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BIOREMEDIATION OF PETROLEUM HYDROCARBON

CONTAMINATED SOIL BY INDIGENOUS BACTERIA IN

ASSOCIATION WITH PLANT

By

Hafiz Muhammad Rafique

M.Sc. (Hons.) Soil Science

A thesis submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

IN

SOIL SCIENCE

Institute of Soil & Environmental Sciences,

FACULTY OF AGRICULTURE,

UNIVERSITY OF AGRICULTURE

FAISALABAD, PAKISTAN

2015

DECLARATION

I hereby declare that contents of the thesis, “Bioremediation of

petroleum hydrocarbon contaminated soil by indigenous bacteria in

association with plant” are product of my own research and no part has

been copied from any published source (except the references, standard

methods/equations/formulae/protocols etc.). I further declare that this work

has not been submitted for award of any other diploma/degree. The

university may take action if the information provided is found inaccurate

at any stage. (In case of any default, the scholar will be proceeded against

as per HEC plagiarism policy).

Hafiz Muhammad Rafique

2001-ag-2581

To

The Controller of Examinations,

University of Agriculture,

Faisalabad.

We, the supervisory committee, certify that the contents and form of thesis

submitted by Hafiz Muhammad Rafique, Regd. No. 2001-ag-2581 have been found

satisfactory and recommend that it be processed for evaluation by the external

examiner(s) for the award of degree.

SUPERVISORY COMMITTEE:

CHAIRMAN:________________________________

(Dr. Hafiz Naeem Asghar)

MEMBER:___________________________________

(Dr. Zahir Ahmad Zahir)

MEMBER:____________________________________

(Dr. Muhammad Shahbaz)

This thesis is dedicated to my Parents and my eldest brother

Muhammad Sharif without their affection and support I would not be

able to reach the goal

ACKNOWLEDGMENT

With profound gratitude and deep sense of devotion, I wish to thank my worthy

supervisor, Dr. Hafiz Naeem Asghar, Associate Professor, Institute of Soil and

Environmental Sciences, for his valuable suggestion, inspiring guidance, skillful

supervision and constructive criticism in completion of the research work. I extend my

thanks to the members of my supervisory committee Dr. Zahir Ahmad Zahir, Professor,

Institute of Soil and Environmental Sciences, and Dr. Muhammad Shahbaz, Assistant

Professor, Department of Botany, for their useful suggestions and guidance throughout

course of the study.

I could not have completed this work without the help and friendship of

Muhammad Imran, Muhammad Yahya Khan, Muhammad Siddique, Muhammad

Arshad, Muhammad Haroon, Dr. Hafiz Faiq Siddique Gul Bakhat and Muhammad

Abdul Qayyum. I am grateful to all whose hands raised to pray for me. My special

thanks to my Brothers, Father-in-Law and my life partner for their support and love.

Finally, I am profuse elated to pay my thanks to Higher Education Commission of

Pakistan for financial support for this study.

(Hafiz Muhammad Rafique)

TABLE OF CONTENTS

No. Title Page

Chapter 1 General Introduction 1

1.1 Justification of study 5

1.2 Objectives of study 5

Chapter 2 Review of Literature

2.1 Petroleum hydrocarbons contamination 6

2.2 Hazards of petroleum hydrocarbon contamination 6

2.3 Remediation strategies 8

2.3.1 Phytoremediation 9

2.3.2 Bioremediation 10

2.3.3 Plant assisted Bioremediation 15

2.4 Plant growth promoting bacteria in plant assisted bioremediation 17

2.5 Bacterial Genera with plant growth promotion activities and

bioremediation capabilities

19

2.6 Role of plant roots for enhancing bioremediation 19

2.7 Degradation of petroleum hydrocarbons 22

Chapter 3 Isolation, Screening and Characterization of Bacteria

3.1 Introduction 24

3.2 Material and methods 25

3.2.1 Collection of soil samples 25

3.2.2 Isolation of bacteria 25

3.2.3 Preservation of bacteria 26

3.2.4 Bioremediation assay (BRA) 26

3.2.5 Particle size distribution of collected soil samples 26

3.2.6 TPH by infrared spectroscopy 27

3.2.7 ACC-metabolism assay of isolates 27

3.2.8 ACC-deaminase activity 28

3.3 Results 29

3.3.1 Sampling sites characteristics 29

3.3.2 TPH in collected samples 30

3.3.3 Microbial isolates 30

3.3.3.1 Isolates from Sheikhupura district 30

3.3.3.2 Isolates from Wazirabad district 31

3.3.3.3 Isolates from Gujranwala district 32

3.3.3.4 Isolates from Muzzafargarh (JIMCO Oil Depot) 35

3.3.3.5 Isolates from Muzzafargarh Thermal Power 35

3.3.3.6 Isolates from Sher Shah Multan (Railway track junction) 35

3.3.3.7 Isolates from Faisalabad soil samples 36

3.3.3.8 Isolates from Pindi Morga Rawalpindi soil samples 37

3.3.4 Quantification of ACC-deaminase activity 41

3.3.5 Summary of categorization of bacterial isolates on the basis of

Bioremediation and ACC-metabolism assay

41

3.4 Discussion 45

Chapter 4 Evaluation of Bacterial Isolates for Plant Growth Promotion

under Axenic Conditions

4.1 Introduction 48

4.2 Methodology 49

4.2.1 Growth pouch experiment 49

4.2.1.1 Preparation of broth culture 49

4.2.1.2 Seed disinfection and inoculation 49

4.2.1.3 Growth conditions 50

4.2.2 Jar experiment (Sand culture) 50

4.2.3 Root colonization assay 50

4.2.4 Root length by Delta T-Scanner 51

4.2.5 Identification of bacteria 51

4.3 Results 52

4.3.1 Root length 52

4.3.2 Shoot length 53

4.3.3 Fresh biomass 58

4.3.4 Oven dried biomass 59

4.3.5 Jar experiment (Sand culture) 62

4.3.5.1 Root length 62

4.3.5.2 Shoot length 63

4.3.5.3 Fresh biomass 67

4.3.5.4 Oven dried biomass 68

4.3.6 Root colonization 69

4.3.7 Identification of bacteria 74

4.4 Discussion 79

Chapter 5 Plant Growth Performance in Petroleum Contaminated Soil as

Affected by Bacterial Inoculation and TPH Removal in

Association with Alfalfa, Maize and Canola

82

5.1 Introduction 82

5.2 Methodology 83

5.2.1 Seed viability 83

5.2.2 Germination test 83

5.3 Pot trial on artificially spiked soil 84

5.3.1 Soil analysis 84

5.3.1.1 Maximum water holding capacity 84

5.3.1.2 Soil pH 84

5.3.1.3 Electrical conductivity 84

5.3.1.4 Soil organic carbon 85

5.3.2 Spiking of soil with crude oil 85

5.3.3 Treatment plan 86

5.3.4 TPH by Infrared spectroscopy 87

5.3.5 Analysis of soil for remaining diesel on GC 87

5.3.6 Statistical analysis of data 87

5.4 Results 88

5.4.1 Plant growth performance in petroleum contaminated soil under

controlled light and temperature

88

5.4.1.1 Physicochemical properties of soil 88

5.4.1.2 Germination 88





5.4.2 Plant growth performance in petroleum contaminated soil under

ambient light and temperature

96





5.4.3 TPH removal by bacteria in association with alfalfa, maize and canola

under controlled conditions of light and temperature

103

5.4.3.1 TPH removal in association with alfalfa 103

5.4.3.2 TPH removal in association with canola 103

5.4.3.3 TPH removal in association with maize 103

5.4.4 TPH removal by bacteria in association with alfalfa, maize and canola

under ambient light and temperature

106

5.4.4.1 TPH removal in association with alfalfa 106

5.4.4.2 TPH removal in association with canola 106

5.4.4.3 TPH removal in association with maize 106

5.4.5.1 TPH removal by bacterial isolates independent of plants association

under controlled conditions of light and temperature

109

5.4.5.2 TPH removal by bacterial isolates independent of plants association

under ambient conditions of light and temperature

109

5.4.6 Plant assisted bioremediation of diesel under controlled conditions of

light and temperature

111

5.4.6.1 Bioremediation of diesel in association with alfalfa 111

5.4.6.2 Bioremediation of diesel in association with canola 111

5.4.6.3 Bioremediation of diesel in association with maize 112

5.4.7 Plant assisted bioremediation of diesel under ambient conditions of


114

5.4.7.1 Bioremediation of diesel in association with alfalfa 114

5.4.7.2 Bioremediation of diesel in association with canola 114

5.4.7.3 Bioremediation of diesel in association with maize 114

5.4.8 Effect of root biomass in enhancing bioremediation of petroleum

hydrocarbons

116

5.5 Discussion 120

Chapter 6 General Discussion 122

Chapter 7 Summary 130

References 132

LIST OF TABLES

No Title Page

a Plant species used for phytoremediation of petroleum hydrocarbons/PAHs 12

b Bacterial genera used in bioremediation of petroleum hydrocarbons/PAHs 13

c Advantages and disadvantages of different biological strategies 14

1 Characteristics and TPH-IR of Sampling Sites in Different Districts of Punjab 42

2 Quantification of ACC-deaminase activity 42

3 Summary of categorization of bacterial isolates on the basis of

Bioremediation and ACC-metabolism assay

44

4 Summary of the traits of bacterial isolates screened for further study 44

5 Effect of bacterial inoculation on root length of alfalfa, maize and canola under

axenic condition in growth pouch assay

55

6 Effect of bacterial inoculation on shoot length of alfalfa, maize and canola

under axenic condition in growth pouch assay

56

7 Effect of bacterial inoculation on fresh biomass of alfalfa, maize and canola

under axenic conditions in growth pouch assay

60

8 Effect of bacterial inoculation on oven dried biomass of alfalfa, maize and

canola under axenic conditions in growth pouch assay

61

9 Effect of bacterial inoculation on root length of alfalfa, maize and canola under

axenic condition in sand culture

65

10 Effect of bacterial inoculation on shoot length of alfalfa, maize and canola

under axenic condition in sand culture

66

11 Effect of bacterial inoculation on fresh biomass of alfalfa, maize and canola

under axenic conditions in sand culture

70

12 Effect of bacterial inoculation on oven dried biomass of alfalfa, maize and

canola under axenic conditions in sand culture

71

13 Root colonization of alfalfa, maize and canola 72

14 Summary table of isolates selected for further study 73

15 Identification of 8 bacterial isolates by 16S rRNA sequencing 74

16 Physicochemical properties of soil 89

LIST OF FIGURES

No. Title Page

1 Schematic diagram of remediation strategies 8

1a Terminal methyl oxidation pathways adapted by alkane degrading

microorganisms

23

1b Degradation of aromatic ring by microorganism showing ortho- and meta-

cleavage of catechol

23

2 Isolates from Sheikhupura district soil samples contaminated with different

petroleum products

33

3 Categorization of isolates from Sheikhupura district on the basis of BRA and

ACC- metabolism assay

33

4 Categorization of isolates from Wazirabad district soil samples on the basis

of BRA and ACC metabolism assay

34

5 Categorization of isolates from Gujranwala district soil samples on the basis


34

6 Categorization of isolates from Muzzafargarh district (JIMCO Oil Depot)

soil samples on the basis of BRA and ACC metabolism assay

38

7 Categorization of isolates from Muzzafargarh Thermal Power soil samples

on the basis of BRA and ACC metabolism assay

38

8 Categorization of isolates from Sher Shah Multan soil samples on the basis


39

9 Categorization of isolates from Faisalabad soil samples on the basis of BRA

and ACC metabolism assay

39

10 Categorization of isolates from Pindi Morga Rawalpindi soil samples on the

basis of BRA and ACC metabolism assay

40

11 Categorization of bioremediation assay into low, medium and high 43

12 Comparison of inoculated and un-inoculated root growth of alfalfa under

axenic conditions

57

13 Phylogenetic tree of bacterium PM32Y 75

14 Phylogenetic tree of bacterium SFD2S2 75

15 Phylogenetic tree of bacterium WZ3S1 76

16 Phylogenetic tree of bacterium MZT72 76

17 Phylogenetic tree of bacterium SP104Y 77

18 Phylogenetic tree of bacterium SM73 77

19 Phylogenetic tree of bacterium WZ3S3 78

20 Phylogenetic tree of bacterium JM44 78

21 Germination percentage at different concentration of crude oil 89

22 Percent increase in root length of alfalfa, maize and canola over their

corresponding un-inoculated control under controlled conditions of light and

temperature

92

23 Percent increase in shoot length of alfalfa, maize and canola over their


temperature

93

24 Percent increase in fresh biomass of alfalfa, maize and canola over their


temperature

94

25 Percent increase in oven dried biomass of alfalfa, maize and canola over

their corresponding un-inoculated control under controlled conditions of


95

26 Percent increase in root length of alfalfa, maize and canola over their

corresponding un-inoculated control under ambient conditions of light and

temperature

99

27 Percent increase in shoot length of alfalfa, maize and canola over their


temperature

100

28 Percent increase fresh biomass of alfalfa, maize and canola over their


temperature

101

29 Percent increase in oven dried biomass of alfalfa, maize and canola over

their corresponding un-inoculated control under ambient conditions of


102

30 TPH removal by bacterial isolates in association with alfalfa under

controlled conditions of light and temperature

105

31 TPH removal by bacterial isolates in association with canola under


105

32 TPH removal by bacterial isolates in association with maize under controlled

conditions of light and temperature

108

33 TPH removal by bacterial isolates in association with alfalfa under ambient


108

34 TPH removal by bacterial isolates in association with canola under ambient


108

35 TPH removal by bacterial isolates in association with maize under ambient


110

36 TPH removal by bacterial isolates independent of plants association under


110

37 TPH removal by bacterial isolates independent of plants association under

ambient conditions of light and temperature

110

38 Bioremediation of diesel in association with alfalfa under controlled


113

39 Bioremediation of diesel in association with canola under controlled


113

40 Bioremediation of diesel in association with maize under controlled


113

41 Bioremediation of diesel in association with alfalfa under ambient conditions

of light and temperature and temperature

115

42 Bioremediation of diesel in association with canola under ambient


115

43 Bioremediation of diesel in association with maize under ambient conditions

of light and temperature and temperature

115

44 Effect of root dry biomass of alfalfa on bioremediation of petroleum

hydrocarbons under controlled and natural conditions of light and

temperature

117

45 Effect of root dry biomass of maize on bioremediation of petroleum


temperature

118

46 Effect of root dry biomass of canola on bioremediation of petroleum


temperature

119

List of Abbreviations

ACC-deaminase ACC-deaminase

ACC ACC

BRA BRA

BTEX BTEX

DF DF

IAA IAA

OD OD

PAHs PAHs

PGPB PGPB

PGPR PGPR

p-INT p-INT

ABSTRACT

Petroleum, the backbone of today's mechanized society, now became a threat to

environment due to extraction and transportation. Due to transportation accidental spills

occur regularly all over the world. Such spills have also occurred at many locations

throughout Pakistan over time. Petroleum and its products contain carcinogenic and

mutagenic compounds and therefore contamination of soil from petroleum hydrocarbons

is a serious problem. Efforts are now focused on seeking potential remediation techniques

for cleanup of petroleum contaminated soils in a cost effective and eco-friendly way.

Plant assisted bioremediation of petroleum contaminated soil is getting attention as

compared to the alone use of either microorganism or plant. The challenging task for such

efforts to be successful is not only the survival of microorganisms upon their inoculation

into xenobiotic environment but also microbe’s positives interaction with plants. Plant

growth promoting bacteria (PGPB) having 1-aminocyclopropane 1-carboxylic acid

deaminase (ACC-deaminase) are considered to helpful for plants in stressed environment

by reducing stress induced ethylene. Over three hundred bacterial isolates were cultured

from collected petroleum contaminated soil samples. Bioremediation and ACC-

metabolism assays screened out 27 from 301 bacterial isolates. These 27 bacterial isolates

possessed medium to high bioremediation potential for PAHs and also high ACC

deaminase activity. Compatibility of these 27 bacterial isolates with alfalfa, maize and

canola was assayed in growth pouches and jar experiment under axenic conditions.

Finally, on the basis of bioremediation, ACC-deaminase and plant growth promotion

activity, 8 bacterial isolates were screened out. Plant -assisted bioremediation of

artificially spiked coarse textured soil bearing 10,000 mg kg-1 (w/w) crude oil

concentration was carried out both under controlled and ambient conditions of light and

temperature. Also growth performance of inoculated plants was compared with un-

inoculated plants for each crop. After 60 days, results revealed that four bacterial isolates

Bacillus subtilis, Bacillus cereus and other Bacillus sp. significantly degraded petroleum

hydrocarbons. The most efficient was Bacillus subtilis which reduced TPH contamination

47%, 37% and 43% in combination with alfalfa, canola and maize, respectively. While

the same bacterium when inoculated alone reduced 33% TPH contamination under

controlled light and temperature while 31% under ambient light and temperature.

Significant increase in growth attributes such as root length, shoot length and fresh

biomass of all three crops was observed as compared to their corresponding un-inoculated

plant control. Comparatively, alfalfa with respect to germination and growth in petroleum

contaminated soil performed better as compared to maize and canola. Study can be

concluded that use of bacteria possessed with dual traits of bioremediation potential and

ACC-deaminase activity in combination with plant can be a good approach for

remediation of petroleum hydrocarbons contaminated soil.

Chapter 1: General Introduction

1

Chapter 1

General Introduction

Petroleum derived from fossil fuels are important part of our today's mechanized society,

however, an inevitable risk to environment is being posed due to extraction and

transportation of these fuels. Contamination of terrestrial environment with petroleum

hydrocarbons occurs due to both human and mechanical error. Accidental spills of

petroleum hydrocarbons into various compartments of environment such as water and soil

are increasing all over the world, however, the exact detail of extent of petroleum

hydrocarbons contamination is difficult to bring into figures because the contamination is

unintentional. Pakistan's coastal line near Karachi faced contamination of 28,000 tons of

crude oil when in July, 2003 a Greek ship naming Tasman Spirit cracked into two pieces.

The citizen of Korangi Town Karachi, Pakistan found oil in their streets, house yards and

shops due to extraordinary rupture in the pipeline of Pak Arab Refinery Corporation

(PARCO) during construction work by Sui Southern Gas Company (Alam, 2008).

Another un-intentional spill of 18 tankers of crude oil happened in September, 2009 in

Sindh Province of Pakistan due to collision of two goods carrying trains (Khan, 2009).

Most recently, another huge oil spill happened when two NATO oil tankers each bearing

60,000 liters gasoline toppled on Khojak Pass near Chaman, Pakistan. Further release of

petroleum hydrocarbons into the environment occurs at well sites, petroleum refineries.

Petroleum refining industries in Karachi, Rawalpindi, Multan, Qasba Gujrat Kot Adu are

sources of contaminants such as phenols, sulphides and oily residues (National

Environmental Policy of Pakistan, 1999). A significant contribution in hazardous waste

level, according to Pakistan Environmental Protection Agency (2005), is from petroleum

and petro-chemical industries.

Contamination of different compartments of the environment i.e. air, water and soil with

petroleum hydrocarbons poses serious threat to health of human beings. For instance,

among benzene, toluene, ethylbenzene and xylene commonly abbreviated as BTEX,

benzene is carcinogenic, toluene damages central human nervous system, ethylbenze

causes skin irritation and long exposure to xylene may lead to aplastic anemia (US EPA,

2011). BTEX are mono-aromatic compounds and are important component of gasoline

and constitute 40% of gasoline's composition (Ribeiro et al., 2012). Similarly, diesel fuel

is composed of branched alkanes, cycloalkane, alkenes and recalcitrant aromatic


2

hydrocarbons (ATSDR 1996). Therefore, remediation measures of such pollutants in the

environment especially in soil are of utmost importance.

A number of remediation techniques have been applied for the cleanup of

petroleum hydrocarbon contaminations including physical, chemical and biological

techniques. Physical remediation measures such as incineration and thermal desorption

and chemical remediation processes such as solvent extraction, encapsulation and

oxidation-reductions are not only expensive but also need substantial use of heavy

machinery and energy. Moreover, bulk of soil has to be shifted to treatment facility which

is ecologically disruptive (Huang et al., 2004). These limitations make biologically based

remediation techniques such as phytoremediation and bioremediation more attractive.

Phytoremediation is the use of plants for removal or containment of contaminants

through various processes such as phytoextraction, phytostabilization,

phytotransformation and phytovolatilization (Glick, 2003; 2010). Phytoremediation, no

doubt, is an appealing strategy for decontamination of environmental xenobiotic

compounds, however, in case of petroleum hydrocarbons it is evident from various

studies that plant growth including root shoot biomass and elongation of root and shoot

was reduced due to petroleum hydrocarbons stress (Masakorala et al., 2013; Peng et al.,

2009; Germaine et al., 2009). Petroleum hydrocarbons are of various nature and have

different characteristics especially solubility in water. For successful phytoremediation,

contaminants must have solubility in water while petroleum hydrocarbons with octanol

water partitioning co-efficient (Kow) greater than 3 are not soluble in water thus not

translocated to shoots. Due to less solubility in water, phytoremediation of such

contaminants becomes difficult (Rojo, 2009).

Another appealing strategy is the use of indigenous bacteria usually called as in-

situ bioremediation which is an established strategy for petroleum hydrocarbons

decontamination. For successful application of this technique, it is necessary to isolate

such bacteria that not only survive in the contaminated conditions but also capable of

degrading the target contaminants (Malik and Ahmad, 2012). As petroleum hydrocarbons

are complex mixture of different compounds having different structure such as n-alkanes,

polyaromatics and cycloalkanes, the in-situ remediation of polyaromatics and

cycloalkanes is sometimes difficult because of failure of bacteria in achieving contact

with such contaminants (Sarma and Prasad, 2015). Moreover, high molecular weight

compounds are not degraded at early stages of inoculation so persistent survival of


3

bacterial inoculants is necessary for degradation of such compounds (Mishra et al., 2001;

Schwartz and Scow, 2001).

Bioremediation assisted by plants is useful to overcome such constraints and

thereby enhances degradation of recalcitrant soil contaminant like polyaromatic

hydrocarbons (Kawasaki et al., 2012). Roots of higher plants play important role in

assisting microbial population to degrade pollutants in many ways such as provision of

molecular oxygen, inorganic nutrients through sloughed off cells, soluble exudates and

lysates (Khan et al., 2013; Segura and Ramos, 2013; Phillips, 2008; Martin et al., 2014).

