G.) Mahavidyalaya Jodhpur 39 Preparation of textile filters for IBC 07.88 Dr. M. Joshi selective filtration of waste waters Indian Institute of Technology (Delhi) Department of Textile Technology New Delhi 40 Effective utilization of sludge from SERB-FT 14.90 Dr. S. textile & paper industry effluent Senthilkumar treatment plant as construction K.S.R. College of materials Engineering Department of Civil Engineering Tiruchengode 41 Treatment of textile wastewater SERB-FT 17.64 Dr. P. Bhunia via ultrasonication & biological Indian Institute of anaerobio-aerobic treatment route Technology (Bhubaneswar) Department of Civil Engineering Bhubaneswar 42 Characterization & utilization of TSDP 34.98 Dr. A. Das selected, deoiled cake as Ponnaiyah adsorbents in textile wastewater Ramajayam treatment Institute of Science & Technology Thanjavur

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Table 4: Research Projects sanctioned by Council of Scientific and Industrial Research (CSIR), New Delhi on development of treatment technologies for dye and textile industrial effluents

No. Title Funding Cost Principal Investigator Agency (in lakhs) (CSIR) Rs. 1 Microbial degradation of EXTRM 01.74 Dr. T. B. Karegoudar Synthetic Organic Department of Compounds Biochemistry Gulbarga University Gulbarga 2 Designing and EXTRM 11.86 Dr. H. S. Saini optimisation of the Department of working parameters of an Microbiology immobilised cell Guru Nanak Dev bioreactor for treatment of University industrial effluents Amritsar 3 Assessment of ozone EXTRM 07.63 Dr. V. Tare treatment as a polishing Department of Civil step for a full-scale Engineering Indian anaerobic (UASB) reactor Institute of Technology effluent (K) Kanpur 4 Studies on the assessment EXTRM 09.38 Dr. Y. Anjaneyulu. of soil leaching potential Centre for Environment and insitu biodegradation Jawaharlal Nehru on soils of hazardous Technological University organics: Development of Hyderabad certain low cost technology for reclamation of industrial hazardous waste dumpsites and polluted groundwaters 5 Detoxification and GS 05.88 Dr. Q. Husain decolorization of textile Department of and other industrial dyes Biochemistry from polluted waste water Aligarh Muslim University by using immobilized Aligarh polyphenoloxidases 6 Use of turbulence to GS 08.22 Dr. S. D. Gupta enhance flux during Department of Chemical membrane separation of Engineering Indian toxic dyes from waste Institute of Technology water (Kh) Kharagpur 7 Treatment of textile GS 11.96 Dr. H. S. Saini. processing industry waste: Department of Pilot scale testing of Microbiology

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Appendices

upflow immobilized cell Guru Nanak Dev bioreactor for effluent University treatment and laboratory Amritsar microcosm studies for reclamation of polluted soils 8 Electrochemical treatment GS 14.31 Dr. I. D. Mall of textile industry Department of Chemical wastewater Engineering Indian Institute of Technology (R) Roorkee 9 Decolorization & GS 13.92 Dr. K. V. B. Rao, degradation of azo dyes by University School of using indigenous bacteria Bioscience & Technology & their mediated Vellore Institute of nanoparticles Technology Vellore

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Table 5: Research Projects sanctioned by All India Council of Technical Education (AIECT), New Delhi on development of treatment technologies for dye and textile industrial effluents

No. Title Funding Cost Principal Investigator Agency (in lakhs) (AICTE) Rs. 1 Development of treatment R&DP 06.50 Dr. P. Manisankar process for dye house effluent Department of by electrochemical methods Industrial Chemistry Alagappa University Karaikudi 2 Photocatalytic degradation of TAPTEC 06.50 Dr. V. Murugesan dyes in wastewater of textile Department of and leather industries Chemistry Anna University College of Engineering Chennai 3 Removal of phenolic R&DP 05.00 Dr. C. Balomajumder, compounds from the industrial Department of effluents in bioactive GAC Chemical Engineering columns Roorkee University Roorkee 4 Studies in reactor for R&DP 05.00 Dr. V. G. Gurjar, electrochemical methods for Department of effluent treatment Chemical Engineering Indian Institute of Technology (B) Bombay 5 Bioremediation of R&DP 08.00 Dr. T. B. Karegoudar, environmental pollutants Department of Biochemistry Gulbarga University Gulbarga 6 Treatment of dyeing mill R&D 05.00 Dr. H. S. Rai, effluents by a low cost Department of Civil treatment system for safe Engineering Guru disposal Nanak Dev Engineering College Ludhiana 7 Treatment of hazardous dye- R&D 05.00 Dr. M. C. S. Reddy, house waste water by low cost G. Pulla Reddy materials Engineering College Kurnool 8 Application of colour TAPTEC 07.00 Dr. C. J. Jahagirdar. measurement technique in Department of textiles & dyes- Chemical Technology

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photochemistry of dyes & Bombay University fibres- studies on ecosystem Mumbai 9 Construction of an eco- TAPTEC 05.00 Dr. T. Ramachandran, friendly working plant for in P.S.G. College of situ electrolytic bleaching of Technology jute based textiles Coimbatore 10 Development of electrodialysis TAPTEC 15.75 Dr. M. Y. membranes for water Kariduraganavar purification, waste Karnatak University management, effluent Dharwad treatment 11 Development of eco-friendly TAPTEC 08.30 Dr. H. G. Prabhu methods for textile industry Alagappa University wet processing & recyclone of Karaikudi water 12 Studies on degradation of R&D 09.97 Dr. M. M. Swamy phenol(s) and kinetics of gaden Sri Jayachamarajendra type in fermentation using College of Engineering imobile whole cells in different Mysore reactors 13 Photocatalytic degradation of TAPTEC 16.15 Dr. B. Arabindoo non- biodegradabie prganic Anna University pollutants in wastewater Chennai 14 Development of polymeric TAPTEC 04.50 Dr. D. Mohan membranes for industrial Department of effluent treatment Chemical Sciences Anna University Chennai 15 Design & investigation of R&D 05.46 Mr. T. Kannadasan hydrocyclone Coimbatore Institute of electrolyser for the treatment Technology of dye-house effluents Coimbatore 16 Design & investigation of R&D 05.46 Mr. T. Kannadasan hydrocyclone Coimbatore Institute of electrolyser for the treatment Technology Civil of dye-house effluents Coimbatore 17 Semibatch membrane TAPTEC 13.60 Dr. S. Sundaramoorthy seperation process for Department of treatment of industrial Chemical Engineering effluents- experiments, model Pondicherry and optimal control Engineering College Pondicherry 18 Preparation, characterization RP 16.50 Mr. S. K. Samdarshi and performance analysis of Tezpur University fixed bed Solar Photocatalytic Tezpur reactor for degradation of

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Appendices

Azodyes 19 Synthesis, characterization & RP 07.50 Dr. S. Dash photochemical studies of some Department of novel polymethine cyanine Materials & Metallurgy dyes University College of Engineering Sambalpur 20 Photocatalytic treatment of RP 11.50 Mr. H. Bhunia Textile effluent Department of Chemical Engineering Thapar Institute of Engineering & Technology Patiala 21 Bioremediation & reuse of RP 04.00 Mr. M. spent reactive dyebaths Thirumarimurugan Coimbatore Institute of Technology Department of Chemical Engineering, Coimbatore

