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EVALUATION OF AZO DYE DEGRADING BACTERIA FOR

BIOMASS PRODUCTION BY ENERGY CROPS GROWN

WITH DYE-CONTAMINATED WATER

MATEEN SHAFQAT

07-arid-1086

Department of Environmental Sciences

Faculty of Forestry, Range Management and Wildlife

Pir Mehr Ali Shah

Arid Agriculture University Rawalpindi

Pakistan

2017

ii

EVALUATION OF AZO DYE DEGRADING BACTERIA FOR

BIOMASS PRODUCTION BY ENERGY CROPS GROWN

WITH DYE-CONTAMINATED WATER

by

MATEEN SHAFQAT

(07-arid-1086)

A thesis submitted in the partial fulfillment of

the requirements for the degree of

Doctor of Philosophy

in

Environmental Science

Department of Environmental Sciences

Faculty of Forestry, Range Management and Wildlife

Pir Mehr Ali Shah

Arid Agriculture University Rawalpindi

Pakistan

2017

iv

For

Mom & Dad

v

CONTENTS

Page

List of Tables ix

List of Figures x

List of Abbreviations xiv

Acknowledgements xvi

ABSTRACT xviii

1. INTRODUCTON 1

2. REVIEW OF LITERATURE 6

2.1 BACTERIAL DECOLORIZATION OF AZO DYES 9

2.1.1 Bacterial Decolorization of Azo Dyes Under Anaerobic

Conditions

10

2.1.2 Bacterial Decolorization of Azo Dyes Under Aerobic

Conditions

12

2.2 FACTORS REGULATING THE PROCESS OF

DECOLORIZATION

13

2.3 PHYTOREMEDIATION OF AZO DYES 16

2.4 BACTERIAL ASSISTED PHYTOREMEDIATION OF

AZO DYES

19

2.5 DECOLORIZATION OF AZO DYES BY PLANT

GROWTH PROMOTING BACTERIA

21

3. MATERIALS AND METHODS 24

3.1 COLLECTION AND ANALYSIS OF TEXTILE

WASTEWATER

24

3.2 ISOLATION AND SCREENING OF DYE-DEGRADING

BACTERIA

24

3.2.1 Dye Chemicals and Culture Medium 25

3.2.2 Isolation of Dye-Degrading Bacteria 25

3.2.3 Screening of Efficient Dye-Degrading Bacterial Isolates 27

3.3 CHARACTERIZATION OF SELECTED BACTERIAL

ISOLATES FOR PLANT GROWTH PROMOTION

28

vi

3.3.1 Indole Acetic Acid Production Assay 28

3.3.2 Phosphate Solubilization Activity 29

3.3.3 ACC-Deaminase Activity 30

3.4 BOX POLYMERASE CHAIN REACTION AND 16S

rRNA GENE ANALYSIS OF BACTERIAL ISOLATES

CARRYING DUAL TRAITS OF DYE-DEGRADATION

AND PLANT GROWTH PROMOTION

31

3.5 BIOAUGMENTATION POTENTIAL OF SELECTED

BACTERIAL ISOLATES (POSSESING DUAL TRAITS)

FOR ACCELERATED DEGRDATION OF TEXTILE

DYES

33

3.5.1 Optimization of Dye Degradation Process 33

3.5.2 Biodegradation Potential of Isolate I-15 for Structurally

Different Azo Dyes

34

3.5.3 Bioaugmentation Potential of Isolate I-15 for Textile

Effluent Treatment

35

3.5.4 Identification and Biodegradation of Secondary

Metabolites

35


REACTIVE BLACK 5 AND PLANT GROWTH

PROMOTION

36

3.6.1 Maize Growth Study in Dye Containing Liquid Medium 37

3.6.2 Maize and Barley Growth Study in Soil Under Greenhouse

Conditions

37

3.7 STATISTICAL ANALYSIS 37

4. RESULTS 39

4.1 PHYSICO-CHEMICAL ANALYSIS OF

WASTEWATER SAMPLES

39

4.2 ISOLATION AND SCREENING OF DYE-DEGRADING

BACTERIA

39

4.3 CHARACTERIZATION OF SELECTED BACTERIAL 42

vii

ISOLATES FOR PLANT GROWTH PROMOTION

4.3.1 Indole Acetic Acid Production Assay 42

4.3.2 Phosphate Solubilization Activity 42

4.3.3 ACC-Deaminase Activity Assay 45

4.4 BOX POLYMERASE CHAIN REACTION AND 16S

rRNA GENE ANALYSIS OF BACTERIAL ISOLATES

CARRYING DYE-DEGRADING AND PLANT

GROWTH PROMOTING TRAITS SIMULTANEOUSLY

45


BACTERIAL ISOLATES (POSSESING DUAL TRAITS)


DYES

48

4.5.1 Optimization of Dye Degradation Process 48

4.5.1.1 Effect of different redox conditions 48

4.5.1.2 Effect of different pH levels 56

4.5.1.3 Effect of different co-substrates 56

4.5.1.4 Effect of different temperature conditions 59

4.5.2 Biodegradation Potential of Isolate I-15 for Structurally

Different Azo Dyes

59

4.5.3 Bioaugmentation Potential of Isolate I-15 for Textile

Effluent Treatment

63

4.5.4 Identification and Biodegradation of Secondary

Metabolites

63


RB5 AND PLANT GROWTH PROMOTION

69

4.6.1 Maize Growth Study in Dye Containing Liquid Medium 69

4.6.2 Maize and Barley Growth Study in Soil Under Greenhouse

Conditions

82

5. DISCUSSION 89

5.1 CHARCTERISTICS OF TEXTILE WATEWATER 89

5.2 ISOLATION AND SCREENING OF BACTERIAL 90

viii

ISOLATES POSSESSING DUAL TRAITS OF DYE

DEGRADATION AND PLANT GROWTH

PROMOTION


BACTERIAL ISOLATES POSSESING DUAL TRAITS


DYES

93


REACTIVE BLACK 5 AND PLANT GROWTH

PROMOTION OF BIOENERGY CROPS

97

SUMMARY 102

CONCLUSIONS 109

FUTURE RECOMMENDATIONS 109

LITERATURE CITED 111

APPENDICES 139

ix

List of Tables

Table No. Page

1. Analysis of textile wastewater and sludge samples collected

from industrial sites of different cities

40

2. Isolation and screening of bacteria capable of decolorizing

RB5 dye in liquid medium under static conditions

41

x

List of Figures

Fig. No. Page

1. Structural formula of azo dye Reactive Black 5 26

2. Concentrations of indole acetic acid produced by isolated

bacteria in the presence of L-TRP

43

3. Size of halo ring formed by isolated bacteria after solubilizing

inorganic from of rock phosphate on solid NBRIP medium

after 7 days incubation

44

4. Photographs showing positive P-solubilizing activity by

different bacterial isolates in the form of halo rings formation

on solid NBRIP medium

46

5. Cluster Analysis of the BOX-PCR profiles of the bacterial

isolates showing their genetic differences (%) among each

other and E. coli

47

6. Molecular Phylogenetic analysis by Maximum Likelihood

method showing resemblance of I-15 with other bacterial

strains

49


method showing resemblance of S-10 with other bacterial

strains

50


method showing resemblance of 7.3 with other bacterial

strains

51


method showing resemblance of 11.4 with other bacterial

strains

52


method showing resemblance of AE-5 with other bacterial

strains

53



54

xi

strains



strains

55

13. Effect of different redox conditions on decolorization of RB5

by different bacterial isolates after 24 h

57

14. Effect of different pH levels on decolorization of RB5 by

different bacterial isolates after 24 h

58

15. Effect of different co-substrates on decolorization of RB5 by


60

16. Effect of different temperatures on decolorization of RB5 by


61

17. Percent decolorization of structurally different azo dyes by

bacterial isolate I-15 after 16 h

62

18. Percent decolorization of textile effluent by the

bioaugmentation of I-15 after 48 h

64

19. Percent decolorization of textile effluent spiked with RB5

(100 mg L-1

) by the bioaugmentation of I-15 after 48 h

65

20. Percent decolorization of textile effluent spiked with mixture

of dyes (20 mg L-1

each) by the bioaugmentation of I-15 after

48 h

66

21. Percent degradation of MSM amended textile effluent spiked

with RB5 (100 mg L-1

) by the bioaugmentation of I-15 after

48 h

67

22. Percent degradation of MSM amended textile effluent spiked

with mixture of dyes (20 mg L-1

each) by the bioaugmentation

of I-15 after 48 h

68

23. LC-MS Spectra showing degradation products of RB5 after

decolorization by isolate I-15

70

24. LC-MS Spectra showing degradation byproducts of RB5 after

decolorization by isolate S-10

71

xii


decolorization by isolate 7.3

72


decolorization by isolate 11.4

73

27. Biodegradation of aniline (20 mg L-1

) in different redox

conditions by selected bacterial isolates after 96 h

74

28. Biodegradation of 1-Amino-2-napthol-4-sulphonic Acid (50

mg L-1

) in different redox conditions by selected bacterial

isolates after 48 h

75

29. Dye decolorization trend of RB5 enriched mineral salt media

by maize plants inoculated with different bacterial isolates

grown under laboratory conditions (after 10 days)

77

30. Dye decolorization trend of RB5 containing water by maize

plants inoculated with different isolates grown under

laboratory conditions (after 10 days)

78

31. Effect of different bacterial isolates on growth of maize plants

grown in RB-5 enriched mineral salt media under laboratory

conditions (10 days duration)

79


grown in RB5 containing water under laboratory conditions

(10 days duration)

81

33. Photographical presentation of simultaneous activities of dye

degradation and plant growth promotion of maize plants by

isolates I-15 and S-10 under laboratory conditions. Controls

represents non-inoculated plants grown in MSM with dye

(Control) and MSM without dye (Control 2)

83


irrigated with dye containing water (28 days harvest)

84

35. Photograph showing relevant difference in growth parameters

of maize plants irrigated with dye contaminated water

decolorized by different bacterial isolates (28 days harvest)

86

36. Effect of different bacterial isolates on plant growth of barley 88

xiii

crop irrigated with dye containing water (28 days harvest)

xiv

List of Abbreviations

% percent

°C degree Centigrade

µ micron

µg microgram

µL microlitre

ACC 1-Aminocyclopropane-1-Carboxylic Acid

AMATS Aquatic Macrophytes Treatment System

AOP Advanced Oxidation Process

BOD Biological Oxygen Demand

cm centimeter

CN Carbon and Nitrogen

COD Chemical Oxygen Demand

DCIP Dichlorophenolindophenol

DNA Deoxyribonucleic Acid

EC Electrical Conductivity

g grams

GEM Genetically Engineered Microorganisms

GPAM Glucose Peptone Agar Medium

GPM General Purpose Medium

h hours

HPLC High Performance Liquid Chromatography

IAA Indole Acetic Acid

kg kilogram

L Litre

LC-MS Liquid Chromatography-Mass Spectroscopy

L-TRP L-Tryptophan

mg milligram

mL millilitre

mm millimeter

xv

MSM Minimal Salt Media

NADH Nicotinamide Adenine Dinucleotide

NADPH Nicotinamide Adenine Dinucleotide Phosphate

NBRIP National Botanical Research Institute's Phosphate growth

medium

NCBI National Center for Biotechnology Information

ng nanogram

nm nanometer

OD Optical Density

PCR Polymerase Chain Reaction

PGPB Plant Growth Promoting Bacteria

ppm parts per million

P-solubilizing Phosphate solubilizing

RB5 Reactive Black 5

ROS Reactive Oxygen Species

RP Rock Phosphate

rpm rounds per minute

TDS Total Dissolved Solids

TOC Total Organic Carbon

v/v volume by volume

w/v weight by volume

xvi

ACKNOWLEDGEMENTS

In the name of Almighty Allah, The most Benevolent, The Merciful, The

Supreme and giving, Whose gratefulness bestowed me the opportunity to compile

this seemingly impossible task. Praises be to our Holy Prophet, Muhammad (ملسو هيلع هللا ىلص)

Who through his heart catching teachings enables the mankind to recognize his

creator, Allah, and put the inspiration to acquire knowledge for the benefit of

mankind.

I find no words to express my gratitude to my supervisor, Dr. Azeem

Khalid, Associate Professor/Chairperson, Department of Environmental

Sciences, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, who

always put an urge into me to do my best during each moment of my research

work. Piles of heartedly thanks to Prof. Tariq Mahmood and the members of

my supervisory committee for their sincere and valuable instructions I got

throughout this humble effort. I am also very thankful to Dr. Mussie Y.

Habteselassie, Associate Professor of Soil Microbiology, University of Georgia

for his support in this research. His enthusiasm, encouragement and faith in me

throughout have been extremely helpful. I must also acknowledge Higher

Education Commission and Pakistan Science Foundation for providing me

financial support and opportunities to complete this task.

It is an opportunity for me to pay special thanks to my friends Luqman

Riaz, Usman Ali, and my senior Dr. Shahid Mahmood, who were always

encouraging and helping and stood with me whenever I needed them. My

xvii

appreciation also extends for the support and love of my Teachers, Friends,

Colleagues and Lab. Fellows. I would specially like to thank Dr. Audil Rashid,

Ms. Aniqa Batool, Muzammil Anjum, Samia Muzammil, Wahab Yasir and

Bashir Ahmed. They all kept me going and this work would not have been

possible without them.

Finally, it was never possible for me to lead this work to the completion

without prayers, support and lot of encouragement of my loving father Shafqat

Ali, mother, my brothers, Mubeen Shafqat and Daud Shafqat, and also my

sisters who kept me on the straight path to work with devotion.

Mateen Shafqat

xviii

ABSTRACT

The release of untreated textile effluents in environmental systems and

their subsequent application to cropland pose serious threat to ecological health.

Irrigation with textile effluents may lead to accumulation of toxic metabolites in

plants, resulting in bioaccumulation in food chain. Various bacterial species have

the ability to degrade azo dyes under anaerobic and aerobic conditions; however,

no work has been done to elucidate the role of dye-degrading bacteria for plant

growth promoting activity. The present study was designed with the aim to

isolate and evaluate the bacterial strains carrying dual traits such as azo dyes

degradation and plant growth promotion simultaneously. About 468 bacterial

isolates were collected from textile effluent, sludge and dye contaminated soil

through enrichment of the MSM with Reactive Black 5 dye (100 mg L-1

) under

static conditions. A total of 23 isolates with potential to completely decolorize the

Reactive Black 5 dye (100% in 12-48 h) were further examined for 1-indole-3-

acetic acid production and P-solubilization. Isolates I-15, S-10, 7.3, 11.4, AE-5,

AE-7 and AE-8 exhibited significant production of 1-indole-3-acetic acid (9-21

µg mL-1

), and also showed halo ring formation (diameter 6-11 mm). The selected

isolates that displayed both dye degradation and plant growth promotion were

further evaluated for their ability to degrade a variety of dyes in liquid medium as

well as to remove color of dyes from textile effluents after bioaugmentation.

Complete decolorization of textile effluent was achieved within 6-12 h after

bioaugmentation with I-15 with when supplemented with 0.4% of yeast extract.

MS spectra of the decolorized medium showed the formation of secondary

xix

products, with molecular weights similar to 1,3,4-oxadiazol-2-ol, aniline, 1-

amino-2-naphthol-4-sulfonic acid and some derivatives of benzidine. Isolate AE-

7 was able to achieve highest aniline degradation rate of 92% (20 mg L-1

) within

96 h under static conditions. 1-Amino-2-naphthol-4-sulfonic acid (50 mg L-1

) was

completely degraded by all isolates within 48 h under both static and shaking

conditions. Inoculation of bacteria carrying dual traits to maize (Zea mays) plants

grown in Reactive Black 5 containing liquid medium resulted into complete

decolorization of Reactive Black 5 in 4-10 days, while significantly improving

plant biomass as compared to non-inoculated maize plants. Under soil (pot)

conditions, the maize plant biomass in the case of inoculation with bacterial

isolate 7.3 was significantly higher compared with non-inoculated plants.

Similarly, barley plants inoculated with isolate 7.3 produced significantly greater

plant biomass than non-inoculated plants. The 16S rRNA gene analysis showed

that the isolates I-15, S-10, 7.3, 11.4, AE-5, AE-7 and AE-8 had highest

similarity (99%) with Pseudomonas japonica, Achromobacter xylosoxidans,

Burkholderia ginsengisoli, Pseudomonas alcaligenes, Comamonas testosteroni,

Aeromonas aquatica and Comamonas testosterone, respectively. The findings of

present study show that application of bacterial isolates possessing dual traits

could be used simultaneously for the treatment of textile effluent as well as

increasing biomass production of bioenergy crops such as maize and barley.

1

Chapter 1

INTRODUCTION

Textile manufacturing and dyeing industry is considered as the main

industrial sector responsible for the production of large quantities of wastewater.

Globally, there are 10,000 types of different dyes and their production exceeded

735 metric tons in recent years (Singh, 2015). The most common dyes used in the

textile industry are azo dyes (Vilaseca et al., 2010; Solís et al., 2012; Ito et al.,

2016). Azo dyes are also applied in other commercial sectors, such as food,

cosmetics and printing industry (Saratale et al., 2011; Gürses et al., 2016). Azo

dyes in textile effluent are of great concern because of the potential risk of

contamination of environmental systems. These dyes can inhibit photosynthesis

and reduce dissolved oxygen in water bodies and are also toxic to flora, fauna and

humans (Mahmood et al., 2016). Moreover, the anaerobic breakdown of azo dyes

generates secondary products such as aromatic amines, which are scientifically

proven to have hazardous properties of being carcinogenic and mutagenic (Fu

and Viraraghavan, 2001; Balakrishnan et al., 2016). Therefore, it is necessary to

treat textile wastewater before discharge into wastewater streams.