Plants improve soil aeration by directly giving off oxygen to the root zone as well as

allowing improved entry of oxygen into the soil by diffusion along old root channels (Nye

and Tinker, 1977). If microbes do not have degradative pathways for petroleum

hydrocarbons and cannot use them as carbon source, even then petroleum hydrocarbons

can be mineralized through the process of co-metabolism (Li et al., 2015). Co-

metabolism is mechanism by which plants assist microbes to co-metabolize a

contaminant in the soil using the root exudates as an energy source. Plant enzymes

degrade the compounds, and then further degradation is carried out by microbes

ultimately into carbon dioxide and water. Some structural analogs of polyaromatic

hydrocarbons such as phenols, terpernes and flavonoids are released by some plant roots

as exudates and thus promote the growth of petroleum hydrocarbons degrading bacteria

(Khan et al., 2013; Rentz et al., 2005; Martin et al., 2014) and can act as trigger of PAHs

degradation-pathway (Singer et al., 2003). Although plant cooperation to microorganism

is true yet organic pollutants present in the system causes impairment in plant growth and

thus limit the benefits of adding vegetation to a contaminated soil (Gartler et al., 2014).

Thus, plant growth impairment is a serious limitation in petroleum hydrocarbons

contaminated soils because plant species are sensitive to contaminants and fails to

produce sufficient biomass and root activities (Huang et al., 2001). Successful plant

assisted microbial remediation of petroleum hydrocarbons contaminated soil is dependent

on elimination of root inhibition. This inhibition in root growth in contaminated soils is

mainly due to stress-induced ethylene; an established stress phytohormones (Arshad et

al., 2007). Synthesis of ethylene in plant at a rate where it exerts inhibitory effects on

plant roots is in response to contaminant-induced stress. Thus, a preferential target is to

regulate/ limit the biosynthesis of ethylene so that normal or extreme root biomass can be

achieved in stressed environment. Some plant growth promoting rhizobacteria (PGPR)

are reported to be equipped with ACC-deaminase enzyme that hydrolize ACC (an


4

immediate precursor of ethylene in plants) into α-ketobutyric acid and ammonia and thus,

regulate the biosynthesis of ethylene when plants are inoculated with such bacteria

(Ajuzieogu et al., 2015). This reveals the fact that just as plant can affect microbial growth,

microorganism can affect and protect plant growth in contaminated soils. Thus

rhizobacteria containing ACC deaminase activity regulate plant growth in both biotic and

abiotic stresses and dramatically increase the biomass of plant especially roots which is a

desirable parameter for plants to be used to aid in the process of biodegradation of

petroleum hydrocarbon contaminated soils.

But, unfortunately, the isolation and subsequent inoculation with bacteria having

ACC-deaminase activity does not fulfill the job of remediating the petroleum

hydrocarbons contamination unless they are acclimated to such contaminants. According

to suggestions made by Glick (2010) for the enhancement of degradation of organic

contaminants in soil that the bacteria inoculated for remediation of organic contaminants

must possess twin nature of plant growth promotion as well as degrader of soil

contaminant. Indigenous bacteria from petroleum hydrocarbons polluted soils can

degrade wide range of petroleum hydrocarbons but their efficiency may be different and

is affected by contaminants when in higher concentrations (Barbeau et al., 1997). But the

isolation and re-inoculation after enrichment may be helpful in overcoming problems

such as tolerance to higher toxicity (MacNaughton et al., 1999).

Another important consideration in establishing successful plant bacterial

cooperation for enhanced remediation of petroleum hydrocarbons contaminated soil is the

selection of suitable plants and assessment of bacterial inoculants behavior to the selected

plants. As a result of differences in morphology, physiology and interaction with bacterial

inoculants, all plant species are not tolerant to petroleum hydrocarbon contamination or

not effective in enhancing remediation of petroleum hydrocarbons contaminated soil

(Walker et al., 2003). The problem is that the plant characteristics that enhance plant

tolerance to oil may have antagonistic effect on biodegradation potential of plants. For

example, plants with high water use efficiency may have high tolerance to oil but plants

equipped with this trait may not contribute to enhance bioremediation of oil. While on the

other hand plants with lower evapotranspiration, small leaf area may reduce the oxygen

supply in soil which is important limiting factor in aerobic bioremediation that may

reduce bioremediation rate (Kim and Beard, 1988). Hence, crops alfalfa, maize and

canola with different morphology were selected to assist in bioremediation process.


5

1.1 Justification of study

Accidental spills of petroleum hydrocarbons used to happen now and then, additionally,

generation of petroleum sludge in refining process of crude oil in oil refineries all around

the country is unavoidably being happened. General practices adapted by refineries are

either dumping of sludge or incineration which are not only costly but also not

ecofriendly. Due to lack of technology, accidental spills do not handle properly. Other

technologies such as bioremediation, phytoremediation and rhizoremediation developed

abroad cannot be imported as there is a significant difference in environmental factors

such as climate, plant species. This research work was therefore focused on assessing of

bacterial potential of bioremediation in association with plants.

1.2 Objectives of the study

The basic objective of the study was to isolate bacteria from petroleum

contaminated soil capable of degrading petroleum hydrocarbons.

As interest is growing in the convergence of bacteria and plants for remediation

of organic contaminants since last few decades due to its multiple benefits, so the

other objective of the study was to screen out such bacteria that have both

biodegradation potential and plant growth promotion activities.

Plants generally face root inhibition due to contamination induced ethylene.

Isolation of bacteria capable of relieving plants from this stress induced ethylene

was another objective of the study.

Assessment of germination and growth performance of alfalfa, maize and canola

in petroleum contaminated soil alone and in association with bacterial isolates

Ultimate goal of the study was the assessment of bioremediation potential of

selected isolates in association with alfalfa, maize and canola.


6

Chapter 2

Review of Literature

2.1 Petroleum hydrocarbons contamination

Petroleum hydrocarbons are extracted out as raw material and refined into various

products such as gasoline, diesel and kerosene to serve as energy source for industrialized

society. Consequently extraction, transportation and refining processes increased chances

of petroleum contamination by multi-folds in all compartments of the environment (Atlas

and Bartha, 1981). Some major accidental spills in last few decades worldwide starting

with Torrey Canyon in 1969; Exxon Valdez in Prince William Sound, Alaska in 1989,

history biggest ever oil spill during Gulf War in 1991 (Fayad and Overton, 1995) and

British Petroleum Deep Water Horizon Oil spill in 2010 happened and damaged aquatic

environment as well as terrestrial environment. In Pakistan scenario, oil pollution has not

been well reported due to one or other reasons except the major accidental spills which

comes under the eyes of electronic or publishing media. Examples of such major

accidental spill in Pakistan may be led by Tasman Spirit, a Greek Ship, which off

grounded in 2003 and contaminated not only aquatic environment but also several

kilometers of coastal area of Karachi. The citizens of the same city, the Karachi, again

faced a major accidental oil spill five years later in 2008 while pipeline of Pak Arab

Refinery Corporation (PARCO) rupture bathed the streets and homes of Korangi Town

Karachi with several tons of crude oil (Alam, 2008) and in the following year collision of

two cargo trains spilled 18 tankers of crude oil in the same province (Khan, 2009). Most

recently, another huge oil spill happened when two NATO oil tankers each bearing

60,000 liters gasoline toppled on Khojak Pass near Chaman border, Pakistan. Other than

accidental spills, sources of contamination may be storage tanks due to non-maintenance

and absence of proper vandalism (Adam, 2001) and oily sludge unavoidably produced by

refining industries throughout the country (National Environmental Policy of Pakistan,

1999). Other source of petroleum hydrocarbon contamination spreaded all around the

country are petrol pumps and service stations which has not been reported by any

environment regulating agency.

2.2 Hazards of petroleum hydrocarbon contamination

Petroleum hydrocarbons contamination affects almost all kind of life adversely. As for as

human health is concerned, petroleum hydrocarbons and its various fractions have

deleterious effects. For instance, upon exposure to gasoline, one of the distillate of crude


7

oil, causes serious health hazards to human being such as ethylbenze cause irritation in

respiratory system, skin and eyes. Upon inhalation, gasoline may cause drowsiness,

nausea and numbness. Moreover, benzene, an important constituent of gasoline may

damage nerves and cause anemia and toluene is depressant for central nervous system of

human being. Previously tetraethyl lead was included in some gasoline formulas may

cause damage to human liver (USEPA, 2011). One of the most persistent distillate of

petroleum hydrocarbons in environment is diesel which causes mutagenicity and

carcinogenicity (CCME, 2001). Benzene ring containing petroleum hydrocarbons

compounds called aromatic hydrocarbons are not only among most prevalent constituents

of petroleum hydrocarbons but also most hazardous to human beings as these compounds

are carcinogenic and mutagenic (Masiol et al., 2012). Mutagenicity of the polyaromatic

hydrocarbons was studied by (Lemiere et al. (2005) on rats which were given

polyaromatic hydrocarbon contaminated food for 2 to 4 weeks. Upon analysis of DNA

and bone marrow, the authors observed mutagenic changes and they concluded that food

contaminated with oil can result in genotoxic changes in consumer. Environmental

protection agency of United States of America listed 16 PAHs as carcinogenic to human

beings (Lampi et al., 2006). After spill of crude oil, birds and animals of the area face

serious consequences of the contamination which lead them to death because of failure in

absorption of nutrients in their digestive tract and this failure is attributed to presence of

black emulsion in their digestive tract (Khan and Ryan, 1991). Ecosystem is severely

affected by the complex component of crude oil. Microbial activities of the site may also

be inhibited by some compounds present in crude oil such as phenolic and quinonic

compounds reduced microbial cell growth (Sikkema et al., 1995) and some compounds

impair oxygen uptake of microorganism and thereby energy transduction (Uribe et al.,

1990). Petroleum hydrocarbons contamination effect on other living entities such as

plants, microorganisms and birds are obvious. Regarding plant, it is generally considered

that stunted plant growth occurs if concentration of contamination is 1% or exceeds it

(Wardley, 1979), however, literature have controversies in this regard. Chaineau and co-

workers (1997) observed that diesel contamination did not affect the germination of seeds

of Scots pine, poplar while the seeds of maize, sunflower, wheat and barley were affected

by hydrocarbon contamination. Plant growth parameters such as plant height, root length

and biomass were suppressed by the toxic effect of petroleum hydrocarbons (Shahsavari

et al., 2013).


8

RemediationStrategies

Physical

Incineration

Vacuum extraction

Soil washing

Chemical

Solvent extraction

Neutralization

Oxidation reduction

Biological

Biopiling and Land farming

Bioremediation

Phytoremediation

Infiltration galleries

Considering the toxicology of petroleum hydrocarbons, scientific community is in search

of the most efficient, cost effective, environment friendly and self-sustainable strategy

since the beginning of industrial era (Shim et al., 2009).

2.3 Remediation strategies

Numerous technologies for the remediation of petroleum hydrocarbons have been

developed. Mainly three types of technologies; physical, chemical and biological have

been used for cleaning up of petroleum contaminated soils. The schematic diagram is

given below

Fig. 1 Schematic diagram of remediation strategies

Physicochemical strategies are means of destroying or separating contaminants

abiotically from the environment. Although these techniques are well established and

have higher efficiency in recovering contaminants, most of them are too costly or add

secondary pollutants in the environment. For example, incineration or thermal destruction

removes contaminants with 99% efficiency (USEPA, 1996) but it is not only costly but

also become the source of secondary pollution (Lingrong et al., 2012) due to incomplete

combustion and releasing volatilized compounds into air. Similarly, solvent extraction, an

ex situ chemical method for the removal of organic and non-aqueous contaminants by


9

separating and concentrating and subsequent incineration has recovery efficiency as high

as 10,000:1 (Donnelly et al., 1994), but costly and generate secondary pollutant in the

environment. Soil washing is done by using scrubbing action of water supplemented

sometimes with surfactants. This technique is only pretreatment which is followed by

successive treatments such as incineration and thermal desorption that make the cleaning

process too costly and laborious. As these techniques bear drawbacks of economics and

public non-acceptance, so the scientific community shifted its attention towards seeking

of some alternatives and biological approaches has been under focus since 1990's

extensively.

2.3.1 Phytoremediation

Phytoremediation is a promising technology for the cleanup of soil or ground water

contamination by plants and their associative microorganism (Fatima et al., 2015). In

phytoremediation, natural synergistic relationship among plants, microorganism and

environment play role through various mechanisms such as phytodegradation,

phytostabilization, phytovolatilization and rhizoremediation for the degradation,

containment of xenobiotic compounds. Plants degrade contaminant either internally in the

cell by taking up contaminants in their cells and subsequent degradation in metabolic

process or externally by releasing enzymes that breakdown contaminants (Mougin, 2002;

Liu et al., 2013). But in case of organic contaminants such as petroleum hydrocarbons

and polycyclic aromatic hydrocarbons (PAHs) metabolic degradation by plants in their

cells is limited due to inability of plants to take up such contaminants (Burken and

Schooner, 1998; Mougin, 2002; Newman and Reynolds, 2004; Martin et al., 2014).

However, hydrophilic organic compounds with octanol-water partition coefficient (Kow)

log Kow≤1 can be taken up, translocated and metabolized by plants (Cunningham and

Berti, 1993; Limmer and Burken, 2014). Extracellular degradation of organic compounds

by the release of several enzymes such as cytochrom P450 a gultathion-s-transferase is

reported by several scientists (Gianfreda and Rao, 2004; Gonzalez et al., 2006). Another

mechanism of phytoremediation is phytovolatilization in which organic contaminants are

taken up, translocated, metabolized and volatilized through stomata into air (Souza et al.,

2014). But due to limited ability of plant to uptake organic contaminants, extensive work

has not been done in this field.

Mechanism of phytoremediation by which plants make contaminants immobilized

and not bioavailable to other organisms is said to be phytostabilization (Germida et al.,

2002). Phytostabilization of contaminants is carried out by plant roots either directly by


10

absorbing them on root surface (Pilon-Smits, 2005) or indirectly through the release of

enzymes which bind contaminant in organic matter; a process called humification (Frick

et al., 1999; Schwab et al. 1998; Binet et al., 2000).

2.3.2 Bioremediation

Bioremediation is a promising and emerging strategy for cleanup of petroleum

hydrocarbons contamination and refers to complete mineralization of contaminants into

carbon dioxide, water and inorganic compound (Kuiper et al., 2004; Barea and Pozo,

2005). Application of microbes for remediation of contaminated soils has several

advantages such as cost effective and friendly to environment as compared to physical

and chemical traditional technologies (Gallego et al., 2001; Bundy et al., 2002; Mulligan

and Yong, 2004; Bento et al., 2005; Joo et al., 2008; Gargouri et al., 2014; Fuentes et al.,

2014; Pizarro et al., 2014). Above all, advantage of using microbial population for

degradation of petroleum hydrocarbons is that bacteria may completely degrade

contaminants into carbon dioxide and water leaving no contaminants any more. Various

genera of the bacteria are reported to have biodegradation ability of organic contaminants,

for example, Pseudomonos sp. (Liu et al., 2013), Bravibacterium and Bacillus (Xiao et

al., 2012), Burkholderia (Caballero-Mellado et al., 2007), Sphingomonos and

Cycloclasticus (Ho et al., 2002) are well known genera used in bioremediation

technology. Different kinds of bioremediation methods have been used which includes

bioreactors, biostimulation, bioaugmentation biopiling and land farming (Zhou and Hua,

2004).

In biostimulation, indigenous microbes are stimulated by providing inorganic

nutrients such as phosphorus and nitrogen to enhance the speed of bioremediation process

(Perfumo et al., 2007) as in most cases sites polluted with organic contaminant are

deficient in phosphorus and nitrogen or theses mineral nutrients are not available to

microorganisms (Malina and Zawierucha, 2007; Ron and Rosenberg, 2014).

Biostimulation by addition of these mineral nutrients has been adopted by many workers

to enhance microbial growth and consequently degradation rates of organic pollutants

(Sarkar et al., 2005). On the other hand, it is observed that the addition of these mineral

nutrients may have negative effect in a sense that these nutrients may inactivate microbial

population and thereby decrease in bioremediation process (Johnson and Scow, 1999).

Bioremediation of hydrocarbons can be enhanced by provision of oxygen to

overcome low redox potential that is characteristics of organic pollutant contaminated soil

(Dibble and Bartha, 1979) and the process is called bioventing. This process makes the


11

bioremediation technology costly. Other processes parallel to bioventing such as

bioslurping and biosparging has been innovated to decrease the cost of bioventing

process. For example, biosparging is used to provide air or gas to enhance biodegradation

process rather than volatilizing the contaminants (Brown et al., 1994). Another strategy of

bioremediation is bioaugmentation which is enhancing the bioremediation process by the

use of pre-acclimated microorganism or genetically engineered microorganism or transfer

of genes relevant to biodegradation so that they may get conjugated in the indigenous

microbial population (El Fantroussi and Agathos, 2005). Bioaugmentation is quite a

simple method for enhancing rate of bioremediation and success has been recorded by

some examples such as Alisi and co-workers (2009) claimed 75% reduction of diesel in a

period of 42 days. However application of exogenous bacteria for bioaugmentation

purpose may fail the strategy due to indigenous microbial population of the contaminated

soil which would compete out exogenous population (Vidali, 2001). Some other factors

that may prone bioaugmentation to failure are provision of oxygen as petroleum

hydrocarbons have very low redox potential and thereby most important electron acceptor

for microbial process is oxygen; inorganic nutrient availability; soil moisture and

bioavailability of contaminant (Suja et al., 2014).


12

Table:- a Plant species used for phytoremediation of petroleum hydrocarbons/PAHs

Plant Contaminant Parameters Measured Initial Conc. Degradation (%) References

Cotton TPH Root growth, Enzymatic activity 0.4% Not available (N/A) (Jingyan et al.,

2013)

Alfalfa Benzo[a]pyrene Growth parameters, Dissipation of

Benzo[a]pyrene

N/A 78% by alfalfa

supplied with organic

fertilizer

(Fu et al.,

2012)

Poplar TPH and

Mineral oil

Plant growth parameters, Net

photosynthesis rate, Reductase

activity, Proline content, Chlorophyll

content

11.039 g Kg-1 and

6.839 g Kg-1

N/A (Fiziološki et

al., 2012)

Cotton,

Ryegrass, Tall

fescue Alfalfa

TPH Effect on growth parameters,

Effect of Urea on degradation

Effect of PGPR in combination with

plants on degradation

5% 90-150% degradation

rate by Tall

fescue+PGPR

(Tang et al.,

2010)

Birdsfoot

trefoil

Italian ryegrass

Diesel Plant biomass, Diesel degradation 10g Kg-1 57% degradation by

Italian ryegrass and

47% by birdsfoot

trefoil

(Yousaf et al.,

2010)

Maize and Tall

Fescue

TPH Germination, Growth parameters,

TPH remainging

3.5% 57.5% by Tall Fescue

and 55.2% by Maize

(Shirdam et al.,

2009)

Barn Yard TPH Microbial activity, TPH degradation 0.6% 100% in rhizosphere (Kim et al.,

2007)

Fescue Grasses Diesel Seed germination, 0.8% Only 8% reduction in

germination of the

crop

(Al-Ghazawi et

al., 2005)

Grass/maize

mixture

Used Motor oil Germination, degradation of used

motor oil

1.5% 38% with plants

67% with plants

+fertilizer

Dominguez-

Rosado and

Pichtel 2004

Tall fescue Kentucky

bluegrass Wild

Creosote Germination

percentage

0.5 to 3% (Huang et al.,

2004)


Table b. Bacterial genera used in bioremediation of petroleum hydrocarbons/PAHs

Bacteria Contaminant Strategy Efficacy of Treatment References

Bacillus sp, Staphylococcus sp,

Micrococcus sp, Pseudomonas

sp, Psychrobacter sp

Crude oil Consortium of bacteria

was applied to

bioremediate crude oil

90% C13 alkane, 77% C15,74%

C18 alkane and 46 to 55% aromatic

hydrocarbons degraded after 24

hour incubation

(Malik and Ahmad, 2012)

Alcanivorax, Pseudomonas,

Bacillus, Bortetela, Brucella,

Acenitobacter, Staphia,

Crude oil Bacterial isolates with

bio-surfactant producing

potential

Straight chain alkanes were

degraded but bacterial isolates were

unable to degrade branched

hydrocarbons

(Susilaningsih et al., 2013)

Pseudomonas aeruginosa,

Bacillus subtilis, Acinetobacter

lwoffi

Crude oil Isolated from crude oil

contaminated soil for

bioremediation

88.5% degradation by mixed

consortium, 77.8%,76.7%,74.3 by

used bacteria

(Al-Wasify and Hamed, 2014)

Bacillus Sp., Staphylococcus

Sp,. Pseudomonas Sp.,

Flaviobacterium Sp.,

TPH Bioremediation study

supplemented with cow

dung

80% of TPHs were biodegraded (Adams et al., 2014)

Alcaligenes faecalis, Bacillus

Sp., Citrobacter murliniae,

Crude oil Saline aquatic culture

isolates used for crude oil

Negative co-relation was observed

between time and amount of TPH

depicting depletion of substrate

(Ichor et al., 2014)

Pseudomonas, Bacillus and

Micrococcus sp.

Crude oil Bioremediation with

consortium of bacteria

90% crude oil degraded in bacterial

consortium. Application of single

bacterium was not effective

(Omm-e-Hanny et al., 2015)

Pseudomonas putida,

Sphingomonas sp.