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Table 6: Research Projects sanctioned by DOEN, MOEN, MNES, MOEF, New Delhi on development of treatment technologies for dye and textile industrial effluents

No. Title Funding Cost Principal Agency (in lakhs) Investigator Rs. 1 Microbial degradation of DOEN- 03.29 Dr. J. Musarrat polycyclin aromatic hydrocarbons ERS Interdisciplinary in soil & subsurface environment Biotechnology Unit in vicinity of Mathura oil refinery Aligarh Muslim University Aligarh 2 Studies on degradation of gelatine DOEN- 02.76 Dr. S. K. Vasija factory effluent by bacteria R&D Department of Biological Sciences Rani Durgavati Vishwavidyalaya Jabalpur 3 Microbial degradation of DOEN- 03.67 Dr. L. Kumari pesticide and application of R&D Madras University immobilized enzymes/ cells in Chennai pesticide effluent treatment 4 Degradation of organic pollutants DOEN- 03.61 Dr. K. D. S. Yadav by ligniases of Phanerocharte R&D Department of chrysosporium Chemistry Gorakhpur University Gorakhpur, 5 Consideration of efficient strains DOEN- 05.29 Dr. A. Mahadevan of bacteria for cleavage of ERS Department of Botany aromatic pollutants and their Madras University applications in industrial effluent Chennai treatment 6 Bio-remediation of textile dye MOEN- 05.14 Dr. P. Ghosh industry waste water ERP Birla Institute of Scientific Research Jaipur 7 Electro-chemical treatment of MOEN- 10.28 Dr. R. Jain dyes in textiles, cosmetics, food ERP School of Studies in and pharmaceutical industrial Chemistry effluents Jiwaji University Gwalior 8 Decolourisation of industrial MOEN- 13.53 Dr. S. M. Gaddad effluents using immobilised ERP Department of micro-organisms Microbiology Gulbarga University Gulbarga 9 Solar photocatalytic treatment for MNES- 10.50 Dr. S. N. Kaul

195

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colour removal from textile waste R&D National water Environmental Engineering Research Institute Nagpur 10 Solar plhotocatalvtic MNES- 04.42 Dr. C. K. Joshi deteoxyfication of industrial R&D Department of wastewater Chemical Engineering Thapar Institute of Engineering & Technology Patiala 11 Development of integrated MOEF- 12.66 D. M. Khare advanced oxidation and microbial ERP Indian Institute of technology for dye wastewater Technology (D) treatment Department of Civil Engineering New Delhi 12 Studies on the impact of industrial MOEF- 02.99 Dr. S. K. Gupta, effluents and sludge on ERP Industrial Toxicology earthworm and the potential of Research Centre tolerant strain in environmental Lucknow restoration 14 Electrolytic recovery of copper MOEF- 05.90 Dr. S. Chellammal and removal of organic pollutants ERP Central from the copper phthlocyanine Electrochemical dye process stream Research Institute Karaikudi 15 Degradation of organic pollutants, MOEF- 18.63 Dr. K. Pandian, colored dyes & pesticides in ERP Department of water resources using polymer Inorganic Chemistry protected metal nanoparticles: A Madras University nanotechnological approach Chennai 16 Microbial decolurisation of MOEF- 13.23 Dr. K. Singh, coloured textile industrial ERP Department of effluents Applied Chemical Science & Technology Guru Nanak Dev University Amritsar DOEN: Deparment of Environment; MOEN: Ministry of Environment; MNES: Ministry of Non-Conventional Energy Sources; MOEF: Ministry of Environmnet and Forest

196

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Table 7: Research Projects sanctioned by Department of Atomic Energy-Board of Research in Nuclear Sciences (DAE-BRNS), New Delhi on development of treatment technologies for dye and textile industrial effluents

No. Title Cost Principal Investigator (in lank) Rs. 1 Development of 07.66 Dr. A. K. Mishra, fluorescence based optical Department of Chemistry Indian sensors for monitoring Institute of Technology (M) pollutants in industrial Channai effluents 2 DAE-YSRA-process 14.50 Mr. V. C. Padmanahan development for the Centre for Biotechnology degradation of textile dyes M.E.T’s. School of Engineering by wild type & radiation Thrissur induced mutants 3 Design & evaluation of 20.61 Dr. J. Jayapriya anaerobic sequential Department of Chemical Microbial Fuel Cell (MFC) Engineering for decolourization of textile Anna University effluent Chennai

197

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History of Dyes

2600 BC The earliest written record of the use of colouring substance (dyestuffs) was found from China, from as early as 2600 BC. 715 BC Around 715 BC Wool dyeing was an established craft in Rome. 331 BC After conquering Susa (capital of Persia) Alexander finds 190 year old purple robes. 327 BC Alexander mentions “beautiful printed cottons” in India. 236 BC An Egyptian Papyrus mentions dyers as “stinking of fish”, with tired eyes and hands working unceasingly. 55 BC Romans found painted people “picti” in Gaul dyeing themselves with Woad (same chemical content of color as indigo). 2nd and 3rd Roman graves found with madder and indigo dyed textiles, Centuries AD replacing the old Imperial Purple (purpura). 3rd Centuary Papyrus found in a grave contains the oldest dye recipe known, for imitation purple called “Stockholm Papyrus”. It is a Greek work. 273 AD Emperor Aurelian refused to let his wife buy a purpura dyed silk garment. It cost its weight in gold. Late 4th Emperor Theodosium of Byzantium issued a decree forbidding Centuary the use of certain shades of purple except by the Imperial family on pain of death. 400 AD Murex (the mollusk from which purpura comes) becoming scarce due to huge demand and over harvesting for Romans. One pound of cloth dyed with Murex worth $20,000 in terms of our money today (Emperor Augustus source). 700's Chinese manuscript mentions dyeing with wax resist technique (batik) 925 ‘The Wool Dyers’ Guilds first initiated in Germany. 1188 The first mention of Guilds for Dyers in London. 1197 King John (of Magna Carta fame) persuaded parliament to regulate dyeing of woolens to protect the public from poor quality goods. 1200's Rucellia, of Florence, rediscovered the ancient art of making purple dye from lichens sent from Asia Minor (similar to Orchils). 1212 The city of Florence had over 200 dyers, fullers and tailors. A directory of weavers and spinners was published as well. 1290 The only blue dye of the period, Woad, began to be raised extensively in Germany. The 3 major dyes were now: woad, madder and weld. 1321 Brazilwood was first mentioned as a dye, source from East Indies and India.