There are several studies reporting physico-chemical methods for the

treatment of azo dyes discharged from the textile units (Pandey et al., 2007;

Saratale et al., 2011). However, use of such methods have inherent

disadvantages, such as incomplete degradation of dyes and their intermediates,

the production of large amounts of sludge (secondary infection) and the high cost

2

of treatment. Considering these limitations in the physico-chemical methods, the

use of microbial technology has attained attention worldwide because of the

ability of microorganisms to degrade a wide range of recalcitrant dyes and

relatively low treatment cost without production of secondary sludge (Saratale et

al., 2011). The mechanisms responsible for the degradation of these complex

compounds by bacteria are based on the enzymatic conversion. These enzymes

are required specifically for the degradation of dyes due to the ability to attack

the dye molecules (Kandelbauer and Guebitz, 2005).

In general, biodegradation of azo dyes by microorganisms can be

achieved under both reduced (anaerobic or facultative) and oxidized conditions

(Stolz, 2001; Popli and Patel, 2015). The azoreductases, peroxidases and laccases

are some of the enzymes that are primarily responsible for the microbial

decomposition of azo dyes and cleavage of the azo bond (–N=N–). The cleavage

of the azo bond and its relevant aromatic groups generates colorless aromatic

amines under anaerobic conditions, which are known to be more toxic than the

parent compounds (Van der Zee and Villaverde, 2005). These aromatic amines

are further degraded under aerobic conditions, resulting in complete degradation

of azo compounds (Joshi et al., 2008). In addition to bacteria, fungi, algae and

yeast, some plants have been used for decolorization of azo dyes (Mbuligwe,

2005; Kagalkar et al., 2009; Nilratnisakorn et al., 2009; Ong et al., 2009; Page

and Schwitzguébel, 2009; Kagalkar et al., 2010). Plant peroxidases and laccases

are well documented for the effective removal of phenolic compounds from

aqueous solutions (Klibanov et al., 1983; Dec and Bollag, 1994). A number of

3

studies have indicated that peroxidases produced by plants could degrade

substantially different synthetic dyes (Akhtar et al., 2005). Although, the

individual role of plants and microorganisms (particularly bacteria) to degrade

dyes has been reported in different studies (Mahmood et al., 2013; Patil and

Jadhav, 2013; Torbati et al., 2014), but none of the studies reported simultaneous

potential of microorganisms to degrade dyes and to promote plant growth.

A wide array of bacterial species from different genera have been studied

for their beneficial role in plants (Härtel and Buckel, 1996; Kang et al., 2009;

Huang et al., 2012; Qurashi and Sabri, 2012). Likewise, different bacteria have

been studied for their potential for decolorization of a wide range of structurally

different azo dyes (Hsueh et al., 2009; Ayed et al., 2011; Balamurugan et al.,

2011). It has been noticed in different studies that species belonging to the same

genus have some common characteristics. The bacterial species that are capable

of degrading azo dyes also showed potential to promote plant growth. For

instance, Ghodake et al., (2011) reported that Acinetobacter calcoaceticus

degraded dye Amarnath, while in another study, Kang et al., (2009) reported that

Acinetobacter calcoaceticus had capacity to produce gibberellins and solubilize

phosphate, which help in plant growth promotion. Similarly, Aeromonas

hydrophila is repeatedly documented with ability to completely decolorize the

dye Reactive Black 5 (RB5) (Hsueh et al., 2009) and also documented its

capability of fixing nitrogen in the soil (Zhang et al., 1996; Sashiwa et al., 2002).

Likewise, Bacillus cereus has been reported to degrade Reactive Red 195 dye

(Modi et al., 2010), whereas, (Huang et al., 2012) reported it for controlling

4

plants diseases. Citrobacter freundii was shown to be capable of degrading a

number of structurally different azo dyes (Khalid et al., 2009), and also reported

for nitrogen fixation and P-solubilization (Thaller et al., 1995; Nilratnisakorn et

al., 2003). In another study, it was reported that Dyella ginsengisoli had the

ability to decolorize Acid Red GR (Zhao et al., 2010) and also hold ACC-

deaminase activity that assist the plants to survive in extreme conditions

(Hardoim et al., 2012). These studies provide unequivocal evidence that

microbial species carrying plant growth promoting and dye degrading traits can

be used for bioremediation of dye-pollutants. Since in developing countries such

as Pakistan dye-contaminated textile wastewater is also used for irrigation

purpose, it is very likely that the use of dual traits microorganisms may assist in

the removal toxic dyes from the effluent of textile industry and reduce the toxic

effects of dyes for plants.

The present study was designed to isolate bacterial species which have the

potential for efficient decolorization of azo dyes and promote plant growth

simultaneously. These bacterial isolates were applied to bioenergy crops such as

maize and barley to demonstrate their effect on both dye degradation and plant

biomass production. This approach may help the plant to survive in toxic

environments grown for the remediation of pollutants and also help to reduce the

risk of toxicity of dyes to human through bioaccumulation. Previous studies have

shown that azo dyes can pose a serious threat to human and other life through

bioaccumulation in the food chain (Shehzadi et al., 2014; Aravind et al., 2016).

The specific objectives of the study include:

5

Isolation of efficient azo dye decolorizing bacteria through enrichment

technique.

Characterization of azo dye decolorizing bacteria for plant growth

promotion.

Evaluation of selected strains of bacteria for degradation of dyes and

enhancing plant growth under controlled conditions.

Enhancing biomass production of energy crops by using dual trait

bacterial strains.

6

Chapter 2

REVIEW OF LITERATURE

Wastewater released from the textile dying industries has antagonistic

impacts on the water quality. Dyes present in textile wastewater are responsible

for increases in chemical and biological oxygen demand (COD & BOD), total

organic carbon (TOC), salinity, suspended solids along with change in color &

pH. Textile waste water also increases the pollution load by introducing certain

resistant organic compounds, such as azo dyes (Kuberan et al., 2011; Saratale et

al., 2011). High load of such organic compounds in the textile wastewater is

recognized by a high BOD to COD ratio that ranged from 0.2 to 0.5 which is bio-

recalcitrant (Yusuff and Sonibare, 2004; Akan et al., 2009). For instance, 1kg of

cotton requires 0.6–0.8 kg of sodium chloride, 30 – 60 g of dyes and 70–150 L of

water. Therefore, the wastewater generated from the textile industry contains

around 20–30% of reactive dyes ~ 2000 ppm along with significantly higher salt

concentration and other coloring supplements (Babu et al., 2007).

Azo dyes are electron deficient xenobiotic compounds with complex

chemical structures , and are composed of assorted groups of aromatic amines

with single or multiple azo (–N=N–) groups which is a characteristic of absorbing

light in the visible spectrum (Chang et al., 2000). The azo compound (–N=N–) is

substituted with either single or double aromatic groups, having a variety of

substituents, such as amino (–NH2), nitro (–NO2), chloro (–Cl), hydroxyl (–OH),

carboxyl (–COOH) and methyl (–CH3), that determine the type of azo-dye

7

(Zollinger, 1991). Almost all the azo dyes are synthetic, except naturally

occurring 4–4dihydroxyazobenzene (Gill and Strauch, 1983). Many byproducts

of azo dyes (i.e. secondary metabolites/aromatic amines) resulting from the

degradation of the parent dyes are known to have more toxic properties

(carcinogenic and mutagenic) than the parent dyes. These metabolites may

contribute to surface and ground water pollution and are cause of serious

concerns s to the public health (Heiss et al., 1992). Furthermore, the release of

textile effluents containing parent dyes and intermediate compounds into the

surface water results in aesthetic complications, hinders light permeation and

ultimately results in the oxygen deficiency in water bodies (Rajaguru et al., 2002;

De Aragão Umbuzeiro et al., 2005). This phenomenon reduces the photosynthetic

activity of aquatic flora, and ultimately result in the death of aquatic flora and

fauna (Garg and Tripathi, 2011). Furthermore, several studies in previous

literature have also reported that textile effluents containing azo dyes retard seed

germination in those plants in particular, which are known to have critical

ecological roles, in soil management and provision of habitat for wildlife (Calow,

2009; Ghodake et al., 2009).

During the last few decades, extensive studies were carried out to

understand the eco-toxicological consequences of azo dyes with distinctive

emphasis on food colors belonging to azo compounds. These investigations were

carried out at different biotic levels ranging from assessing the effects of dyes on

the bacterial activity in wastewater treatment systems, to their toxicity in

mammals (Clarke and Anliker, 1980). Exposure to azo dyes may result in acute

8

reactions in human, such as the severe allergic reactions i.e.,contact dermatitis or

eczema (Specht and Platzek, 1995). Furthermore, free amino groups in azo dye

are known to possess carcinogenic and mutagenic activity and therefore, workers

in dye processing and manufacturing industries are at serious health risk (Zee,

2002).

Enormous studies have been carried out to investigate the efficiency of

different physico-chemical processes for the treatment and color removal from

azo containing effluents (López-Grimau and Gutiérrez, 2006). Numerous studies

have reported physical techniques for the treatment of textile effluent, e.g.

coagulation/flocculation; however, such techniques resulted in constrained

industrial application as it produces large amounts of sludge, which further

requires treatment for its safe disposal (Pandey et al., 2007). These restrictions

lead to the development of advanced oxidation processes (AOP’s) and

alternatives biological methods as. In AOP’s, strong oxidizing agents are used, as

a source of OH- radical for the breakdown of recalcitrant and hazardous

pollutants (Alaton et al., 2002; Al-Kdasi et al., 2004). Since the legislations have

become strict for waste discharge, biological methods of textile effluent treatment

has attained much intention. Biological treatments methods are described as

economically feasible, ecological friendly nature and possibility of their

application to the structurally diverse group of recalcitrant dyes (Saratale et al.,

2011). Microorganisms like bacteria, fungi and algae have successfully been

optimized for the textile wastewater treatment systems. Microbial degradation of

pollutants comprises approaches like acclimatization in which microbes adjust

9

themselves to the xenobiotics and naturally develop resilience in new strains.

This results in the transformation of toxic and complex compounds into smaller

and less toxic forms. The overall process that carries out the breakdown of such

persistent compounds facilitated by the bacterial strains is based on the enzymatic

processes responsible for biotransformation (Saratale et al., 2007). These

enzymatic actions of biotransformation are specifically required for the

degradation of dye effluents because of their specific nature to specifically attack

the dye molecules (Kandelbauer and Guebitz, 2005). Moreover, aquatic

macrophytes treatment system (AMATS) has become a well-established and cost

effective technique for the phytoremediation of numerous pollutants, including

azo dyes present in the industrial wastewaters (Priya and Selvan, 2014).

2.1. BACTERIAL DECOLORIZATION OF AZO DYES

Microbial decolorization of azo compounds could be achieved using

bacterial strains belonging to different taxonomical groups, under either aerobic,

anaerobic or facultative conditions (Taskin and Erdal, 2010). The chemical

structures and their reducing capacities (i.e. electron withdrawing) makes the azo

dyes highly recalcitrant to the oxidizing agents and enzymatic actions of

microbial oxidases (Grekova-Vasileva et al., 2009). Moreover, type and

arrangement of the functional group, as well as a number of azo-bonds present in

the compound also influence the process of degradation of dyes. Hence the

variations in decolorization potential of different bacterial strains can be

attributed to the complex chemistry of dyes, differences in bacteria culture media

as well as genetic variations in bacterial cell (Rani et al., 2009).

10

The azo-reductases, bacterial peroxidases, laccases are some of the

enzymes, which cause reductive split of azo bond (–N=N) and are the main

derivatives of different mechanisms for the microbial breakdown of azo-dyes

(Stolz, 2001). However, the breakdown of azo-bonds and their related aromatic

groups may form colorless compounds termed as aromatic amines, which are

known to be more toxic than the parent compound (Van der Zee and Villaverde,

2005). The resulting aromatic amines (intermediate metabolites) are further

degraded via aerobic or anaerobic microbial processes to achieve complete

degradation of the azo compounds (Joshi et al., 2008). In past, substantial

investigations have been conducted to determine the effectiveness of

decolorization mechanisms of bacterial strains, which belong to various groups

until complete biotransformation of the diverse group of azo dyes could be

achieved in future.

2.1.1. Bacterial Decolorization of Azo Dyes Under Anaerobic Conditions

Anaerobic decolorization of azo dyes is generally considered favorable

because of its potential to cleave the azo-bond, that leads to decolorization of

dyes (Luangdilok and Panswad, 2000). Dye decolorization process under

anaerobic conditions usually demands a source of organic carbon (co-substrate)

like acetate, ethanol, glucose and sometimes more complex organic co-substrates

such as yeast extract, tapioca starch or peptone, etc. Depending upon the ambient

environmental conditions and types of the bacteria; different biochemical

mechanisms could be responsible for reductive decolorization of azo dyes. In

general, the mechanism usually involve the reductive cleavage of azo bond into

11

their respective amines with the aid of enzyme azoreductases (Chang et al.,

2000). Electrons are transferred by a redox mediator which behave as an electron

shuttle between the intracellular reductase and the extracellular dye (Chacko and

Subramaniam, 2011). Methyl viologen is considered the most efficient redox

mediator with reduced shuttling potential, discreetly increase the extent of Acid

Red 27 azo dye reduction by bacterial strain BN6 (identified as Sphingomonas

sp.) (Kudlich et al., 1997). Similarly, Bacteroides thetaiotaomicron has increased

reduction of Acid Yellow-23 by 4.5 times, competing to other redox mediators

with higher potential (Chung et al., 1978). Reduced electron shuttles have

potential to transmit electrons to azo dyes which serve as electron-deficient

compounds (Hong and Gu, 2009; Van der Zee and Cervantes, 2009), resulting in

the production of colorless compounds, i.e., aromatic amines (Chang et al.,

2004). Electron shuttling compounds are of important concern as they can cross

the cell membranes. Bacterial cell membranes form a barrier for redox mediators

and dyes and prevent them from entering into the cell; therefore, azoreductase

activity could be reduced as compared to the azoreductase activity of cell extract

(Kudlich et al., 1997; Russ et al., 2000). Moreover, it has also reported that

reduction mechanism of E. coli, could be based on the dissemination over cell

membrane carried out by the combination of intracellular enzymes and a

mediator. Such mediator-reliant azoreductase enzyme was distinguished as two

cytosolic oxygen-intensitive-nitroreductases, that act as quinone reductases. The

mediator involved was laws one which is identified as the only quinone having a

capacity to get reduced by the enzymatic actions (Rau and Stolz, 2003).

Lawsone's obvious capability to cross the cellular membrane is most likely

12

identified with its little size and its relative lipophilicity. Based on the tendency

of intracellular reductases identified from different strains, there could be other

shuttle compounds carrying membrane crossing potential that serve as effective

mediators for efficient azo dye reduction. Therefore, it could be concluded that

azoreductase activity of different bacterial cells is primarily related to the

expression of different genes, representing that azoreductase enzymatic

mechanism could be linked to additional reductase enzymes, subjected to the

class of microorganism as well as culture conditions.

2.1.2. Bacterial Decolorization of Azo Dyes Under Aerobic Conditions

During last decade, extensive research has been carried out to study

bacterial strains capable of decolorizing azo dyes aerobically. Most of the

bacterial strains are likely to carry out decolorization activity only in the

presence of organic carbon source, probably due to incapability of consuming

carbon or nitrogen directly from the dye(Stolz, 2001). Oxygenase enzymes are

involved in the catalytic incorporation of the oxygen atom in aromatic ring of azo

dyes before splitting the ring (Sarayu and Sandhya, 2010). Moreover, there is a

limited number of known bacteria which are capable of cleaving azo bonds

through reduction and consume aromatic amines as carbon source for their

growth (Lin et al., 2010). Pigmentiphaga kullae K24 as well as Xenophilus

azovorans KF46 are reported to have potential to consume carboxy-orange II and

carboxy-orange-I, respectively under aerobic condition (Kulla et al., 1983).

However, these strains lack the potential to utilize similar compounds like

sulfonated dyes. The mechanism of aerobic decolorization involves the transfer

13

of electrons to the final electron acceptor by electron transport chain; the acceptor

is azo dye (reduced compound) which is consequently, decolorized through

reduction and re-oxidized flavin nucleotide (Robinson et al., 2001). For

intracellular dye reduction diffusion of molecule across the cellular membrane is

compulsory. However, presence of the sulfonate group could reduce the transport

of a molecule through the cell membrane (Doble and Kumar, 2005). It was also

reported that use of co-factors like Nicotinamide Adenine Dinucleotide

Phosphate (NADPH)/ NADH by aerobic azo reductase enzymes can potentially

cleave both carboxylated growth substrates and the sulfonated structural

analogues (Nachiyar and Rajakumar, 2005). Two Pseudomonas strains KF46 and

K22 are known to possess such kind of flavin-free system of azoreductase

enzymes (Zimmermann et al., 1984).

2.2. FACTORS REGULATING THE PROCESS OF

DECOLORIZATION

Although the rate of microbial decolorization generally depends on the

structure of azo dyes, as well as the added organic carbon source (Stolz, 2001).

Furthermore, operational conditions also influence bacterial potential of dye

decolorization, such as the presence of high salt concentrations, varying dye

concentration, temperature, oxygen and pH (Solís et al., 2012).