TPH Bioremediation with

artificial consortium of

Pseudomonas putida,

Sphingomonas sp. and

rhizoremediation with

these bacteria

All P. Putida sp. and

Sphingomonas sp. survived in

concentration up to 30 g Kg-1 but

rhizoremediation was found more

effective than bioremediation alone

(Pizarro-Tobías et al., 2015)

Pseudomonas putida TPH Rhizoremediation with

alfalfa

99% removal of TPH (Gouda et al., 2015)


14

Table:-c Advantages and disadvantages of different biological strategies

Strategy Disadvantages Advantages

Land Farming and

Biopiling

1. Inadequate oxygen, reduced contact between

microorganisms and petroleum hydrocarbons and

inadequate nutrients (Hansen et al., 2004)

2. Regulatory issues as more than 80-85%

concentration difficult to reduce by land farming

(Singh et al., 2009)

3. Require extensive area to be operative (Van

Hamme et al., 2003)

4. Uncontrolled growth conditions of temperature

and moisture for biodegrading agent (Van

Hamme et al., 2003)

1. Designing and implementation is easy (Pope

and Matthews, 1993)

2. Cost effective as compared to chemical and

thermal strategies (Singh et al., 2009)

3. Minimum maintenance measures are required

4. Less monitoring effort is needed

Bioremediation 1. Slow process. It requires months or more time

(Barin et al., 2014)

2. Heavy metals are not biodegradable (Ali et al.,

2013)

3. Anaerobic bioremediation process is too slow so

in-situ application with low permeable soil is

difficult

4. All contaminants cannot be removed

5. Strong specialized knowledge is required for

successful application of bioremediation (Kumar

et al., 2011)

1. Low cost and publically accepted technology

(Kumar et al., 2011)

2. Unlike chemical treatment secondary

pollution is not produced (Baghel and Pandey,

2013)

3. Can be used as both in-situ and ex-situ

technology

4. Ecologically not disruptive (Ali-Elredaisy

2010)

Phytoremediation 1. Slow process. It cannot be used where human

health is in danger

2. Depends on season for application

3. High level of contaminants can be too toxic for

plants to grow

4. May not meet regulatory parameters regarding

time and final residues of contaminants as 100%

removal is not possible

1. It is solar energy driven green technology and

aesthetically gains public acceptance

2. Low cost (60-80%) as compared to physico-

chemical technologies (Morikawa, 2003)

3. Preserving the natural structure of soil and

permanent in situ remediation (USEPA 2000).

4. It can be used for treatment of broad range

contaminants


15

2.3.3 Plant assisted bioremediation

Plants and microorganisms both are able to degrade petroleum hydrocarbons

independently of each other, however, hydrophobicity of high molecular weight

compounds with high octanol water co-efficient (Kow>4) limits the ability of plant to

degrade petroleum hydrocarbons (Pilon-Smits 2005). Bacterial degradation in association

with plants or in the rhizosphere of plants is said to be rhizoremediation (Kuiper et al.,

2004). In search of the cost effective, publically accepted and at meaningful rate

degradation lead to find alternative which is bioremediation in association with plants or

rhizoremediation as the synergistic use of plant and microbes showed higher degradation

rate as compared to bioremediation or phytoremediation alone (Gurska et al., 2009). For

successful establishment of the strategy, tolerance to petroleum hydrocarbons

contamination must be possessed by both plants and bacteria (Germida et al., 2002 and

Bona et al., 2011). Alfalfa (Medicago sativa L.) a perennial grass with long, dense and

fibrous root system make the crop tolerant to stress conditions such as drought and

pollutants. Therefore, alfalfa has been suggested as an ideal candidate to be used in

rhizoremediation process (Villacieros et al., 2003; Fan et al., 2008). Wiltse et al. (1998)

studied 20 genotypes of alfalfa for their potential to degrade crude oil at concentration of

20 g/ kg and observed reduction of 56% in one year. In a phytoremediation of kerosene

based jet fuel at concentration of 1700 mg kg-1 with alfalfa and horseradish, Karthikeyan

et al. (1999) observed up to 90% removal of the jet fuel in watered mesocosm study

which lasted for 150 days. Alfalfa and Ditch reed was assessed for their potential to

degrade paraffin and naphthene (79.9 g kg-1) and PAHs at concentration of 80 mg kg-1 by

Muratova et al. (2003) and recorded 82% removal of hydrocarbons with both plants in a

time period of 27 months, however, most of the removal was done in first 18 months. On

the basis of enumeration of rhizosperic bacteria, they concluded that most of the PAHs

removal was the result of rhizoremediation mechanism. Similarly, alfalfa was declared

tolerant to organic pollutants (Kaimi et al., 2007 and Marti et al., 2009). A study

conducted by Fu et al. (2012) in environmentally controlled conditions to assess the

degradation of benzo [a] pyrene in an aged soil by the effect of alfalfa revealed that

alfalfa in association with microorganism succeeded to dissipate 11.8% benzo [a] pyrene

as compared to sterilized treatments. The author concluded that the main mechanism in

the degradation of contaminant was due to combined effect of alfalfa and

microorganisms. Some part of organic contaminants like petroleum hydrocarbons are

phytoremediated either by taking up of plants through absorption process and broken


16

down within plants or by releasing enzymes through roots to degrade outside before taken

up (Afzal et al., 2010; Newman and Reynold, 2005). The uptake and transfer of organic

contaminant depends upon on the lipophilic nature of compounds and size of molecules

and it is difficult for plants to absorb and transport organic compounds with octanole

water co-efficient (Kow) greater than 3 (Kaimi et al., 2007). But presence of

microorganisms can enhance uptake of such organic compounds due to catabolic action

of these microorganisms (Huang et al., 2005). The efficiency of plants for contaminants

increased if they possess extensive root system and high biomass production (Olson,

2007). Bacteria with biodegrading potential and plant growth promoting activity increase

plant growth and aid plants in producing higher biomass which ultimately lead into more

uptake/biodegradation of organic compounds (Arshad et al., 2007). In contaminated soils,

root growth is severely affected (Zhuang et al., 2007) due to production of ethylene and

bacteria with ACC-deaminase activity lower or regulate plant ethylene level (Arshad et

al., 2007). Due to reduced level of ethylene and other plant growth promoting activity,

intensive root growth occurs and this higher biomass of root facilitates bacterial

proliferation and resultantly considerable reduction of organic contaminants. Plants draw

contaminant towards their rhizosphere via transpiration stream where microorganisms

induced degradation happens (Barac et al. 2004). Plant roots exude low molecular weight

organic substances which serve as alternative source of energy for microorganisms in

case the microorganisms are unable to degrade some recalcitrant molecules such as

PAHs. This alternative source of energy accentuates process of co-metabolism of higher

molecules of petroleum hydrocarbons and thus interactive action of plants and

microorganism enhances degradation of petroleum hydrocarbons (Juhasz and Naidu

2000). Graj et al. (2013) inoculated alfalfa and other three crops with pre-isolated bacteria

instead of indigenous bacteria to assess the impact of crop plants in combination with

applied consortia of bacteria on diesel degradation. The recorded plant growth attributes

such as germination index, root shoot length and biomass of the alfalfa was increased

significantly as compared to un-inoculated control.

Maize (Zea mays L.) is C4 plant which uses water more efficiently than C3 plants.

Plants with high water use efficiency have high tolerance to petroleum contamination.

Chaineau et al. (2000) assessed phytoremediation potential of maize and observed 20%

reduction in petroleum hydrocarbons as compared to soil where maize was not grown.

The study lasted for 120 days and the concentration of petroleum hydrocarbons was 330

mg kg-1 of soil. Chouychai et al. (2007) found corn the least sensitive among four crops


17

ground nut, mung bean, cowpea and corn and concluded that the corn is the most suitable

plant to grow on soils contaminated with PAHs. Variable reports on the tolerance limit of

maize to petroleum hydrocarbons has been published, for example, 95% reduction in

maize yield and impedance in germination occurred while grown on 10.6% (w/w) crude

oil contaminated soil (Adam and Duncan, 2002 and Sharifi et al., 2007) while on the

other hand, Ayotamuno and Kogbara (2007) claimed survival and 60% gain of fresh

biomass by maize grown on soil contaminated with 21% oil. Zand et al. (2010) evaluated

phytoremediation potential of maize and tall fescue in a soil contaminated with 3.5% of

petroleum hydrocarbons and concluded that tall fescue remediated soil by removing 96%

oil and germination of the tall fescue was not affected by oil contamination while that of

maize was suppressed by 21% as compared to un-inoculated control.

Canola (Brassica napus L.) is well known hyper accumulator of heavy metals and

also able to tolerate petroleum hydrocarbon contamination (Frick et al., 1999) but

exhibited poor growth (Bailey and McGill, 1999). Graj et al. (2013) tested

bioaugmentation effect by growing three crops alfalfa, rapeseed and Brassica napus as

these are high biomass producing crops which are required consideration in

rhizoremediation process. The authors observed that Brassica napus displayed highest

germination index at 3000 mg kg-1 concentration. Similarly, Asghar et al. (2013) assessed

biophytoremediation potential of canola inoculated with bacteria having ACC deaminase

activity at 1%, 2% and 3% contamination of mixed petroleum hydrocarbons and

concluded that tolerance of canola to petroleum hydrocarbon was improved by the

inoculation of ACC-deaminase containing bacteria.

2.4 Plant growth promoting bacteria in plant assisted bioremediation

In plant assisted bioremediation process, biomass production in considerable

quantity is of key importance as root biomass provide multi-fold benefits to bacteria in

facilitating the degradation of petroleum hydrocarbon compounds. And shoot biomass

provide cover to soil which not only accentuates degradation process but also

aesthetically more pleasant. To aid plants in production of higher biomass both under

normal and stress conditions, bacteria which are usually called plant growth promoting

bacteria (PGPB) play their role directly or indirectly (Glick, 1995). Plant growth

promoting bacteria benefit directly by solublizing of mineral nutrients such as phosphorus

(Hussain et al., 2013), fixing atmospheric nitrogen symbiotically or in rhizosphere of

plants in associative manner (Shaharoona et al., 2006), siderophore production,

phytohormones production (Vessey, 2003) or regulating stress induced ethylene (Arshad


18

et al., 2007), production of volatile compounds (Ryu et al., 2003; Blom et al., 2011).

Indirect mechanisms of plant growth promotion by PGPB include inducing defense

mechanism of plant (Ryu et al., 2004), biocontrol by producing antibiotic against

pathogenic microorganism and induced systemic resistance (Ryu et al., 2004).

Plant assisted bioremediation occurs naturally, however it can be accentuated by

exploiting suitable and proficient plant and microbe synergism especially PGPB with

bioremediation potential of pollutant may be beneficial as these bacteria not only degrade

pollutants of the interest but also get rid of the plants from toxic effect of pollutants

(Kuiper et al., 2004). In case of plant assisted bioremediation of mixture of petroleum

hydrocarbons, plants are unable to take up petroleum hydrocarbons, however, PGPB

equipped with bioremediation potential break these large molecular weight compounds by

their catalyzing action (Huang et al., 2005). Bacteria with ability to utilize 1-

aminocyclopropane-1-carboxylate (ACC) as sole nitrogen source are of crucial

importance when used in combination with plants under stress conditions (Arshad et al.,

2007). Huang et al. (2005) conducted study to evaluate different processes such as

bioremediation, phytoremediation, land farming and combination of plant and growth

promoting bacteria having ACC-deaminase activity for remediation of PAHs polluted

contaminated site. The results revealed to them that the most efficient process was

multiprocess system rather than bioremediation or phytoremediation alone and the

authors concluded that success of the multiprocess system was due to tolerance of plants

to contaminants and PGPR that enhanced the tolerance of plants by reducing stress

induced ethylene. ACC-deaminase containing bacteria play role in germination of seed

and elongation of roots as immediate precursor of ethylene, ACC is exuded to roots or

seed surface. To maintain the equilibrium of ACC inside and outside of the roots or seed,

ACC move outside the roots or seed as exuded ACC is being cleaved by the bacteria into

ammonia and α-ketobutyrate and thus lowers ethylene production inside plant which

results into better germination and root growth (Glick, 2005 and Glick et al., 2007b).

However, the survival and proliferation of inoculated ACC-deaminase bacteria in the

polluted environment is of special consideration. Glick et al. (2010) suggested bacteria

with twin nature of plant growth promotion and biodegradation potential as better option

instead of only plant growth promoting bacteria to be used in plant assisted

bioremediation process.


19

2.5 Bacterial genera with plant growth promotion activities and bioremediation

capabilities

Bacteria from various genera such as Bacillus, Pseudomonas, Burkholderia,

Azospirrillum and Serratia are reported to have enormous plant growth promoting

activity (Saxena and Matta, 2005). Genera Bacillus and Pseudomonas have been most

extensively studied because of possessing dual mechanism of direct and indirect plant

growth promotion. Various traits such as spore formation and secretion of extracellular

enzymes make the bacteria from Bacillus genus ideal candidate to be used in adverse

environmental conditions as plant growth promoting agent (Richardson et al., 2009).

Bacillus species occur as free living, in the rhizosphere, in rhizoplane and within root

tissue as endophytic bacteria. For example, Moore et al. (2006) isolated strains of

Bacillus from the tissues of plants and found the isolated strain capable of degrading

aromatic hydrocarbons. Among 368 isolates of Genus Bacillus isolates from 38 samples

collected from desert in Kuwait, two isolates degraded 80-90% of crude oil (Sorkhoh et

al., 1993). Similarly, Al-Maghrabi et al. (1999) found the ability of bacillus strains

capable of degrading 20% (v/v) crude oil concentration at temperature of 40-45°C.

Kvesitadze et al. (2012) applied biosurfactant along with Bacillus and Psuedomonas

inoculation on alfalfa to study degradation of petroleum hydrocarbons. Results of the

study revealed that un-inoculated alfalfa degraded 63% petroleum hydrocarbons while

inoculated alfalfa in presence of biosurfactant removed petroleum hydrocarbons by 82%

and thus allowed the authors to conclude that application of bacteria in association with

plant could enhance bioremediation of petroleum hydrocarbons. Bacteria are attracted

towards roots due to signals in the form of root exudates. Dutta et al. (2013) assessed the

effect of root exudates of various crops on cell wall change of non-rhizospheric Bacillus

cereus and concluded that bacteria from non-rhizosphere could also be used for plant

growth promotion. They observed that root exudates of tobacco changed the components

of cell surface and thus bacteria and plants were able to establish interaction. This change

in cell surface facilitated the root colonization and thereby plant growth promotion.

2.6 Role of plant roots for enhancing bioremediation

Plants are primary producers of food for all kind of life and roots are feeders for

these primary producers because plant roots play crucial role in exploring soil for water

and nutrients for plants. Anchorage is provided by roots for acquisition of water, nutrients

and other substances for growth (Schnoor et al., 1995). For enhanced degradation of

organic contaminants there are some special circumstances to be considered such as soil


20

texture and structure, soil aeration, soil moisture, provision of nutrients to

microorganisms and plants, contamination type and bioavailability of contaminants (Frick

et al., 1999). Roots play important role in improvising soil structure which directly affects

all other parameters such as availability of oxygen, nutrients etc. Soil oxygen is important

for aerobic degradation of petroleum hydrocarbons which is depleted due to respiration of

microorganism and replenished by the plants by two mechanism either through

parenchyma (Shimp and Erickson, 1993) or through the roots which are sink of oxygen in

petroleum hydrocarbons contaminated soil or roots cause diffusion of oxygen by

dewatering of soil pore (Rentz et al., 2004). Moreover, roots can provide oxygen to soil

microorganism through root exudates (Frick et al., 1999). Soil oxygen may be improved

by bioventing or by addition of oxygen releasing compounds; however, adapting these

amendments make the remediation strategy costly.

Soil moisture availability is necessary for survival of plant bacterial interaction,

because bacteria and plants are 70-90 % water by weight. Beside this, transport of

nutrients to plants and bacteria is only possible through water. Importance of moisture

availability to plants and bacteria in petroleum hydrocarbon contaminated soil is

increased multitudinously because hydrocarbons are repellent of water. Study conducted

by Parker et al. (1984) yielded results that growth and biomass of bacteria decreased

rapidly with the drying of soil. Plant roots can regularize the moisture content of the soil;

roots can drain out extra moisture of soil by dewatering after heavy rainfall or heavy

irrigation and also can retain the moisture when it is less in soil. For example, Jing et al.

(2008) observed that roots of grasses grown in petroleum contaminated soil were able to

retain 5% soil moisture content. Adam (2001) argued that hydrophobicity of petroleum

hydrocarbons initially may affect plant roots but later on by the development of roots soil

organic matter would be increased and consequently the organic matter should hold

moisture in the rhizosphere.

Mineral nutrients are essential for both bacteria and plants and essentiality

increases under stress condition of pollutants such as petroleum hydrocarbons (Frick et

al., 1999). Living plant roots bring nutrients by mining soil and secreting high organic

molecule called mucigel and protein to microorganisms and thus nutrients, microbes and

contaminant come into contact (Cunningham et al., 1996). In response to any

environmental challenge, plant roots always secrete root exudates and this adaptive

response helps the plants to survive in the stress conditions (Walker et al., 2003).

Narasimhan et al. (2003) attributed the enhanced degradation of an organic contaminant


21

polychlorinated biphenyl to the nutritional bias favored by root exudate phenylpropanoid.

Similarly, Lee et al. (2008) reported potential nutrient supply by plant roots to

rhizospheric microorganism in the form of amino acids and soluble proteins.

Rhizoremediation study in nitrogen deficient soil using Australian native grasses by

Gaskin (2008) revealed that the grasses secreted such root exudates which became the

source of nitrogen for rhizospheric microorganism and resultantly increased

rhizoremediation of petroleum hydrocarbons occurred as compared to the bulk soil or soil

without plants.

Another benefits of plant roots in plant assisted bioremediation or

rhizoremediation of petroleum hydrocarbons or high molecular weight organic compound

is the ability to release co-metabolites in the form of root exudates. Another process of

co-metabolism is that the contaminant induced two sets of enzyme in bacteria; one

enzyme target compound of interest and other enzyme degrade another compound

(Jenkins, 1992). The process of co-metabolism is important in petroleum hydrocarbons

degradation because high molecular compounds of fraction F3 (C16-C35) and PAHs are

hydrophobic in nature and cannot degraded intracellularly either by plants or by

microorganisms. For instance, benz [a] pyrene degradation was occurred co-metabolically

by bacteria Sphingomonas yanoikuyae JAR02 while the carbon and energy source was

root exudates for the degrading bacteria (Rentz et al., 2005). Hydrophobicity of

hydrocarbons is natural and increases with increasing molecular weight in straight chain

alkanes and benzene ring in PAHs compounds. Degradation of petroleum hydrocarbons is

positively correlated with solubility in water (Stroud et al., 2007). Plant root have role in

solublizing hydrocarbons by excreting surfactants in the form of root exudates.

Nevertheless plants differ in releasing kind and amount of surfactants and this difference

may be due to the morphology of plant roots (Phillips, 2008). Parrish et al. (2005) found

Fescue roots to degrade more available PAHs as compared to legumes while the sorbed

compounds were degraded more by leguminous crops and author attributed it to lipid

contents exuded by legume roots. The root lipid contents are correlated with the uptake of

lipophilic aromatic hydrocarbons (Gao and Zhu, 2004). Plants being phototrophic

organisms do not need to mineralize petroleum hydrocarbons for energy purpose but

break down to innocuous intermediates. Being sparingly soluble in water, petroleum

hydrocarbons require specific enzymes for initial oxidation to breakdown.

Monooxygenase also called hydroxylases initially oxidize n-alkane hydrocarbons and

later in successive step beta-oxidation occur likewise fatty acids (Jenkins, 1992).


22

2.7 Degradation of petroleum hydrocarbons

Crude oil is complex mixture of aliphatic and cyclic hydrocarbons. On refining

crude oil yield different compounds such as gasoline, kerosene, diesel and lubricating oil.

Straight chain alkane and alkene are called aliphatic hydrocarbons while cyclic

hydrocarbons are of two kinds; cycloalkanes (saturated hydrocarbons) and aromatic

hydrocarbons (unsaturated hydrocarbons). Alkane compounds are of three types; linear

alkane (n-alkanes), branched alkanes and cycloalkane. One or more rings of carbon atoms

are present in cycloalkanes; however, these rings are not benzene rings because the

hydrocarbon molecules characterized by the presence of one or more benzene or aromatic

rings comprises separate class which is called aromatic hydrocarbons. These compounds

are further categorized into mono, di and polyaromatic hydrocarbons. In crude oil the

major portion is linear alkane or n-alkanes if biodegradation has not happened earlier

(Ollivier and Magot, 2005). Generally it is considered that alkanes are degraded more

rapidly and wide range of microorganisms is capable of biodegrading alkanes both short

and long chain. The alkanes are degraded monoterminally by addition of oxygen and

converted into alcohols, aldehyde and fatty acids. The resistance to degradation increases

with increasing length of chain (Atlas and Bartha, 1981). Three mechanisms of alkane

degradation are proposed by Atlas and Bartha (1981) given as Fig. 1a.

Cycloalkanes are recalcitrant constituent of petroleum hydrocarbons and found

abundantly in petroleum products. These are mostly degraded through co-metabolism by

alkane degraders. This process of co-metabolism initiates by conversion of cycloalkanes

into alcohol or ketone by monooxygenase (Sayyed and Patel, 2011).

Degradation of aromatic hydrocarbons especially of PAHs is slow as compared to

alkane because one or more oxidation steps are required to convert into catechol which is

further opened by oxidation on ortho or meta points of the ring (Atlas and Bartha, 1981).

The initial oxidative step is mediated by monooxygenase or hydroxylating dioxygenase

and this dioxygenase mediated opening of catechol resulted into the production of cis,cis

muconate and unsaturated dicarboxylic acid. Finally acetyl-CoAs are produced from this

product through beta-oxidation.


23

a) alkane primary alcohol aldehyde

O2H

b) alkane

c) alkane alkylhydroperoxide fatty acid

Fig. 1a Terminal methyl oxidation pathways adapted by alkane degrading

microorganisms (From Atlas and Bartha, 1981)

Naphthalene naphthalene 1,2 diol 4 hydroxy-2-oxo-4-2-hydroxyphenly butyric acid

O2 O2 H2O O2

Catechol Salicyclic acid salicylaldehyde Pyruvic acid

O2

2-hydroxy cis,cis muconic

semialdehyde

H2O cis,cis muconic acid

2-keto-4-pentenoic acid O2

Beta-ketoadipic acid CoA

Pyruvic acid

Succinic acid

Aldehyde +

Acetyl-CoA

Fig. 1b Degradation of aromatic ring by microorganism showing ortho- and meta-

cleavage of catechol (From Atlas and Bartha, 1981)

Hydra

tion Dehydrogenation

-2H alkene

Hydra

tion

Oxidation Dehydrogenation

Beta-oxidation


24

Chapter 3

Isolation, Screening and Characterization of Bacteria

3.1 Introduction

Our modern industrialized society depends on fossil fuels energy for its existence.