198

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1327-1377 Edward III, “Royal Wool Merchant” offered protection to all foreigners living in England and to all who wanted to come to help improve the textile industry. Early 15th Cennino Cennini of Padua, Italy described the printing of cloth Centuary (block printing) in his treatise called ‘Method of Painting Cloths by Means of Moulds’. 15th Centuary Aztecs under Montezuma conquered the Mayans. 11 Mayan cities paid a yearly tribute of 2000 decorated cotton blankets and 40 bags of Cochineal (insect dye) each. 1429 The 1st European book on dyeing ‘Mariegola Dell'Arte de Tentori’ was published in Italy. 1464 Pope Paul II introduced the so called “Cardinals' Purple” which was really scarlet from the Kermes insect. This became the first luxury dye of the Middle Ages just as Imperial Purple (Murex) had been for the ancient world. 1472 Edward IV incorporated the Dyers' Company of London. 1507 In Europe, Germany, Holland and France begin the cultivation of dye plants (as an industry). 1519 Pizarro and Cortez find that there is cotton in Central and South America. They send back brightly printed fabrics showing that the Indians knew about block printing prior to the Conquest. Cochineal from Mexico and Peru now being shipped back to Spain. 1614 Dyeing cloth “in the wood” was introduced in England: logwood, fustic, etc. 1630 Drebbel, a Dutch chemist, produced a new brilliant red dye from cochineal and tin. It was used at Goblein (Paris) and the Bow Dyeworks (England). 1631-33 The East India Company began importation of calico from Calicut, India to England; at first they thought the fabric was linen, not cotton. Mid-1600's English Logwood cutters in Honduras lead a dangerous life (danger from Spaniards, hurricanes, swamps, disease) in the Bay of Campeachy, but could get very rich. 1688 James II, of England, prohibited exportation of un-dyed cloth from England to help bolster the home industry for English dyers over that of the Scottish dyers. 1689 The first calico printworks was begun in Germany at Augsburg and was later to grow into a large industry. 18th Centuary English dyehouse gets contract to dye the Buckingham Palace Guards coats with cochineal, this contract continued into the 20th Centuary still using cochineal. 1708 William III, signed a law prohibiting the importation of printed silks, this only made calicos and silks more popular.

199

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1716 There were now more than 30 laws in England prohibiting the importation of calico and cotton; prints became more popular than ever. 1727 A method of bleaching linen with kelp (seaweed) was introduced in Scotland. 1745 Indigo begins to be grown in England, after the Revolution when it became cheaper to import from the East Indies. 1766 Dr. Cuthbert Gordon patents Cudbear (derived from his mother's name) Cuthbert was prepared from a variety of lichens. Only one of 2 natural dyes ever credited to an individual (other is quercitron to Bancroft). 1769 Arkwright's spinning frame in England (aka the Spinning Jenny) 1774 Swedish chemist, Scheele, discovered chlorine destroyed vegetable colors by observing a cork in a bottle of hydrochloric acid. 1774 Prussian Blue and Sulphuric acid available commercially. Prussian blue formed from prussite of potash and iron salt (copperas). Actually one of the early chemical dyes. 1775 Bancroft introduced the use of quercitron bark as a natural dye. One of only 2 natural dyes whose discoverer is known, it yields a yellow, brighter than fustic, and is from the inner bark of American oak. 1786 Bertholet, France, recommended chlorine water for commercial bleaching. Other oxidizing agents began to be used, viz. hydrogen peroxide, sodium peroxide and sodium perborate. 1785 Bell, England, who had invented printing from plates, developed roller printing 1788 Picric acid available (yellow dye and disinfectant) could be dyed from acid dyebath on wool 1790 Acid discharge of mordant printing developed. 1794 Three Frenchmen set up first calico printing. 1796 Tennant developed bleaching process. 1797 Bancroft develops a process for steam fixation of prints. 1798 Oberkampf (in Jouy, France) pleased Napoleon by showing him a roller printer made from a cannon Napoleon had seized from the Pope. This began the famous ‘Toiles de Jouy’ production. 1802 Sir Robert Peel brought out a resist method; he had purchased the idea for from a commercial traveller for equivalent of $25. It consisted of a wax or other resist on the background, actually a batik technique done on large scale. 1823 Mercer discovered chromate discharge of indigo. 1825 Mathias Baldwin began the first American production of engraved metal rollers for calico printing which were used in the Philadelphia area and could produce 300 yards of fabric per day.

200

Appendices

1834 Runge, a German chemist, noticed that upon distilling coal tar, aniline would give a bright blue color if treated with bleaching powder. This helped to pave the way to the development of aniline (basic) dyes 22 years later. 1844 The process was called “mercerization”. John Mercer discovered that treating cotton with caustic soda (lye) while under tension improved its strength, luster, dyeability, absorbency. 1856 William Henry Perkin discovered the first synthetic dye stuff “Mauve” (aniline, a basic dye) while searching for a cure for malaria and a new industry was begun. It was a brilliant fuchsia type color, but faded easily so our idea of the color mauve is not what the appearance of the original color was. 1858 Griess discovered diazotisation and coupling on/in the fiber. 1858-59 Magenta (fuchsin) discovered by Verguin the 2nd basic dye and more widely used than Mauve. 1861 Methyl violet, basic dye developed by Lauth. 1862 Hofmann's Violet, developed by Hofmann, was one of the great dye chemists of all time. 1862 Bismarck Brown developed by Martius and Lightfoot, first soluble azo dye. 1863 Aniline Black, developed by Lightfoot, a black produced by oxidation of aniline on the cotton fiber. 1866 Methyl Violet, basic dye was developed. 1868 Graebe and Liebermann, German chemists, produced alizarin (synthetic madder). This was the first time a synthetic substitute for a vegetable dye had been manufactured. W. H. Perkin also synthesized it about the same time, but independently. 1872 Methyl Green, the basic dye was developed by Lauth and Baubigny, still in use. 1873 Cachou de Laval, 1st sulphur dye, a brown, by Groissant and Bretonniere, France. 1875-76 Caro and Witt prepared Chrysoidine, 1st important member of azo class of dye. 1876 Caro, an important dye chemist, discovered Methyl Blue. 1877 Malachite Green, basic dye developed by Dobner and Fisher. 1878 Biebrich Scarlet invented, a very pure red acid dye, rivalling cochineal in brightness. 1878 von Baeyer synthesized synthetic indigo. It was not marketed until 1897. 1880 Thomas and Holliday, England, synthesized the first azo dye formed on the fabric by coupling. Vacanceine red formed by treating fabric with napthol and then dipping in a diazolized amine, a very fast category of dyes.