Presence of salt remnants in the textile wastewater, primarily the

concentration of sodium ions, affects the rates of bacterial degradation because

they can cause plasmolysis of bacterial cells, ultimately restraining bacterial

activities (Anjaneya et al., 2011; Gopinath et al., 2011). However, there are

14

certain bacterial strains which carry enhanced dye decolorization mechanisms in

higher proportions of salts (Meng et al., 2012). Two isolated strains from sea

water namely, Psychrobacteralim entarius and Staphylococcus equorum are

reported to decolorize different dyes (RB5, Reactive Blue BRS and Reactive

Golden Ovifix) in the prevalence of broad concentrations of salt (0 – 100 g NaCl

L−1

) (Khalid et al., 2012).

The concentrations of azo dyes present in the system is also a contributing

factor for microbial decolorization of dyes, possibly due to the toxicity of dyes to

the bacteria or inappropriate cell to dye ratio, in addition to obstruction of active

sites of azoreductases by dye molecules with diverse structures (Saratale et al.,

2009). For instance, Lysinibacillus sp. completely decolorize Metanil Yellow

dye up to 200 ppm but decolorization rate was limited to only 62% 1000 ppm of

dye concentration (Anjaneya et al., 2011).

The pH conditions of a medium in which microbes are applied, contribute

significantly to decolorization of azo-dyes. Previous literature studies have

reported the optimum range of pH which is from pH 6.0 to 10.0 (Kılıç et al.,

2007; Gou et al., 2009). The microbial decolorization rates are accelerated at

optimum pH, and decline sharply at high alkalinity or strong acidic conditions. It

is believed that transportation of dye molecules across the cell membrane may be

related to the varying effects pH, and is reflected by the controlling phase for

bacterial decolorization process (Chang et al., 2001; Kodam et al., 2005).

Microbial cleavage of azo bond may raise pH due to production of secondary

metabolites like aromatic amines which are more basic in nature than the parent

15

compound (Willmott, 1997). Usually, pH variations within range of 7.0–9.5 have

little or no influence on dye decolorization process. Nevertheless, dye

decolorization rates were accelerated approximately 2.5 times as pH raised from

acidic (5) to neutral, although the decolorization rate became unresponsive to

further rise in pH up to 7.0 – 9.5 (Chang et al., 2001). However, there are certain

strains that behave independent of the pH variation, for instance, complete

decolorization was achieved by the application of Galactomyces geotrichum and

Bacillus sp. in consortium to degrade Brilliant Blue G (azo-dye) and showed no

pH dependency within a range of 5 – 9 (Jadhav et al., 2008). Where, Citrobacter

sp., completely decolorized Reactive Red 190 and showed no dependency on

relatively wider pH range of 4 – 12 (Wang et al., 2009; Wang et al., 2009). The

ability of bacteria to tolerate higher pH conditions could be appropriate candidate

for their potential application in the effluent treatment systems of dye processing

units (Aksu and Dönmez, 2003; Wang et al., 2009).

Temperature is an important parameter in the operational condition of

different processes related to microbial activity. Investigations concerned with

the activation energy required during the bacterial decolorization of azo dye have

been conducted in past. Such studies helped in determined the temperature

ranges which are necessary for the continuation of the process (Dos Santos et al.,

2007). Furthermore, it also known that the temperature modification results in an

abrupt shift in the activation energy of bacterial functioning (Yu et al., 2001). In

addition, influence of temperature on the stages of microbial growth and reaction

kinetics have additionally been testified (Angelova et al., 2008). Furthermore, it

16

has also been witnessed that the rates of decolorization accelerated proportionally

until reaching optimum temperature range, and then started declining along with

the slight reduction in temperature. The decline in decolorization rate at a high

temperature is attributable either to the cell destruction or the denaturation of

azoreductase enzyme (Saratale et al., 2009). Efficient decolorization of the dyes

is generally attributed to the optimum incubation temperature specified for every

microbial species (Asad et al., 2007; El Ahwany, 2008; Bardi and Marzona,

2010).

Finally, it is worth emphasizing the remarkable potential of some dye

decolorizing bacteria for operating in extreme temperatures. For instance, a

bacterial strain (B. licheniformis LS04) having laccase activity was found to be

resistant to extreme temperature and high pH. These bacteria with the laccase

enzyme efficiently decolorized a variety of structurally diverse dyes such as

Indigo Carmine, Reactive Blue 19 and RB5 under normal to alkaline conditions.

Bacterial strains with such rare and extraordinary inherent properties should be

further investigated for widespread industrial application (Lu et al., 2012).

2.3. PHYTOREMEDIATION OF AZO DYES

Phytoremediation has gained much attention because of being as an

effective technique for the decolorization of textile effluents containing azo-dye.

This process is known to be cost-effective with aesthetic advantages, applicable

for long terms, and can be applied directly at the contaminated sites (Pletsch,

2003). The decolorization of dyes by plants may be carried out by following

ways: (i) release of photosynthetic carbon by rhizo-deposition results in the

17

increased growth of microorganisms in the dye-contaminated water, (ii) release

of dye degrading enzymes like lignin peroxidase or manganese dependent

peroxidase and laccase by the root exudates, (Vyas and Molitoris, 1995), (iii)

formation of aerobic and anaerobic microenvironments in the rhizospheric zone

which may stimulate or promote biodegradation of azo dyes (Ong et al., 2009),

(iv) adsorption of azo dye from wastewater by the plants, which could increase

the interaction of azo dye with the microorganisms (Zhou and Xiang, 2013).

Plant peroxidases and laccases are well documented for their efficient

removal of phenolic compounds from aqueous solutions (Klibanov et al., 1983;

Dec and Bollag, 1994). In addition to plants, the Laccases enzymes are also

extensively present among fungi and bacterial species (Mayer and Staples, 2002;

Claus, 2003). Several different studies have revealed that peroxidases synthesized

by plants could effectively breakdown diverse forms of synthetic dyes (Akhtar et

al., 2005). Moreover, polyphenol oxidases of the plants origin are also effective

in the decolorizing mixture of different dyes (Khan and Husain, 2007).

Decolorization of Acid Blue 92 by duckweed plant (Lemna minor) have been

studied, and results indicated that superoxide dismutase enzyme converts the

toxic superoxide radicals into H2O2, which are further detoxified by antioxidant

enzymes like chloramphenicol acetyl-transferase and peroxidase. Plant exposure

to 20 mg L-1

of Acid Blue 92 for 7 days resulted in increasing enzymes activity

up to 116% and 67% respectively for chloramphenicol acetyl-transferase and

peroxidase in contrast to control (Khataee et al., 2012). The amount of the

reactive oxygen species (ROS), generated increases in the presence of

18

xenobiotics, which probably stimulate the synthesis of chloramphenicol acetyl-

transferase and peroxidase (Geoffroy et al., 2004). in particular, a higher dye

concentrations could result in enhanced synthesis of ROS and stimulation of

enzyme activity, which enhances the plant tolerance against dyes. Similarly,

plant-plant coalition of Aster amellus Linn along with Glandulariapulchella

(Sweet) Tronc. facilitate the complete removal of color of Remazol Orange 3R in

36 h (h). After degradation of dye, A. amellus showed introduction in the enzyme

activities of dichlorophenolindophenol (DCIP) reductase enzyme and veratryl-

alcohol oxidase enzyme while G. pulchella exhibited introduction of tyrosinase

and laccase, signifying their contribution to dye degradation. Association of both

plants exhibited stimulation in the enzymatic actions of different enzymes like

veratryl alcohol oxidase, lignin peroxidase, tyrosinase, laccase, and DCIP

reductase. These two dissimilar combinations of enzymes produced by A.

amellus and G. pulchella perform synergistically in combination subsequently

enhancing dye decolorization (Kabra et al., 2011). In the root cells of Typhonium

flagelliforme, Tagetespatula, riboflavin reductase and azo reductase enzymes

have also shown remarkable induction in their activities along with veratryl

alcohol oxidase, tyrosinase, lignin peroxidase, laccase and DCIP reductase. Such

enzymes also exhibited efficient degradation of Brilliant Blue R and Reactive

Blue 160 (Kagalkar et al., 2010; Telke et al., 2011; Patil and Jadhav, 2013).

A limited number of studies reporting the biodecolorization of azo dyes

19

by the plants are available so far. For example, researchers have used Phragmites

australis to investigate its potential for degradation Acid Orange-7 (Ong et al.,

2009). Where Typha angustifolia L. was also examined for the decolorization of

reactive dyes (Nilratnisakorn et al., 2009). Furthermore, 75% color reduction was

reported in wetlands grown with coco yam plants (Mbuligwe, 2005). Plant and

tissue cultures of Blumea malcolmii H and Typhonium flagelliforme were

effective in degrading the Reactive Red 2, Red HE8B, Brilliant Blue Malachite

Green, Methyl Orange and Direct Red 5B (Kagalkar et al., 2009; Kagalkar et al.,

2010). Studies on Rumex acetosa provided promising results for decolorization

of secondary metabolites like sulfonated aromatic compounds (Page and

Schwitzguébel, 2009).

2.4. BACTERIAL ASSISTED PHYTOREMEDIATION OF AZO DYES

The individual role of plants and microorganisms in the dye degradation

has been reported in different studies (Mahmood et al., 2013; Patil and Jadhav,

2013; Torbati et al., 2014). However, the combined applications of different

bacterial strains and plant species have also been used separately to study

decolorization and degradation of azo-dyes (Tony et al., 2009; Saratale et al.,

2010; Watharkar and Jadhav, 2014). The combination of soil rhizobacteria with

dye degrading plants have exhibited a promising result in the degradation of

textile dyes (Watharkar et al., 2013). Microbial colonization in the plant

rhizospheric zone is usually attributed to the synergistic relationship between the

two organisms (Doran, 2009). Synergism among various species belonging to

different classes has been reported in scientific literature for their beneficial roles

20

carried out for each other. Plants in synergism with bacterial strains have shown

extraordinary metabolic capabilities, absorption potential along with transport

systems that can uptake contaminants from soil and water (Chaudhry et al.,

2005). Moreover, some rhizobacteria can affect the plants in a positive way by

enhancing growth and root development (Glick, 2010). The consortium of

Petunia grandiflora Juss. and rhizospheric bacteria Bacillus pumilus was efficient

in decolorization of reactive Navy Blue RX with less toxic metabolites produced.

The consortium also revealed enhanced stimulation of the enzymatic actions of

riboflavin reductase, tyrosinase, laccase and lignin peroxidase in tissue cultured

plant roots while tyrosinase, laccase and riboflavin reductase in the bacterial cells

(Watharkar et al., 2013). Similarly, Remazol Black B, a highly sulfonated

reactive azo dye was efficiently degraded individually by Zinnia angustifolia

(Kunth) as well as with its rhizospheric assisted bacterium Exiguobacterium

aestuarii strain-ZaK. However, their association was more effective and

demonstrated substantial induction of DCIP reductase, lignin peroxidase, laccase

and tyrosinase during dye decolorization process. In addition, the application of

plant and bacteria in the consortium led to the formation of non-toxic metabolites

after dye degradation (Khandare et al., 2012). In another study, synergism of

plant (Portulaca grandiflora) and bacteria (Pseudomonas putida) exhibited

complete decolorization of Direct Red 5B, whereas Portulaca grandiflora and

Pseudomonas putida failed to decolorize completely within the given time in lab

conditions. Significant production of tyrosinase, 2,6-dichlorophenol indophenol

reductase, lignin peroxidase, and riboflavin reductase enzymes was detected in

roots of P. grandiflora during dye decolorization; while enzymatic reactions of

21

veratryl alcohol oxidase, 2,6-dichlorophenolindophenol reductase and laccase

found to be induced in P. putida (Khandare et al., 2013).

2.5. DECOLORIZATION OF AZO DYES BY PLANT GROWTH

PROMOTING BACTERIA

The use of PGPB's in order to enhance the nutrient uptake and for

diseases prevention in plants is under practice for years. Phytoremediation is

novel and well known approach for the removal of contaminants from

environmental systems. However, use of solitary plants for bioremediation

purpose creates numerous constraints. Lately, the introduction of PGPB-assisted

phytoremediation has been investigated for the removal of contaminants from the

soil. Of all the existing pollutants, the intense impacts of organic pollutants and

heavy metals have raised concerns worldwide (Zhuang et al., 2007). Bacteria-

assisted phytoremediation of textile effluents containing azo dyes have been

successfully investigated (Khandare et al., 2013; Watharkar et al., 2013;

Shehzadi et al., 2014), however, only a few studies have been conducted so far

using PGPB in association with plants for this purpose (Kadam et al., 2014). The

addition of PGPB could intensify the removal efficacy of organic contaminants

probably, by increasing the plant germination and its endurance in highly

contaminated soil along with promoting the plants to grow rapidly and enhancing

biomass production (Huang et al., 2004). Ethylene is identified as an important

hormone for plant (Deikman, 1997), while under stress conditions extreme

production of ethylene can depress the plant growth during various stages of

development (Morgan and Drew, 1997). PGPB’s have an optimistic influence on

22

plant growth by utilizing amino-cyclopropane carboxylic-acid (ACC), the instant

precursor of ethylene production, through production of 1-aminocyclopropane-1-

carboxylate-deaminase (ACC deaminase) therefore, drops the ethylene release in

plants under stress (Glick, 2010). This supportive mechanism of PGPB could

help the plant to survive in toxic environments grown for the remediation of

pollutants. A number of bacterial strains from different genera have been

investigated to probe their beneficial role in the growth of plants (Härtel and

Buckel, 1996; Kang et al., 2009; Huang et al., 2012; Qurashi and Sabri, 2012).

Other studies also evaluated their potential to decolorize a wide variety of

structurally different azo dye by (Hsueh et al., 2009; Ayed et al., 2011;

Balamurugan et al., 2011). There are numerous other scientific studies that have

reported bacterial species which are not only capable of degrading different azo-

dyes but also possess traits for plant growth promotion separately. For instance,

Acinetobacter calcoaceticus is known to degrade dye Amarnath (Ghodake et al.,

2011) and for gibberellins production and P-solubilization (Kang et al., 2009).

Similarly, Reactive Black 5 degradation (Hsueh et al., 2009) and nitrogen

fixation (Zhang et al., 1996; Sashiwa et al., 2002) can be accomplished by spp.

Aeromonas hydrophila; Reactive Red 195 decolorization (Modi et al., 2010).

Likewise, the control of disease (Huang et al., 2012) facilitated by Bacillus

cereus; and the decolorization of Disperse Orange 3, Reactive Black 5, Direct

Red 81 and Acid Red 88 (Khalid et al., 2009) and nitrogen fixation/P-

solubilization (Thaller et al., 1995; Nguyen et al., 2003) by Citrobacter freundii;

is also documented in literature. Similarly, the decolorization of Acid Red GR

(Zhao et al., 2010) and ACC-Deaminase activity (Hardoim et al., 2012) by

23

Dyella ginsengisoli. Using Klebsiella oxytoca, decolorization of Methyl Orange

(Yu et al., 2012) and ACC-Deaminase activity was also reported in different

studies (Babalola et al., 2003; Wu et al., 2011). Studies regarding decolorization

of RB5, Acid Red 88, Disperse Orange 3, Direct Red 81, (Khalid et al., 2008)

and chitinase production (Faramarzi et al., 2009) by Massilia timonae are also

described in scientific literature. Moreover, decolorization of Reactive Black 5,

Reactive Red 141 by Pseudomonas putida (Telke et al., 2008; Mahmood et al.,

2013) and indole acetic acid (IAA) (Çakmakçi et al., 2007), gibberellic acid

production (Humphry et al., 2007) by Pseudomonas putida and Rhizobium

radiobacter, respectively is also reported.

Therefore, proper treatment of textile effluents contaminated with azo

dyes and their secondary metabolites is considered an extremely important step

prior to their discharge into aquatic environmental systems. This review focuses

on different cost-effective and amicable biological mechanisms by which azo

dyes and their intermediate compounds could be detoxified, or be degraded into

relatively, less toxic forms. Possible opportunities are also discussed to reuse azo

dye contaminated wastewater (treated with PGPB’s) for irrigation of non-edible

crops (energy crops) to enhance biomass production by using dye degrading

plant growth promoting bacteria.

24

Chapter 3

MATERIALS AND METHODS

The present study was conducted to isolate bacterial species capable of

decolorizing textile dyes, while having the ability to promote plant growth. The

effectiveness of these bacterial isolates was also tested in the laboratory and

greenhouse conditions to improve biomass production of bioenergy crops, such

as maize and barley, grown with dye-contaminated water.

3.1. COLLECTION AND ANALYSIS OF TEXTILE WASTEWATER

Several samples of wastewater and sludge were collected from textile

dyeing units of Rawalpindi, Faisalabad, and Sheikhupura districts, Punjab,

Pakistan. Samples were taken from wastewater streams of textile industrial units

located at different sites in each district. All samples were transported

immediately after collection in pre-sterilized jars (250 ml) and stored at 4 oC

before analysis. These wastewater samples were analyzed for pH, electrical

conductance (EC), total dissolved solids (TDS) and color intensity (dye level).

The pH, EC and TDS were analyzed using the Multimeter (Crison MM-40+).

The color intensity of the wastewater samples was measured as absorbance with a

spectrophotometer (BMS-VS-1100) at 600 nm.

3.2. ISOLATION AND SCREENING OF DYE-DEGRADING

BACTERIA

Several bacterial isolates capable of azo dye degradation were isolated

25

from the textile wastewater and sludge collected from wastewater streams of

textile units. Additionally, some bacterial isolates were isolated from the

rhizosphere soil of maize crop grown with the dye-contaminated wastewater in

the premises of in the premises of textile units.