Due to its extensive exploration, transportation and refining, the petroleum hydrocarbons

have become the most prevalent contamination in all compartments of the environment

either by intended but uncontrolled anthropogenic activities or by accidental spills due to

human error or mechanical failure (Pena-Castro et al., 2006). Petroleum hydrocarbons go

under attack of various microorganisms present in soil (Bundy et al., 2004) but bacteria

are key players in the process of dissipating petroleum hydrocarbons from soil. Bacterial

population naturally present in almost all kind of ecosystem are capable of converting

toxic substances in the environment into innocuous products due to enzymatic action

(Atlas and Barhta, 1981; Leahy and Colwell, 1990). Among other biotic and abiotic

factors affecting bioremediation regarding success, rate and sustainability, selection of

microbial inoculums is of crucial importance. A selected microorganism should have high

growth rate, longtime survival, competency, ability to tolerate higher concentration of

contaminant and diverse metabolic pathways to degrade different kind of contaminants

(Mrozik and Piotrowska-Seget, 2010). It is unfortunate that in establishing bioremediation

strategy, the survival and ecology of microorganism is often ignored (Thompson et al.,

2005; Vogel and Walter, 2001) and consequently questions are raised about credibility of

the strategy. Another technique called biostimulation refers to increase the activity of

resident bacteria to enhance bioremediation of contaminated sites and this technique

seems to be effective as resident bacteria are adapted to xenobiotic compounds (Rahman

et al., 2003). However, scarcity of bacterial population at contaminated sites or

concentration of xenobiotic above high threshold level lead to failure of biostimulation

strategy (Ueno et al., 2007). Therefore, isolation of bacteria acclimated to petroleum

hydrocarbons contamination is imperative so that upon inoculation there should be

minimum lag period and maximum survival and growth of bacteria. Moreover, isolation

and screening of bacteria not only from already contaminated soil but also on such media

supplemented with those compounds as a source of carbon and energy that are to be

degraded (Wang et al., 2011) will lead to a successful bioremediation.


25

3.2 Material and Methods

3.2.1 Collection of soil samples

Soil samples (10 from each site) were collected from district Shaikhupura

(31.720126°N and 73.980046°E), Wazirabad, Muzzafargarh (30.093893°N and

71.162486°E, 30.096125°N and 71.342528°E) Multan (30.183971°N and 71.517333°E),

Rawalpindi and Faisalabad (31.51052°N and 73.06830°E, 31.42732°N and 73.09336°E,

31.51781°N and 73.15859°E, 31.45477°N and 73.15852°E). Soil samples were collected

using tube augar. The depth of the soil samples was from surface soil up to 90 cm. Soil

samples were used to isolate the bacteria and composite samples were analyzed for total

petroleum hydrocarbon (TPH). The rationale behind the isolation of bacteria from

contaminated soil was that enriched bacterial population, when inoculated, often faces

difficulty to find niche in the soil and face competition by the indigenous soil bacteria.

Moreover, the bacterial population has to acclimate themselves to xenobiotic compounds

like high molecular weight organic compounds such as PAHs. So to overcome the

problem, bacteria were isolated from already petroleum contaminated soil hypothesizing

that the isolates would be acclimated to PAHs and would be more effective in

bioremediation process. The collected soil samples were stored in zipped plastic bags at

4°C in dark prior to use for isolation of bacteria and assessment of TPH contamination in

the samples.

3.2.2 Isolation of bacteria

For isolation of bacteria an enrichment culture system was used. Bacterial isolates

were cultured on basal salt mineral medium (per liter distilled water: 0.64 g K2HPO4, 0.31

g KH2PO4, 0.5 g NH4Cl, 0.2 g MgSO4.7H2O, 0.005 g FeSO4.7H2O) with 1% crude oil as

sole source of carbon. Briefly, 10 g of each sample was added in separate sterilized 250

mL Erlenmeyer flask containing 95 mL sterilized water. The flasks were shaken

vigorously using flatbed end to end shaker to homogenize the soil water suspension. For

further dilution, 1mL soil water suspension from 250 mL flask was transferred to 10 mL

test tube that contained 9 mL of sterilize water. Following the same procedure, dilutions

up to 10-5 were prepared. Sterilized water was used in all the dilution procedure to prepare

soil exact as the intention was to avoid contamination of isolation from other sources than

the target soil samples. Basal salt mineral medium lacking carbon source was sterilized

and solidified in petri plates. Crude oil filtered through 0.2 µm membrane was automized

on solidified agar plates as a sole carbon source. From each dilution 1 mL was


26

transferred to petri plates and thoroughly spread on plates with the help of sterilized

inoculating needle. These petri plates were incubated at 28±1oC for 72 hours. Growth was

checked after different intervals and up to 72 hours maximum growth was observed and

longer 72 hours incubation would have started death phase. On the basis of colony color,

shape and size, further purification was done by streaking on the media as mentioned

above.

3.2.3 Preservation of bacteria

For preservation of bacterial isolates, broth cultures were prepared by using the

same media except agar. Well grown colonies were picked with the help of inoculating

needle and inserted in the flask having 20 mL of broth. The inoculated flasks were

incubated for 72 hours at 28±1oC in rotary shaking incubator at 180 rpm. Broth cultured

isolates were preserved by pouring 400 µL glycerol and 600 µL broth culture in 1mL

Eppendorf. These were kept at -40oC for long preservation.

3.2.4 Bioremediation assay (BRA)

Bioremediation Assay was carried out to check the PAHs degrading ability of

isolates. For this purpose, 24 wells microtitre plates were used. Mixture of PAHs was

used as test compound. First two lanes to serve as control were filled with only 40 µL

PAHs mixture (10 g phenanthrene 1g anthracene and 1g flourene dissolved per litre of

pentane). All chemicals used for bioremediation were purchased from Fluka, Japan and

have purity > 97%). In these wells there was neither Bushnell Hass medium nor bacterial

inoculation. In next two lanes in addition to 40 µL PAHs mixture, 720 µL of Bushnell

Hass and 80 µL bacterial broths was poured. Lane 5 and 6 were kept as another control.

Each well of these lanes were added with PAHs mixture and Bushnell Hass medium but

without bacterial inoculation. One row in each plate was attributed to one bacterial

isolates. These plates were then incubated at 28±1oC for three weeks. After incubation

period (21 days), two hundred microliter of indicator p-Idonitrotetraazolium (p-INT) was

added in each well. Due to the oxidation of PAHs, the wells inoculated with bacterial

isolates turned red revealing that bacteria have PAHs biodegradation capability. The

intensity of the color was attributed to have higher, medium and lower capability. The

most intense colored wells were marked as (+++) showing the higher biodegradation

potential of bacteria.

3.2.5 Particle size distribution of collected soil samples

Textural analysis of collected soil samples was conducted by hydrometer method

with few modifications. Ten milliliter of 30% H2O2 was added in 10 g of soil sample


27

taken in 250mL Erlenmeyer flask to remove organic matter followed by heating of

sample at 80°C for 30 minutes to remove residual peroxide as proposed by Sheldrick and

Wang (1993) in their pipette method. Soil was dispersed by dispersing agent Sodium

hexametaphosphate to make soil and water suspension. This suspension was transferred

to 1L graduated cylinder and the reading was taken after 40 seconds for silt plus clay and

for clay only hydrometer reading was taken after two hours. The sand fraction was

determined by sieving the same suspension through a 50 µm sieve until the water passing

the sieve is clear. The sand was transferred to a pre-weighed beaker. The sand and beaker

was then weighed again and heated at 105°C until constant weight. The textural class was

determined using texture triangle according to international scheme.

3.2.6 TPH by infrared spectroscopy

Infrared spectroscopy measures energy absorbed by a molecule in electromagnetic

spectrum. Horiba-350 oil content analyzer (Horiba Ltd, Japan) was used to assess TPH in

collected soil samples. TPH oil content analyzer principally measure TPH using infrared

light. Five gram of soil was taken in china dish and five gram of sodium sulfate was

added to absorb moisture in the soil. These were mixed thoroughly and 40 mL of RDH-

CCl4 was added in mixture of soil and sodium sulfate. This mixture was shaken for 30

minutes and filtered using Whatman No.40 (11cm). During filtration, 100 mesh size silica

was placed in funnel to absorb moisture and biogenic hydrocarbons. Filtrate was filled in

1.5 cm cell and placed in instrument to get the reading in mg/kg. Three replicates for each

sample was carried out and average of three replicate was taken. Dilutions were made

using RDH-CCl4 to bring the concentration into range of the standards and instrument.

Prior to measurement of TPH, the instrument was calibrated. The calibration of the

instrument was carried out by preparing working standards of 200, 400, 600, 800 and

1000 ppm by using same crude oil that was used in the experiment by dissolving in RDH-

CCl4. Regression line was drawn and calculation was carried by using the regression

equation.

3.2.7 ACC-metabolism assay of isolates

To assess the presence of ACC metabolism in screened bacteria an assay was

carried out in which bacterial strains were allowed to grow on two different nitrogen

sources (ACC and Ammonium sulphate). Modified method as described by Jacobson et

al. (1994) was followed for the assessment of ACC-metabolism in bacterial strains.

Bacterial isolates from contaminated soil positive for ACC-metabolism were grown in

Tryptic Soy Broth. Ten mL of Tryptic Soy Broth was prepared by inoculating with


28

bacteria. This broth was diluted ten times with sterilized MgSO4 in autoclaved inoculating

boats. Ninety six well microtitre plates were used for this assay. In all wells of microtitre

plates 122 µL DF salt minimal medium were poured. Fifteen microlitres of 3 mM ACC,

0.1 M (NH4)2SO4 and 0.1 M MgSO4 each in three consecutive lanes, respectively. Four

replicates were kept in a manner that in lanes 1, 4, 7 and 10 nitrogen source was ACC and

in lanes 2, 5, 8 and 11 nitrogen source was (NH4)2SO4 and MgSO4 as a control was kept

in lane 3, 6, 9 and 12. All wells were inoculated with 22 µL of broth culture that was

already diluted 10 times by magnesium sulphate prior to inoculation. One plate was kept

as un-inoculated control in which all ingredients were same as that in inoculated plates

except bacterial inoculation. All material used in the assay were sterilized at 120°C for 15

minutes except ACC as ACC is heat labile. Therefore, ACC was filtered through 0.2 µm

membrane to ensure microbial decontamination and stored at -20°C until used in assay.

Optical density (OD590) was measured after 0, 24, 48 and 72 hours to observe the growth

of bacteria on two different nitrogen sources. Value of ACC and (NH4)2SO4 well was

compared with MgSO4 to determine the ability of bacteria to metabolize ACC. The

bacteria were categorized as High, Medium and Low depending on the observed OD

value. Bacteria with OD value greater than 0.75 were attributed as High, OD value

between 0.5 and 0.75 were Medium and Low were having OD value less than 0.50.

3.2.8 ACC-deaminase activity

Quantification of ACC-deaminase activity was accomlished by following the

modified method of Honma and Shimomura (1978) and Penrose and Glick (2003) which

measures the amount of α-ketobutyrate produced when the enzyme ACC-deaminase

cleaves ACC into ammonia and α-ketobutyrate. A standard curve of α-ketobutyrate

ranging between 0.1 and 1.5 µmol was constructed for comparison of the absorbance of

standard and samples at 540 nm to determine the amount of α-ketobutyrate (nmol)

produced by the cleavage of ACC due to the action of bacteria having ACC-deaminase

activity. For preparation of working standards, a stock solution of 1mM was prepared in

0.1 M Tris-HCl (pH 8.5) and stored at 4°C. Just prior to use, sub-stock solution of 10µM

was prepared by diluting the stock solution with the 0.1 M Tris-HCl (pH 8.5) buffers.

From this sub-stock solution, working standards ranging between 0.1 and 1.5 µmol with

the same buffer solution were prepared. In each series of standard containing 2 mL of

known concentration of α-ketobutyrate, 3 mL of 2,4-dinitrophenylhydrazine reagent

(0.2% 2,4-dinitrophenylhydrazine in 2 M HCl) was added and the contents were vortexed

and incubated at 30°C for 30 minutes, during which time the α-ketobutyrate was


29

derivatized as phenylhydrazone. The color of the phenylhydrazone was developed by the

addition of 20 mL 2 M NaOH to each standard; after mixing the absorbance of the

mixture was measured at 540 nm wavelength. ACC deaminase activity was measured in

bacterial extracts prepared by the following manner. Bacterial cell pellets were prepared

by harvesting them at 45000 g centrifugation for 15 minutes and these pellets were twice

dissolved in 0.1M phosphate buffer solution having pH 7.0. The prepared cell pellets

were suspended in 10 mL of 0.1 M Tris-HCl (pH 7.6) and transferred to 15mL centrifuge

tube. The contents of the tube were centrifuged at 13500 g for 5 minutes and the

supernatant was removed. The pellet was suspended in 6 mL 0.1 M Tris-HCl (pH 8.5).

Cell suspension was toluenized by adding 3 mL of toluene and this toluenized cell

suspension was immediately assayed for ACC deaminase activity. Each sample was

assayed for three times. In a fresh 15 mL centrifuge tube, 2 mL of the toluenized cells and

0.2 mL of 0.5 M ACC taken, vortexed, and then incubated at 30°C for 15 min. Following

the addition of 10 mL of 0.56 M HCl, the mixture was vortexed and centrifuge for 5

minutes at 13500g at room temperature. Ten milliliters of the supernatant was vortexed

together with 8 mL of 0.56 M HCl. Thereupon 3 mL of the 2,4-dinitrophenylhdrazine

reagent was added to the glass tube, the contents were vortexed and then incubated at

30°C for 30 min. Following the addition and mixing of 20 mL of 2 N NaOH, the

absorbance of the mixture was measured at 540 nm. Two series were run for the

absorbance of assay. In the first series, reagents included ACC, bacterial cells and assay

reagents with their control containing ACC and assay reagents. Second series included

bacterial cell and assay reagents with its control containing assay reagents only. The assay

was carried out on digital spectrophotometer (Evolution 300, Thermo Electron, England)

so the control value was subtracted automatically from the treatment values. Values of the

first series were subtracted from the values of the second series for respective bacterial

inoculation, to estimate the amount of α-ketobutyrate in nmol from the standard curve.

Value of ACC-deaminase activity of strains was further estimated on the basis of per

gram biomass of bacterial cell.

3.3 Results

3.3.1 Sampling sites characteristics

Soil samples were used to isolate the bacteria and composite samples were

analyzed for TPH. Soil sampling sites were of diverse nature regarding type of

contaminants, soil texture, and temperature of the area and aging of petroleum


30

hydrocarbons. Soil samples collected from Muzaffargarh, Thermal Power and JIMCO Oil

Depot, were of sandy and loam in nature, respectively, and the contamination of

petroleum hydrocarbons replenished approximately on daily basis. In contrast to

Muzzafargarh, soil samples taken from Sher Shah Multan (Railway Station) were of clay

in nature and the contamination was aged. Main contributors to the contamination were

diesel and engine oil at Sher Shah Multan. However, the average annual temperature of

these both areas was approximately same. Samples collected from Sheikhupura had

contamination with different petroleum products such as kerosene, gasoline and high

octane petroleum and jet fuel. Petroleum sludge was collected from Oil Refineries in

Pindi Morga, Rawalpindi. Samples from Faisalabad were collected from petrol pumps,

car washing centers and repairing shops located in different areas of the Faisalabad city,

therefore the texture of the collected soil samples were different but most of them were

loam to clay loam.

3.3.2 TPH in collected samples

Collected soil samples were analyzed for TPH concentration by infra-red

spectroscopy. The results revealed that maximum polluted site was Sher Shah Multan

railway station, where TPH concentration of 791 mg kg-1 was observed (Table. 1). Other

noticeable contamination was found in JIMCO Oil Depot Muzzafargarh, Thermal Power

Muzzafargarh, Shiekhupura and Pindi Morga Rawalpindi where it was 674, 651, 650 and

565 mg kg-1, respectively. The reason for maximum contamination at railway station was

that repair and filling of fuel. That is why the main contaminants at railway station were

diesel and engine oil. Other places were contaminated due to the same reasons; storage,

delivery and service of oil to vehicles were carried on these places except in Pindi Morga

Rawalpindi where source of contamination was petroleum sludge resulted from refining

of crude oil.

3.3.3 Microbial isolates

3.3.3.1 Isolates from Sheikhupura district

Soil samples contaminated with different petroleum hydrocarbons such as diesel,

kerosene, aged diesel and engine oil, gasoline and high octane were collected from

different sites of the district. A total of 91 bacterial isolates were obtained through

culturing on agar plates supplemented with 1% 0.2µm membrane filtered crude oil

through dilution plate technique. As shown in pie graph, maximum numbers of isolates

were obtained from samples contaminated with diesel (36) followed by 22 isolates each

from aged diesel, engine oil and kerosene oil. Samples with aged diesel and engine oil


31

were collected from Shiekhupura Railway station. Isolates from samples contaminated

with gasoline and high octane were very low 4 and 7, respectively. Isolates collected from

Sheikhupura district were coded as SFD2S6, HOBC1S2, SST3S6, SST3S7, SFD6S3,

SF1S5, SFD5S1, SFDS3, SFD6S2, SF1S4, SFD5S4, SST3S2, SST3S5, SST3S8, R2S2,

SFD6, R2S1, HOBCS6, HOBC1S4, SFD1S2, SFD1S4, SFD1S6, SFD2S1, SFD2S3,

SFD5S3, SFD2S7, SFD5S5, SFD6S6, SFM5S3, SFM5S4, SFMS6, FSD1S1, SFD1S3,

SFD6S5, SFD1S5, SFM4S2, SFD2S2, SFDS1, SFD2S5, SFM6S1, SFM4S5, HOBCS2,

SFM5S2, SFM6S2, SFDS4, SFM5S1, SFMS7, HOBC1S1, SFM6S3, HOBCS4,

HOBCS3, SFKIS2, SST6S1, SST1S1, SFD5S4, SST1S3, SFK2S4, SFD9S3, SST2S1,

SST4S1, SFM4S2, SST5S4, SFD9S4, SST5S3, SFM4S1, SFMS1, SFD5S2, SFDS4,

SST2S3, SFD8S3, SFK1S3, SFM4S3, SFK2S1, SFK2S3, SST2S2, SFD9S1, SST4S4,

SST1S9, SFK2S2, SST4S3, SFD1S1, SFD9S2, SFD8S1, SST1S2, SST4S2, SFD8S2,

SST6S3, GCD2S3, SST6S2, SST5S2.

These isolates were further characterized for their potential to use aromatic

hydrocarbons and ability to hydrolyze 1-aminocyclopropane-1-carboxylate (ACC) by the

action of ACC-deaminase enzyme. On the basis of the results of BRA and ACC

metabolism assay, the bacterial isolates were categorized into low, medium and high.

Firstly, all 91 bacterial isolates were assayed for their potential to degrade aromatic

hydrocarbons. Among 91 isolates, 69 were found to have ability to oxidize aromatic

hydrocarbons. Intensity of the color further categorized these 69 isolates into medium and

low category; 60 were low and 9 produced medium intense color. Secondly, bacterial

isolates were assayed for their ability to hydrolyze ACC by the action of ACC-deaminase

enzyme by recording OD590 to assess their growth on ACC as sole source of nitrogen.

Among 69 isolates, 27 isolates were able to hydrolyze ACC to use it as sole source of

nitrogen for their growth (Fig. 3). Bacterial growth determined by measuring OD has

direct relation to their ACC-deaminase activity in absence of any other nitrogen source

except ACC; more the ACC-deaminase activity more will be bacterial growth and thereby

OD. Eleven bacterial isolates showed prolific growth as indicated by optical OD>0.75

while 15 bacterial isolates were medium (Fig. 2) and one isolate showed less growth as

optical density recorded was OD<0.50.

3.3.3.2 Isolates from Wazirabad district

Soil samples collected from Wazirabad district were contaminated with gasoline

and diesel simultaneously. Twenty eight isolates were obtained from soil samples

collected from Wazirabad district by culturing on agar plates. These isolates were tested


32

for their ability to degrade aromatic hydrocarbons in BRA. The results of the BRA

yielded 16 bacterial isolates coded as WZ4S4, WZ3S4, WZ3S2, WZR1, WZR51,

WZR23, WZR53N, WZR53S, WZR52, WZR41, WZR31, WZ1S1, WZ2S1, WZ1S2,

WZ2S5 and WZ4S1 as negative because they were failed to oxidize aromatic

hydrocarbons. While bacterial isolates named WZR21Y, WZR32S, WZR24, WZR42,

WZR34, WZR11, WZR22, WZR12, WZ2S2, WZ3S3, WZ4S3, and WZ3S1 were found

positive in BRA. Among these 12 bacterial isolates with bioremediation ability, WZ3S3

and WZ3S1 produced intense color and categorized as high and remaining BRA positive

isolates were less efficient in oxidizing aromatic hydrocarbons. An isolate WZR32S was

in medium range. ACC metabolism assay revealed that out of 12 bacterial isolates, 10

bacterial isolates were able to grow on ACC as sole source of nitrogen and among these

10 isolates WZ3S3, WZ3S1 and WZR32S were most efficient in utilizing ACC and

recorded OD for these isolates was 0.77, 1.10 and 0.76, respectively (Fig. 4). Bacterial

isolates lying under medium category regarding utilization of ACC were WZ2S2,

WZR41, WZR24 and WZR21Y with OD 0.54, 0.56, 0.59, 0.74, respectively. Slow

growth on ACC was shown by the bacterial isolates WZR1, WZ4S3 and WZ4S4 and

hence attributed as bacteria having low ACC metabolism.