201

Appendices

1884 Congo Red by Bottiger, first of the direct cotton dyes. 1885 Benzopurpurine, early direct dye by Duisberg, bright and highly substantive. 1885 Para Red dye brought out by von Gallois and Ullrich. (β-napthol and nitraniline). 1885-89 Chardonnet, France, made the first successful rayon and showed it at the Paris Exposition of 1889. 1887 Alizarin Yellow GG (1st azo mordant dye) and Rhodamine B (brilliant red violet, basic dye) was synthesized. 1890 Direct Black BH, first direct black dye was developed. 1891 Diamine Green B, first green azo dye was introduced. 1891 Chardonnet built his first commercial plant at Besancon for manufacturing rayon, by the Chardonnet process. 1891 Sky Blue FF (a direct dye) with good light fastness was first synthesized and for many years was used as blue colour dye. 1893 Vidal Black (2nd sulphur dye) was synthesized. 1895 Viscose method of making rayon invented by Cross and Bevan, England was begun. This is now the most common process for manufacture of rayon. 1898 Direct Black E, a black dye of major importance was first synthesized. 1900 When Mozaffer ed Din became Shah of Persia one of his first edicts was to prohibit the use of analine dyes for rugs. All analine dyes were seized and publicly burned. Penalties included jail and fines equal to double the value of the merchandise. 1901 Rene Bohn patented his invention of Indanthrene Blue RS, the first anthraquinone vat dye, a category of dyes with extremely good fastness to light and washing. 1901 Bohn developed 2nd vat dye, Flanthrene a yellow 1902 Thesmar, Baumann, Descamps, and Frossard brought out hydrosulfite and sulfoxylateformaldehyde. 1905 Thio indigo Red, by Freidlander, 1st indigoid dye was developed. 1908 Hydron Blue, a rival to indigo, developed by Cassella. 1914 USA importing 90 % of its dye stuffs, a problem during WWI, as many came from Germany. 1915 Neolan dye, 1st metallized chrome dye, dyed from strong acid bath. 1921 Bader developed soluble vat colors, the Indigosols. 1922 The AATCC (American Association of Textile Chemists and Colorists) formed its first subcommittee to study washfastness of printed and dyed cottons, formulate testing procedures, standards of fastness.

202

Appendices

1924 Indigosol-0 devloped by Baeyer and Sunder, 1st commercial indigosol dye. 1928 Dupont began the fundamental research that would lead to discovery of nylon. 1936 First pair of stockings knit with a new synthetic fiber from DuPont called “nylon” for which Carothers received the patent. 1938 Nylon formally introduced to the public. 1948 Textiles became second largest industry in USA. The average consumer consumption per capita of fibers: 27 lbs cotton, 6.3 lbs rayon, 4.9 lbs wool. 1950 DuPont introduced first commercial availability of Orlon, a new acrylic “wool substitute” 1951 Irgalan dyes introduced by Geigy, 1st neutral pre metallized dyes (did not require a lot of acid as Neolans did) Cibalans are the same type. 1951 DuPont announced that a plant in N.Carolina would begin to manufacture Dacron polyester. 1951 A new acrylic, Acrilan was introduced by Chemstrand Corporation. 1953 Cibalan Brilliant Yellow 3GL, a dye which leads the way to discovery of the fiber reactive dyes was introduced. 1954 Celanese Corporation announced first commercial production of an American triacetate, Arnel. 1956 ICI in England introduced Procion, first range of fiber reactive dye this dye was to have a major impact on industry as well as textile artists around the world. 1956 Eastman Kodak introduced Verel, a modified acrylic. 1956 American Cyanamid introduced a new acrylic, Creslan. 1956 One person working out of every 7 in the USA received his income from work performed in textile or apparel industries! 1957 CIBA introduces Cibacrons, a new range of reactive dyes and the first to compete with ICI's Procion series. 1958 Eastman Kodak introduced Kodel polyester. 1964 First permanent press finishes used. 1968 DuPont introduces Qiana, a fancy nylon with “silk” feel and drape. 1968 For the first time manmade fibers topped natural fibers for US consumption. 5 billion pounds vs 4.6 billion pounds, the use of polyester was growing the most quickly. 1970s Late in the 70s, CIBA Geigy introduced Cibacron F series.

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Appendices

Table A: List of Relevant International Patents for developing novel technologies in treatment of dye and textile effluents

Patent No. Patent Title

CA-2745053-C Treatment of textile materials

CN-101041532-A Printing and dyeing waste water treatment method based on film technology

CN-101139155-A Non-excess activated sludge discharged printing and dyeing wastewater processing equipment and operation method thereof CN-101139156-A Textile printing and dyeing wastewater advanced treatment method

CN-101172741-A Dyeing and printing waste clearing, synthetic wastewater advanced treatment circulation production and recycle technique CN-101224357-A Two-stage dynamic membrane filtering method for printing and dyeing wastewater recycling CN-101234814-A Printing waste water advanced treatment and reusing method adapted for medium and small-sized printing plant CN-101279803-A Processing system and method for printing and dyeing wastewater

CN-101293714-A Advanced treatment method and equipment for printing and dyeing wastewater CN-101293726-A Method for processing and separate-recycling printing and dyeing wastewater CN-101343131-B Multi-stage combined degradation and recycle method for printing and dyeing wastewater CN-101353215-A Dyeing waste water comprehensive processing and reclaiming process

CN-101412571-A Technique for reclaiming waste water of azoic dye production

CN-101423292-A Anaerobic bioreactor for treating high concentration printing and dyeing wastewater and method thereof CN-101503268-A Zero discharge processing EBM method for dyeing waste water

CN-101514057-A Method for recycling textile, printing and dyeing wastewater

CN-101525202-A Advanced dyeing wastewater treatment and reclaimed water reuse system and method thereof CN-101538107-A Method for treating wastewater in textile printing and dyeing industry

CN-101538107-B Method for treating wastewater in textile printing and dyeing industry

CN-101613167-A Method for treating recycled printing and dyeing sewage

CN-101633541-A Integrated technology for deep purification treatment for printing and dyeing waste water CN-101638279-A Method for treating sulfur-containing printing and dyeing wastewater

CN-101654314-A Dye waste water treatment method

204

Appendices

CN-101659500-A Dye waste water treatment system

CN-101670266-A Method for removing organic cationic dyes from waste water by magnetic nano-adsorption material CN-101700943-A Printing and dyeing waste water advanced treatment recovery method

CN-101700948-A Wastewater treatment technology for textile dyeing and finishing industry

CN-101704594-A Device and method for purifying printing and dyeing advanced treatment wastewater CN-101705626-A Textile printing method with little amount of water of cotton fabric by utilizing reactive dye CN-101767917-B Reprocessing device and method of textile sewage of conventional secondary treatment CN-101798159-A Textile printing and dyeing wastewater treatment device and technology

CN-101857328-A Spinning printing and dyeing waste water reclamation method, device and application thereof CN-101891319-A Alkaline printing and dyeing wastewater materialization pre-treatment method and system CN-101948220-A Method for treating printing and dyeing wastewater

CN-101955282-A Method for realizing zero emission of dyeing wastewater with high salinity in printing and dyeing enterprises CN-101955303-A Treatment method of dye wastewater

CN-102040289-A Method and equipment for regenerating and recycling textile dyeing wastewater CN-102050555-A Device and method for treating and recycling printing and dyeing wastewater CN-102092879-A Dye wastewater cyclic utilization device and method based on electrolysis and lamination technologies CN-102145965-A Textile dyeing wastewater advanced treatment recycling technology

CN-102190412-A Method for recycling textile dyeing and finishing wastewater

CN-102198998-A Advanced treatment and reuse integrated equipment for textile dyeing and finishing wastewater CN-102259964-A Contaminant components situ inherent synergistic dyeing wastewater treatment reinforced composite chemicals CN-102399038-A Secondary sewage treatment device for textile dyeing