3.2.1. Dye Chemicals and Culture Medium

Reactive Black 5 (RB5) was used as a primary dye for the isolation and

biodegradation, owning to its complex chemical structure and recalcitrant

properties (Fig. 3.1). Four other textile dyes, including Direct Blue 3R, Direct

Pink B, Sunfix Red S3B and Sunfix Yellow were also used in this study. The

dyes were obtained from BASF Pakistan. Mineral salt medium (MSM) was used

to grow the bacterial cultures and for the decolorization studies (Khalid et al.,

2008). The MSM contains (g L-1

) sodium chloride 1.0, calcium chloride 0.1,

magnesium sulfate 0.5, monopotassium phosphate 1.0, disodium phosphate 1.0

and yeast extract 4.0. For the isolation of dye-degrading bacteria, azo dye RB5 at

a concentration of 100 mg L-1

was added to the medium. All chemicals used in

MSM except yeast extract were purchased from Sigma-Aldrich and VWR, USA.

Yeast extract was purchased from Thermo Scientific UK.

3.2.2. Isolation of Dye-Degrading Bacteria

Bacteria were isolated through enrichment of the medium (MSM) with

RB5 azo dye using dilution plate technique. For this purpose, MSM broth was

prepared in conical flasks (100 ml capacity) and autoclaved at 121 oC for 20

minutes. After cooling, the medium was spiked with RB5 (at rate of 100 mg L-1

)

26

Figure 1: Structural formula of azo dye Reactive Black 5 (Source: Sigma-

Aldrich)

27

azo dye in a clean air-cabinet. About 10 ml of textile wastewater or equivalent

amount (10 g) of sludge or rhizospheric soil was added to each conical flask

containing dye-amended broth. The flasks were incubated at 30±1 °C for a period

of 5 days under static conditions.

By following dilution plate technique, bacterial colonies were obtained

from each flask through dilution plate technique on agar medium containing 100

mg L-1

of RB5. The agar plates were incubated at 30 °C for 24 h before selecting

bacterial colonies. Bacterial colonies showing prolific growth on the agar

medium were selected for testing their ability to decolorize azo dyes in liquid

medium. Overall, 468 bacterial isolates (134 from wastewater, 196 from sludge

and 138 from rhizosphere soil) were collected and initially kept on agar plates at

4 oC before using for decolorization experiments.

3.2.3. Screening of Efficient Dye-Degrading Bacterial Isolates

The bacterial isolates capable of efficiently degrading RB5 azo dye in

liquid medium were screened by inoculating the autoclaved broth medium

containing 100 mg RB5 L-1

. The initial screening was done in 2 mL sterilized

Eppendorf tubes. The medium (dye solution) to inoculum ratio was 100:1 (v/v).

Eppendorf tubes were incubated at 30±1 °C. After 24 and 48 h, color intensity

was measured by spectrophotometer at 597 nm wavelength. Percent color

removal was calculated using the formula given below.

Percent color removal = (Initial absorbance – Final absorbance) × 100

Initial absorbance

28

Based on color removal efficiency, 23 bacterial isolates (out of 468) were

selected to confirm their decolorization efficiency in 20 mL glass vials. Inoculum

for each selected isolate was prepared in MSM and a uniform cell density of

0.8±0.02 (optical density measured at 600 nm) was maintained for each isolate

before using them for inoculation. Population density at this OD was between

108-10

9 cfu mL

-1. The inoculum was added to sterilized glass vials at a ratio of

100:1 (medium to inoculum ratio, v/v), making total volume as 20 mL. Azo dye

RB5 was used at rate of 100 mg L-1

. The vials were incubated at 30 ºC for 48 h

under static conditions. Aliquots were collected after every 12 h interval. The

decolorized medium was centrifuged at 9800 rpm for 10 minutes at 4 ºC to get a

cell free supernatant. Percent decolorization was determined as described above

by measuring the absorbance of the dye medium.

3.3. CHARACTERIZATION OF SELECTED BACTERIAL ISOLATES

FOR PLANT GROWTH PROMOTION

After confirming the decolorization potential, all these 23 bacterial

isolates were further tested for their plant growth-promoting characteristics, such

as indole acetic acid production, P-solubilization and ACC-deaminase activity.

3.3.1. Indole Acetic Acid Production Assay

Indole acetic acid (IAA) production by the selected bacterial isolates was

determined by colorimetry method. General purpose agar medium (GPAM) was

used as bacterial culture for the detection of auxin production (Wollum, 1982).

GPAM contains (g L-1

) glucose 0.75, ammonium sulphate 0.25, potassium

29

hydrogen phosphate 0.25, peptone 0.25, magnesium sulphate hepta-hydrate 0.05.

Selected isolates were inoculated on glucose peptone liquid medium in

Erlenmeyer flasks containing 0.1% (w/v) filtered sterilized L-TRP solution. Flasks

were incubated at 30±1 °C for 24 h with shaking. The flask contents were then

filtered through Whatman filter paper No. 2, prior to quantify auxin production as

IAA equivalents (Khalid et al., 2004). To quantify IAA production, protocol

described by (Sarwar et al., 1992) was used. Briefly, 3 mL of the filtrate added

with 2 mL Salkowski reagent (2 mL of 0.5 M FeCl3 + 98 mL 35% HClO4). The

tubes containing the sample and reagent solution were kept in dark for 30 minutes

for color development, of which the absorbance was measured

spectrophotometrically at 535 nm (detection limits 0.2–45 µg mL-1

). Similarly,

standard solutions of known IAA concentrations were also analyzed to establish

the standard curve by analyzing their color intensity.

3.3.2. Phosphate Solubilization Activity

Inorganic phosphate-solubilizing (P-solubilizing) activity of dye

decolorizing bacteria was qualitatively determined by following the protocol

documented by (Nautiyal, 1999). According to the protocol, rock phosphate (RP)

(3Ca3 (PO4) 2CaF2) was added to the agar medium instead of calcium phosphate

as an insoluble form of inorganic phosphate. Qualitative assay was carried out by

using general purpose medium (g L-1

): glucose 0.75; ammonium sulphate 0.25;

dipotassium phosphate 0.25; peptone 0.25; magnesium sulfate heptahydrate 0.05

and RP 5.0. Bacterial culture was placed on newly prepared medium (National

Botanical Research Institute's phosphate growth medium, NBRIP) on agar plates

30

and incubated for 7 days at 30 °C. A clear halo formation around grown colonies

after 7 days indicated the positive P-solubilizing bacterial activity. The size of

halo ring formed around colonies was determined (Nautiyal, 1999) by measuring

its diameter for the qualitative efficacy of bacterial isolates to solubilize RP (Baig

et al., 2012). The assay was performed thrice for all isolates.

3.3.3. ACC-Deaminase Activity

ACC (1-aminocyclopropane-1-carboxylate) deaminase activity was

determined by using Dworkin Foster (DF) minimal salt medium (Dworkin and

Foster, 1958) containing ACC as sole nitrogen source. ACC was prepared with a

concentration of 3 mM (millimolar) and was filter sterilized using membrane

filter sizing 0.2 µm. DF minimal salt media contains (g L-1

) monopotassium

phosphate KH2PO4 4.0, disodium phosphate 6.0, magnesium sulfate heptahydrate

0.2, glucose 2.0, gluconic acid 2.0 and citric acid 2.0. Trace elements were also

added in DF minimal salt media with the following proportions L-1

: iron II sulfate

heptahydrate 1 mg, boric acid 10 µg, manganese (II) sulfate monohydrate

11.19 µg, zinc sulfate heptahydrate 124.6 µg and copper (II) sulfate pentahydrate

78.22 µg. Ammonium sulfate (2 g L-1

) was alternately used with ACC as nitrogen

source for positive controls. DF minimal salt media containing flasks (50 mL

culture medium in 250 mL flask) inoculated with bacterial isolates were

incubated on orbital shaker (120 rev min-1

) at 28 ±1º C for two days. After

incubation, 1 mL aliquot was taken from the culture and was added to newly

prepared ACC (300 µL) containing DF minimal salt media as nitrogen source

(instead of ammonium sulfate) and were incubated on orbital shaker (120 rev

31

min-1

) at 28 ±1º C for another two days. After incubation, 10 µL of aliquots from

each flask were plated into solid DF minimal salt medium (with ACC as nitrogen

source) and incubated at the same temperature as previous incubations for five

days.

3.4. BOX POLYMERASE CHAIN REACTION AND 16S rRNA GENE

ANALYSIS OF BACTERIAL ISOLATES CARRYING DUAL

TRAITS OF DYE-DEGRADATION AND PLANT GROWTH

PROMOTION

Based on concurrent ability of selected bacterial isolates to effectively

decolorize azo dyes and exhibiting plant growth promoting characteristics, seven

isolates were selected for subsequent experiments and identified through gene

analysis. Cell pellets of the selected bacterial isolates were obtained through their

sedimentation by centrifuging at 10,000 rpm for 9–10 minutes. Extraction of the

genomic Deoxyribonucleic acid (DNA) of each isolate was done following

manufacturer instructions of DNAzol Kit (MRC, Inc). Post extraction DNA

concentration was confirmed with Nanodrop prior to setting reaction. BOX-

polymerase chain reaction (BOX PCR) was performed to avoid repetition of

bacterial isolates in further studies (Rademaker et al., 2004). BOX A1R primer

(5′-CTA CGG CAA GGC GAC GCT GAC G-3′) was obtained from Georgia

Genomic facility, USA. BOX-PCR amplification was achieved on DNA

thermocycler, with temperature profile which included initial denaturation of 4

minute at 94 °C (1 cycle), followed by 3 seconds at 94 °C, 45 seconds at 92 °C, 1

minute at 50 °C, and 6 minutes at 65 °C (29 cycles) (Rademaker et al., 2004).

32

About 45 ng (nanogram) of DNA was mixed with 5 ng loading dye, was set for

PCR in the thermocycler. High resolution agarose gel (2% for w/v) was used for

electrophoresis of reaction products in which temperature and voltage was

maintained to 4 °C and 70, respectively for 14 h. Ethidium bromide with

concentration of 5 ng L-1

was used to stain the gel for 30 minutes (Zhang et al.,

2013). Gel was imaged under ultraviolet light using a digital camera equipped

with charged coupled device (for UV imaging). The banding pattern analysis of

the BOX-PCR was carried out to calculate similarity among DNA of selected

bacterial isolates.

Conventional PCR of genomic DNAs of selected bacterial isolates was

also performed to target sections of their 16S rRNA using universal bacterial

primer pair 968 F (5′-AAC GCG AAG AAC CTT AC-3′) and 1401R (5′-CGG

TGT GTA CAA GAC CC-3′) (Felske et al., 1996). DNA thermocycler was used

to get PCR amplification, with temperature profile included initial denaturation of

2 minutes at 94 °C (1 cycle), followed by 1 minute at 94 °C, 1 minute at 58.5 °C,

and 2 minutes at 72 °C (35 cycles) and final extension for 10 minutes at 72 °C

(Felske et al., 1996). For the purpose of sequencing with forward primer 968F,

the PCR reaction products were filtered using PCR purification kit (Promega),

and samples were submitted to Georgia Genomics Facility, USA for sequencing.

The sequence data obtained were submitted to the GenBank database for

comparison against existing sequences of the National Center for Biotechnology

Information (NCBI) (http://blast.ncbi.nlm.nih.gov/). Partial sequence data of 16S

ribosomal RNA gene for closely related strains were downloaded from NCBI for

http://blast.ncbi.nlm.nih.gov/

33

molecular phylogenetic analysis by maximum likelihood method based on the

Tamura-Nei model (Tamura and Nei, 1993) using MEGA7 (Kumar et al., 2016).

The analysis involved 16 nucleotide sequences including the sequence of selected

bacteria. All positions containing gaps and missing data were eliminated before

final analyses. The phylogenetic trees with the highest log likelihood are shown

in the results section. The percentage of trees in which the associated taxa

clustered together is shown next to the branches. Initial tree(s) for the heuristic

search were obtained automatically by applying Neighbor-Join and BioNJ

algorithms to a matrix of pairwise distances estimated using the Maximum

Composite Likelihood (MCL) approach, and then selecting the topology with

superior log likelihood value.

3.5. BIOAUGMENTATION POTENTIAL OF SELECTED

BACTERIAL ISOLATES (POSSESING DUAL TRAITS) FOR

ACCELERATED DEGRDATION OF TEXTILE DYES

Bacterial isolates were selected for testing their bioaugmentation potential

of dye degradation under laboratory conditions. Prior to testing bioaugmentation

potential of selected isolates, effects of various environmental/ incubation

conditions were studied to optimize dye decolorization process under laboratory

conditions.

3.5.1. Optimization of Dye Degradation Process

Azo dye RB5 (100 mg L-1

) was subjected to bacterial decolorization by

bacterial isolates under different operational conditions to achieve efficient

decolorization rates. RB5 containing dye medium inoculated with different

34

bacteria was incubated under varied redox conditions (static and shaking), pH

levels (6–9), and carbon sources (glucose, peptone and yeast extract with

concentrations of 4 g L-1

). For temperature optimization, bacterial isolates were

monitored for dye decolorization at 24, 28, 32 and 36 °C for duration of 24 h.

3.5.2. Biodegradation Potential of Isolate I-15 for Structurally Different

Azo Dyes

Most commonly used azo dyes in textile dyeing sector of Pakistan were

subjected to decolorization by I-15. These dyes include Direct Blue 3R, Direct

Pink B, Sunfix Red S3B R, Sunfix Yellow, and RB5 (100 mg L-1

) in liquid

media. Additionally, a blend of these dyes (20 mg each dye L-1

medium) was also

tested as most of the effluents from these industries contain mixture of different

dyes. For this purpose, different sets of culture tubes containing sterilized MSM

broth (10 mL) amended with different dyes (100 mg L-1

) were prepared. The dye

containing decolorization medium was then inoculated with uniform cell density

(108-10

9 CFU mL

-1) (OD of 0.8 at 535 nm) of bacterial isolates. The culture tubes

were sealed firmly and incubated at 35 °C under stationary condition.

Decolorization was measured after 4, 8, 12, 16 and 24 h at wavelengths according

to type of azo dye by spectrophotometer. Biodegradation monitoring with

spectrophotometer was performed at respective wavelengths of the dyes used

(Direct Blue-3R at 405 nm, Direct Pink-B at 620 nm, Sunfix Red S3B-R at 635

nm, Sunfix Yellow at 550 nm, RB5 at 597 and their mixture at 580 nm).

3.5.3. Bioaugmentation Potential of Isolate I-15 for Textile Effluent

Treatment

35

Efficient dye degrading bacterial isolate I-15 was examined for its

bioaugmentation potential to decolorize azo dyes present containing textile

effluents in the existence of indigenous micro flora. For this purpose, effluents

containing structurally different azo dyes were subjected to bioaugmentation with

the isolated bacteria. Samples like fresh textile effluent, textile effluent spiked

with RB5 azo dye (100 mg L-1

), textile effluent spiked with mixture of

structurally different azo dyes (20 mg each dye L-1

), MSM induced textile

effluent spiked with RB5 and MSM induced textile effluent spiked with mixed

azo dyes were used to examine decolorization of azo dyes through

bioaugmentation. Samples were taken in 100 mL conical flasks and augmented

by I-15 and incubated at 35 ± 2 ºC under stationary condition.

3.5.4. Identification and Biodegradation of Secondary Metabolites

Metabolites resulting from the breakdown of azo compounds (under static

conditions) were identified by liquid chromatography-mass spectroscopy (LC-

MS) technique. The mobile phase used was acetonitrile and water with a ratio of

50/50 (v/v) with maintaining a flow rate of 0.8 mL Min-1

and injection volume of

10 µL.

Major intermediate compounds of bacterial degradation of RB5, i.e.,

aniline and 1-amino-2- naphthol-4-sulfonic acid were subjected to be degraded

by selected bacterial isolates both in static and shaking conditions (120 rpm). For

this purpose, flasks containing MSM amended with aniline (20 mg L-1

) as well as

1-amino-2- naphthol-4-sulfonic acid (50 mg L-1

) separately (in triplicates) were

inoculated with uniform cell density of selected bacterial isolates. After

36

incubation at 32 ºC for 96 h, 1.5 mL sample was acquired from each replicate and

purified by centrifugation at 10,000 rpm for 10 minutes to get cell free

supernatant. The supernatant was further purified with syringe filter (0.2 µ pore

size). Degradation of intermediate compounds by bacterial isolates was

determined by analyzing the purified samples in high performance liquid

chromatography (HPLC). For HPLC determination of aniline degradation, the

mobile phase used was methanol/water (40/60, v/v) in isocratic eluent with flow

rate maintained at 0.6 mL/minute with a total injection volume of 20 µL (Song et

al., 2007). The oven temperature was upheld to 40 ºC and the detection was

monitored at 235 nm. Similarly, for the determination of percent degradation of

1-amino-2- naphthol-4-sulfonic acid, the mobile phase used was methanol/water

(50/50, v/v) in isocratic eluent with a constant flow rate maintained at 0.6

mL/minute with a total injection volume of 20 µL. The oven temperature was

upheld to 40 ºC and the detection was monitored at 235 nm (Khalid et al., 2008).

3.6. BACTERIAL ASSISTED PHYTOREMEDIATION OF REACTIVE

BLACK 5 AND PLANT GROWTH PROMOTION

Isolated bacterial isolates were tested for simultaneous processes of dye

decolorization and plant growth promotion in liquid and soil medium. At first, all

the selected bacterial isolates were inoculated in the liquid medium used for

growing pre-germinated maize plants. Subsequently, better performing isolates

were used to inoculate soil for growing both barley and maize in order to evaluate

their potential for plant growth promotion being irrigated with dye containing

water.