3.3.3.3 Isolates from Gujranwala district

Bacterial isolates coded as GC24, GC13, GCS11, GC22W, GC22Y, GCDS8,

GCDS9, GCDS4 and GCD1S2 were isolated from soil samples contaminated with jet fuel

collected from Gujranwala Cantonment and these bacterial isolates were found unable to

degrade aromatic hydrocarbons while the bacterial isolates GC14, GC23Y, GC23W,

GCDS4, GCDS2, GCDS6, GCD4S2, GCDS11, GCD1S1, GCD2S1, GCD2S2, GCD2S4

and GCD2S3 from same soil samples have potential to degrade aromatic hydrocarbons as

determined by BRA using aromatic hydrocarbons as sole source of carbon. Out of 22

bacterial isolates from Gujranwala Cantonment soil samples, 13 bacterial isolates able to

degrade aromatic hydrocarbons were further assayed to determine the presence of ACC

metabolism in the isolates. Out of these 13 bacteria, 7 isolates possessed metabolic

pathway to utilize ACC (Fig. 5). Bacterial isolates GCDS11 and GC23Y showed prolific

growth and categorized as high (OD 0.83 and 0.91, respectively) followed by GCD1S2,

GCD2S1 and GCD1S1 as medium category with OD 0.58, 0.61 and 0.60, respectively.


33

Fig. 2 Isolates from Sheikhupura district soil samples contaminated with different

petroleum products

Fig. 3 Categorization of isolates from Sheikhupura district on the basis of BRA and ACC-

metabolism assay

High

(11)

Medium

(15)

Low

(1)

Chapter 3 Isolation, screening and characterization of Bacteria

34

Fig. 4 Categorization of isolates from Wazirabad district soil samples on the basis of

BRA and ACC metabolism assay

Fig. 5 Categorization of isolates from Gujranwala district soil samples on the basis of

BRA and ACC metabolism assay

Low

(3)

Medium

(4)

High

(3)


35

3.3.3.4 Isolates from Muzzafargarh (JIMCO Oil Depot)

Bacterial isolates coded as JM61, JM53S, JM41, JM63, JM33, JM34, JM23,

JM74, JM22, JM52, JM103, JM71 and JM14 were successfully grown on agar plates

supplemented with crude oil as sole source of carbon but these bacterial isolates were

lacking mechanism to degrade aromatic hydrocarbons as indicated in BRA because they

were found negative in the assay while the isolates JM11, JM13, JM12, JM31, JM53,

JM62, JM44, JM43, JM32, JM12, JM51, JM72, JM11, JM21, JM24, JM64, JM42, JM73

and JM54 were positive in assay indicating their potential to degrade aromatic

hydrocarbons. Hence out of 32 isolates obtained from soil samples collected from

Muzzafargarh (JIMCO Oil Depot), 13 were lacking PAHs bioremediation potential while

19 isolates were having this ability (Fig. 6). Assessment of ACC metabolism of these

bacterial isolates revealed that out of 32 isolates, 16 were able to utilize ACC as sole

source of nitrogen. Further categorization of isolates on the basis of OD revealed that

only one isolate JM44 (OD 0.97) has high rate of ACC metabolism, 5 isolates naming

JM103, JM21, JM24, JM32 and JM61 (OD 0.51, 0.59, 0.68, 0.59 and 0.53, respectively)

have medium ability to hydrolyze ACC while ten isolate were found to have low ACC

metabolism.

3.3.3.5 Isolates from Muzzafargarh Thermal Power

Nineteen isolates were collected from soil samples taken from Muzzafargarh

thermal power station. Code names given to isolates were MZT4, MZT3, MZT21,

MZT72, MZT71, MZT13, MZT22, MZT23, MZT23, MZT12, MZT11, MZT14, MZT73,

MZT6, MZT43, MZT74, MZT62, MZT32 and MZT63. All isolates were assayed for

both bioremediation and ACC-metabolism. Among 19 isolates, 6 isolates were negative

in PAHs bioremediation assay and 13 isolates were positive. Out of 19 isolates, five

isolates were able to hydrolyze ACC and among these five isolates MZT72 was

categorized as “High” because that isolate showed high prolific growth in ACC-

metabolism assay and thereby high OD (OD>0.75). Two isolates MZT13 and MZT6

were categorized as “Medium” and MZT62 and MZT14 was considered as “Low” in its

ability to utilize ACC as sole nitrogen source. MZT14 was found to have had low ACC

metabolism but was lacking PAHs bioremediation ability (Fig. 7).

3.3.3.6 Isolates from Sher Shah Multan (Railway track junction)

Railway transportation is one of prominent sources of organic especially of

aromatic hydrocarbons contamination. This contamination becomes multifold at railway

track junctions due to storage and change of engine lubricator. Railway track junction at


36

Sher Shah Multan is one of the biggest and busiest junctions and was therefore selected

for collection of petroleum contaminated soil samples to isolate bacteria capable of

degrading petroleum hydrocarbons. Out of 38 isolates from soil samples taken from Sher

Shah Multan railway track junction, 14 isolates with code names SM92, SM52S1, SM54,

SM74, SM61, SM43, SM13, SM101, SM42, SM11, SM94, SM33, SM34 and SM32 were

found to lack enzymatic activity for degradation of aromatic hydrocarbons as the wells

inoculated with these bacterial isolates did not turn red during BRA. Twenty four

bacterial isolates coded as SM102, SM93, SM84, SM53, SM44, SM71, SM52, SM62,

SM72, SM51, SM91, JM13, JM12, SM63, SM65, SM73, SM64, SM41, SM112, SM11,

SM14, SM31, SM82 and SM12 were positive in BRA. Upon their assessment for

presence of capability of metabolizing ACC it was found that 21 isolates failed to grow

on ACC as sole source of nitrogen. Recorded ODlaid 4 isolates into "High" category, 5

isolates into "Medium" and 8 isolates in “Low” category.

3.3.3.7 Isolates from Faisalabad soil samples

Faisalabad is industrial city of Pakistan and petroleum is back bone of any

industry. Soil samples were drawn from different petroleum contaminated locations

within and surroundings of the city. A total of 45 isolates collected from soil samples

through enrichment culture technique using crude oil as sole source of carbon were

assayed in 24 well microtitre plates bearing aromatic hydrocarbons for assessment of

PAHs degrading potential. While following 29 bacterial isolates with code name SP11Y,

SP41, SP73W, SP61Y, SP52Y, SP84, SP11S2, SP53Y, SP94, SP84, SP14, SP72W,

SP3S2, SP42, SP13, SP113, SP72S2 were failed to show biodegradation potential for

PAHs. Isolates coded as SP61W, SP53Y, SP14W, SP71, SP11W, SP82, SP103, SP74Y,

SP22, SP102W, SP14, SP3S2, SP91, SP23W, SP62W, SP24W, SP11S1, SP54, SP83,

SP54W, SP102, SP112, SP92, SP64W, SP81, SP122, SP102S1, SP2, and SP104Y were

positive in BRA. All 45 bacterial isolates were also assayed for the presence of ACC

deaminase enzyme and this assay revealed that out of 45 isolates, 17 were able to grow on

ACC (Fig. 9). The bacterial isolates SP61W, SP53Y, SP14W, SP71, SP11W, SP82,

SP103, SP74Y, SP22, SP102W, SP14 and SP3S2 were positive in BRA but did not

succeed to utilize ACC as a sole source of nitrogen. Among 16 ACC metabolism

possessing bacteria, 5 isolates with code name SP91, SP23W, SP62W, SP54, SP104Y

and SP112 with OD 1.04, 0.81, 0.80, 0.87, 0.82 and 0.82, respectively were categorized

as "High" while 10 bacterial isolates SP24W, SP11S1, SP83, SP54W, SP102, SP92,

SP64W, SP81, SP122, SP102S1 and SP2 with OD 0.59, 0.55, 0.52, 0.62, 0.66, 0.59, 0.69,


37

0.68, 0.64, 0.66, 0.50 were categorized as "Medium" as their recorded OD lies between

0.50 to 0.75. One isolate SP11S2 which was negative in BRA also had low ACC

metabolism as indicated by its growth on ACC.

3.3.3.8 Isolates from Pindi Morga Rawalpindi soil samples

Petroleum sludge contaminated soil samples were collected from Pindi Morga

Rawalpindi for isolation of bacteria. A total of 26 isolates were obtained through dilution

plate technique on agar plates using crude oil as a sole source of carbons. Bacteria thus

isolated were assayed for their potential to degrade aromatic hydrocarbons. The assay

revealed that out of 26 isolates, 12 bacterial isolates with code name PM32W, PM34W,

PM51, PM34Y, PM54, PM41W, PM12, PM41Y, PM33Y, PM52, PM and PM42 were

lacking metabolic pathway for the biodegradation of aromatic hydrocarbons. Bacterial

isolates with code name PM1W, PM12W, PM13W, PM11W, PM42, PM32Y, PM34Y,

PM82W, PM13Y, PM33Y, PM63, PM62Y, PM62W and PM12S were positive in BRA.

All 26 bacterial isolates were further characterized for their ability to metabolize ACC for

their growth by the action of ACC deaminase. The assay revealed that bacterial isolate

PM32Y had maximum ability to grow on ACC (OD 1.23) while PM63 and PM62W with

OD 0.59 and 0.63, respectively were categorized as "Medium" and PM12S and PM13W

were "Low" as OD was 0.29 and 0.25, respectively (Fig. 10).


38

Fig. 6 Categorization of isolates from Muzzafargarh district (JIMCO Oil Depot) soil

samples on the basis of BRA and ACC metabolism assay

Fig. 7 Categorization of isolates from Muzzafargarh Thermal Power soil samples on the

basis of BRA and ACC metabolism assay

High

(1)

Medium

(5)

Low

(10)

High

(1)

Medium

(2)

Low

(2)


39

Fig. 8 Categorization of isolates from Sher Shah Multan soil samples on the basis of BRA

and ACC metabolism assay

Fig. 9 Categorization of isolates from Faisalabad soil samples on the basis of BRA and

ACC metabolism assay

High

(4)

Medium

(5)

Low

(7)


40

Fig. 10 Categorization of isolates from Pindi Morga Rawalpindi soil samples on the basis


High

(1)

Medium

(2)

Low

(2)


41

3.3.4 Quantification of ACC-deaminase activity

Out of 103 ACC positive bacterial isolates, 29 showed prolific growth indicating

that isolates had relatively high ability to utilize ACC as a sole source of nitrogen for their

growth. But two isolates out of these 29 isolates were discarded as those were lacking

bioremediation potential. Therefore out of these 29 bacterial isolates, 27 were further

tested for the quantification of ACC-deaminase activity. The results revealed that out of

27 isolates 23 isolate hydrolyzed more than100 nmol g-1 biomass hr-1 ACC into α-

ketobutyrate while only 4 isolates were having hydrolysis ability more than 95 nmol g-1

biomass hr-1. Maximum hydrolysis of ACC per unit biomass per unit time was observed

from the isolate PM32Y which showed 1334.15 nmol g-1 biomass hr-1 while minimum α-

ketobutyrate production was observed from the isolate WZR32S that was isolated from

the samples collected from Wazirabad district (Table 2).

3.3.5 Summary of categorization of bacterial isolates on the basis of bioremediation

and ACC-metabolism assay

Over three hundred bacterial isolates collected from petroleum contaminated soil

samples taken from different locations of Punjab. These bacterial isolates were

qualitatively assayed for their potential to degrade PAHs, the most recalcitrant portion, of

petroleum hydrocarbons. Out of 301 bacterial isolates, 189 were positive in BRA. And

these bioremediation positive isolates were categorized into High, Medium and Low on

the basis of intensity of color produced as indicated in the Fig. 11. Out of 189

bioremediation positive bacterial isolates, 8 were "High", 19 were "Medium" and

remaining 162 isolates were categorized as "Low". Similarly, bacterial isolates were

categorized into High, Medium and Low on the basis of their potential to grow on ACC

in the absence of any other nitrogen source. Bacterial growth on ACC was measured by

recording OD. Bacterial isolates having OD>0.75 were categorized as "High", isolates

with OD ranging from 0.50 to 0.75 were "Medium" and OD below 0.50 were categorized

as "Low". Out of 103 ACC metabolism containing isolates, 29 bacterial isolates were

"High", 46 were "Medium" and 28 bacterial isolates showed slow growth on ACC and

categorized as "Low".


42

Table 1 Characteristics and TPH-IR of sampling sites in different districts of Punjab

Table 2 Quantification of ACC-deaminase activity

No Isolate

Name


(OD)

ACC-deaminase activity

)1-biomass hr1 -ketobutyrate nmol g-(α

1 PM32Y 1.23 1334.15

2 SFD2S2 1.14 1027.28

3 SFD5S3 1.2 377.77

4 WZ3S1 1.1 268.76

5 SFD1S1 0.97 251.88

6 JM44 0.97 212.08

7 SM14 0.97 211.34

8 SFD9S4 0.92 178.22

9 MZT72 0.93 175.46

10 GC23Y 0.91 172.06

11 SM31 0.89 163.78

12 SST6S1 0.89 157.95

13 FSD1S1 0.88 149.77

14 SFK2S2 0.88 143.83

15 SP54 0.87 139.48

16 SM112 0.87 138.84

17 SFKIS2 0.85 130.67

18 GCDS11 0.83 124.19

19 SP112 0.82 121.96

20 SP104Y 0.82 112.51

21 SST4S3 0.8 111.88

22 SP62W 0.8 98.93

23 SM73 0.79 96.91

24 SST1S3 0.78 95.00

25 WZ3S3 0.77 107.63

26 SST5S2 0.76 112.88

27 WZR32S 0.76 96.91

Name of Area Texture Dominant Contaminants TPH

(mg kg-1)

Gujranwala Clay Loam Jet fuel 24±2.4

Wazirabad Loam Diesel, Gasoline 442±3.6

Faisalabad Various Diesel, Gasoline 421±4.5

Sheikhupura Clay Loam High Octane, Kerosene, Diesel 650±3.4

Pindi Morga Loam Petroleum sludge 565±2.2

Sher Shah Multan Loam Aged Diesel and Engine Oil 791±3.2

JIMCO Oil Depot

Muzzafargarh Loam Diesel, Gasoline 674±4.5

Thermal power Muzzafargarh Sandy Furnace oil 651±3.5


43

High

Low

Fig. 11 Categorization of bioremediation assay into low, medium and high

Medium


44

Table 3 Summary of categorization of bacterial isolates on the basis of Bioremediation

and ACC-metabolism assay

Table 4 Summary of the traits of bacterial isolates screened for further study

Isolates

Bioremediation assay ACC metabolism assay ACC deaminase activity

Medium High Optical density> 0.75 (α-ketobutyrate nmol g-1

biomass hr-1)

PM32Y +++ 1.23 1334.15

SFD2S2 +++ 1.14 1027.28

SFD5S3 ++ 1.2 377.77

WZ3S1 +++ 1.1 268.76

SFD1S1 ++ 0.97 251.88

JM44 +++ 0.97 212.08

SM14 ++ 0.97 211.34

SFD9S4 ++ 0.92 178.22

MZT72 +++ 0.93 175.46

GC23Y ++ 0.91 172.06

SM31 ++ 0.89 163.78

SST6S1 ++ 0.89 157.95

FSD1S1 ++ 0.88 149.77

SFK2S2 ++ 0.88 143.83

SP54 +++ 0.87 139.48

SM112 ++ 0.87 138.84

SFKIS2 ++ 0.85 130.67

GCDS11 ++ 0.83 124.19

SP112 ++ 0.82 121.96

SP104Y +++ 0.82 112.51

SST4S3 ++ 0.8 111.88

SP62W ++ 0.8 98.93

SM73 +++ 0.79 96.91

SST1S3 ++ 0.78 95.00

WZ3S3 +++ 0.77 107.63

SST5S2 ++ 0.76 112.88

WZR32S ++ 0.76 96.91

Name of Area Total

Isolates

PAHs

degrader

Bioremediation assay


Low Medium High Low Medium High

Gujranwala 22 13 11 2 Nil 2 3 2

Wazirabad 28 12 9 1 2 3 5 3

Faisalabad 45 29 25 3 1 1 10 6

Sheikhupura 91 69 60 9 Nil 1 15 11

Pindi Morga 26 14 11 2 1 2 2 1

Sher Shah

Multan 38 26 22 2 2 7 5 4

JIMCO Oil

Depot 32 13 12 Nil 1 10 5 1

Thermal power

Muzzafargarh 19 13 12 Nil 1 2 2 1

Grand total 301 189 162 19 8 28 47 29


45

3.4 Discussion

For successful bioremediation, the most crucial step is to find out bacterial isolates

that are able to degrade petroleum hydrocarbons. Soils with a history of contamination

possess inherent bacterial population with capability to degrade hydrocarbons as bacterial

population present in the soils has already adjusted to contamination (Leahy and Colwell,

1990). Hydrocarbon degrading bacteria are present in almost all kind of habitats such as

in marine and soil habitats (Atlas and Bartha, 1973). It is believed that presence of high

number of bacteria from certain contaminated environment reflect the fact that these

bacteria are active degrader of that pollutant (Okerentugba and Ezeronye, 2003). For

successful bioremediation strategy, the key is to find out bacterial population whether

indigenous or exogenous equipped with suitable enzymes (Gargouri et al., 2014). For the

isolation of native population acclimatized to petroleum hydrocarbons, soil samples were

collected from petroleum polluted sites in different districts of Punjab, Pakistan.

Soil samples were used to isolate the bacteria and samples were analyzed for TPH.

Soil sampling sites were of diverse nature regarding type of contaminants, soil texture,

and temperature of the area and aging of petroleum hydrocarbons. Soil samples collected

from JIMCO Oil Depot Muzaffargarh were of sandy nature and the contamination of

petroleum hydrocarbons replenished approximately on daily basis. In contrast to

Muzzafargarh, soil samples taken from Sher Shah Multan Railway Station were of clay

nature and the contamination was aged. Main contributors to the contamination were

diesel and engine oil at Sher Shah Multan. Samples collected from Sheikhupura have

contamination with different petroleum products such as kerosene, gasoline, and high

octane petroleum and jet fuel. Petroleum sludge was collected from Oil Refineries in

Pindi Morga, Rawalpindi.

Maximum numbers of isolates (91) were cultured from the soil samples collected

from Sheikhupura district. The reason for presence of such higher population may be that

the sites were contaminated with various petroleum products such as kerosene, high

octane and diesel. A controversy apparently invalidate the reason given for higher

population in the samples collected from Sheikhupura district when comparatively low

number of isolates collected from Wazirabad (28 isolates) and Muzzafargarh JIMCO oil

depot (32 isolates) are considered as in the later mentioned sites there are also

contamination with different petroleum products. But this controversy may be counter

attacked by considering other factors that support bacterial population. As for as low


46

number of bacterial isolates from Wazirabad sites is concerned, it may be low due to the

reason that contamination concentration at this site is far low than that in Sheikhupura

district samples. Contamination level in samples from Sheikhupura district is 650 mg kg -

1 while that in Wazirabad samples is 442 mg kg-1 (Table 1) revealing that although the

contamination type is approximately similar, concentration is low in the later one; so less

microbial population was present in Wazirabad samples. This concentration of petroleum

hydrocarbons at Wazirabad may be the lower than the threshold level to support the

microbial population. Capareilo and La-Rock (1975) in their study noted higher oxidizing

activity of bacteria with increasing concentration of [14C] hexadecane and n-alkane

mixture.

On the other hand, number of microbial isolates collected from soil samples of

Muzzafargarh Thermal Power were also low (19 isolates) as compared to Sheikhupura

district though the contaminant concentration in collected soil samples were not too

different (Table 1). As the texture of the soil collected from Muzzafargarh Thermal Power

was sandy and the nutrients such as nitrogen, phosphorus are generally deficient in sandy

soils. The sandy textured soils are generally low in organic matter. Higher microbial

population is mostly associated with high organic matter and clay content of the soil. A

similar argument has been given by Al-rumman et al., 2015) to support their results which

revealed higher microbial carbon in soils with high organic matter and clay content. The

authors argued that in aqueous phase organic matter and clay content tend to reduce

hydrocarbon toxicity to microbial population as compared to soils with low organic

matter and sandy nature. Kristensen et al. (2010) study on degradation of petroleum

hydrocarbons in vadose zone and slurry bioreactors also supports the concept that soils

dominated by fine particles generally possess high number of microorganisms than coarse

textured soils until the sufficient aeration is allowed to prevail and air-filled porosity is

high. It can also be postulated that despite of considerable amount of substrate (Carbon

source in form of petroleum hydrocarbons) availability, low microbial population was the

result of deficiency of nutrients such as nitrogen and phosphorus due to sandy texture of

soil. Generally, the abundance of microbial population and diversity are governed by

environmental factors and availability of water, substrate concentration, electron acceptor

and inorganic nutrients (Holden and Fierer, 2005). Inorganic nutrients especially nitrogen

and phosphorus and in some case iron become limiting factors in increasing or sustaining

microbial textured soils as fine textured population and subsequently bioremediation

(Malina and Zawierucha, 2007; Ron and Rosenberg, 2014). As general rule, soils with


47

abundant fine textured particles harbor larger number of microorganisms that coarse soils

provide high organic matter, nutrients and larger surface area for microorganisms to cling

on (Young and Crawford, 2004). Additionally, bacterial population may be unable to

thrive in Muzzaffargarh soils due to low water holding capacity of coarse texture of soil

as the loss of water will reduce substrate diffusion to microorganisms clung on soil

particles. Afzal et al. (2010) analyzed abundance of bacterial population and

biodegradation of petroleum hydrocarbons in different type of soils and observed less

microbial population in sandy soils as compared to other soil types. The authors

concluded that low water holding capacity of soil, deficient inorganic nutrients supported

less microbial population in sandy soil and consequently less degradation of petroleum

hydrocarbons as compared to other soil types.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3100517/#R62

Chapter 4 Plant Growth Performance under Axenic Conditions

48

Chapter 4

Evaluation of Bacterial Isolates for Plant Growth Promotion under

Axenic Conditions

4.1 Introduction

Initially isolates were screened out on the basis of their potential for bioremediation

and presence of high ACC deaminase activity but the study was aimed at discovering the

bacteria that not only have bioremediation potential but also have plant growth promoting

activity. Therefore, in the next step plant growth promoting activity of preliminarily

screened isolates under axenic conditions both in growth pouches and in soil was assessed

for canola, maize and alfalfa. Bioremediation in association with plants at meaningful rate

is only possible when plants and bacterial inoculants have positive relationship with each

other. Plant growth promoting bacteria in association with plants are being used in

biodegradation process since last two or three decades. In polluted soils, plants facing

inhibition in growth especially of roots due to stress induced ethylene are reported to

relieve from this inhibition by inoculation with bacteria encoding ACC-deaminase

enzyme (Gerhardt et al., 2006). But under normal conditions there are various

mechanisms by which bacteria promote plant growth; indirectly by acting as bio-control

agent against pathogens and directly by producing siderophore, phytohormones such as

auxin and cytokinin and solublization of mineral nutrients like phosphorus (Glick et al.,

2007b). To be applied for bioremediation process in convergence with plants, it is

necessary to assess that the bacteria capable of biodegrading hydrocarbons do not possess

any pathogenicity towards plants as bacteria isolated from contaminated soils where there

is no vegetation, inoculation with these bacteria ma pathogenic or beneficial to plants

upon inoculation. One of the factors affecting successful establishment of plan bacterial

system for enhanced bioremediation is the compatibility of bacteria with specific plants

(Yousaf et al., 2010). For assessment of compatibility of bacterial isolates with selected

plants alfalfa, maize and canola growth pouch assay and sand culture experiments under

axenic conditions were conducted. Plants with fibrous and extensive root system and

adapted to local ecological conditions are considered ideal candidates for use in

phytoremediation. Maize (Zea mays L) has fibrous and extensive root system and adapted

to local ecological conditions of Pakistan. Additionally, maize has been reported to

release such root exudates which have positive effect on microbiological activity in the

rhizosphere (Benizri et al 1995; Schnoor et al 1995; Ayotamuno et al 2006). Alfalfa has


49

been used by various researchers in phytoremediation studies due to its perennial nature,

fibrous root system and thereby increased surface area for microbiological activity,

diffuse distribution all over the world and well adapted to different climatic conditions

(Ouvrard et al., 2011; Cook and Hesterberg, 2013; Zhang et al., 2013). Canola is well

recognized hyper-accumulator and has been widey used in the phytoremediation of soils

contaminated with inorganic pollutants. When the aim is to extract contaminants from

deeper zone of soil, the tap root characteristics of canola make it suitable candidate for

use in phytoremediation. Moreover, petroleum hydrocarbons being hydrophobic in nature

pose water stress to growing plants in petroleum hydrocarbons contaminated soil. Canola

requires less water and therefore may be a suitable candidate for use in phytoremediation

of petroleum contaminated soils.