CN-102531248-A Effluent decolouring method and effluent decolouring device

CN-102627350-A Non-woven fabric dynamic membrane bioreactor for printing and dyeing waste water treatment CN-102674497-A Method for remediating azo dye pollution in water body by utilizing nano- mushroom fungus biological adsorbent CN-102674640-A Washing bleaching and dyeing wastewater treatment method

205

Appendices

CN-102701500-A Printing and dyeing wastewater zero-discharge reuse treatment method

CN-102718344-A Recycling treatment process of printing and dyeing wastewater

CN-102730907-A Deep treatment method for printing and dyeing industry production waste water CN-102849873-A Textile sewage treatment and recycling system

CN-102923903-A Textile printing and dyeing wastewater processing technology

CN-103011516-A Reactive printing wastewater treatment process and device

CN-103011524-A Recycling and processing method for printing and dyeing wastewater

CN-103043861-A Textile wastewater treatment method

CN-103058335-A Novel graphene-Ti electrode printing and dyeing wastewater treatment electrochemical reactor CN-103102048-B Textile wastewater treatment method and device

CN-103304071-A Printing and dyeing wastewater heat energy recovery and anti-dirty treatment method CN-103359878-A Treatment method for realizing zero emission of printing and dyeing wastewater CN-103359897-B Process and device for treating high-concentration sulphate radical textile- dyeing wastewater CN-103466768-A Floc reflux coagulation process for treating biochemical printing and dyeing effluent CN-103508632-A Textile sewage treatment system

CN-103523996-A Treatment device and method of printing and desizing mixed wastewater

CN-103739064-B Application of a dyeing wastewater aerobic activated sludge treatment process CN-103755021-B An improved ammoniation upflow anaerobic reactor and a method of treating wastewater of high organic nitrogen for dyeing CN-103755092-B A novel textile dyeing Wastewater Treatment and Reuse Technology

CN-103755093-A Fenton fluidized bed-IBAC combined method used for advanced treatment of textile dyeing and finishing waste water CN-103771662-A Printing and dyeing wastewater treatment process

CN-103964531-A Reverse micelle dye extraction and recycling method for textile dyeing waste water CN-104030427-A Supercritical water oxidation treatment system for printing and dyeing wastewater and sludge CN-104030437-A Biological combined reactor used for printing and dyeing waste water processing, device and method CN-104030437-B Dyeing composition for biological wastewater treatment reactor, the apparatus and method CN-104030532-B Textile dyeing wastewater treatment process

206

Appendices

CN-104108775-A Composite reagent for treating recycled water of printing and dyeing wastewater and application method of composite reagent CN-104150643-B Method for applying the bleaching agent treated sewage Dyeing

CN-104176892-A Textile waste water treatment system

CN-104193119-A Process for deeply treating printing and dyeing wastewater in presence of attapulgite catalyst CN-104213356-B A textile dyeing process water treatment system

CN-104313824-A Wastewater treatment device for textile suppress washing machine

CN-104326622-A Treatment method of chemical fibre textile dyeing wastewater

CN-104445819-B A multi-series substance dyeing wastewater treatment method

CN-104478174-A High-salt-content dyeing wastewater treatment recovery zero discharge integration method CN-104529018-A Process for treating and recycling printing and dyeing wastewater by virtue of electro-coagulation CN-104609680-A Textile dyeing wastewater treatment and reclamation technique

CN-104743714-A Combination technology for processing difficultly-degraded dye waste water

CN-104743737-B A method of treatment of industrial wastewater dyeing

CN-104843949-A Textile printing and dyeing waste water treatment process

CN-105130092-B A processing apparatus and method and the degradation of organic wastewater of high concentration dyeing denitrification CN-105148891-A Congo red decolouriser for treating textile printing and dyeing wastewater

CN-105152334-A Textile mill dyeing wastewater treatment method and system

CN-105601029-B Kinds of printing and dyeing wastewater treatment process

CN-105617742-A Textile wastewater suspension treatment apparatus

CN-105692826-A Flocculating agent composition for treating textile wastewater and using method thereof CN-105692952-B Treatment of textile wastewater

CN-105727902-A Adsorption composition for treating textile wastewater

CN-105776712-A Method for deep treatment and salt recovery of textile dying wastewater and used system CN-105776776-A Treatment method of cotton fabric printing and dyeing wastewater

CN-105854359-A Textile sewage segment treatment structure and sewage treatment method

CN-105859055-A Complex printing and dyeing wastewater quality-divided treating and reusing integrated technology CN-105967448-A Treatment method and treatment system for wastewater of textile industry

207

Appendices

CN-105967453-B A composite textile printing and dyeing wastewater treatment process

CN-106115807-A Environment-friendly treatment agent for textile wastewater

CN-106348551-A Toxicity reduction and recycling system and treatment method for biotreated effluent in printing and dyeing industry CN-106430810-A Printing-dyeing textile wastewater and domestic wastewater mixed treatment system CN-106542703-A Textile printing and dyeing wastewater treatment method

CN-106565053-A Textile printing and dyeing wastewater and domestic sewage mixed treatment system CN-106830172-A Compound degradation agent for degrading rhodamine B dye and preparation method of compound degradation agent CN-106882880-A Treatment method of rhodamine B dye wastewater

CN-107200382-A Sludge-free deep treatment method for dyeing wastewater

CN-107261605-A Advanced purification processing equipment for textile waste water treatment CN-107261637-A Textile sewage treatment system

CN-107311411-A Textile printing and dyeing sewage system

CN-107352590-A Efficient-recycling chemical-fibre textile wastewater treatment system

CN-107352606-A Textile dyeing waste water treatment system

CN-1073663-A Washing and dyeing wastewater treatment and renovation

CN-107522322-A Textile sewage treatment and purification system

CN-107522364-A Textile printing and dyeing wastewater treatment system

CN-107555684-A Recycling method and equipment of textile, printing and dyeing wastewater

CN-107619149-A Printing and dyeing textile wastewater treatment process

CN-107792994-A A woven dyeing wastewater treatment method

CN-107963727-A A method for textile dyeing wastewater treatment

CN-108046450-A Textile waste water recycling seed treatment apparatus

CN-108083552-A Kinds of textile industrial wastewater treatment methods

CN-1335274-A Treatment method of textile printing industry effluent

CN-1680193-A Apparatus and process for wastewater treatment of dyeing and finishing textile CN-1733625-A Textile printing and dyeing waste water processing system and method

CN-1765779-A Printing and dyeing wastewater recovery and disposal method

208

Appendices

CN-1778722-A Dyeing waste-water decolorizing degradation, recovery and utilization

CN-1785844-A Biological anaerobic reactor of textile printing dyeing waste water

CN-1785854-A Treatment and reuse method of high temperature dyeing waste water

CN-1827535-A Dye waste water treatment method based on membrane technology

CN-1830823-A Treatment agent for textile dyeing waste water

CN-1931751-A Combined process of treating azo dye effluent with high salinity

CN-1978337-A Printing-dyeing waste water treatment process

CN-201343478-Y UASB-MBR integrated system for treating dyeing wastewater

CN-201400610-Y Textile wastewater treatment and recycling system

CN-201560154-U Advanced-treated dyeing wastewater purifier

CN-201908025-U Printing and dyeing wastewater treatment recycling device based on nano- catalysis electrolysis and membrane technology CN-201923914-U Printing and dyeing wastewater recycling device based on electrolysis and laminating technologies CN-201971709-U Sewage treatment system in regeneration textile industry