37

3.6.1. Maize Growth Study in Dye Containing Liquid Medium

Maize (Zea mays) plants were grown and inoculated with selected

bacterial isolates and consortium (mixture of I-15 and S-10) (1%) in dye-

containing liquid medium (100 mg of RB5 L-1

). MSM and water amended with

dye were used as both decolorization and growth medium. Pre-germinated seeds

were placed with support in 30 mL of liquid medium in 60 mL culture tube

wrapped with black paper to avoid sunlight penetration. Plant containing tubes

were placed in plant growth chambers (Temperature: 28 ºC, RH: 61%, Light

intensity: 240 µM). Dye degradation rate (%) was assessed every 2 days. Plant

biomass, root and shoot lengths were measured after 10 days of growth period.

3.6.2. Maize and Barley Growth Study in Soil Under Greenhouse

Conditions

Maize (Zea mays) and barley (Hordeum vulgare) were grown in pots

under controlled conditions. Both crops were irrigated with RB5 contaminated

water (100 mg L-1

) by the interval of 2 days for the duration of 28 days. Surface

sterilized seeds inoculated by dipping in selected bacterial isolates were grown in

containers with sieved soil. Controls were maintained as non-inoculated seeds

and pre-sterilized soil.

3.7. STATISTICAL ANALYSIS

Experiments were performed in replications. Results regarding

38

degradation of dyes are presented in percent over a period of time. Standard

deviation (SD) was calculated using Microsoft Excel. ANOVA was performed to

find significant difference in effectiveness among different isolates in lab and pot

trials, while individual means were compared by LSD. Level of significance was

tested at p ≤ 0.05.

39

Chapter 4

RESULTS

4.1. PHYSICO-CHEMICAL ANALYSIS OF WASTEWATER

SAMPLES

The physico-chemical examination of textile effluent and sludge samples

taken from different sampling sites exhibited wide variations in pH levels (7.82–

13.12), EC (0.46–37.33 mS cm-1

) and TDS (0.229–23.87 g L-1

) (Table 1).

Highest pH (13.12) was observed in wastewater samples taken from an outlet of

industrial unit in Faisalabad region. However, highest EC (37.3 mS cm-1

) and

TDS (23.8 g L-1

) were found in the samples taken from another textile unit in

Faisalabad. High color intensity of 2.46 was recorded in the samples taken from a

dying unit in Faisalabad region that carries high concentrations of dyes.

4.2. ISOLATION AND SCREENING OF DYE-DEGRADING

BACTERIA

A total of 468 bacterial cells were isolated through enrichment technique

and purified. The isolates exhibited wide variations in decolorization rates of

Reactive Black 5 (RB5) within 48 h (Table 2). These isolates were screened on

the basis of their RB5 decolorization efficiency. Bacterial isolates exhibiting dye

degradation rate of more than 90% were selected for their further

characterization. Highest dye degradation rate (100%) was exhibited by the

isolate I-15 within 8 h of incubation under static conditions.

40

Table 1: Analysis of textile wastewater and sludge samples collected from industrial sites of different cities

Sample Code

Physico-chemical Characteristics

pH ± SEa

EC ± SE

(mS cm-1

)

TDS ± SE

(g L-1

)

Absorbance ± SE

a

(600nm)

Faisalabad A 8.48 ± 0.01 7 ± 0.06 4.49 ± 0.04 2.41 ± 0.07

B 8.36 ± 0.01 8.6 ± 0.04 5.49 ± 0.02 2.46 ± 0.001

D 13.12 ± 0.01 37.33 ± 0.55 23.87 ± 0.35 0.12 ± 0.004

E 10.2 ± 0.06 21.45 ± 0.12 12.24 ± 0.15 1.22 ±0.003

F 8.39 ±0.05 4.57 ± 0.07 2.9 ± 0.04 0.12 ± 0.003

G 8.19 ±0.02 7.66 ± 0.01 4.9 ± 0.01 0.18 ± 0.002

HV 8.49 ± 0.01 7.47 ± 0.02 4.75 ± 0.01 0.14 ± 0.002

I 8.12 ± 0.02 3.20 ± 0.01 2.05 ± 0.01 0.17 ± 0.003

Sheikhupura J 7.82 ± 0.02 3.4 ± 0.01 2.17 ± 0.01 0.16 ± 0.001

K 7.83 ± 0.01 2.06 ± 0.01 1.32 ± 0.01 0.13 ± 0.002

NWX 12.27 ± 0.1 29.33 ± 0.16 20.1 ± 0.09 1.92 ± 0.002

M 8.32 ± 0.02 0.79 ± 0.01 0.51 ± 0.01 0.25 ± 0.001

L 8.26 ± 0.07 0.46 ± 0.01 0.29 ± 0.01 0.17 ± 0.001

Rawalpindi O 8.72 ± 0.02 2.10 ± 0.01 1.34 ± 0.01 0.13 ± 0.002

S 8.56 ± 0.02 2.12 ± 0.01 1.35 ± 0.01 0.19 ± 0.001

8.55 ± 0.05 2.11 ± 0.01 1.35 ± 0.01 0.17 ± 0.003 aStandard Error

41

Table 2: Isolation and screening of bacteria capable of decolorizing RB5 dye in liquid medium under static conditions

Sample

Source

Sample Code

No. of

Bacteria

Isolated

Isolates capable of Dye

Degradation (within 48 h)

(≥ 90%)

Range

Meana ± SE

24 h 48 h

*WW A 18 - 22-66 27.5 ± 2.5 30 ± 5

WW B 72 - 25-86 54.1 ± 6.8 59.6 ± 8.1

WW D 8 - 13-48 41.7 ± 8.3 41.7 ± 8.3

WW E 13 - 32-88 50 ± 18.2 60 ± 22.2

WW G 7 - 25-75 52 ± 10.1 66 ± 4

WW HV 16 - 25-75 37.5 ± 8.5 62.5 ± 8.5

**S I 27 2 (7%) 100 100 ± 0 100 ± 0

S J 33 - 14-45 37.5 ± 7.2 37.5 ± 7.2

S AE 32 4 (13%) 56-100 85.9 ± 3.2 96.4 ± 1.5

S L 26 - 21-85 75 ± 0 81.7 ± 4.4

S M 48 4 (8%) 59-100 66.2 ± 5.7 92.2 ± 2.4

S NWX 30 - 17-56 56.3 ± 6.3 66.3 ± 9.9

***RS 5 6 - 37-75 50 ± 10.2 50 ± 10.2

RS S 10 1 (10%) 58-100 62.5 ± 12.5 72.5 ± 13.1

RS 7 12 3 (25%) 28-100 67 ± 11.7 87 ± 9.6

RS K 10 1 (10%) 38-100 42 ± 10.1 65.8 ± 6.07

RS 9 28 2 (7%) 37-100 54.8 ± 7.4 88.8 ± 7.43

RS 11 22 3 (14%) 47-100 61.6 ± 12.7 81.8 ± 15.2

RS 13 50 3 (6%) 12-100 55.9 ± 6.12 88.3 ± 5.9

b468

c23

a Percent Degradation;

bTotal No. of bacteria Isolated;

c No. of bacteria selected for characterization of plant growth promoting traits

*Wastewater; **Sludge; ***Rhizospheric soil

42

Depending on enhanced decolorization rates of isolated bacteria (minimum:

100% in 48 h), a sum of 23 isolates were selected for their further

characterization of plant growth promoting activities.

4.3. CHARACTERIZATION OF SELECTED BACTERIAL ISOLATES

FOR PLANT GROWTH PROMOTION

4.3.1. Indole Acetic Acid Production Assay

A wide range of auxin concentrations (stated as IAA equivalent in µg mL-

1) were produced by bacterial isolates on GPAM when added with L-TRP (Figure

2). The results revealed that 8 isolates were able to produce notable

concentrations of auxins ranging from 9 to 21 µg mL-1

. Isolate S-10 was able to

produce higher IAA concentration of 21 µg mL-1

compared to 16, 14 and 14 µg

mL-1

produced by isolates I-15, 7.3 and 11.4 respectively. Moreover, isolates AE-

8, AE-4, AE-7 and AE-5 also produced notable IAA concentrations of 13, 12, 11

and 9 µg mL-1

respectively, in liquid medium. However, remaining isolates did

not produce IAA more than 4 µg mL-1

(ranges from 1–4 µg mL-1

).

4.3.2. Phosphate Solubilization Activity

The rock phosphate solubilizing (P-solubilizing) activity of the isolates

was monitored by measuring the diameter of halo rings formed around bacterial

colonies (after 7 days) grown on NBRIP medium. A total of 17 out of 23 (74%)

isolates showed positive P-solubilizing activity and developed halo zones (3–11

mm) and are given in Fig. 3. Maximum P-solubilization activity was exhibited by

two isolates, I-15 and S-10 with halo zones formation of 11 mm. Isolates 7.3

43

Figure 2: Concentrations of indole acetic acid produced by isolated bacteria in

the presence of L-TRP. The values are presented as mean of three

replicates ± SD

0

5

10

15

20

I-15

I-21

AE

-4

AE

-5

AE

-7

AE

-8

M-1

2

M-1

7

M-2

3

M-4

7

1.4

1.5

3.1

4.2

S-1

0

7.3

K-3

K-7 9.3

10.1

11.4

14.5

17.2

IAA

con

cen

trati

on

(µ

g m

L-1

)

Bacterial Isolates

44

Figure 3: Size of halo ring formed by isolated bacteria after solubilizing

inorganic from of rock phosphate on solid NBRIP medium after 7

days incubation. The values are presented as mean of three replicates

± SD

0

2

4

6

8

10

12

I-1

5

I-2

1

AE

-4

AE

-5

AE

-7

AE

-8

M-1

2

M-1

7

M-2

3

M-4

7

1.4

1.5

3.1

4.2

S-1

0

7.3

K-3

K-7 9.3

10

.1

11

.4

14

.5

17

.2

Ha

lo z

on

e d

iam

eter

(m

m)

Bacterial Isolates

45

and 11.4 formed halo zones of 9 and 10 mm, respectively. Moreover, isolates

AE-4, AE-5, AE-7 and AE-8 also formed halo zones of 6, 8, 7 and 6 mm,

respectively. However, isolates 14.5, 10.1, 17.2, 1.5, M-12 and M-17 did not

form halo zones which indicate lack of their potential to solubilize rock

phosphate as a source of phosphate. Photographs representing positive P-

solubilization activity by different bacterial isolates are shown in Fig. 4.

4.3.3. ACC-Deaminase Activity Assay

The selected bacterial isolates were also tested for the presence of ACC

Deaminase activity. The results revealed that isolated bacteria exhibited negative

growth in the presence of ACC as a source of nitrogen.

4.4. BOX POLYMERASE CHAIN REACTION AND 16S rRNA GENE

ANALYSIS OF BACTERIAL ISOLATES CARRYING DYE-

DEGRADING AND PLANT GROWTH PROMOTING TRAITS

SIMULTANEOUSLY

BOX Polymerase Chain Reaction (PCR) was performed to understand the

genetic similarity between the isolated bacteria. The results showed 72% of

genetic similarity between isolates AE-5, S-10, AE-8 and the E. coli (positive

control), whereas, 69% of genetic relatedness was found between E. coli and

bacterial isolates AE-5, S-10, AE-8. The maximum relatedness of bacterial

isolate with E. coli was 75 % and it was of I-15, AE-7 (Fig. 5). No banding

pattern was observed for bacterial isolate AE-4, therefore not selected for further

analyses. Sequence data of isolates I-15, S-10, 7.3, 11.4, AE-5, AE-7 and AE-8

46

Figure 4: Photographs showing positive P-solubilizing activity by different

bacterial isolates in the form of halo rings formation on solid NBRIP

medium

47

Figure 5: Cluster Analysis of the BOX-PCR profiles of the bacterial isolates showing their genetic differences (%) among each other

and E. coli

57

69

75

72

69

84

BOXA1R

10

0

50

BOXA1R

0 20

40

60

80

10

0

100 BP Ladder

AE-4

11.4

7.3

AE-7

Escherichia coli

I-15

AE-8

S-10

AE-5

48

were submitted to Bankit, NCBI with accession numbers of KX553914,

KX553915, KX553916, KX553917, KX553918, KX553919 and KX553920

respectively. Advance nucleotide blast search revealed that the isolates were

closely related (similarity index ≥ 99%) to previously described spp.

Pseudomonas japonica (I-15), Achromobacter xylosoxidans (S-10), Burkholderia

ginsengisoli (7.3), Pseudomonas alcaligenes (11.4), Comamonas testosteroni

(AE-5), Aeromonas aquatica (AE-7) and Comamonas testosteroni (AE-8).

Phylogenetic trees of isolated bacteria were constructed by Maximum Likelihood

method and are shown in Fig. 6-12.

4.5. BIOAUGMENTATION POTENTIAL OF SELECTED

BACTERIAL ISOLATES (POSSESING DUAL TRAITS) FOR


4.5.1. Optimization of Dye Degradation Process

Prior to testing bioaugmentation potential, of selected isolates, effects of

various environmental/ incubation conditions were studied to optimize dye

decolorization process under laboratory conditions.

4.5.1.1. Effect of different redox condition

Different operational/process conditions for efficient decolorization of

RB5 by selected bacterial isolates were optimized, and the outcome revealed

significant difference in the dye decolorization rates under various redox

(static/shaking) conditions. In static condition, decolorization of RB5

49

Figure 6: Molecular Phylogenetic analysis by Maximum Likelihood method showing resemblance of I-15 with other bacterial strains

50

Figure 7: Molecular Phylogenetic analysis by Maximum Likelihood method showing resemblance of S-10 with other bacterial strains

51

Figure 8: Molecular Phylogenetic analysis by Maximum Likelihood method showing resemblance of 7.3 with other bacterial strains

52

Figure 9: Molecular Phylogenetic analysis by Maximum Likelihood method showing resemblance of 11.4 with other bacterial strains

53

Figure 10: Molecular Phylogenetic analysis by Maximum Likelihood method showing resemblance of AE-5 with other bacterial strains

54


55


56

from 67–100% after 24 h. Bacterial isolates AE-5, AE-7, AE-8, I-15 and S-10

exhibited complete decolorization (100%) of the dye under static conditions after

24 h. However, isolate 7.3 and 11.4 were able to decolorize the dye up to 77 and

67% respectively, after 24 h under static conditions. Under shaking conditions,

highest decolorization rate (56%) of RB5 was exhibited by isolate AE-5, while

isolate 11.4 exhibited only 25% decolorization of azo dye under shaking

conditions (Fig. 13).

4.5.1.2. Effect of different pH levels

Selected isolates were also evaluated for their efficiency in different pH

levels. The results indicated that selected bacterial isolates performed efficiently

in decolorization medium with a pH level maintained at 7 after 24 h under static

conditions (Fig. 14). Moreover, bacterial isolates also demonstrated their

remarkable potential of dye degradation at pH levels of 8 and 9, as the bacterial

isolates AE-5, AE-7, AE-8, I-15 and S-10 decolorized the dye efficiently (91–

97%). However, isolates 7.3 and 11.4 showed decolorization rates of 56 to 78%,

respectively. At slightly acidic pH (pH=6) higher decolorization rates was 77%

carried out by I-15 after 24 h.

4.5.1.3. Effect of different co-substrates

Different co-substrates such as glucose, peptone and yeast extract were

supplemented in dye decolorization medium to examine their function in the

biodegradation process. Highest decolorization percentage (99%) was observed

with the supplementation of yeast extract by isolates AE-5, AE-7, AE-8, I-15 and

57

Figure 13: Effect of different redox conditions on decolorization of RB5 by

different bacterial isolates after 24 h. The values are presented as

mean of three replicates ± SD

0

10

20

30

40

50

60

70

80

90

100

AE-5 AE-7 AE-8 I-15 S-10 7.3 11.4

Dec

olo

riza

tion

(%

)

Bacterial Isolates

Static Conditions Shaking Conditions

58

Figure 14: Effect of different pH levels on decolorization of RB5 by different

bacterial isolates after 24 h. The values are presented as mean of

three replicates ± SD

0

20

40

60

80

100

AE-5 AE-7 AE-8 I-15 S-10 7.3 11.4

Dec

olo

riza

tion

(%

)

Bacterial Isolates

pH 6 pH 7 pH 8 pH 9

59

S-10 (Fig. 15). Bacterial isolates 7.3 and 11.4 showed the relatively lower

degradation potential (77% and 69% respectively) after 24 h. On the other hand,

decolorization rates of RB5 ranged from 48-86% when peptone was used as co-

substrate. Bacterial isolates exhibited least decolorization efficiency when

supplemented with glucose as a co-substrate (decolorization rates from 23–64%).

4.5.1.4. Effect of different temperature conditions

Incubation temperature also had significant effect on biodegradation of

RB5 by different bacterial isolates. The rise in temperature up to 32 ºC resulted in

substantial increase in the biodegradation process when 96–99% of the dye was

decolorized by isolates AE-5, AE-7, AE-8, I-15 and S-10 (Fig. 16). Whereas,

isolates 7.3 and 11.4 achieved dye decolorization rates of 78% and 70%,

respectively. Decolorization efficiency of all the isolates inhibited substantially

on either side of 32 ºC.

4.5.2. Biodegradation Potential of Isolate I-15 for Structurally Different

Azo Dyes

The most efficient bacterial isolate I-15 was further examined for its

potential to degrade structurally varied dyes that are currently under excessive

use by textile industry of Pakistan. Biodegradation studies of I-15 exhibited

complete decolorization (100%) of diverse dyes in 8-16 h. Azo dyes Direct Pink-

B, Sunfix Red S3BR and Sunfix Yellow and dyes mixture were completely

decolorized within 8 h of incubation period. Complete decolorization of RB5 and

Direct Blue 3R was achieved after 16 h (Fig. 17 a-b).