4.2 Methodology

4.2.1 Growth pouch experiment

4.2.1.1 Preparation of broth culture

Growth promotion experiment under axenic condition was conducted in pre-

sterilized growth pouches in growth room. For preparation of broth, DF salt minimal media

(Dworkin and Foster, 1958) and flasks were sterilized at 120±1oC for 15 minutes and

allowed to cool under laminar flow hood for half an hour. Erlenmeyer flasks each

containing 100 mL of sterilized DF salt minimal media were inoculated with pre-screened

isolates. After inoculation, flasks were incubated for 72 hours at 28±1oC in rotary shaking

incubator at 100 rpm. A uniform cell density of 108 to 109 was maintained by recording OD

of 0.5 at 535 nm wavelength.

4.2.1.2 Seed disinfection and inoculation

Seeds of alfalfa, canola and maize were surface disinfected by dipping them in 95%

ethanol solution for 30 seconds followed by thorough washing with distilled water and

dipping in 0.2% HgCl2. Another thorough washing with sterilized distilled water was

employed prior to inoculation of seed (Russell et al., 1982). Surface disinfected seeds of

alfalfa, maize and canola were inoculated by dipping them in 10 mL of broth culture

supplemented with 5 mL of 10% sugar solution in petri plates for five minutes. Each petri

plate devoted to separate bacterial isolate was triplicated to inoculate the seeds of alfalfa,

maize and canola separately and to make the experimental procedure uniform for each crop.

For un-inoculated control, seeds were dipped in sterilized broth supplemented with 5 mL of

10% sugar solution lacking bacterial inoculation. Sugar solution was added to aid bacterial


50

population for initial proliferation. Sugar solution was helpful in establishment of microbial

population in stressed environment as it provided initial easy source of carbon for isolates.

4.2.1.3 Growth conditions

The treatments were triplicated and four inoculated seeds per replicate were placed

in each growth pouch. For nutrition,10 mL of half strength sterilized Hoagland’s solution

(Hoagland and Arnon,1950) per pouch was used two times in a week and in remaining

days of the week sterilized distilled water was used to keep the growth pouch moist to

avoid dehydration of seedlings. Growth pouches were placed in growth room and

temperature was adjusted at 28±1oC. After fifteen days of sowing, data regarding root and

shoot length, total fresh biomass and oven-dried biomass were recorded.

4.2.2 Jar experiment (sand culture)

A jar experiment for each crop was conducted in growth room for further

verification of pouch experiment results. Same strains were used in jar experiments under

axenic conditions in sand culture. Dimension of the jar used in this experiment was 13×6

cm where 13 cm was length of jar and 6 cm was internal diameter. These jars were filled

with quartz sand which was passed through 2 mm sieve to remove gravel and debris. The

sand was gently shaken to compact it. The sand filled in plastic jars was soaked with 50 mL

half strength Hoagland solution. For sterilization, jars were wrapped with papers and

autoclaved at 120±1oC for 20 minutes. To ensure complete sterilization, the jars were

autoclave three times. All jars were transferred to laminar flow hood and allowed the sand

in jars to cool down. During this cooling period, ultra-violet lights of the laminar flow kept

on to ensure disinfection. Broth culture preparation was done as explained earlier in the

growth pouch experiment. For inoculation, seed dressing was carried out with inoculated

peat mixed with 10 % sugar solution. In case of the un-inoculated control, the seeds were

coated with the same but without inoculum suspension. Four seeds per treatment of alfalfa,

canola and maize each were sown in jars and each treatment was triplicated. Mineral

nutrition was applied twice a week in the form of sterilized Hoagland solution and in

remaining days of the week sand was kept moist by applying sterilized distilled water. The

Jars were placed in growth room and temperature was adjusted at 28±1oC. After 30 days of

sowing, data regarding root and shoot length, total fresh biomass and oven dried biomass

were recorded.

4.2.3 Root colonization assay

Root colonization assay was carried out according to the protocol described by

Simons et al. (1996). At the time of harvest of growth pouch assay after 15 days, 0.2 g of


51

root tips were cut and shaken in 5 mL sterilized distilled water and shaken vigorously in

rotary shaking incubator at 100 rpm. Dilutions ranging from 10-1 to 10-5 were made. DF salt

minimal medium was sterilized and solidified in petri plates. One milliliter of each dilution

was poured in petri plates containing solidified DF salt minimal medium and incubated at

28±1°C for 48 hours. Bacterial colonies were counted using digital colony counter (Suntex

Model 570, Taiwan) and CFU g-1 of root was calculated.

4.2.4 Root length by Delta T-Scanner

Roots length of alfalfa, canola and maize plants was measured by using Delta T-

Scan root analyzer. The sand particles of rhizoplane were thoroughly washed to avoid

interference in measurement and damage to delicate glass of scanner. Washed roots were

placed on scanner and the roots were scanned to get the image of roots. The image was

converted to monochrome. This image was then loaded to adobe photo shop of delta T-

scanner and the image was made as bitmap image. The image was then loaded to delta T-

scan and root length was measured in centimeter. The same procedure was repeated to get

the length of root of each plant.

4.2.5 Identification of bacteria

Initially selected bacteria on the basis of high bioremediation activity, high ACC-

deaminase activity and plant growth promotion were identified using Biolog ®

identification system (MicrologTM System Release 4.2, Hayward, CA, USA) on the basis

of carbons source utilization. Ninety-six wells Biolog® plates containing 32 different

carbon sources with three repeats of each carbon source were inoculated and incubated

for assessment of growth on different source of carbons. For inoculation of Biolog®

plates, bacterial isolates were grown on glucose peptone agar medium. Sterilized

inoculating fluid impregnated with the well grown colonies of bacteria by picking them

with the help of wool swab and rubbing them on walls of test tubes up to the turbidity

level of standard fluid. The inoculated fluid was put in sterilized inoculating boats.

Microtitre plates were inoculated with 120 μL with the help of Biolog® pipette. Each

microtitre plate was inoculated with one bacterial isolate. For control, an un-inoculated

fluid filled microtitre plate was kept. Inoculated plates were incubated at 28±1oC for 24

hours prior to reading the plates on Biolog® for identification. Biolog identification

software has a data base library and after reading plates it matches the data with library

and gives identification of bacteria.

Screened bacteria (8 isolates) were further identified by 16S rRNA sequencing.

Samples were analyzed by using DNA sequencing service provided by Macrogen Inc.


52

Seoul, Korea, by using following procedure. Colonies were picked up with a sterilized

toothpick, and suspended in 0.5 mL of sterilizes saline in a 1.5 mL centrifuge tube,

centrifuged at 10,000 rpm for 10 minutes. After removal of supernatant, the pellets were

suspended in 0.5 mL of Insta Gene Matrix (Bio-Rad, USA). Incubated at 56°C for 30 min

and then heated at 100°C for 10 min. After heating, supernatant was used for PCR. One

mL of template DNA was taken in 20 mL of PCR reaction solution. Primers

27F(AGAGTTTGATCMTGGCTCAG) and (TACGGYTACCTTGTTACGACTT)1492R

(Lane, 1991) were used and then performed 35 amplification cycles at 94°C for 45 sec,

55°C for 60 sec, and 72°C for 60 sec. DNA fragments were amplified about 1,400 bp, and

E. coli genomic DNA was used as a positive control in the PCR. Purification of PCR

products was done by using Montage PCR Clean up kit (Millipore). The purified PCR

products of approximately 1,400 bp were sequenced. Sequencing was performed by using

Big Dye terminator cycle sequencing kit (Applied Bio Systems, USA). Sequencing

products were resolved on an Applied Biosystems model 3730XL automated DNA

sequencing system (Applied BioSystems, USA). Results were interpreted by using the

database BLAST (Altschul et al. 1997).

4.3 Results

4.3.1 Root length

Bacterial inoculation significantly improved the growth of roots of all three

inoculated crops alfalfa, maize and canola. Bacterial isolates PM32Y, SFD2S2, SFD5S3,

WZ3S1, JM44, SFK2S2, WZ3S3 and WZR32S increased root length of alfalfa most

significantly (Table 5). Percent increase in root length as compared to un-inoculated

control was 70%, 67%, 67% and 66% caused by WZR32S, PM32Y, WZ3S3 and

SFD5S3, respectively. Other noticeable increase in root length of alfalfa was by WZ3S1,

SFD2S2 and SFK2S2 which increased root length by 61%, 60% and 59%, respectively.

Bacterial isolates with code name SST4S3 and GCDS11 were non-significant to un-

inoculated control and caused minimum increase in root length. Bacterial isolate SST4S3

caused 10% increase while GCDS11 increased root length by 11% as compared to un-

inoculated control.

Bacterial inoculation caused significant increase in root length of maize. As

shown in (Table 5) the effect of inoculation on maize was more pronounced as compared

to alfalfa when grown under stress free conditions. Root elongation of maize was

prominent in treatments receiving PM32Y, WZ3S1, WZ3S3, SFD2S2, SM112 and


53

WZR32S inoculation which caused percent increase of 90%, 78%, 84%, 74%, 73% and

70%, respectively as compared to un-inoculated control. Bacterial isolates coded as

MZT72, SP62W, JM44, SFK1S2, and SM73 also elongated root significantly. The

percent increase by these bacterial isolates was 68%, 66%, 65%, 62% and 61%,

respectively. Results revealed that SST6S1 inoculation had negative effect on maize root

elongation and other bacterial isolates were significant to un-inoculated control in

increasing root length.

Canola roots showed maximum response to bacterial inoculation as compared to

alfalfa and maize. As indicated by the data (Table 5), the most promising increase in root

length of canola was caused by the inoculation of PM32Y which increase root of canola

by 116% as compared to un-inoculated control revealing that the PM32Y was consistent

in improving root length of all three crops. Other considerable increase of 82%, 81%,

79%, 68%, 63%, 63% and 62% in root length of canola was caused by bacterial isolates

SFD2S2, WZ3S3, SFK2S2, WZR32S, SFK1S2, SM112 and SFD5S3, respectively.

Compatible with other two crop alfalfa and maize, the bacterial isolate WZ3S1 failed to

maintain its consistency in increasing root length of canola. Bacterial isolate GCDS11

caused minimum increase in root length which was 13% more than un-inoculated control.

4.3.2 Shoot length

Shoot elongation of alfalfa was greatly improved by bacterial isolate PM32Y

which increased maximum shoot height among all the bacterial isolates. The PM32Y

increased shoot length of alfalfa by 67% followed by an increase of 66% caused by

SFD5S3 inoculation (Table 6) The PM32Y was statistically at par with WZ3S1, SFD2S2

and SFD9S4 which elongated shoot of alfalfa by 60%, 59% and 56%, respectively as

compared to un-inoculated control. Bacterial isolates WZ3S3, WZR32, SM14, SST6S1,

GCDS11, SM73, JM44 and MZT72 also caused considerable increase of 51%, 48%,

47%, 46%, 46%, 44%, 41% and 40% in shoot length of alfalfa. Minimum increase in

shoot length of alfalfa was caused by SP54 which increased 5% shoot height as compared

to un-inoculated control.

In case of maize, bacterial isolate PM32Y caused a maximum increase of 80% in

shoot elongation. While bacterial isolates SFD2S2 and WZ3S3 also showed their

compatibility with maize each caused 75% increase in shoot length in growth pouch assay

under axenic conditions. Other bacterial isolates which caused considerable increase in

plant height of maize were WZR32S, WZ3S1, SM112, SP62W and JM44 which caused

66%, 52%, 45%, 43% and 39% increase in shoot length as compared to un-inoculated


54

control. Bacterial isolates SP54 and SST1S3 each caused 8% increase in shoot length of

maize which was lowest increase as compared to un-inoculated control.

Likewise root length, shoot length of canola was most significantly affected by

bacterial inoculation grown under stress free condition in growth pouches. The most

consistent bacterial isolate with all three crops was PM32Y which increased shoot length

of canola by 107% as compared to un-inoculated control. This maximum increase was

followed by other noticeable increase of 86%, 83%, 81%, 78% and 68% in shoot length

caused by bacterial isolates SFD2S2, SM31, SFD5S3, WZ3S3 and WZR32S,

respectively. Bacterial isolates SP112, SST4S3 and SST1S3 were statistically at par with

un-inoculated control with minimum increase of 11%, 15% and 15% in shoot length,

respectively.


55

Table 5 Effect of bacterial inoculation on root length of alfalfa, maize and canola under

axenic condition in growth pouch assay

Means sharing same letter (s) are non-significant at P< 0.05

Treatment Root length (cm)

Alfalfa Maize Canola

Control 7.29±0.21k 6.08±0.22L 7.13±0.58L

PM32Y 12.18±0.22a 11.55±0.69a 15.43±0.51a

SFD2S2 11.69±0.28ab 10.58±0.30ad 13.00±0.26b

SFD5S3 12.07±0.15a 9.38± 0.24ei 11.53±0.35cd

WZ3S1 11.75±0.49ab 11.83± 0.44ac 9.23±0.17fj

SFD1S1 10.53±0.58df 8.20±0.28jk 9.83± 0.54fh

JM44 11.09±0.28bd 10.03± 0.55cf 9.96±0.13fg

SM14 10.24 ±0.17df 8.68±0.22hk 10.13±0.12ef

SFD9S4 10.29 0.25df 8.24± 0.50jk 9.60±0.34fh

MZT72 10.31±0.23df 10.20±0.17bf 8.33±0.25ik

GC23Y 10.23±0.40df 7.70±0.29k 8.50±0.29ik

SM31 10.21±0.53df 9.57±0.26di 12.40±0.44bc

SST6S1 9.10±0.56gi 6.03±0.75L 8.360± 0.46ik

FSD1S1 9.04±0.36hi 7.60± 0.42k 11.03±0.24de

SFK2S2 11.59±0.22ac 9.60± 0.55di 12.73±0.46b

SP54 8.67±0.27ij 7.77±0.41k 8.230±0.29jk

SM112 9.71±0.27fh 10.50±0.29ad 11.60±0.51cd

SFK1S2 8.78±0.22ij 9.87± 0.19cg 11.63±0.41cd

GCDS11 8.11±0.13jk 9.40±0.26ei 8.10± 0.41kl

SP112 10.70±0.26ce 8.18±0.47jk 9.30±0.50fl

SP104Y 8.77±0.26ij 9.13±0.23fj 9.23± 0.39fj

SST4S3 8.05±0.13jk 8.93± 0.18gj 8.50± 0.29ik

SP62W 8.62±0.32ij 10.06±0.43cf 9.93± 0.23fg

SM73 9.97±0.29eg 9.77±0.15ch 9.06±0.43gk

SST1S3 8.78±0.43ij 7.67±0.44k 8.20±0.22jk

WZ3S3 12.18±0.19a 11.20±0.42ab 12.87±0.30b

SST5S2 8.970.35hj 8.63± 0.29ik 8.80±0.29hk

WZR32S 12.37±0.37a 10.33± 0.10be 12±0.10bd


56

Table 6 Effect of bacterial inoculation on shoot length of alfalfa, maize and canola under

axenic conditions in growth pouch assay

Treatment Shoot length (cm)


Control 4.90±0.06k 9.13±0.35j 8.20±0.34r

PM32Y 8.20±0.17a 16.40±0.65a 16.00±0.22a

SFD2S2 7.79±0.29ab 16.00±0.26ab 14.93±0.22b

SFD5S3 8.15±0.28a 10.87±0.43gi 14.47±0.52bc

WZ3S1 7.82±0.17ab 13.88±0.22c 12.33±0.58ei

SFD1S1 6.50±0.14eg 11.38±0.34fg 10.93±0.60jm

JM44 6.90±0.13de 12.67±0.38d 12.70±0.30eg

SM14 7.19±0.34be 10.70±0.37gi 12.07±0.58fj

SFD9S4 7.67±0.11ac 10.11±0.23hj 11.67±0.12gk

MZT72 6.84±0.20de 12.32±0.23d 11.50±0.27hl

GC23Y 5.81±0.48gj 10.83±0.44gi 11.23±0.29im

SM31 6.12±0.32fh 12.53±0.36de 14.70±0.51b

SST6S1 7.17±0.50be 10.07±0.44hj 10.93±0.51jm

FSD1S1 5.35±0.26ik 11.43±0.35fg 12.53±0.55eh

SFK2S2 5.76±0.16hj 12.75±0.43d 11.43±0.55hl

SP54 5.15±0.31jk 9.90±0.45hj 10.47±0.36lo

SM112 6.74±0.29df 13.25±0.38cd 13.17±0.33df

SFK1S2 6.72±0.20df 12.20±0.36df 12.00±0.73fj

GCDS11 7.16±0.29be 10.05±0.43hj 10.33±0.29lp

SP112 6.04±0.06fi 10.92±0.44gh 8.867±0.67qr

SP104Y 5.80±0.19gj 10.67±0.22gi 10.13±0.22mp

SST4S3 5.44±0.23hk 10.57±0.35gi 9.23±0.20pr

SP62W 5.85±0.14hk 13.10±0.32cd 10.7±0.32kn

SM73 7.06±0.17ce 11.46±0.29ef 9.50±0.43oq

SST1S3 6.10±0.15fh 9.83±0.36ij 9.2±0.29pr

WZ3S3 7.41±0.15bd 16.02±0.45ab 14.27±0.34bd

SST5S2 5.93±0.27gi 11.48±0.37ef 9.53±0.52nq

WZR32S 7.26±0.33bd 15.17±0.38b 13.47± 0.44ce



57

Fig. 12 Comparison of inoculated and un-inoculated root growth of alfalfa under axenic

conditions


58

4.3.3 Fresh biomass

In growth pouch assay under axenic conditions, alfalfa produced maximum fresh

biomass in response to PM32Y inoculation (Table 7). This maximum increase in fresh

biomass of alfalfa was 154% as compared to un-inoculated control. Other bacterial

isolates efficient in increasing biomass of alfalfa were SFD5S3, SM112, SFK1S2,

WZ3S3, SFD2S2, MZT72, SM31 and WZR32S which increased biomass of alfalfa by

97%, 94%, 80%, 71%, 70%, 69%, 67% and 62%, respectively. Bacterial isolates with

code name FSD1S1, SP54, GCDS11, SST4S3, SST1S3 and SST5S2 did not increase

biomass significantly as compared to un-inoculated control. The SP54 isolate caused least

increase in biomass which was 7% more than un-inoculated control.

Response of maize to bacterial inoculation regarding increase in biomass was also

pronounced. The PM32Y was the best among all the bacterial isolates in increasing

biomass of maize as it increased 116% over un-inoculated control (Table 7). Bacterial

isolate SFD2S2 also prominently increased fresh biomass of maize and was statistically at

par with PM32Y. The SFD2S2 increased biomass by 102% as compared to un-inoculated

control. Bacterial isolates WZ3S1, WZ3S3, JM44, SFK2S2, SM112, WZR32S, MZT72,

and SM73 also caused noticeable increase of 87%, 62%, 50%, 48%, 48%, 48%, 47% and

46%, respectively in fresh biomass of maize. Bacterial isolates SFD1S1, SM14, SST6S1,

SP54, GCDS11, SP112, SST4S3, SST1S3 and SST5S2 were statistically non-significant

to increase plant biomass as compared to un-inoculated control. Minimun increase of 4%

in fresh biomass as compared to un-inoculated control was the result of SST6S1

inoculation.

Bacterial isolate PM32Y maintained its consistancy in plant growth promotion

with all three crops alfalfa, maize and canola as it caused maximun increase in fresh

biomass of canola like alfalfa and maize. The PM32Y caused an increase of 69% in fresh

biomass of canola followed by 54% increase resulted from the inoculation of SM31 as

second best bacterial isolate regarding increase in fresh biomass of canola. Other

noticeable increase of 53%, 47%, 47%, 39%, 39%, 36% and 36% in fresh biomass was

caused by SFD2S2, WZ3S1, SM112, JM44, SFD5S3, SST1S3 and WZR32S,

respectively. Out of 27 bacterial isolates tested for plant growth promotion, 3 bacterial

isolates with code name SST6S1, SP54 and SFK1S2 were statistically non-significant to

un-inoculated control in improving fresh biomass of canola. Minimum increase in fresh

biomass of canola was 6% caused by inoculation of SST6S1 as compared to un-

inoculated control.