CN-202415328-U Dyeing and finishing sewage treatment system used for textile process

CN-202688095-U Efficient bioreactor for treating printing and dyeing wastewater

CN-202729944-U Treatment device of printing and dyeing wastewater

CN-202785890-U Advanced treatment and recycling device for textile printing and dyeing wastewater CN-203112652-U Activated printing wastewater treatment equipment

CN-203173940-U Textile wastewater treatment device

CN-203474587-U Printing and dyeing textile wastewater treating and recycling device

CN-203754570-U Textile wastewater treatment recycling system

CN-204097290-U Textile wastewater treatment system

CN-205011579-U Sewage treatment plant is used in textile mill

CN-205603431-U Strengthening biological degradation preliminary treatment printing and dyeing destarch waste water PVA's device CN-205803125-U Water pollution dyes little bioremediation device

CN-206127055-U Printing and dyeing wastewater processing system based on IC MBR technique CN-206219384-U Printing and dyeing wastewater processing system who combines MBR membrane

209

Appendices

CN-206447728-U Textile printing and dyeing wastewater processing apparatus

CN-206616120-U Textile waste water treatment system

CN-206814600-U Printing and dyeing wastewater's processing system

CN-206843222-U Printing and dyeing wastewater degree of depth decolouration treatment process system CN-206970410-U Dyeing and finishing effluent disposal system

CN-206985964-U Processing system of textile industry waste water

CN-207108747-U A woven dyeing wastewater treatment system

CN-207243529-U An efficient back fibre textile dyeing wastewater treatment system with

CN-207243625-U Sewage treatment plant of textile dyeing apparatus

CN-207259343-U A woven dyeing wastewater treatment system resources

CN-207276403-U Printing a woven waste water treatment apparatus

CN-207375908-U Textile waste water treatment plant species

CN-207404859-U Recovery type printed textile wastewater treatment

CN-207435226-U Textile waste recycling equipment

CN-207468267-U Precision seed dosing system for the wastewater treatment of textile

CN-207483489-U Kinds of textile wastewater pre-treatment system

CN-207511963-U Kinds of textile sewage purification system

DE-10132546-C1 Textile material used in biological waste water treatment plant includes effect structures and base structure which is independent of load bearing capability DE-10143600-A1 Biological treatment of dye-containing waste water from the textile and leather industry comprises adding ozone and optionally hydrogen peroxide to the treated water and recycling it as process water DE-10164623-A1 Combined sterilization/contaminant removal treatment of textile industry or laundry waste water involves addition of small amounts of chemicals DE-19610345-C1 Catalysts used with peracid/s or hydrogen peroxide to remove dyes from textile effluent DE-19654028-A1 Oxidative treatment of textile dyeing plant effluent

DE-19716939-A1 Treatment of waste water containing azo- and sulphur-based dyes from textiles and leather industry DE-19725096-C2 Process for the elimination of reactive dyes and their hydrolysates from sewage DE-2347329-A1 Textile-treatment plant effluent working up - and re-cycling the treated water to the plant DE-4125319-C1 Textile for support of microorganisms or catalyst in (an)aerobic sewage

210

Appendices

treatment - comprises strips joined with catalyst or microorganisms, with wide non-supporting spacer strips between them EP-0440482-A1 Textile treatment

EP-1831115-B1 Biological neutralization of highly alkaline textile industrial wastewater

EP-1921045-A1 Use of Cunninghamella elegans Lendner in methods for treating industrial wastewaters containing dyes ES-2238933-A1 Electrochemical treatment of textile fibre dyeing effluent comprises decolorizing, for ultraviolet irradiation and re-utilization of the process products ES-2395318-B1 Treatment process and reusing textile effluents by electrochemical techniques. FR-2429283-A1 Treatment of textile packages, e.g. dyeing, or bleaching and/or drying - in two=phase process involving pressurising and depressurising JP-2007209890-A Treatment method and apparatus for organic wastewater

JP-2013006174-A Flocculant composition and flocculation method for purifying dye wastewater JP-3064723-U Wastewater treatment for textile materials blocks and waste water treatment apparatus JP-4536158-B1 Colored wastewater treatment apparatus for use in coloring wastewater treatment method and the method JP-5068277-B2 Purification simulation method for the bioremediation

JP-H07124569-A Decolouration treatment of printing/dyeing waste water

JP-H0714520-B2 Processing method of textile dyeing wastewater

JP-H08281271-A Treating device of waste dyeing water and treatment of the same

KR-0177549-B1 How to remove the color of the dye by the player Wu Lotus sp and the strain with the color of the dye Removal KR-100271849-B1 Biological treatment method and apparatus for dyeing waste water using two-stage anaerobic treatment KR-100541844-B1 The processing method of textile-waste water by the combined process

KR-100614561-B1 Biological treatment of dye waste-water using moving-bed bioreactor

KR-100961667-B1 A method for treating dyeing wastewater by using porous polyurethane foam media comprising carbonaceous materials obtained from carbonization of sludges and white-rot fungi KR-19990038487-A Textile dyeing wastewater treatment

KR-20010056962-A Method of treating dye containing waste water

KR-20010092575-A Novel soil Streptomyces ND002 which have new fuchin dye-decolorizing activity and 2,4-dichlorophenol oxidation activity, and isolation method thereof KR-20100093433-A Apparatus and method for treatment of textile wastewater using anaerobic- aerobic biofilter and post ozone process

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US-2002151038-A1 Process for removing dye from dye containing water or soil using white rot- lignin-modifying fungus Flavadon flavus US-2004009726-A1 Multi-functional protective textiles and methods for decontamination

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US-5639379-A Process for removing colour and odour from aqueous effluent contaminated with textile dye US-5876461-A Method for removing contaminants from textiles

US-5961838-A Amphoteric polymer/polyamine combinations for colour removal and clarification of paper mill waste water US-5976197-A Dyeing process and dyes

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US-6540920-B2 Wastewater treatment system utilizing textile filter media

US-7658849-B2 Use of iRhizopus stolonifer (Ehrenberg) vuillemin in methods for treating industrial wastewaters containing dyes US-8017374-B2 Processes for decolorization of colored effluents

WO-0242228-A1 Bioadsoprtion process for the removal of colour from textile effluent

WO-2004038089-A2 Textile treatment agent

WO-2008089529-A1 Process and apparatus for reuse of liquid effluents generated in the textile finishing, laundering, dyeing and stamping process WO-2011063769-A1 Purification device and method for advancedly treating printing and dyeing wastewater WO-2011086567-A1 Magnetic dye-adsorbent catalyst

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Summary of Patent generated through one of the DBT supported research project and patent funding supported by DBT.

Title: A Process for Treating Dyes Wastewater

Inventor: Dr. K.P.Sharma, Department of Botany Dr. Subhasini Sharma, Department of Zoology, University of Rajasthan, Jaipur

Abstract: This invention relates to a process for treating dyes wastewater. The process comprises a primary step of chemical treatment followed by secondary and tertiary steps of biological treatment performed in bio-reactors.