60

Figure 15: Effect of different co-substrates on decolorization of RB5 by different

bacterial isolates after 24 h. The values are presented as mean of three

replicates ± SD

0

20

40

60

80

100

AE-5 AE-7 AE-8 I-15 S-10 7.3 11.4

Dec

olo

riza

tion

(%

)

Bacterial Isolates

Yeast Extract Peptone Glucose

61

Figure 16: Effect of different temperatures on decolorization of RB5 by different

bacterial isolates after 24 h. The values are presented as mean of three

replicates ± SD

0

20

40

60

80

100

AE-5 AE-7 AE-8 I-15 S-10 7.3 11.4

Dec

olo

riza

tio

n (

%)

Bacterial Isolates

24 °C 28 °C 32 °C 36 °C

62

Time (h)

0 2 4 6 8 10 12 14 16

Deco

loriz

ati

on

(%

)

0

20

40

60

80

100

Dirct Blue 3R

Direct Pink B

Sunfix Red S3B-R

Control

Time (h)

0 2 4 6 8 10 12 14 16

Dec

olo

riza

tio

n (

%)

0

20

40

60

80

100

Sunfix Yellow

Reactive Black-5

Mixture of Dyes

Control

Figure 17: Percent decolorization of structurally different azo dyes by bacterial

isolate I-15 after 16 h. The values are presented as mean of three

replicates

a)

b)

63

4.5.3. Bioaugmentation Potential of Isolate I-15 for Textile Effluent

Treatment

The bioaugmentation studies revealed that I-15 possess efficient potential

to decolorize the dyes in textile effluent within 6-12 h when supplement of 0.4%

yeast extract was added. After augmentation of textile effluent with isolate I-15,

complete color removal of original textile effluent containing dyes was observed

within 6 h when yeast extract was added, while the dye decolorization rate was

inhibited to 21% without carbon source supplement (Fig. 18).

Textile effluent spiked with RB5 (100 mg L-1

) was also completely

decolorized (100%) by I-15 within 6 h with yeast extract, while without yeast

extract supplementation decolorization rate decreased to 24% in 48 h (Fig. 19).

Moreover, I-15 also exhibited its potential for complete decolorization of textile

effluent spiked with mixture of dyes within 6 h when the yeast extract was added.

However, decolorization rate did not exceed to 24% in the absence of co-

substrate (Fig. 20). Furthermore, addition of other mineral salts in the dye

medium also resulted into complete decolorization within 12 h as compared to

complete decolorization in 6 h with yeast extract supplement for textile effluent

spiked with RB5 (Fig. 21). Furthermore, addition of such minerals in textile

effluent spiked with mixture of dyes resulted in completely decolorization at the

same time (i.e., 6 h) with or without yeast extract supplementation (Fig. 22).

4.5.4. Identification and Biodegradation of Secondary Metabolites

Intermediate compounds formed as a result of biodegradation process

64

Time (h)

0 6 12 18 24 48

Dec

olo

riza

tion

(%

)

0

20

40

60

80

100

with Yeast Extract

without Yeast Extract

control

Figure 18: Percent decolorization of textile effluent by the bioaugmentation of I-

15 after 48 h. The values are presented as mean of three replicates

65

Time (h)

0 6 12 18 24 48

Dec

olo

riza

tio

n (

%)

0

20

40

60

80

100

with Yeast Extract


control

Figure 19: Percent decolorization of textile effluent spiked with RB5 (100 mg L-

1) by the bioaugmentation of I-15 after 48 h. The values are presented

as mean of three replicates

66

Time (h)

0 6 12 18 24 48

Dec

olo

riza

tio

n (

%)

0

20

40

60

80

100

with Yeast Extract


control

Figure 20: Percent decolorization of textile effluent spiked with mixture of dyes

(20 mg L-1

each) by the bioaugmentation of I-15 after 48 h. The values

are presented as mean of three replicates

67

Time (h)

0 6 12 18 24 48

Deco

loriz

ati

on

(%

)

0

20

40

60

80

100

with Yeast Extract


control

Figure 21: Percent degradation of mineral salt media amended textile effluent

spiked with RB5 (100 mg L-1

) by the bioaugmentation of I-15 after 48

h. The values are presented as mean of three replicates

68

Time (h)

0 6 12 18 24 48

Dec

olo

riza

tio

n (

%)

0

20

40

60

80

100

with Yeast Extract


control

Figure 22: Percent degradation of mineral salt media amended textile effluent

spiked with mixture of dyes (20 mg L-1

each) by the bioaugmentation

of I-15 after 48 h. The values are presented as mean of three replicates

69

were analyzed using LC-MS. The spectra analyses indicating different molecular

weights and retention times (ranging from 0–300 m/z) of secondary metabolites

generated during biodegradation process carried out by different bacterial

isolates. The secondary metabolites were identified as 1,3,4-oxadiazol-2-ol (MW

= 86.05), aniline (MW = 93.13), 1-amino-2-naphthol-4-sulfonic acid (MW =

239.25) and some derivatives of benzidine. Some of the compounds detected in

MS spectra remained unidentified (Fig. 23–26).

Two of the major degradation byproducts of RB5, aniline and 1- amino-2-

naphthol-4-sulfonic acid (1-NSA) were subjected for biodegradation by selected

bacterial isolates. For aniline degradation, bacterial isolate AE-7 was able to

achieve degradation rate of 92% after 96 h under static conditions. Isolate 11.4

exhibited aniline degradation up to 76% after 96 h under static conditions.

Remaining isolates degraded the toxic compound with degradation rates of 28–

60% after 96 h under static conditions. However, relatively limited degradation of

aniline was observed in shaking conditions. Under shaking conditions, isolate I-

15 was able to degrade aniline up to67% after 96 h. Aniline degradation

efficiency of rest of the isolates ranged from 40–63% after 96 h under shaking

conditions (Fig. 27). For 1-NSA (50 mg L-1

), all the isolates completely degraded

the aromatic compound (99–100%) after 48 h of incubation under both static and

shaking conditions (Fig. 28).

4.6. BACTERIAL ASSISTED PHYTOREMEDIATION OF RB5 AND

PLANT GROWTH PROMOTION

4.6.1. Maize Growth Study in Dye Containing Liquid Medium

70

Figure 23: LC-MS Spectra showing degradation products of RB5 after decolorization by isolate I-15

71

Figure 24: LC-MS Spectra showing degradation products of RB5 after decolorization by isolate S-10

72

Figure 25: LC-MS Spectra showing degradation products of RB5 after decolorization by isolate 7.3

73

Figure 26: LC-MS Spectra showing degradation products of RB5 after decolorization by isolate 11.4

74

Time

96 h (St.) 48 h (St.) 0 h 48 h (Sh.) 96 h (Sh.)

Deg

rad

ati

on

(%

)

0

20

40

60

80

100 AE-5

AE-7

AE-8

I-15

S-10

7.3

11.4

Figure 27: Biodegradation of aniline (20 mg L-1) in different redox conditions by

selected bacterial isolates after 96 h. The values are presented as mean

of three replicates

(St.) Stationary Conditions

(Sh.) Shaking Conditions

75

Figure 28: Biodegradation of 1-Amino-2-napthol-4-sulphonic Acid (50 mg L-1)

in different redox conditions by selected bacterial isolates after 48 h.

The values are presented as mean of three replicates

0

20

40

60

80

100

48 Hours0

48 HoursStatic

ConditionsHours

Shaking

Conditions

% D

egra

da

tio

n

AE-5 AE-7 AE-8 I-15 S-10 7.3 11.4

76

Simultaneous processes of dye degradation and plant growth promotion

by the selected bacteria were observed by growing maize plants in dye-containing

liquid medium. RB5 containing liquid medium (MSM) with maize seedlings

inoculated with isolate AE-8 was completely decolorized within first 4 days after

inoculation. Liquid medium with maize plants inoculated with AE-5, AE-7, I-15

and consortium were decolorized within 6 days. Moreover, liquid medium with

plants inoculated with S-10 and 7.3 were decolorized after 8 days. Similarly, 11.4

completely decolorized the dye after 10 days of inoculation in plant containing

liquid medium. Whereas, non-inoculated plant containing liquid medium

(control) was also decolorized up to 19% after 10 days (Fig. 29). In another set of

experiments, dye decolorization rate was also observed in plant grown in dye

contaminated water (RB5 containing water) (Figure 30). Plants inoculated with

bacterial isolate S-10 exhibited decolorization rate of 28% after 10 days. On the

other hand, non-inoculated plant (control) also achieved decolorization rate of 9%

within first 2 days and overall decolorization rate of 15% after 10 days. Minimum

decolorization efficiency in dye containing water was exhibited by plants

inoculated with isolates I-15 and 11.4 (10% dye decolorization) after 10 days.

Plants inoculated with isolates AE-5, AE-7, AE-8, 7.3 and 11.4 were able to

achieve decolorization rates ranging from 11–18% after 10 days.

Plant growth parameters were also observed in this study. In dye amended

MSM liquid medium, plants inoculated with consortium of I-15 and S-10

produced maximum root length (7 cm) which was significantly greater than

plants inoculated with other isolates as well as control (Fig. 31).

77

Time

0 Day 2 Day 4 Day 6 Day 8 Day 10

Dec

olo

riza

tio

n (

%)

0

20

40

60

80

100

Control

AE-5

AE-7

AE-8

I-15

S-10

7.3

11.4

Consortium

Figure 29: Dye decolorization trend of RB5 enriched mineral salt media by

maize plants inoculated with different bacterial isolates grown under

laboratory conditions (after 10 days). The values are presented as

mean of five replicates ± SD

78

Time


Dec

olo

riza

tion

(%

)

0

5

10

15

20

25

30 Control

AE-5

AE-7

AE-8

I-15

S-10

7.3

11.4

Consortium

Figure 30: Dye decolorization trend of RB5 containing water by maize plants

inoculated with different isolates grown under laboratory conditions

(after 10 days). The values are presented as mean of five replicates ±

SD

79

Figure 31: Effect of different bacterial isolates on growth of maize plants grown

in RB-5 enriched mineral salt media under laboratory conditions (10

days duration). The values are presented as mean of five replicates ±

SD (LSD values are 2.05, 5.05 and 0.4 for root length, shoot length

and plant biomass, respectively, at p = 0.05)

0

0.2

0.4

0.6

0.8

1

1.2

0

4

8

12

16

20

Pla

nt

Bio

mass

(g)

Root

& S

hoot

Len

gth

(cm

)

Bacterial Isolates

Root Length (cm) Shoot Length (cm) Plant Biomass (g)

80

Root lengths of plants inoculated with isolates I-15, S-10, 11.4 and AE-7 were

measured 5.75, 5.5, 5.25 and 5.25 cm which were significantly greater than non-

inoculated control (4.5 cm). Moreover, root lengths of plants inoculated with

isolates AE-5 and 7.3 were measured to 4.75 and 4.5 cm, respectively. Whereas,

root lengths of plants inoculated with isolate AE-8 and non-inoculated control

were measured the same 4.5 cm after 10 days of growth period. In shoot length

measurements, plants inoculated with isolates AE-7 and AE-8 gained highest

shoot lengths of 19.25 and 18.5 cm respectively, which were significantly higher

than control, AE-5, I-15, S-10, 7.3, 11.4 and consortium (6.25, 15.25, 10.75, 8.75,

9.0 and 10.75 cm respectively). Moreover, plants inoculated with isolates I-15

developed significantly higher shoot lengths as compared to plants inoculated

with AE-5, S-10, 7.3, 11.4, consortium and control. In plant biomass

observations, maximum biomass observed was 1.07 g (grams) by plants

inoculated with isolate AE-7. Biomass of plants inoculated with isolates AE-8, I-

15, S-10, 7.3, 11.4 and consortium (1.05, 0.97, 0.97, 0.91, 0.89 and 0.89 g,

respectively) were significantly higher than control and AE-5 inoculated plants.

However, no significant difference was observed in biomass of plant inoculated

with isolate AE-5 and non-inoculated control.

Better growth rates were achieved by inoculated plants grown in dye

containing water (Fig. 32). Plants inoculated with isolates I-15, 11.4 and AE-5

found to have higher biomass production with no significant difference with each

other (1.55, 1.46 and 1.51 g, respectively) after 10 days. However, significant

difference was observed when compared with biomass produced by plants

81

Figure 32: Effect of different bacterial isolates on growth of maize plants grown

in RB5 containing water under laboratory conditions (10 days

duration) The values are presented as mean of five replicates ± SD

(LSD values are 6.37, 6.35 and 0.29 for root length, shoot length and

plant biomass, respectively, at p = 0.05)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0

5

10

15

20

25

30

35

40

Pla

nt

Bio

mass

(g)

Root

& S

hoot

Len

gth

(cm

)

Bacterial Isolates


82

inoculated with isolates AE-7, AE-8, S-10, 7.3, consortium and control (1.17,

1.01, 1.16, 1.33, 1.26 and 1.07 g, respectively). Similarly, significantly higher

root lengths were observed (24.75cm) by plants inoculated with consortium (I-15

and S-10). Plants inoculated with I-15 had root lengths of 21.5 cm, which is

significantly greater than root lengths of plants inoculated with isolates AE-5,

AE-7, AE-8, S-10, 7.3, 11.4 and control (avg: 16.5, 19.5, 18.5, 15.25, 15.75,

13.75 and 11.5 cm, respectively). No significant difference was found when root

lengths of plants inoculated with AE-8 and control were compared. Photograph

depicting potential of dye degradation and plant growth promotion of maize

plants by isolates I-15 and S-10 under lab conditions is shown in Fig. 33.

4.6.2. Maize and Barley Growth Study in Soil Under Greenhouse

Conditions

Growth was monitored for maize plants grown in soil irrigated with dye

containing water. Comparison of growth parameters was performed among plants

inoculated with selected bacterial isolates, plants grown in the presence of

indigenous soil microflora (Control 1) and plants grown in sterilized soil (Control

2). The data regarding plant biomass, root and shoot lengths of maize plants are

presented in Fig. 34. Plants inoculated with bacterial isolate 7.3 found to have

significantly higher biomass production (8 g) when compared with non-

inoculated plants (3.56 g) as well as plants grown in sterilized soils (3.51 g).

Biomass of plants inoculated with isolate 7.3 was also significantly higher than

biomass produced by non-inoculated plants as well as plants inoculated with

isolates I-15, S-10 and 11.4 (3.92, 4.21, 4.21 g/plant, respectively). Similarly,

83

Figure 33: Photographical presentation of simultaneous activities of dye

degradation and plant growth promotion of maize plants by isolates I-

15 and S-10 under laboratory conditions. Controls represents non-

inoculated plants grown in mineral salt media with dye (Control) and

mineral salt media without dye (Control 2)

2

84

Figure 34: Effect of different bacterial isolates on growth of maize plants

irrigated with dye containing water (28 days harvest). The values are

presented as mean of five replicates ± SD

0

1

2

3

4

5

6

7

8

9

0

10

20

30

40

50

60

70

80

90

Control 1 Control 2 I-15 S-10 7.3 11.4

Pla

nt

Bio

mass

(g)

Len

gth

(cm

)

Bacterial Isolates

Shoot Length Root Length Plant Biomass

85

plants inoculated with isolate 7.3 also had higher root lengths (56 cm) as

compared to all other treatments (Control 1 = 29 cm; Control 2 = 31 cm; I-15 =

49 cm; S-10 = 41 cm; 11.4 = 52 cm). In case of shoot length, significantly higher

rates were observed in plants inoculated with isolates11.4 (75 cm) and isolate 7.3

(74 cm). Moreover, plants inoculated with isolates I-15 and S-10 had high

average shoot lengths of 62 and 66 cm, as compared to 55 and 61 cm, average

shoot lengths measured in non-inoculated plants and plants grown in sterilized

soil. Photographical representation of effect of different bacteria on growth of

maize plants is shown in Fig. 35.

Effect of selected bacterial isolates on growth of barley plants were also

tested in comparison with non-inoculated plants (Control 1) as well as plants

grown in sterilized soil (Control 2) (Figure 36). Plants inoculated with isolate 7.3

observed to have high biomass production of 0.87 g, which is relatively higher

than plants inoculated with isolates S-10 and 11.4 (0.69 and 0.79 g, respectively).

No significant difference was found when biomass of non-inoculated plants and

plants inoculated with isolate I-15 were compared. However, plants grown in

sterilized soil observed to have lower plant biomass (0.22 g). In case of root

lengths, plants inoculated with isolate 11.4 and I-15 observed to have root lengths

of 15.33 and 14.67 cm, which are significantly greater than root lengths of plants

inoculated with S-10 and 7.3 (13.33 and 13.67 cm, respectively). Non-inoculated

plants also gained root length of 14.67 cm, which is significantly greater than

plants grown in sterilized soil (10.33 cm). In case of shoot length measurements,

maximum length was gained by plants inoculated with isolates I-15 and 11.4

86

Figure 35: Photograph showing relevant difference in growth parameters of

maize plants irrigated with dye contaminated water decolorized by

different bacterial isolates (28 days harvest)

87

(28.67 cm both). Plants inoculated with isolates S-10 and 11.4 had average shoot

lengths of 24.67 and 26.67 cm, respectively. On the other hand, non-inoculated

plants had average shoot length of 26.33 cm, which is relatively higher than

plants grown in sterilized soil (20.33 cm).