59

4.3.4 Oven dried biomass

Maximum increase in oven dried biomass of alfalfa was observed in response to

PM32Y bacterial inoculation. This increase was 171% over un-inoculated control (Table 8).

Other bacterial isolate which effectively increased oven dried biomass of alfalfa were

SFD5S3, SFD2S2, SM112, SM31, WZ3S1, SFK1S2, SM14, SF1D1S1, JM44, SM73,

MZT72, SFD9S4, WZ3S3, SFK2S2 and WZR32S 127%, 120%, 108%, 107%, 105%,

102%, 100%, 95%, 94%, 81%, 79%, 78%, 78%,75% and 75%, respectively. While

bacterial isolates statistically at par with un-inoculated control regarding increase in oven

dried biomass of alfalfa were SST6S1, SST4S3, SST1S3 and SST5S2. The bacterial

isolates SST1S3 and SST6S1 caused minimum increase of 8% each as compared to un-

inoculated control.

Bacterial isolate SFD2S2 increased 151% oven dried biomass as compared to un-

inoculated control in case of maize which was followed by increase of 148% over control

caused by PM32Y. Bacterial isolates WZ3S1, WZ3S3, SFK2S2, WZR32S, SM73, SM112,

MZT72 and JM44 were also effective as these bacterial isolates increased oven dried

biomass of maize by 82%, 76%, 71%, 54%, 51%, 45%, 44%, 41%, respectively, as

compared to un-inoculated control. Statistically non-significant to un-inoculated control

bacterial isolates were SFD1S1, SM14, GC23Y, SST6S1, FSD1S1, SP54, GCDS11,

SST4S3, SST1S3 and SST5S2. Among all 27 bacterial isolates, SST5S2 was the isolate

whose inoculation showed the least effect (1%) on maize regarding increase in oven dried

biomass as compared to un-inoculated control.

Canola also showed great response to bacterial inoculation as out of 27 bacterial

isolates, 11 bacterial isolates prominently increased oven dried biomass of canola

comparative to un-inoculated control. These prominent bacterial isolates were PM32Y,

SM31, SFD2S2, SM112 , SM73, WZ3S3, WZ3S1, WZR32S, SFD5S3, JM44 and SFD1S1

which increased oven dried biomass of canola over un-inoculated control by 101%, 87%,

81%, 64%, 64%, 64%, 62%, 62%, 53% and 47%, respectively. The bacterial isolate

PM32Y was found to be the most efficient in increasing oven dried biomass. While

bacterial isolates coded as SP112 and SST5S2 were non-significant to un-inoculated control

with minimum increase of 4% and 11%, respectively.

In short, bacterial isolates PM32Y, SFD2S2, WZ3S1, SM112, JM44, WZ3S3 and

WZR32S were all consistent with all three crops alfalfa, maize and canola in increasing

oven dried biomass comparative to un-inoculated control, however, the percent increase by

these consistent bacterial isolates over un-inoculated control varied from crop to crop.


60

Table 7 Effect of bacterial inoculation on fresh biomass of alfalfa, maize and canola under

axenic conditions in growth pouch assay

Means sharing same letters don't differ significantly at 95% level of confidence

Treatment Fresh biomass (g)


Control 0.60±0.02l 0.92±0.08j 0.72±0.01m

PM32Y 1.52±0.04a 1.99±0.09a 1.22±0.01a

SFD2S2 1.02±0.06de 1.86±0.05ab 1.10±0.01b

SFD5S3 1.18±0.04b 1.14±0.07eh 1.00±0.01c

WZ3S1 0.93±0.02fh 1.72±0.09b 1.06±0.01b

SFD1S1 0.86±0.03hi 1.02±0.04gj 0.95±0.03cd

JM44 0.86±0.03hi 1.38±0.10cd 1.00±0.01c

SM14 0.88±0.05gi 1.03±0.05gj 0.90±0.02de

SFD9S4 0.82±0.03ij 1.16±0.02eg 0.80±0.04il

MZT72 1.01±0.05df 1.35±0.05cd 0.83±0.01fi

GC23Y 0.84±0.03hi 1.10±0.02ei 0.79±0.02il

SM31 1.00±0.03df 1.14±0.03eh 1.11±0.01b

SST6S1 0.73±0.02jk 0.96±0.03ij 0.75±0.01lm

FSD1S1 0.65±0.02kl 1.18±0.03ef 0.85±0.02eh

SFK2S2 0.87±0.07hi 1.36±0.02cd 0.86±0.02eg

SP54 0.64± 0.01l 1.03±0.04fj 0.77±0.02km

SM112 1.16±0.02bc 1.36±0.03cd 1.06±0.02b

SFK1S2 1.08±0.02cd 1.24±0.02de 0.77±0.02jm

GCDS11 0.68±0.05kl 1.04±0.03fj 0.84±0.01fi

SP112 0.74±0.02jk 1.01±0.02hj 0.82±0.01gj

SP104Y 0.81±0.03ij 1.15±0.03eh 0.74±0.01eh

SST4S3 0.67±0.04kl 1.01±0.03hj 0.86±0.02eh

SP62W 0.82±0.03ij 1.15±0.03eg 0.88±0.01ef

SM73 0.88±0.02hi 1.35±0.05cd 0.73±0.02de

SST1S3 0.67±0.03kl 0.97±0.02 ij 0.98±0.01c

WZ3S3 1.03±0.02 de 1.49±0.09c 0.81±0.02hk

SST5S2 0.68±0.02kl 1.06±0.05fj 0.90±0.01e

WZR32S 0.97±0.04eg 1.36±0.06cd 0.98±0.01c


61

Table 8 Effect of bacterial inoculation on oven dried biomass of alfalfa, maize and canola

under axenic conditions in growth pouch assay


Treatment Oven dried biomass (g)


Control 0.27±0.01L 0.34±0.01k 0.30±0.01j

PM32Y 0.73±0.03a 0.84±0.03a 0.60±0.01a

SFD2S2 0.59±0.01bc 0.85±0.02a 0.54±0.01b

SFD5S3 0.61±0.02b 0.40±0.02fi 0.48±0.02cd

WZ3S1 0.55±0.02cd 0.62±0.01b 0.48±0.02cd

SFD1S1 0.53±0.02df 0.34±0.01jk 0.44±0.01e

JM44 0.52±0.05dg 0.48±0.01de 0.46±0.02de

SM14 0.54±0.04de 0.34±0.01jk 0.40±0.01f

SFD9S4 0.48±0.01fh 0.39±0.02gi 0.38±0.01fg

MZT72 0.48±0.03fh 0.49±0.01cd 0.39±0.01f

GC23Y 0.44±0.02hi 0.36±0.02ik 0.36±0.03gh

SM31 0.56±0.01cd 0.44±0.02ef 0.56±0.01b

SST6S1 0.29±0.02kl 0.34±0.01jk 0.38±0.01fg

FSD1S1 0.32±0.02k 0.36±0.02ik 0.36±0.02gh

SFK2S2 0.47±0.01gh 0.62±0.01b 0.36±0.03gh

SP54 0.33±0.02k 0.38±0.01hk 0.33±0.01hi

SM112 0.56±0.09cd 0.49±0.02cd 0.49±0.01c

SFK1S2 0.55±0.03cd 0.41±0.02fh 0.34±0.03hi

GCDS11 0.33±0.02k 0.34±0.01jk 0.31±0.01ij

SP112 0.39±0.02ij 0.38±0.01gj 0.35±0.01gh

SP104Y 0.39±0.01j 0.41± 0.01fh 0.39±0.02f

SST4S3 0.31±0.02kl 0.36±0.01ik 0.36±0.01gh

SP62W 0.33±0.01k 0.42±0.02fg 0.34±0.01hi

SM73 0.49±0.01eh 0.51±0.01cd 0.49±0.02c

SST1S3 0.29±0.01kl 0.36±0.02ik 0.33±0.01hi

WZ3S3 0.48±0.02fh 0.62±0.01b 0.49±0.01c

SST5S2 0.31±0.01kl 0.34±0.01jk 0.33±0.01hj

WZR32S 0.47±0.02h 0.52±0.02c 0.48±0.06cd


62

4.3.5 Jar experiment (sand culture)

Initially plant growth promoting activity of 27 bacterial isolates was assessed in

growth pouch assay and the results were discussed as above. As sand was to be used in

bioremediation trials, so a jar experiment of 30 days was also conducted to assess the

bacterial efficiency in increasing plant growth in sand. This experiment was conducted to

have final verification of the compatibility of selected plants and bacterial inoculants with

each other to get help in further screening of the bacterial isolates to be used in further

study. The methodology adapted for the experiment is as given in section (4.2.2). Plant

growth parameters such as root length, shoot length, fresh biomass and oven dried biomass

were recorded at harvesting after 30 days of sowing.

4.3.5.1 Root length

Bacterial isolate PM32Y maintained its consistency in increasing root length of

alfalfa in sand culture experiment as indicated by maximum increase of 91% over un-

inoculated control. Other noticeable increase in root length of alfalfa was observed in

treatments receiving inoculation of bacterial isolates SM14, SM112, SFK1S2, WZ3S3,

JM44, SFK2S2, SM73, MZT72, WZR32S and SFD2S2 which caused root elongation by

71%, 68%, 68%, 59%, 57%, 55%, 54%, 53%, 53%, 45% and 45%, respectively, as

compared to un-inoculated control. Out of 27, plant growth promoting activity regarding

root elongation of 4 bacterial isolates with code name GCDS11, SST4S3, SP62W and

SST5S2 did not yield statistically significant result as compared to un-inoculated control.

The percent increase by these bacterial isolates was 4%, 3% and 3%, respectively. Other

bacterial isolates such as GC23Y, SM31, SST6S1, SP112 and SP104Y also significantly

increased root length of alfalfa compared to un-inoculated control.

As for as the root elongation of maize in sand culture experiment is concerned,

unlike canola and alfalfa, the maximum increase in root length was observed in the

treatment receiving inoculation of SFD2S2 bacterial isolate that increased roots of maize by

105% in comparison with un-inoculated control (Table 9) . This maximum increase was

followed by 79% increase in root length caused by the isolate PM32Y. Increase in root

length by WZ3S1, WZ3S3, MZT72, SP104Y, SM73 and JM44 could not be neglected as

these isolates caused considerable increase of 76%, 65%, 58%, 56%, 55% and 50%,

respectively, as compared to un-inoculated control. Non-significant increase in root length

to un-inoculated control was observed in treatments receiving inoculation of GCDS11 and

SST5S2 isolates. These two bacterial isolates increased root length by 11% and 7%,

respectively, which was recorded as the least increase among all 27 bacterial isolates.


63

Similarly, root length of canola was also affected by bacterial inoculation

significantly in sand culture under axenic conditions. Maximum increase of 72% over un-

inoculated control was observed in response to inoculation of bacterial isolate PM32Y. Out

of 27 bacterial isolates other than PM32Y, 12 bacterial isolates improved roots of canola

considerably in sand culture experiment. These bacterial isolates with code name WZ3S3,

SM31, SFK2S2, SM73, SFD2S2, SM112, JM44, WZ3S1, SP104Y, MZT72, SFK1S2 and

WZR32S increased root length by 71%, 71%, 67%, 66%, 63%, 58%, 56%, 55%, 55%, 50%

and 50%, respectively. Minimum increase of 6% in root length was caused by GCDS11

which was statistically non-significant to un-inoculated control.

Overall observation regarding root elongation in all three crops revealed that

bacterial isolates PM32Y, SFD2S2, WZ3S1, WZ3S3, JM44, MZT72, and SM73 were

consistent in increasing root length of all three crops alfalfa, maize and canola. Some

bacterial isolates such as SFK2S2, SM112, SFK1S2 and WZR32S were among the

prominent isolates in increasing root length of alfalfa and canola but these isolates did not

performed well in increasing root length of maize to the same extent as for alfalfa and

canola. Similarly, bacterial isolate SP104Y prominently increased root length of maize and

canola by increasing 56% root length of maize and 55% increase in case of canola roots as

compared to un-inoculated control but in case of alfalfa the performance of SP104Y was

slightly less (36%).

4.3.5.2 Shoot length

Plant height of alfalfa was prominently increased by bacterial isolate PM32Y,

SFD2S2, SM14, SFK1S2, SFK2S2, WZ3S1, WZ3S3, SM112, JM44, SP104Y and MZT72

with an increase of 72%, 52%, 51%, 50%, 48%, 47%, 46%, 44%, 40%, 40% and 39%,

respectively. Minimum increase in shoot length was the result of bacterial inoculation with

FSD1S1 which increased shoot length by 2% compared to un-inoculated control.

Response of maize regarding increase in shoot length to bacterial inoculation was

significant. The PM32Y was the most efficient as it increased maximum shoot length

among all 27 bacterial isolates test for plant growth promotion. This maximum increase of

67% in shoot length caused by PM32Y was followed by 57% resulted by inoculation of

SFD2S2. Other effective bacterial isolates regarding increase in shoot length of maize were

WZ3S3, MZT72, SP104Y, WZR32S and WZ3S1 which caused increase of 53%, 44%,

42%, 41% and 41% over un-inoculated control, respectively. Out of 27, six bacterial

isolates with code name SFD9S4, GC23Y, SST6S1, FSD1S1, SST4S3 and SST5S2 were


64

statistically at par with un-inoculated control. Minimum 2% increase in root length over un-

inoculated control was caused by GC23Y.

Plant height of canola was significantly increased by PM32Y inoculation.

Maximum increase of 56% by PM32Y was followed by an increase of 55% as second

largest increase in shoot length of canola by the bacterial isolate MZT72. Bacterial isolates

with code name WZR32S, SM14, GC23Y, SM31, SFD2S2, WZ3S3, SM73, WZ3S1, and

JM44 were also efficient in improving shoot length of canola by 53%, 52%, 51%, 49%,

46%, 44%, 43%, 42% and 40% increase over un-inoculated control. While bacterial

isolates SST1S3, SST4S3 and SST5S2 were failed to increase shoot length significantly as

compared to un-inoculated control with minimum increase of 6%, 5% and 0%,

respectively.

Analyzing overall performance of bacterial isolates with all three crops regarding

increase in shoot length revealed that bacterial isolates PM32Y, SFD2S2, WZ3S1, JM44,

SP104Y and MZT72 were efficient with all three crops alfalfa, maize and canola as these

bacterial isolates caused over 40% increase in shoot length of all three crops. Bacterial

isolate SM73 caused increase of more than 40% in shoot length of alfalfa and canola;

however, in case of maize the SM37 caused increase of 36% over un-inoculated control.


65

Table 9 Effect of bacterial inoculation on root length of alfalfa, maize and canola under

axenic condition in sand culture


Treatment

Root length (cm)


Control 15.03±0.09j 19.77±0.15n 10.41±0.47L

PM32Y 28.67±0.49a 32.93±0.29a 17.86±0.30a

SFD2S2 21.77±0.72ef 31.10±0.49b 16.93±0.23ac

SFD5S3 21.37±0.59fg 22.33±0.73jk 13.57±0.21gi

WZ3S1 23.83±0.29cd 27.83±0.47cd 16.16±0.32be

SFD1S1 21.07±0.58fg 22.87±0.32ij 14.00±0.29gh

JM44 23.27±0.41de 27.43±0.88ce 16.23±0.35bd

SM14 25.63±0.56b 21.67±0.53jm 14.50±0.58fg

SFD9S4 19.47±0.84h 20.80±0.29kn 14.50±0.35fg

MZT72 23.07±0.30de 28.50±0.61c 15.60±0.12cf

GC23Y 20.63±0.70fh 20.20±0.17mn 12.50±0.63ij

SM31 20.17±1.10gh 26.00±0.18eg 17.80±0.72a

SST6S1 20.47±0.15fh 20.67±0.63ln 14.40±0.29fg

FSD1S1 19.43±0.70h 20.43±0.34mn 13.67±0.97gi

SFK2S2 23.2±0.49de 26.57±0.25df 17.33±0.67ab

SP54 19.33±0.56h 21.80±0.74jm 11.80±0.44jk

SM112 25.2±0.64bc 27.43±0.38ce 16.40±0.33bd

SFK1S2 25.27±0.46bc 24.40±0.82gi 15.67±0.38cf

GCDS11 15.7±0.32ij 21.53±0.56jm 11.00±0.38kl

SP112 20.43±0.52fh 24.10±0.52hi 15.53±0.21df

SP104Y 20.47±0.47fh 28.07±0.43cd 16.10±0.55be

SST4S3 15.87±0.30ij 21.27±0.44jn 12.90± 0.60j

SP62W 15.43±0.30ij 25.17±0.73fh 14.83±0.44eg

SM73 23.00±0.29de 26.83±0.42de 17.23±0.50ab

SST1S3 16.7±0.92i 22.20±0.88jl 11.90±0.44jk

WZ3S3 23.57±0.35d 30.33±0.65b 17.83±0.40a

SST5S2 15.43±0.43ij 20.77±0.45kn 13.70±0.54gi

WZR32S 21.83±0.84ef 27.83±0.44cd 15.63±0.84cf


66

Table 10 Effect of bacterial inoculation on shoot length of alfalfa, maize and canola under

axenic conditions in sand culture


Treatment Shoot length (cm)


Control 14.93±0.32k 19.77±0.15n 19.37±0.47k

PM32Y 25.73±0.42a 32.93±0.29a 30.23±0.30a

SFD2S2 22.67±0.29b 31.10±0.49b 28.37±0.23bd

SFD5S3 19.40±0.26fg 22.33±0.73jk 24.60±0.21fg

WZ3S1 21.93±0.18bc 27.83±0.73cd 27.47±0.32ce

SFD1S1 19.43±0.35fg 22.87±0.47ij 24.00±0.29gh

JM44 20.83±0.29de 27.43±0.32ce 27.13±0.35de

SM14 22.50±0.18b 21.67±0.88jm 29.50±0.58ab

SFD9S4 18.73±0.26gh 20.80±0.53kn 24.57±0.35fg

MZT72 20.77± 0.26de 28.50±0.29c 30.00±0.12a

GC23Y 20.10±0.33ef 20.20±0.61mn 29.30±0.63ab

SM31 19.47±0.18fg 26.00 ±0.17eg 28.83±0.72ac

SST6S1 18.17±0.60h 20.67±0.88ln 21.00±0.29j

FSD1S1 15.17±0.29k 20.43±0.63mn 21.43±0.97ij

SFK2S2 22.03±0.17bc 26.57±0.34df 23.23±0.62gh

SP54 17.20± 0.72ij 21.80±0.25jm 21.07±0.67j

SM112 21.50±0.30cd 27.43±0.74ce 22.67±0.44hi

SFK1S2 22.37±0.22bc 24.40±0.38gi 24.17±0.33fg

GCDS11 18.07±0.23hi 21.53±0.82jm 22.70±0.38hi

SP112 17.03±0.38j 24.10±0.56hi 25.47±0.38f

SP104Y 20.93±0.40de 28.07±0.52cd 26.90±0.21e

SST4S3 16.70±0.50j 21.27±0.43jn 20.40±0.55jk

SP62W 17.97±0.35hi 25.17±0.44fh 24.17±0.60fg

SM73 22.13±0.65bc 26.83±0.73de 27.67±0.44ce

SST1S3 16.77±0.40j 22.20±0.42jl 20.60±0.50jk

WZ3S3 21.87±0.46bc 30.33±0.88b 27.80±0.44ce

SST5S2 16.80±0.62j 20.77±0.65kn 19.43±0.54k

WZR32S 19.60±0.35fg 27.83±0.44cd 29.67±0.84ab


67

4.3.5.3 Fresh biomass

Significant increase in fresh biomass of alfalfa was observed in response to

inoculation of bacterial isolate PM32Y which caused highest increase of 153% over un-

inoculated control. Bacterial isolates SFD2S2, WZ3S3, SFK1S2, SM112, WZ3S1, JM44,

MZT72, SM73, SM31, SP104Y and WZR32S were also found efficient in increasing fresh

biomass of alfalfa as the increase in fresh biomass was 148%, 140%, 120%, 112%, 97%,

67%, 63% 62%, 60%, 55% and 53%, respectively, as compared to un-inoculated control.

The bacterial isolates non-significant to un-inoculated control were SFD1S1, SST6S1,

FSD1S1, SP54, GCDS11, SST4S3, SST1S3 and SST5S2. Inoculation with SP54 and

SST5S2 caused minimum increase of 8% each as compared to un-inoculated control.

Maize also showed positive response to bacterial inoculation regarding increase in

fresh biomass in sand culture experiment. Maximum biomass as compared to un-inoculated

control was attained by maize in response to inoculation with bacterial isolate PM32Y

which increased biomass by 97% over control. Among rest of the bacterial isolates, 8 were

also found efficient in increasing biomass of maize. These effective bacterial isolates were

SFD2S2, WZ3S1, JM44, SP104Y, WZ3S3, SM73, MZT72, SFK1S2, WZR32S and

SM112 which enhanced biomass by 77%, 61%, 55%, 48%, 45%, 41%, 40%, 40%, 39%

and 39% over un-inoculated control, respectively. Out of 27, six bacterial isolates with code

name SFD9S4, SST6S1, SP54, SP112, SST4S3 and SST1S3 were found statistically non-

significant to un-inoculated control (Table 11).

Bacterial isolate PM32Y increased fresh biomass of canola most significantly which

was 80% as compared to un-inoculated control. The inoculation with bacterial isolate

SM31 caused second highest increase (69%) in fresh biomass of canola. Other effective

bacterial isolates were SFK1S2 WZ3S1, JM44, SM112, SFD2S2, WZ3S3 and MZT72

which caused 68%, 60%, 60%, 60%, 49%, 42% and 39% increase, respectively, in fresh

biomass of canola as compared to un-inoculated control (Table 11). Among 27 bacterial

isolates tested for their plant growth promoting activity regarding increase in fresh biomass

of canola, 7 bacterial isolates SFD9S4, GC23Y, SST6S1, FSD1S1, SP54, GCDS11 and

SP112 were statistically at par with un-inoculated control and minimum 5 % increase in

fresh biomass was recorded in treatment receiving SP54 inoculation.

Briefly, bacterial isolates with code name PM32Y, SFD2S2, WZ3S1, JM44,

MZT72, SM73 and WZ3S3 were effective in increasing fresh biomass of all three crops.