Full Text: This invention relates to a process for treating dyes wastewater.

Prior Art The activated sludge is the most common biological method for treating dye wastewaters, in which the reduction of BOD (90%) and COD (40-507) was comparatively higher than the colouring matter (10-30%, Oil is et. al. 1991. Li and Zhams 1996 and Ahmed and Oil is 1984). The reduction in colouring matter in the activated sludge process has been ascribed, primarily due to its adsorption on the sludge, suggesting a little degradation of dyes during the treatment process (Srimuli and Karthikeyan 1999). Now a day, there is a greater emphasis on the isolation of a noble microorganism capable of degrading a variety of dye/s. A large number of bacterial species capable of degrading azo compounds are Proteus spp., Enterococcus spp., Streptococcus spp., Bacillus subtilis (Zissi et. al., 1997), Closteridium spp., Pseudomonas spp. (Bumpus, 1995), Klebsiella pneumonia and Acetobacter liquefaciens (So et. al., 1990, Wang & Yuen 1996), Streptomyces spp. (Pasti et. al., 1991), Sphingomonas spp. (Kudhch et. al. 1997), Trametes versicolur, Bjerkandera adust (Heinfling et. al. 1997). Azo dyes do not degrade readily in presence of oxygen (Johnson et. al. 1978), whereas in the anaerobic conditions, these are reduced to colourless, toxic and carcinogenic amines (Zimmerman el. al. 1982). Only recently azo dyes have been reported to degrade aerobically (Bumpus 1995, Heinfling et.al. 1997, Pasti et al. 1995, Zissi et al. 1997), F'sedomonas sp. K-l (Wong Yuen 1996), Psedomonas S-42 (Bumpus 1995), Acetobacter liquefaciens (So et. al 1990) and Klebsiella pneumonia RS-13 (Wong & Yuen 1996) are the bacterial species which co-metabolically degrade the dyes in aerobic condition.

Object of the Invention An object of this invention is to propose to process for treating dyes wastewater such as azo dyes, aniline dye and reactive dyes aerobically under the identical conditions. Another object of this invention is to propose to process for treating dyes wastewater which is cheap and requiring very little inputs.

Description of the Invention According to this invention there is provided a process for treating dye wastewater comprising if, a. primary step of chemical treatment as herein described followed by secondary and tertiary steps of biological treatment as herein described performed in bioreactors followed by the step of polishing as herein described in and a wetland for treating the dyes under aerobic conditions. Important findings, which are reported for the first time for the treatment of azo dyes, are

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Appendices described below. The acidic azo wastewaters having a pH 2-4 are first neutralized using lime. This decreased their COD (20-70%), conductivity (40-70"n) and Cu (20-60%) as a result of precipitation of dissolved matters. This neutral wastewater is then fed continuously for example in 24 h into two serially arranged fixed film upflow bioreactors. The bioreactors consisted of biofilm of consortium of aerobic microbes (from Institute of Microbial Technology, chandigarh, Report of which is enclosed) developed on the surface of grits. The aerobic conditions in the bioreactor are maintained by continuous aeration, using an aquarium pump in the reactor. Microbially treated water is then passed through a constructed wetland haing Phragmites and Typha at a loading rate of for example 15 cm/day. The wetland has down flow pattern of hydraulic loading. The retention period of the wastewater in the bioreactors and constructed wetland is for example two days only, one day each in the bioreactors and constructed wetland. The reduction in COD levels as well as its load after treatment of azo dye waslewater’s at successive steps in the bioreactors and constructed wetlands. During spectroscopic studies marked reduction in the size of peaks in the UV region after passing of dye wastewater through bioreactors and constructed wetland were found. The chromatographic studies revealed absence of dyes in the treated wastewater, which were present in the untreated wastewater. The GC mass studies also confirmed degradation of azo dyes during the treatment process, 36 peaks detected in the neutral azo wastewater decreased to 22 after treatment during winter, when micorbial activities are slow on account of low temperature. The toxicity of treated wastewater was constantly monitored during study period, growing Gambusia fishes in a 15 L bucket having submerged plants like Hydrilla and Ceratophyllum. The fishes grow and breed normally during more than six months of the study, establishing the fact that treated water is non toxic and can be safely discharged into the water courses. It was found that treated wastewater can be recycled back in the same industry. The treated wastewater also had no adverse effect on the growth of Cymopsis plants. These had well developed root nodules similar to control plants which were unhealthy and few in number in the plants growing in dye wastewater irrigated soils. The degradation of dissolved methyl red dye (50 ppm) was also studied in the system during summer. The optical density of methyl red decreased after treatment in the system, also noted a marked reduction in size of peaks in both UV Visible range. The GC mass studies of methyl red solution in methanol detected 64 peaks including that one of the solvent. The number of compounds decreased to two in the outflow after microbial treatments in the bioreactors and constructed wetland, affirming degradation methyl red in the treatment system. Thus we have achieved significant degradation of dyes at two days retention period. Further sludge production is minimum in our system. We have also studied degradation of methyl red by both pure culture of two different bacterial isolates from our system and also using their consortium. The complete decolorisation of methy red was achieved within 24 hours after inoculation of both pure isolates of bacteria as well s their mixed consortium. The treatment of dye wastewater released in the silicate process was also studied. The silicate process uses reactive dyes for printing, which are fixed in the concentrated solution of sodium silicate. We have developed method for the removal of sodium silicate in the dye wastewater, thereafter, the wastewater was inoculated with both pure isolates as well contort turn of the microbes. The complete decolourisation of the wastewater was achieved within 48 hours, being relatively faster in the contortium in comparison to the purt isolates, which establishes that the process system can treat a variety of dye wastewaters.

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Appendices

Claim 1. A process for treating dye wastewater comprising in a primary step of chemical treatment as herein described followed by secondary arid tertiary steps of biological treatment as herein described performed in bioreacours followed by the step of polishing as herein described in and a wetland for treating the dyes under aerobic conditions. 2. A process as claimed in claim 1 wherein the step of chemical treatments consists in the step of neutralization. 3. A process as claimed in claim 1 Wherein the aerobic conditions are maintained by continuous aeration; 4. A process as claimed in claim 1 wherein said bioreactors are upflow bioreactors, 5. A process for treating dyes wasU.-wat.er substantially as herein described and illustrated.