88

Figure 36: Effect of different bacterial isolates on plant growth of barley crop

irrigated with dye containing water (28 days harvest). The values are

presented as mean of five replicates ± SD

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0

5

10

15

20

25

30

35

Control

1

Control

2

I-15 S-10 7.3 11.4

Pla

nt

Bio

mass

(g)

Len

gth

(cm

)

Bacterial Strain

Shoot Length Root Length Plant Biomass

89

Chapter 5

DISCUSSION

A number of studies have shown that various microorganisms are able to

degrade synthetic textile dyes in liquid medium and can be used for the treatment

of textile effluent (Stolz, 2001; Khalid et al., 2008; Solís et al., 2012; Ito et al.,

2016; Mahmood et al., 2016). However, no work has been done to devise a

strategy for reducing the dye-pollutants in the rhizosphere if wastewater

containing dyes is used for growing crops. Due to the severe shortage of water in

arid and semi-arid areas, the use of wastewater for agricultural production could

be critical. The direct use of industrial wastewater could be harmful to crops, but

microbial cultures capable of degrading dyes can minimize adverse effects of

dyes. Dye-degrading bacterial cultures may have practical application for the

recycling of industrial wastewater which might be used as an irrigation source to

produce plant biomass. The present study has been focused on the isolation of

bacteria that degrade not only synthetic dyes but also possess traits that promote

plant growth.

5.1 CHARCTERISTICS OF TEXTILE WATEWATER

Textile wastewater samples were collected from different sites and

analyzed for various physico-chemical characteristics. The results reveal that the

wastewater discharged from the textile industry and other dye-stuff contains a

large amount of various salts, resulting in high EC and TDS. The analysis of

various wastewater samples showed a pH in the alkaline range, and the pH was as

90

high as 13.1. These results clearly indicate that wastewater, in most cases, is not

treated at an industrial level and exceeds permissible limits (NEQS, 2000;

Rajaguru et al., 2002). This premise is also supported by the fact that the

absorbance of colored wastewater was also high, indicating the presence of high

amounts of dyes in textile wastewater. These results suggest that the wastewater

from textile processing units, in addition to salts, also carry dyes that do not bind

to the fiber and discharged directly (no treatment) in wastewater streams. These

dyes are known to cause mutagenicity and carcinogenicity (Kousha et al., 2012)

and could be of great concern to public health, especially when a dye-containing

wastewater is used for irrigation purposes in the vicinity of industrial areas or go

directly to the main water stream and contaminate the surface and ground water

resources. This groundwater is commonly used by people for drinking in a

country like Pakistan, eventually deteriorating health of people living around the

textile industrial units. Therefore, textile wastewater must be treated before being

discharged into water bodies to prevent ecological damage and risks to human

health. The dyes and their intermediates could be more harmful for human after

bio-accumulation and entering into food chain through irrigation and soil

contamination.

5.2 ISOLATION AND SCREENING OF BACTERIAL ISOLATES

POSSESSING DUAL TRAITS OF DYE DEGRADATION AND

PLANT GROWTH PROMOTION

First of all, bacterial strains were isolated from textile effluent,

wastewater sludge and rhizospheric soil by enrichment of the growth medium

91

with RB5 dye, in order to isolate efficient bacterial strains capable of degrading

azo dyes. Some bacterial isolates exhibited 100% decolorization of RB5 dye (100

mg L-1

) in the liquid medium within 12 h. Bacterial isolate I-15 (out of seven

isolates selected on the basis of dye degradation) showed complete decolorization

of all the five textile dyes within 8-16 h incubation under static condition. Despite

degrading individual dyes, the isolate I-15 was also able to degrade a mixture of

five different dyes within 8 h. Since textile effluent contains several dyes, the

bacterial isolate I-15 could be effective for the treatment of textile wastewater

containing different dyes. From these results, it is concluded that the selected

isolate possess an effective enzymatic system to degrade a variety of structurally

different textile dyes. The decolorization efficiency achieved in present study was

relatively greater than some previously reported studies. For instance, complete

decolorization of RB5 (200 mg L−1

) and Direct Blue-6 (100 mg L−1

) was

achieved in 24 h (Lucas et al., 2006) and 72 h (Kalme et al., 2007), respectively,

either using pure cultures or consortium.

The selected bacterial isolates that are able to degrade dyes efficiently in

liquid medium were characterized for their growth promoting traits. It was

observed that the selected bacterial isolates showed growth promoting

characteristics such as auxin production and P-solubilization activity, however,

none of the selected isolates showed ACC-deaminase activity. Four isolates (I-15,

S-10, 7.3 and 11.4) produced significantly high amounts of IAA (ranging from 14

to 21 µg mL-1

) in the presence of substrate (L-TRP), which could be vital for plant

growth under dye-induced stress conditions. The IAA is considered as one of the

92

major phytohormones reported for reducing exogenous stress on plant by

regulating plant physiological responses (Leinhos and Bergmann, 1995). The

bacterial ability to synthesize IAA has been important consideration to screen

plant growth promoting bacteria (Khalid et al., 2004; Wahyudi et al., 2011). In

literature, there are enough evidence that different species of bacteria, including

Azospirillum, Bacillus, Paenibacillus, Pseudomonas and Providencia are capable

of synthesizing IAA in varying amounts (Kuss et al., 2007; Beneduzi et al., 2008;

Hernández-Rodríguez et al., 2008; Rana et al., 2011; Hassen et al., 2016). The

plant growth promoting bacteria having ability to produce IAA have been

reported to enhance germination rate, plant biomass, root and shoot weights, and

proliferation of lateral roots and root hairs (Khalid et al., 2006; Fatima et al.,

2009; Joseph et al., 2012).

In addition to IAA production, the selected dye-degrading bacterial

isolates were also positive for P-solubilization, while four isolates (I-15, S-10, 7.3

and 11.4) showed the highest P-solubilization activity (halo ring size ranged from

9-11 mm). Phosphorus is considered as one of the primary plant nutrient which

limits the plant growth because of its less bioavailability and remain in insoluble

form in soil (Abd‐Alla, 1994). P-solubilizing bacteria produce organic acids and

enzymes (phosphatases) which transform the inorganic phosphate into monobasic

and dibasic ions, resulting in bioavailability of phosphates in the rhizospheric

soils (Kumar et al., 2014). Therefore, inoculation with P-solubilizing bacteria in

soil could result into enhance phosphorus uptake by the plant (Subba Rao, 1982;

Rashid et al., 2004; Kumar et al., 2014). It is well described in literature that P-

93

solubilizing ability of bacteria is significantly affected by stress conditions (Pal,

1998; Zaidi et al., 2009). However, studies also have shown that some bacteria

have evolved mechanism(s) to solubilize inorganic phosphate in stressed

conditions, including high alkalinity, high temperature and wide range of pH

(Nautiyal et al., 2000; Chaiharn and Lumyong, 2009). Therefore, the ability of

dye degrading bacteria with auxin production and P-solubilization activities

concurrently can play a dual beneficial role to support plant growth in dye

associated stressed environment.


BACTERIAL ISOLATES POSSESING DUAL TRAITS FOR


One of the most efficient isolates I-15 having the ability to degrade dyes

in liquid medium and possessing plant growth-promoting characteristics, such as

IAA production and P-solubilization was selected to evaluate bioaugmentation

potential to degrade various azo dyes. Isolate I-15 was able to completely remove

color of dyes present in textile effluent in a period of 6-24 h under static

conditions when supplemented with co-substrate (yeast extract). Though, the

biodegradation of azo dyes involve various metabolic pathways under both

aerobic and anaerobic conditions, which affect decolorization rates and nature of

metabolites generated from parent molecule. Enhanced dye decolorization under

anoxic/static (anaerobic) as compared to shaking (aerobic) condition confirms the

possibility of the involvement of reductase enzymes for the breakdown of azo

bonds, which is the initial step of dye decolorization. Azoreductases are

94

considered as primary enzymes synthesized by azo dye decolorizing bacteria,

which catalyze the azo bonds in the reductive conditions. Previously,

azoreductases synthesis has been documented in different species of Aeromonas,

Enterococcus, Pseudomonas and Staphylococcus (Chen et al., 2005; Zhuang et

al., 2007; Hsueh et al., 2009). This implies that I-15 carries an efficient

enzymatic system for the cleavage of azo bond of a variety of textile dyes in the

presence of indigenous microorganisms.

The effectiveness of individual strains could be accelerated by adding

other efficient dye-decolorizing strains (Chen et al., 2006; Bafana et al., 2009).

Therefore, it could be surmised that enzymes produced by mixed bacterial

cultures are more effective than the enzymes produced by individual strains.

However, dye decolorization rates can also be accelerated at times by

augmentation with bacterial sp. possessing nonessential traits to influence

treatment efficiency. For example, E. coli DH5a had the ability to enhance

decolorization potential of Pseudomonas luteola even though E. coli DH5α is not

considered as an active bacterium for the decolorization of azo dyes. Specifically,

Escherichia coli DH5α expressed some extracellular metabolites which

stimulated decolorization activity of Pseudomonas luteola (Chen et al., 2003;

Chen et al., 2006). Genetically engineered microorganisms (GEM) have also

been reported for their effectiveness in biodegradation and their application in

bioaugmentation systems. Introduction of genetically modified E. Coli changed

microbial community structure indistinctively with time which resulted to

enhance decolorization of Direct Blue 71 (Jin et al., 2009). However, testing

95

GEM’s for remediation purposes in field is still a major obstacle due to

ecological and environmental concerns as well as regulatory limitations (Das and

Chandran, 2010).

In the present study, all the tested bacterial isolates exhibited distinctive

decolorization rates of RB5 at varying pH levels of 6-9 (4 levels). However,

results revealed that bacterial isolates decolorized the dye efficiently at pH levels

of 7-8. Bacterial capability to tolerate and perform in varying pH levels (low and

high pH) is considered important for their practical application for the treatment

of azo dye contaminated textile effluents (Narsinge and Hamde, 2013).

Bioaugmentation of E. coli for the treatment of Direct Blue 71 at different pH

levels revealed that no decolorization was achieved at pH 5.0, which suggests

exposure to lower pH levels affects enzymatic performance of microbial cultures.

However, the bioaugmented reactor achieved efficient dye decolorization rates at

pH 9.0, suggesting that inoculation of E. coli enhanced biodegradability of the

alkaline dye containing wastewater.

Likewise, temperature is considered as significant condition for bacteria

to efficiently biodegrade azo dyes as microbial growth rate and their specific

enzymatic mechanisms are directly related to temperature. The results showed

that selected bacterial isolates effectively decolorized the dye in temperature

ranging from 32 - 36 °C. Further increase or decrease in temperature showed

negative influence on bacterial efficiency for dye decolorization. The drop in dye

decolorization rate by increasing process temperature could be attributed to the

thermal deactivation of azoreductase enzyme (Mahmood et al., 2011). Moreover,

96

the tested bacterial isolates probably belong to mesophilic group as all tested

isolates showed the highest dye decolorization rates at a temperature range of 32 -

36 °C.

The concomitant deviation in dye decolorizing rates after augmenting or

decreasing co-substrate (carbon source) refers to the fact that dyes are either

carbon deficient, and/or not bioavailable (Chen et al., 2003; Wang et al., 2009).

For the purpose of optimizing co-substrate supplementation to achieve enhanced

dye decolorization rates, yeast extract, glucose and peptone were added in

decolorization medium to test their role as carbon source availability for selected

bacteria. Among these, culture media with yeast extract had positive outcomes in

terms of RB5 decolorization, while peptone and glucose had comparatively lower

decolorization rates by selected bacteria under anaerobic conditions. In scientific

literature, glucose is reported to be suitable for various bacterial species

(facultative anaerobes and fermenting bacteria) under aerobic conditions.

Addition of glucose enhanced dye decolorization rate of Mordant Yellow 3 by

Sphingomonas xenophaga Strain BN6, while significant drop in decolorization

rate was observed for P. leuteola and some other consortia (Chang et al., 2001;

Chen et al., 2003). In present study, improved rates of dye decolorization with

yeast extract as a co-substrate could be attributed to boosted enzymatic

mechanisms of applied bacteria when supplemented with yeast extract. It could

be comprehended that yeast extract may act as an efficient electron donor for the

reduction of azo bonds present in RB5 by the selected bacterial isolates.

97

LC-MS analysis of the decolorized RB5 dye (under static conditions)

identified formation of secondary metabolites like aniline and 1-amino-2-

naphthol-4-sulfonic acid as degradation byproducts. These secondary metabolites

(aniline and 1-amino-2- naphthol-4-sulfonic acid) have been reported previously

as biodegradation products of RB5 (Pearce et al., 2006; Xu et al., 2007).

Normally, cleavage of azo bond by azoreductase enzyme under reduced condition

is described as the initial step in breakdown of azo compound (Khalid et al.,

2008), resulting the formation of aromatic amines as decolorization byproducts.

In this study, formation of 1-amino-2- naphthol-4-sulphonic acid as secondary

metabolite confirmed the involvement of reduction reaction as an initial step in

dye decolorization process. These aromatic amines are subsequently metabolized

in aerobic conditions (Panswad et al., 2001). This identifies buildup of aromatic

amines in high concentration that could be toxic for bacterial growth. Aniline is

reported to be one of the degradation byproducts of RB5 (Biswas et al., 2007).

Other than aniline and 1-amino-2- naphthol-4-sulfonic acid, 1,3,4-oxadiazol-2-ol

and some derivatives of benzidine were also detected. Some of the compounds

detected in MS spectra remained unidentified

5.4 BACTERIAL ASSISTED PHYTOREMEDIATION OF REACTIVE

BLACK 5 AND PLANT GROWTH PROMOTION OF

BIOENERGY CROPS

In this study, simultaneous activities of dye degradation and plant growth

promotion by the selected bacteria were monitored by their application on maize

plants grown in dye-containing liquid medium. The results showed that plants

98

inoculated with bacteria had positive effect on growth parameters such as plant

biomass, root and shoot length when compared to growth parameters of non-

inoculated plants. The present results suggested that individual bacterial isolates

(AE-7, AE-8, I-15, S-10, 7.3, 11.4) and consortium (I-15 & S-10) possessed traits

which have assisted the plants to survive and uphold growth in dye induced

toxicity and stress conditions. It is well understood that accumulation of azo

compounds in roots can result in significant reduction of plant growth (Khandare

et al., 2013). However, inoculation of PGPB are reported to form symbiotic

association with plants that may promote plant growth either by facilitating

resource acquisition or regulating plant hormones (Glick, 2010; Hassen et al.,

2016). PGPB with specific growth enhancing traits can reduce stress symptoms

in plants being used for phytoremediation of soil and water (Arshad et al., 2007).

Additionally, varying composition and concentration of dyes also have an

hindering effect on root and shoot length of plants (Watharkar et al., 2013). In

current study, growth hindrance was observed in plants grown without bacterial

assistance, and could be referred to the lack of external support (plant growth

promoting activities of bacteria) as well as toxicity of the RB5, as it may inhibit

nutrient uptake and other possible metabolic activities (Chandra et al., 2009).

Furthermore, maize plants also exhibited decolorization of RB5

containing water (up to 16% in 10 days) without bacterial inoculation. This

implies that maize plants may possess enzymatic mechanisms, laccases and/or

peroxidases (Bryan et al., 2016; Tiecher et al., 2016) which may have involved in

the degradation of RB5 over time. It has already been reported that plant

99

peroxidases are well known for their role in degradation of textile dyes

(Watharkar et al., 2013). In addition to plants, the laccases enzymes are also

extensively present among fungi and bacterial species (Mayer and Staples, 2002;

Claus, 2003). While inoculated plants achieved the decolorization rate up to 28%,

which clearly suggest there are certain enzymes produced by bacteria which

enhanced the process rates. In another study, different enzymes like laccase and

azoreductase were combined and resulted into enhanced dye decolorization rates

as well as significant drop in the toxicity of azo dye intermediate

compounds(Mendes et al., 2011). Relying on the current results, it is proposed

that synergistic action of enzymes produced by maize plants and bacteria lead to

enhanced dye decolorization process.

Maize and barley plants were grown in soil (in both sterilized and non-

sterilized) inoculated by selected bacteria and were irrigated with RB5 containing

water for a period of 28 days. Highest biomass production was observed by

plants inoculated with 7.3 and 11.4 for maize, and I-15 and 11.4 for barley grown

in non-sterilized soil. Increased biomass production in plants inoculated with

bacterial strains could be attributed to accelerated production of plant growth

promoting hormones which stimulates specific enzyme mechanisms capable of

integrating starch in plants (Arshad et al., 2008). The general phenomenon of

plant growth promotion may involve either provision of growth promoting

substances (IAA) synthesized by bacteria or facilitating nutrient (phosphate,

nitrogen, potassium etc.) uptake from rhizosphere (Almaghrabi et al., 2014).

Some bacteria can promote plant growth either by synthesizing hormones like

100

IAA (Burd et al., 2000) or by stimulating certain metabolic mechanisms like P-

solubilization (Zaidi et al., 2009; Kumar et al., 2014). Scientific findings reported

in multiple studies confirms the effect of IAA and P-solubilization on enhanced

biomass production, increased shoot and root lengths of plants when inoculated

with PGPB (Subba Rao, 1982; Barazani and Friedman, 1999; San-Francisco et

al., 2005; Kang et al., 2009; Khalid et al., 2009). Application of treated RB5

containing textile wastewater had significant enhanced growth of maize, pea and

tomato plants when compared to untreated wastewater (Khalid et al., 2013).

Availability of nitrogen (a vital nutrient) in azo compounds may have impact on

plant growth (Khan et al., 2011), and subsequently application of such treated

wastewater enhances plant growth. Azo dyes after cleavage under anaerobic

treatment release aromatic compounds that can also serve as carbon and energy

source for degrading community (Karaguzel et al., 2004) and thus textile waste

water treated with PGPB may have optimistic influence on plant growth.