Few Relevant Indian patents with their abstract

(a) Title: An Improved Treatment Plant for Textile Wastewaters and an Improved Process Thereof

Abstract An improved treatment plant for textile wastewaters, which comprises a screen chamber for removing particles and floating bodies above 50 mm diameter, having wastewater inlet, characterized in that the outlet of the said screen chamber being connected through a grit chamber so as to re me inorganic silt having specific gravity ≥2.65, and a conventional parshall flume so as to regulate and measure wastewater flow, to the inlet of an equalisation basin having a plurality of improved high speed floating aerators and for providing a constant wastewater level of 0.5 to 1.0 m below the inlet and retention time of 5 to 7 hours, the outlet of the said equalisation basin being connected to a flash mixer being provided with separate dosing tanks of ferrous sulphate, polyelectrolyte and lime for neutralising and coagulating the wastewater. the outlet of said mixer being connected to a centre feed radial distribution system of a circular clariflocculator so as to provide retention time of 4 to 6 hours and having a separated clarification zone, sludge withdrawal outlet, and peripheral outlet, the said sludge outlet being connected to sludge drying beds having an outlet for filtrate connected to the inlet of equalization basin, the said peripheral outlet being connected in parallel to a plurality of bays of aeration tank having a plurality of improved high speed floating aerators, the output of the said aeration tank being connected to a secondary clarifier so as to provide retention time of 3 to 5 hours and having a sludge withdrawal outlet, a peripheral outlet and treated effluent outlet, the said sludge outlet being connected to the said sludge drying beds and the peripheral outlet being connected to the inlet of aeration tank.

(b) Title: A process for decomposition of organic synthetic dyes using semiconductor-oxides nanotubes via dark catalysis

Abstract In the present invention nanotubes-based dye-adsorbent powder is used for adsorption and decomposition of synthetic dye under dark-condition without the use of any external power- source such as the applied potential-difference, microwave, ultrasonicator, and others to save the energy consumption, which results in a complete dye-decomposition on the powder-surface. The 216

Appendices surface-cleaned dye-adsorbent powder has very high specific surface-area and dye-adsorption capacity comparable with those of the original dye-adsorbent powder and can be recycled for the next cycles of dye-adsorption and dye-decomposition under the dark-condition. In the present method, the nanotubes-based dye-adsorbent powder is stirred under the dark-condition in an aqueous dye solution containing a strong oxidizer to get simultaneous dye-adsorption and dye- decomposition on the powder-surface, or getting dye adsorption in one solution and dye decomposition in a separate solution by said adsorbent powder under dark condition.

(c) Title: Novel method for removal of organic and inorganic contaminants caused by textile dye effluents in the agricultural soil by bio-electrokinetics

Abstract Textile dye contaminated agricultural soil contains large amount of organic and inorganic impurities. The present invention relates to the development of bio-electrokinetic process for the removal of pollutants from the soil. Starch with aromatic degrading bacteria Pseudomonas spp and cellulose degrading bacteria {Bacillus cereus and both cellulose and laccase positive Bacillus subtilus, and Bacillus tequilensis) were used as anolyte, which move towards cathode, via electroosmosis process in electrokinetic technique. The reduction of COD was in the range of 70% to 82%. The inorganic impurities of chloride, sulphate and trace metal ions were removed by electromigration process. Significant reduction of conductivity and TDS can be achieved by bio- electrokinetic process. The injection of starch with bacteria and removal of pollutants improve the fertility of the agricultural soil.

(d) Title: Processes for decolorization of colored effluents

Abstract The present invention relates to a novel process for decolorization of colored effluents. More particularly it relates to a process for decolorization of colored effluents of textile mills, dye- making industries, paper and pulp industries and molasses spent wash from alcohol distilleries using an unidentified white-rot marine fungus NIOCC #2a isolated from mangrove wood and deposited on Sep. 7, 2004 in the microbial type culture collection (MTCC) of the Institute of Microbial Technology, Chandigarh, India, under the accession number MTCC 5159. Further, this invention relates to decolorization of these effluents using the fungus directly, its cell-free culture supernatant or immobilized fungus or extracellular polymeric substances produced by the fungus. Furthermore, the decolorization of effluents can be carried out from 30ºC to 60ºC and at pH 3 to 6. The decolorization of various colored effluents occurs in the presence of sea water with 25 parts per thousand salinity. Several synthetic dyes are also decolorized under similar conditions of temperature and pH by using free mycelia or immobilized fungus or extracellular culture fluids or extracellular polymeric substances.

217

Prof. Datta Madamwar, has completed his B.Sc. and M.Sc. from Nagpur University, and Doctorate from Birla Institute of Technology and Science, Pilani. He is presently UGC-BSR Faculty Fellow at Department of Biosciences, Sardar Patel University, where he was former Dean of Faculty of Science. He has received several reputed awards and is fellow of several national and international societies. He has a vastresearch experience as a postdoctoral fellow at TIFR, Mumbai, Universistat Frankfurt, Germany, Universitstat Konstanz, Germany. His current main focus on research is on Non-aqueous Enzymology, Industrial Liquid Waste Management and Cyanobacterial Phycobiliproteins. He has vast expertise in industrial waste management. His lab has developed and enriched more than 125 bacterial consortia (mixed cultures) and isolated bacterial pure cultures from polluted soil, sediments, water, industrial and CETP efuents exhibiting effective degradation of various dyes and dye intermediates, aromatics, polyaroamtic hydrocarbons (PAH), distillery spent wash and detoxication of heavy metals under different environmental conditions (anaerobic, microaerophilic, aerobic, sequential microaerophilic, aerobic and anaerobic-microaerophilic). He has made signicant contribution in developing different types of bioreactors (anaerobic biphasic xed lm, sequential anoxic-oxic batch process, sequential anaerobic-microaerophilic process, periodic discontinuous batch operation) for the treatment of industrial wastewater. His contribution in the eld is well reected by his publications (250), citations (8927) h-index (51) and i10-index (162). (Email: [email protected] )

Dr. Onkar Tiwari, is Scientist in the Department of Biotechnology (DBT), Ministry of Science & Technology, Government of India. DBT is an Indian Government Department, under the Ministry of Science & Technology responsible for administrating development and commercialization in the eld of modern biology and biotechnology in India. Presently Dr. Onkar is programme ofcer for the DBT R&D programmes in the areas of Forest Biotechnology, Bioresources & Secondary Agriculture and Environmental Biotechnology. He is involved in Science & Technology Policy Research, R&D Projects Management, IPR, Technology Transfer issues etc. His main role is to plan the strategy and management of R&D programme in areas of Environmental Management, Forest & Conservation Biotechnology and Value-added Biomass & Products from Natural Resources to meet the national need for R & D and Technology planning. Major programmes presently being handled by him are DBT River Cleaning R&D programme, Pan India network on Plant Biodiversity Conservation, Bioresources Bioprospecting etc. Besides Ph.D. in Environmental Biology and Masters in Biotechnology, he also holds Bachelor's degree in Laws. (Email: onka r . [email protected] )

Kunal Jain, Ph.D, is currently a Research Associate at Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar (Gujarat). He is an alumnus of VP & RPTP Science College, Vallabh Vidyanagar, where he has graduated in Microbiology. He has earned his post-graduate and doctorate degree from Sardar Patel University, Vallabh Vidyanagar, in Microbiology. He has been working in Environmental Microbiology since a decade, trying to understand microbial behaviour in an anthropogenic ecosystem. He is trying to develop possible energy-efcient biological methods for eliminating xenobiotic compounds from environmental (terrestrial and aquatic) compartments by providing scientic support for the transfer of these methods into practice. (Email: [email protected] )

Department of Biotechnology Ministry of Science & Technology Sardar Patel University Government of India Vallabh Vidyanagar Block-2, CGO Complex, Lodhi Road, New Delhi - 110003