Additionally, maize and barley plants were also grown in sterilized soil to

understand the synergistic relationship between inoculated bacterial strains and

indigenous microflora. The results showed relative growth hindrances in plants

grown in sterilized soil as compared to plant grown in the presence of indigenous

microflora. In the rhizospheric zone, more than 4000 species are present per gram

of soil (Montesinos, 2003). Some of these species are able to influence plant

growth and can have the potential to protect plant roots from phytopathogens.

Such microflora is dynamically associated with biochemical cycling of nutrients

like carbon, nitrogen, phosphorus and sulfur as well as removal of toxins and

101

production of phytohormones (Bhattacharyya and Jha, 2012). These bacteria may

have dependency on other bacterial species for nutrient availability as one species

may convert root exudates into a specific form that may available to the other

species (Mayak et al., 2004). Therefore, it is suggested that microbial interactions

could result into enhanced plant biomass, root and shoot length of maize and

barley plants.

From the present results, it could be comprehensively concluded that

efficient dye degrading bacteria with additional traits of IAA production as well

as P-solubilization could be used simultaneously for azo dye degradation and

plant growth promotion for enhanced biomass production of energy crops.

102

SUMMARY

In Pakistan, textile sector has a vast impact on the country’s economy,

contributing 57% to the export and 30% employees of the total workforce. On the

other hand, textile manufacturing and dyeing processing units are considered as

one of the leading industrial sectors responsible for generating enormous amounts

of liquid effluent. Textile effluents are classified as most polluting wastewater

among the industrial wastewater. Azo dyes present in textile effluents are of

important concern because of their potential risk involved in polluting the

environment systems and affecting human health. The release of untreated

effluents into the environmental systems and its later application for irrigating

croplands pose serious threat to ecological health. It is not easy at farmers’ level

to avoid the use of such contaminated wastewater for irrigation purpose in the

surrounding of industrial area. So, there is a dire need to develop a strategy to

minimize the harmful effects of dye-polluted water on crops and ultimately

human health. One way is to treat this wastewater at industrial level by using

various approaches which is, at moment, not possible to implement 100% at

industrial scale in Pakistan. Therefore, this study was designed to isolate, identify

and characterize bacteria that have the potential to decolorize azo dyes effectively

and also promote plant growth simultaneously. The application potential of the

isolated bacteria was further tested by their inoculation to maize (Zea mays) and

barley (Hordeum vulgare) crops for simultaneous processes of both dye

degradation and plant growth promotion. The use of bio-energy crops could

eliminate the impact of dye or dye-associated by-products on human through

103

bioaccumulation in food where textile wastewater is being used for irrigation

purpose. The key findings of study are summarized as under.

The physico-chemical characterization of textile effluent and sludge

samples taken from different sampling sites showed a wide variation in

pH (7.55 – 13.12), EC (0.46 - 37.33 mS cm-1

) and TDS (0.29 - 23.87 g L-

1). Highest pH (13.12) was observed in samples taken from a dye

processing unit in Faisalabad. Higher EC (37.3 mS cm-1

) and TDS (23.8 g

L-1

) were found in the samples collected from dying unit located in

Faisalabad. High color intensity of 2.46 was also recorded in the samples

taken from wastewater outlet of dying unit in Faisalabad, indicating high

concentrations of dyes.

A total of 468 bacterial isolates were isolated and 23 isolates were

screened depending on their decolorization efficiency.

Selected bacterial isolates were then characterized for plant growth

promoting traits. Among the isolated bacteria, eight isolates were able to

produce potential concentrations of IAA ranging from 9 to 21 µg mL-1

.

Isolate S-10 was able to produce highest auxin concentration of 21 µg

mL-1

compared to 16, 14 and 14 µg mL-1

produced by isolates I-15, 7.3

and 11.4, respectively. Moreover, isolates AE-8, AE-4, AE-7 and AE-5

also produced notable auxin concentrations of 13, 12, 11 and 9 µg mL-1

,

respectively.

Out of 23 isolates, 17 showed P-solubilizing ability and developed halo

zones (diameter ranged from 3–11 mm). Highest P-solubilization activity

104

was observed in two isolates, I-15 and S-10. Bacterial isolates 7.3 and

11.4 formed halo zones on agar plates with diameter measurements of 9

and 10 mm, respectively. Moreover, isolates AE-4, AE-5, AE-7 and AE-8

also developed halo zones with diameter of 6, 8, 7 and 6 mm,

respectively.

BOX PCR was performed to understand the similarity index between the

selected isolated bacteria that had simultaneous capacity to decolorize azo

dye and promote plant growth. The results showed 72% of genetic

similarity between isolates AE-5, S-10, AE-8 and the E. coli (positive

control), whereas, 69% of genetic relatedness was found between E. coli

and bacterial isolates AE-5, S-10, AE-8. The maximum relatedness of

bacterial isolate with E.coli was 75 % and it was of I-15, AE-7 (Fig. 5).

No banding pattern was observed for bacterial isolate AE-4, therefore

dismissed for further analyses.

Advance nucleotide blast search revealed that the isolates were closely

related (similarity index ≥ 99%) to previously described spp. The bacterial

isolates I-15, S-10, 7.3, 11.4, AE-5, AE-7 and AE-8 had maximum

similarity index with Pseudomonas japonica, Achromobacter

xylosoxidans, Burkholderia ginsengisoli, Pseudomonas alcaligenes,

Comamonas testosteroni, Aeromonas aquatiac and Comamonas

testosteroni, respectively.

Biodegrdation and bioaugmentation potential of selected isolates was

tested to remove color of dyes in liquid medium and effluent collected

from textile industry. Before these studies, effect of different conditions

105

like oxygen, pH, temperature and co-substrate was tested to optimize the

dye degradation process. Bacterial isolates showed maximum dye

decolorization (100% color removal by AE-5, AE-7, AE-8, I-15 and S-10)

in static culture conditions at pH 7 and temperature 30 ºC. Moreover,

yeast extract enhanced the decolorization rate compared to the medium

without yeast extract.

Under optimized conditions, isolate I-15 completely removed the color of

five different dyes in liquid medium within 8-16 h. In the case of textile

effluent, isolate I-15 after augmentation completely removed the dyes

color in original textile effluent within 6-12 h in the presence of co-

substrate (yeast extract). The color removal was only 21% without yeast

extract.

Metabolic products and by-products were analyzed on LC-MS, and the

MS spectra of the decolorized medium showed the formation of

secondary products, with molecular weights similar to 1,3,4-oxadiazol-2-

ol, aniline, 1-amino-2-naphthol-4-sulfonic acid and some derivatives of

benzidine. Molecular weights of some products did not show close

similarity to any expected dye products/ by-products.

The degradation of aromatic amines such as aniline and 1-amino-2-

naphthol-4-sulfonic acid was also tested under static and shaking

conditions. Isolate AE-7 was able to achieve highest degradation of 92%

in the case of aniline (20 mg L-1

) within 96 h under static conditions.

Isolate 11.4 was able to degrade aniline up to 76% within 96 h of

incubation at static conditions. Other isolates degraded the toxic

106

compound with an efficiency ranged from 28–60% within 60 h duration

in static conditions. For 1-NSA (50 mg L-1

), all the isolates completely

degraded the aromatic compound (99 – 100%) within first 48 h of

incubation under both static and shaking conditions.

It was observed that the selected isolates carrying dual traits of dye

degradation and plant growth promotion were able to degrade dyes as

well as promote plant biomass. The dye RB5 in MSM completely

decolorized in 4 days when maize seedlings were inoculated with isolate

AE-8. Whereas, isolate AE-5, AE-7 andI-15 and consortium of these

isolates decolorized the same dye under same above conditions within 6

days. Moreover, the growth medium having plants inoculated with isolate

S-10 and isolate 7.3 was decolorized after 8 days of growth period.

Similarly, isolate 11.4 completely decolorized the dye after 10 days of

inoculation in plant growth liquid medium. In the case of non-inoculated

plants, up to 19% decolorization was observed after 10 days.

Simultaneous processes of dye degradation and plant growth promotion

by the selected bacteria were observed by growing maize plants in dye-

containing water. Plants inoculated with bacterial isolate S-10 exhibited

highest dye decolorization rate of 28% after 10 days. On the other hand,

non-inoculated plants also achieved decolorization rate of 15% after 10

days. Plant growth parameters were also observed in this study. Plants

inoculated with isolates I-15, 11.4 and AE-5 found to have higher biomass

production after 10 days. Significant difference was observed when

biomass of plants inoculated with I-15 and 11.4 were compared with

107

biomass produced by plants inoculated with AE-7, AE-8, S-10, 7.3,

consortium and control. Similarly, significantly higher root lengths were

achieved when plants were inoculated with consortium of I-15 and S-10.

After testing bacterial isolates in liquid medium, maize and barley plants

were grown in soil and were irrigated with dye contaminated water. The

results showed that plants inoculated with bacterial isolate 7.3 found to

have significantly higher biomass production (8 g) when compared with

non-inoculated plants (3.56 g) as well as plants grown in sterilized soils

(3.51 g). Biomass of plants inoculated with isolate 7.3 was also

significantly higher than biomass produced by non-inoculated plants as

well as plants inoculated with isolates I-15, S-10 and 11.4. Similarly,

higher roots lengths were observed with plants inoculated with isolate 7.3

when compared to all other treatments. In case of shoot length,

significantly higher rates were observed in plants inoculated with

isolates11.4 (75 cm) and isolate 7.3 (74 cm).

Influence of selected bacterial isolates on growth of barley plants were

also studied in comparison with non-inoculated plants as well as plants

grown in sterilized soil. Results demonstrated that plants inoculated with

isolate 7.3 had higher biomass production, isolates 11.4 and I-15 with

higher root and shoot lengths. Moreover, plants grown in sterilized soil

have relatively lower growth as compared to plants grown in the presence

in soil indigenous microflora.

The findings of present study demonstrate that efficient dye degrading

bacteria with additional traits of IAA production as well as P-solubilization could

108

be used simultaneously for azo dye degradation and plant growth promotion for

enhanced biomass production of energy crops.

109

CONCLUSIONS

Textile effluent analysis showed color, pH, EC and TDS values higher

than permissible limits which require attention for proper treatment prior

to its discharge into the environment.

The dye contaminated water and soil contain indigenous bacteria capable

of degrading dyes efficiently in liquid medium.

Selected dye degrading isolates also possessed plant growth promoting

traits and exhibited tremendous potential to improve plant growth of

bioenergy crops irrigated with dye contaminated water.

These bacteria also showed a great potential to simultaneously degrade

dyes and improve plant growth under controlled conditions (glass tube

experiment)

LC-MS analysis indicated that the textile dyes are degraded by selected

bacterial isolates.

The selected bacterial isolates with dual traits (dye degradation and plant

growth promotion) could have a practical application and can be used for

increasing plant biomass of bioenergy crops using dye-contaminated

industrial wastewater for irrigation.

FUTURE RECOMMENDATIONS

Testing under field condition is required to evaluate the potential of dual

trait strains for improving plant growth of crops grown using wastewater

in the premises of textile industry

110

Genes and enzymes responsible for dual activity i.e. the dye degradation

and plant growth promotion in such bacteria need to be characterized

Phytotoxicity induced by azo dyes before and after treatment with the dye

degrading bacterial strains could be studied for agricultural crops

111

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APPENDICES

Appendix I: Decolorization of structurally different dyes by isolated bacterial

strains after 24 h. The values are presented as mean of three

replicates ± SD

RB5: Reactive Black 5

DPB: Direct Pink B

SRS3BR: Sunfix Red 3BR

0

20

40

60

80

100

AE-5 AE-7 AE-8 I-15 S-10 7.3 11.4

Dec

olo

riza

tion

(%

)

Bacterial Strains

RB5 DPB SRS3BR

140

Appendix II: Decolorization of structurally different dyes by isolated bacterial

strains after 24 h. The values are presented as mean of three

replicates ± SD

SY: Sunfix Yellow

DB3R: Direct Blue 3R

Mix: Mixture of five different dyes used earlier (20 mgL-1

each)

0

20

40

60

80

100

AE-5 AE-7 AE-8 I-15 S-10 7.3 11.4

Dec

olo

riza

tion

(%

)

Bacterial Strains

SY DB3R Mix

141

Appendix III: Dye decolorization trend of RB5 enriched MSM (no-pants) by

different bacterial strains grown under Lab Conditions (after 10

days). The values are presented as mean of five replicates

Dye Degradation in MSM without Plant

Time


% D

ye

De

gra

da

tio

n

0

20

40

60

80

100

Control

AE-4

AE-5

AE-7

AE-8

I-15

S-10

7.3

11.4

Consortium

142

Appendix IV: Effect of different bacterial strains on growth of maize plants

grown in MSM (without RB5 dye) under Lab Conditions (10

days duration) The values are presented as mean of five

replicates ± SD. Overlapping error bars represent non-significant

difference with other error bars and vice versa

0

0.2

0.4

0.6

0.8

1

1.2

0

2

4

6

8

10

12

14

16

18

Pla

nt

Bio

ma

ss (

g)

Ro

ot

& S

ho

ot

Len

gth

(cm

)

Bacterial Isolates


143

Appendix V: Genome sequences of 16S rRNA genes of isolated bacteria and

their subsequent submission to NCBI with accession numbers

Sequence No Strain Sequence Number Accession Number

Seq1 I-15 BankIt1936516 Seq1 KX553914

Seq2 S-10 BankIt1936516 Seq2 KX553915

Seq3 7.3 BankIt1936516 Seq3 KX553916

Seq4 11.4 BankIt1936516 Seq4 KX553917

Seq5 AE-5 BankIt1936516 Seq5 KX553918



Seq1:

GTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCA

GGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGC

TAGTTGGTGAGGTAATGGCTCACCAAGGCTACGATCCGTAACTGGTCT

GAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCT

ACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGAT

CCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTT

TAAGTTGGGAGGAAGGGCAGTAAGCGAATACCTTGCTGTTTTGACGTT

ACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCG

Seq2:

TTGGCAGTGCTCGCAAGAGAACTGGAACACAGGTGCTGCATGGCTGT

CGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCA

ACCCTTGTCATTAGTTGCTACGAAAGGGCACTCTAATGAGACTGCCGG

TGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCT

TATGGGTAGGGCTTCACACGTCATACAATGGTCGGGACAGAGGGTCG

CCAACCCGCGAGGGGGAGCCAATCCCAGAAACCCGATCGTAGTCCGG

ATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCG

CGGATCAGCATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCG

A

Seq3:

CCGAAAGGGAGCCATAACACAGGTGCTGCATGGCTGTCGTCAGCTCG

144

TGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCC

CTAGTTGCTACGCAAGAGCACTCTAGGGAGACTGCCGGTGACAAACC

GGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAG

GGCTTCACACGTCATACAATGGTCGGAACAGAGGGTCGCCAACCCGC

GAGGGGGAGCCAATCCCAGAAAACCGATCGTAGTCCGGATCGCACTC

TGCAACTCGAGTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAG

CATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCG

Seq4:

CAGAGATGGATTGGTGCCTTCGGGAACTCAGACACAGGTGCTGCATG

GCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGA

GCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAA

GGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAG

TCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGG

TACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCG

ATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAAT

CGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGTC

TTGTACACACC

Seq5:

GAGATGGTTTCGTGCTCGAAGAGAACCGTAACACAGGTGCTGCATGG

CTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAG

CGCAACCCTTGCCATTAGTTGCTACATTCAGTTGAGCACTCTAATGGG

ACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTC

ATGGCCCTTATAGGTGGGGCTACACACGTCATACAATGGCTGGTACA

AAGGGTTGCCAACCCGCGAGGGGGAGCTAATCCCATAAAGCCAGTCG

TAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCNGAATCGCT

AGTAATCGTGGATCAGAATGTCACGGTGAATACGTTCCCNGGTCTTGT

ACACACCGAGA

Seq6:

CGGGAGTGCCTTCGGGAATCAGAACACAGGTGCTGCATGGCTGTCGT

CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC

CCCTGTCCTTTGTTGCCAGCACGTAATGGTGGGAACTCAAGGGAGACT

GCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATG

GCCCTTACGGCCAGGGCTACACACGTGCTACAATGGCGCGTACAGAG

GGCTGCAAGCTAGCGATAGTGAGCGAATCCCAAAAAGCGCGTCGTAG

TCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGT

AATCGCAAATCAGAATGTTGCGGTGAATACGTTCCCGGGTCTTGTACA

CACCGA

Seq7:

GAGATGGTTTGGTGCTCGAAAGAGAACCTGCACACAGGTGCTGCATG

145

GCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA

GCGCAACCCTTGCCATTAGTTGCTACATTCAGTTGAGCACTCTAATGG

GACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCT

CATGGCCCTTATAGGTGGGGCTACACACGTCATACAATGGCTGGTACA

AAGGGTTGCCAACCCGCGAGGGGGAGCTAATCCCATAAAGCCAGTCG

TAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCT

AGTAATCGTGGATCAGAATGTCACGGTGAATACGTTCCCGGGTCTTGT

ACACACCGA

146

Appendix VI: List of research papers under review for publication

No Title Status

1 Evaluation of bacteria isolated from textile

wastewater and rhizosphere to simultaneously

degrade azo dyes and promote plant growth

Accepted

2 Bioaugmentation of textile effluents by dye

degrading bacterial strains and their plant growth

promoting impact on maize and barley

Submitted

147

Appendix VII: List of Presentations

No Title Conference

1 Bioaugmentation of selected strains for the

degradation of azo dye contaminated textile

effluents

Soil Science Congress

2014

NARC, Islamabad

evaluation of azo dye degrading bacteria for …prr.hec.gov.pk/jspui/bitstream/123456789/8271/1/ph.d...

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