However, in case of alfalfa SFD5S3, SFK1S2, SM112 and SM31 had also promising effect

on fresh biomass. The SP104Y was effective in both alfalfa and maize but showed slightly


68

low performance in canola where it increased 28% more fresh biomass than un-inoculated

control.

4.3.5.4 Oven dried biomass

Significant increase in oven dried biomass of alfalfa was observed in response to

inoculation with PM32Y and SM14. Both bacterial isolates caused maximum increase

(234%) in oven dried biomass as compared to un-inoculated control. Some other bacterial

isolates also had pronounced positive effect on oven dried biomass of alfalfa such as

SFD2S2, WZ3S3, SFK1S2, SFD5S3, WZ3S1, WZR32S, SP104Y, SFD1S1, SM31,

SM112, MZT72, JM44 and SM73 caused 227%, 207%, 178%, 150%, 120%, 108%, 101%

96%, 96%, 96%, 94% , 93% and 87% increase, respectively, as compared to un-inoculated

control. While the bacterial isolates non-significant compared to un-inoculated control were

GC23Y, SST6S1, FSD1S1, SP54, GCDS11, SP112, SST4S3, SP62W, SST1S3 and

SST5S2. Minimum 7% increase in oven dried biomass as compared to un-inoculated was

the result of inoculation with bacterial isolate SST1S3.

In case of maize, maximum increase of 136% over control in oven dried biomass

was recorded in treatment receiving SFD2S2 inoculation, which was followed by 110% as

second highest increase in oven dried biomass resulted from the inoculation of PM32Y.

Other noticeable bacterial isolates were WZ3S1, WZ3S3, SFk2S2, SM73, JM44, SM112,

WZR32S and MZT72 that enhanced oven dried biomass by 71%, 64%, 54%, 52%, 50%,

49%, 43% and 41%, respectively. Bacterial isolates SFD1S1, SM14, GC23Y, SST1S3 and

SST5S2 were statistically non-significant compared to un-inoculated control regarding

increase in oven dried biomass of maize.

Similarly, response of canola to bacterial inoculation regarding increase in oven

dried biomass was promising with inoculation of PM32Y (96% increase compared to

control). Other bacterial isolates SFD2S2, JM44, SM31, WZR32S, SFD5S3, SM73,

WZ3S3, SM112 and SP104Y caused 87%, 73%, 72%, 54%, 52%, 51%, 48%, 46% and

44% increase in oven dried biomass, respectively, as compared to un-inoculated control.

Out of 27, bacterial isolates SP54, SFK1S2, GCDS11, SP112, SST4S3, SP62W, SST1S3

and SST5S2 did not yield significant increase in oven dried biomass of canola. Minimum

increase (5%) was observed with inoculation of SST5S2 as compared to un-inoculated

control.

Overall, inoculation of isolates PM32Y, SFD2S2, WZ3S1, JM44, MZT72, SM73

and WZ3S3 significantly increased oven dried biomass of three crops alfalfa, maize and

canola.


69

4.3.6 Root colonization

Root colonization assay revealed that roots of alfalfa harbored the highest number

of approximately each of 27 bacterial isolates as compared to canola and maize. Bacterial

isolate MZT72 colonized roots of alfalfa most extensively followed by PM32Y which

formed most colony forming unit of 4.56 х 106 CFU g-1 and 3.90x106 CFU g-1, respectively

(Table 13). Other bacterial isolates which colonized the root of alfalfa extensively under

axenic and controlled conditions of light and temperature were JM44, SFD2S2, SM73,

WZ3S3 and WZ3S1 which results CFU g-1 of root 2.54 х 106, 1.56 х 106, 1.27x106, 5.34

x105 and 4.83 x 105, respectively. These bacterial isolates also colonized the roots of

maize efficiently with maximum population by SM73 which formed 2.83 х 106 units per

gram of maize root. Similarly, canola roots were also colonized by these bacterial isolates

efficiently comparative to rest of bacterial isolates, however, canola roots were relatively

less colonized by these bacterial isolates when compared with root colonization of alfalfa

and maize.


70

Table 11 Effect of bacterial inoculation on fresh biomass of alfalfa, maize and canola under

axenic conditions in sand culture


Treatment Fresh biomass (g)


Control 2.49±0.06m 8.42±0.57m 7.23±0.24n

PM32Y 6.33±0.30a 16.60±0.21a 12.93±0.32a

SFD2S2 6.20±0.38a 14.88±0.13b 10.71±0.41cd

SFD5S3 5.31±0.26c 10.57±0.23g 9.54±0.29eh

WZ3S1 4.93±0.41c 13.53±0.24c 11.53±0.23bc

SFD1S1 2.91±0.11jm 10.08±0.56gi 9.07±0.12fj

JM44 4.19±0.20d 13.02±0.09cd 11.53±0.54bc

SM14 3.42±0.22fj 9.30±0.12il 9.10±0.21ej

SFD9S4 3.37±0.07fk 9.08±0.46km 8.23±0.15jn

MZT72 4.07±0.33d 11.78±0.17ef 10.15±0.43df

GC23Y 3.63±0.09dh 9.83±0.09gk 8.30±0.26in

SM31 3.99±0.11de 10.17±0.44gh 12.19±0.31ab

SST6S1 2.98±0.07im 8.55±0.19lm 7.72±0.31kn

FSD1S1 2.72±0.10lm 9.73±0.27hk 8.13±0.12jn

SFK2S2 3.50±0.07ei 9.61±0.26hk 9.07±0.30fj

SP54 2.70±0.06lm 8.75±0.15lm 7.58±0.50mn

SM112 5.29±0.09c 11.69±0.16f 11.52±0.78bc

SFK1S2 5.50±0.05bc 11.76±0.24ef 12.12±0.62ab

GCDS11 2.83±0.22km 9.75±0.24hk 7.62±0.23ln

SP112 3.14±0.16hl 8.77±0.09lm 7.66±0.39ln

SP104Y 3.87±0.30df 12.49±0.56de 9.25±0.49ej

SST4S3 2.83±0.06km 9.07±0.11km 8.73±0.31hl

SP62W 3.27±0.33gl 9.917±0.06gj 8.43±0.29hm

SM73 4.06±0.24de 11.87±0.20ef 8.83±0.44gk

SST1S3 3.04±0.11im 8.55±0.21lm 9.44±0.26eh

WZ3S3 6.01±0.11ab 12.20±0.29ef 10.20±0.29de

SST5S2 2.71±0.13 lm 9.27±0.16jl 9.36±0.71ei

WZR32S 3.82±0.06dg 11.69±0.27f 9.95±0.53dg


71

Table 12 Effect of bacterial inoculation on oven dried biomass of alfalfa, maize and canola

under axenic conditions in sand culture


Treatment Oven dried biomass


Control 1.21±0.01j 3.44±0.03p 3.43±0.09L

PM32Y 4.04±0.34a 7.22±0.06b 6.71±.25a

SFD2S2 3.96±0.33a 8.11±0.05a 6.42±0.12ab

SFD5S3 3.02±0.30cd 3.91±0.03ln 5.20±0.06cd

WZ3S1 2.66±0.35de 5.89±0.15c 4.83±0.15cf

SFD1S1 2.37±0.33ef 3.56±0.02op 4.69±0.10dg

JM44 2.34±0.32ef 5.15±0.03de 5.94±0.11b

SM14 4.03±0.34a 3.62±0.01np 4.47±0.15eh

SFD9S4 2.01±0.33fi 3.90±0.03ln 4.37±0.12fi

MZT72 2.35±0.01ef 4.85±0.07fh 4.90±0.12cf

GC23Y 1.51±0.32hj 3.56±0.02op 4.04±0.06hk

SM31 2.38±0.34ef 4.76±0.13gi 5.90±0.13b

SST6S1 1.35±0.34j 3.82±0.015lo 4.19±0.18gj

FSD1S1 1.47±0.15ij 4.03±0.06kl 4.00±0.06hk

SFK2S2 2.04±0.41fh 5.29±0.07d 4.07±0.50hk

SP54 1.35±0.07j 3.84±0.11lo 3.70±0.11jl

SM112 2.37±0.33ef 5.13±0.10df 4.99±0.43ce

SFK1S2 3.36±0.57bc 4.22±0.04jk 3.76±0.31jl

GCDS11 1.35±0.10j 3.87±0.07ln 3.64±0.14jl

SP112 1.71±0.16gj 4.04±0.32kl 3.89±0.16il

SP104Y 2.43±0.30ef 4.61±0.23hi 4.95±0.13ce

SST4S3 1.40±0.28j 3.96±0.07km 3.94±0.26hl

SP62W 1.53±0.29hj 4.50±0.06ij 3.93±0.06 hl

SM73 2.26±0.30eg 5.23±0.09d 5.16±0.14cd

SST1S3 1.30±0.01j 3.62±0.07np 3.93±0.20hl

WZ3S3 3.72±0.30ab 5.65±0.07c 5.09±0.10cd

SST5S2 1.60±0.16hj 3.70±0.02mp 3.60±0.28kl

WZR32S 2.52±0.05df 4.92±0.04eg 5.27±0.09 c


72

Table 13 Root colonization of alfalfa, maize and canola

Bacterial Isolates CFU g-1 of root (growth pouch assay)

Canola Alfalfa Maize

PM32Y 2.89 х 105 3.90x106 3.45 х 105

SFD2S2 2.34 х 104 1.56 х 106 4.12 х 105

SFD5S3 2.13 х 103 3.56 х 104 2.30 х 103

WZ3S1 3.67 х 104 4.83 х 105 3.60 х 105

SFD1S1 2.89 х 102 1.93 х 103 2.70 х 104

JM44 4.56 х 105 2.54 х 106 3.30 х 105

SM14 2.67 х 102 1.70 х 103 3.33 х 102

SFD9S4 3.45 х 103 2.62 х 103 2.90 х 102

MZT72 3.81 х 104 4.56 х 106 3.23 х 105

GC23Y 4.72 х 103 2.46 х 104 3.39 х 104

SM31 2.91 х 104 4.35 х 105 3.67 х 104

SST6S1 1.73 х 102 2.36 х 103 1.23 х 104

FSD1S1 2.69 х 103 2.56 х 104 2.78 х 103

SFK2S2 1.82 х 103 7.63 х 104 2.43 х 104

SP54 6.70 х 102 3.34 х 103 6.34 х 103

SM112 3.55 х 103 4.60 х 105 4.50 х 104

SFK1S2 3.25 х 104 3.79 х 104 3.40 х 103

GCDS11 1.23 х 102 2.38 х 105 4.36 х 102

SP112 5.12 х 102 2.36 х 103 2.13 х 104

SP104Y 4.56 х 103 6.30 х 104 3.67 х 104

SST4S3 2.44 х 102 2.67 х 103 6.89 х 102

SP62W 3.41 х 103 4.78 х 102 3.76 х 103

SM73 5.61 х 105 1.27 х 106 2.83 х 106

SST1S3 2.13 х 102 3.23 х 104 8.20 х 102

WZ3S3 3.82 х 104 5.34 х 105 3.45 х 105

SST5S2 1.22 х 102 2.67 х 102 4.78 х 103

WZR32S 3.47 х 104 3.82 х 104 6.56 х 104


73

Table 14 Summary table of isolates selected for further study

Isolates

Bioremediation assay ACC metabolism assay ACC deaminase activity Root colonization

Medium High Optical density> 0.75 (α-ketobutyrate nmol g-1

biomass hr-1)


PM32Y +++ 1.23 1334.15 3.90 x106 3.45 х 105 2.89 х 105

SFD2S2 +++ 1.14 1027.28 1.56 х 106 4.12 х 105 2.34 х 104

SFD5S3 ++ 1.2 377.77 3.56 х 104 2.30 х 103 2.13 х 103

WZ3S1 +++ 1.1 268.76 4.83 х 105 3.60 х 105 3.67 х 104

SFD1S1 ++ 0.97 251.88 1.93 х 103 2.70 х 104 2.89 х 102

JM44 +++ 0.97 212.08 2.54 х 106 3.30 х 105 4.56 х 105

SM14 ++ 0.97 211.34 1.70 х 103 3.33 х 102 2.67 х 102

SFD9S4 ++ 0.92 178.22 2.62 х 103 2.90 х 102 3.45 х 103

MZT72 +++ 0.93 175.46 4.56 х 106 3.23 х 105 3.81 х 104

GC23Y ++ 0.91 172.06 2.46 х 104 3.39 х 104 4.72 х 103

SM31 ++ 0.89 163.78 4.35 х 105 3.67 х 104 2.91 х 104

SST6S1 ++ 0.89 157.95 2.36 х 103 1.23 х 104 1.73 х 102

FSD1S1 ++ 0.88 149.77 2.56 х 104 2.78 х 103 2.69 х 103

SFK2S2 ++ 0.88 143.83 7.63 х 104 2.43 х 104 1.82 х 103

SP54 ++ 0.87 139.48 3.34 х 103 6.34 х 103 6.70 х 102

SM112 ++ 0.87 138.84 4.60 х 105 4.50 х 104 3.55 х 103

SFKIS2 ++ 0.85 130.67 3.79 х 104 3.40 х 103 3.25 х 104

GCDS11 ++ 0.83 124.19 2.38 х 105 4.36 х 102 1.23 х 102

SP112 ++ 0.82 121.96 2.36 х 103 2.13 х 104 5.12 х 102

SP104Y +++ 0.82 112.51 6.30 х 104 3.67 х 104 4.56 х 103

SST4S3 ++ 0.8 111.88 2.67 х 103 6.89 х 102 2.44 х 102

SP62W ++ 0.8 98.93 4.78 х 102 3.76 х 103 3.41 х 103

SM73 +++ 0.79 96.91 1.27 х 106 2.83 х 106 5.61 х 105

SST1S3 ++ 0.78 95.00 3.23 х 104 8.20 х 102 2.13 х 102

WZ3S3 +++ 0.77 107.63 5.34 х 105 3.45 х 105 3.82 х 104

SST5S2 ++ 0.76 112.88 2.67 х 102 4.78 х 103 1.22 х 102

WZR32S ++ 0.76 96.91 3.82 х 104 6.56 х 104 3.47 х 104

The signs “+”,“++” and +++ indicate ability of bacterial isolate to oxidize PAHs was low, medium and high, respectively


74

4.3.7 Identification of bacteria

Among 27 bacterial isolates tested for their plant growth promotion activity and

root colonization with alfalfa, maize and canola, 8 bacterial isolates coded as PM23Y,

SFD2S2, WZ3S1, JM44, MZT72, SP104Y, SM73 and WZ3S3 were found best suited for

use in bioremediation trial in association with alfalfa, maize and canola. The suitability

basis were bioremediation potential for PAHs displayed in BRA, ACC-metabolism assay,

root colonization assay and plant growth promoting activity summarized in Table 13. Some

bacterial isolates such as SFK1S2, SFK2S2, SM112, WZR32S showed plant promoting

activity more than SM73, WZ3S3, JM44, MZT72 and SP104Y but these were excluded for

further study as these have either comparatively less bioremediation potential or ACC-

deaminase activity and root colonization as compared to SM73, WZ3S3, JM44, MZT72

and SP104Y (Table 13). Finally, keeping in view different traits, 8 bacterial isolates were

identified on the basis of carbon source utilization by using Biolog® identification system

and by 16S rRNA. Phylogenetic trees of the bacterial isolates are given in Fig. 13-Fig. 20.

Table 15 Identification of 8 bacterial isolates by 16S rRNA sequencing

Isolate Name Identification by Sequencing Accession No

PM32Y Bacillus subtilis strain Lk939130

SFD2S2 Bacillus Sp. Lk939131

WZ3S1 Bacillus cereus Lk939132

MZT72 Bacillus cereus Lk939133

SP104Y Bacillus Sp. Lk939134

SM73 Bacillus Sp. Lk939135

WZ3S3 Bacillus Sp. Lk939136

JM44 Bacillus Sp. Lk939137


75

Fig. 13 Phylogenetic tree of bacterium PM32Y

Fig. 14 Phylogenetic tree of bacterium SFD2S2


76

Fig. 15 Phylogenetic tree of bacterium WZ3S1

Fig. 16 Phylogenetic tree of bacterium MZT72


77

Fig. 17 Phylogenetic tree of bacterium SP104Y

Fig. 18 Phylogenetic tree of bacterium SM73


78

Fig. 19 Phylogenetic tree of bacterium WZ3S3

Fig. 20 Phylogenetic tree of bacterium JM44

JM44


79

4.4 Discussion

Among microorganisms present in terrestrial environment, bacteria are the most

abundant and of most diverse nature. Bacteria that benefit plants directly or indirectly are

called plant growth promoting bacteria. These bacteria have association with many plant

species and are present in diverse kind of environments (Glick, 2010). The initial purpose

when intention to use plant growth promoting bacteria is to find out best strain for desired

effect on the target crops (Bashan et al., 2014). Bacteria with high ACC metabolism

(OD> 0.75) were assessed for their growth promotion activity for alfalfa, maize and

canola to screen out the best strains. Among 27 bacterial isolates, PM32Y proved best

strains as this bacterial isolate was consistent with all three crops (alfalfa, maize and

canola). Later on, 16S rRNA indemnification revealed PM32Y as Bacillus subtilis. Root

colonization assay revealed that Bacillus subtilis colonized the roots of alfalfa, maize and

canola most extensively. This extensive root colonization may be the cause of enhanced

plant growth promotion. Khalid et al. (2004) observed efficient root colonization and

consequently improved metabolism of plants by Bacillus subtilis and Pseudomonas

fluorescens. Other efficient bacterial isolates were SFD2S2 (Bacillus Sp.), WZ3S1

(Bacillus cereus), JM44 (Bacillus Sp.), SFK2S2 and SM114 that improved plant growth

promotion. Genus Bacillus mostly contains endophytic species, however, not form

specific structures like nodules made by rhizobium. But most recently, Huang et al.

(2011) found nodule like structure on roots of leguminous plant Robinia pseudoacacia

resulted with inoculation of Bacillus subtilis GXIM08. One of the reasons for plant

growth promotion by identified bacillus species may be that they intrude plants roots and

made symbiotic relation with plants and consequently improved plant growth.

Metagenomic analysis carried out by Sessitsch et al. (2012) of most abundant endophytic

bacteria revealed shared mechanisms among endophytic bacteria, among these

mechanisms plant growth promoting were ACC-deaminase activity, biological nitrogen

fixation, phytohormones and volatile organic compounds production. Bacterial isolates

tested for plant growth promotion may have such traits other than ACC-deaminase

activity by which they promoted plant growth. Many researchers reported increased

growth of plants under stress free conditions by bacteria containing ACC deaminase

activity (Belimov et al., 2002 and Ghosh et al., 2003). Bacillus Sp. have been used in

bioremediation of petroleum hydrocarbons by many workers and reported considerable

potential of biodegradation of petroleum hydrocarbons. Al- Wasify and Hamed (2014)


80

conducted experiment to assess the bioremediation potential of Pseudomonas aeruginosa,

Bacillus subtilis,and Acinetobacter lwoffi individually and in mixed consortium to

degrade crude oil. These bacteria were isolated from soil and water contaminated with

petroleum hydrocarbons. The authors observed more degradation by mixed consortium of

these bacterial isolates as compared to individual bacterium, however, 77.8% and 76.7%

degradation of crude oil was observed in the experimental units receiving sole inoculation

of Pseudomonas aeruginosa and Bacillus subtilis, respectively. Bio-surfactant production

has been observed in many Bacillus Sp. especially in Bacillus subtilis which implies good

potential for bioremediation of organic pollutants such as petroleum hydrocarbons due to

its emulsifying properties (Mulligan et al., 2001). Ghazali et al., (2004) also reported

effectiveness of Bacillus strains in remediation of petroleum hydrocarbons. They used

different species from different genera such as Pseudomonas, Micrococcus and Bacillus

and concluded important role of species from genus bacillus. As they observed more

degradation of petroleum hydrocarbons (57%) by consortium dominated by bacillus

species as compared to consortium comprised of Pseudomonas and Micrococcus species.

Many species have been reported for their plant growth promotion. Plant growth

promotion mechanism include solublization of inorganic nutrients, regulation of plant

growth hormones, inhibition of plant stress hormone such as ethylene and inducing

systematic resistance to pathogens (Richardson et al., 2009). Plant growth promotion of

Bacillus sp. have been observed by Sheng et al. (2007) on maize, rape seed and tomato

grown on metal contaminated soil. The Bacillus sp. was able to produce siderophore and

indole acetic acid and increased growth of maize and tomato under metal stress

conditions. They concluded that plant growth promotion was attributed to production of

indole acetic acid and siderphore. The plant-microbe interaction for degradation of

organic pollutant has been emphasized in many studies (Ho et al., 2007; Kidd et al.,

2008). Activity of microorganisms in the rhizosphere of plant is facilitated by organic

compounds in the form of plants exudates (Phillip et al., 2008). Plants with active growth

secrete more organic nutrients in the form of root exudates which support growth of more

microbial population (Elsas et al. 2007). Moreover, bacteria face shortage of water in

petroleum hydrocarbons soils due to extreme hydrophobicity of high molecular weight

petroleum compounds and low water holding capacity of petroleum contaminated soil

resulting in their limited growth even if they use petroleum hydrocarbons as substrate.

Vegetation improves water holding capacity of soils contaminated with petroleum

hydrocarbons and thereby improve survival and growth of degrading bacteria (Gurska et


81

al., 2008). Hence selection of bacterial isolates that can degrade a given contaminants

along with plants has potential advantage for application in plant assisted bioremediation

strategy to clean up petroleum contaminated soils.

Chapter 5 Plant Growth and TPH Removal in Association with Alfalfa, Maize and Canola

82

Chapter 5

Plant Growth Performance in Petroleum Hydrocarbon Contaminated

Soil as Affected by Bacterial Inoculation and TPH Removal in

Association with Alfalfa, Maize and Canola

5.1. Introduction

One of the main considerations in establishing successful plant assisted

bioremediation is tolerance of plants to petroleum hydrocarbons (Tara et al., 2014). The

initial step in establishing plant assisted bioremediation is germination of plant seeds

followed by successful growth. And ideal plant is that which show minimal growth

suppression in response to petroleum hydrocarbon contamination (Merkl et al., 2004a).

Germination alone could not be set as tolerance parameter because there are various reports

in the literature that suggest successfully germinated plant under hydrocarbons

contamination subsequently failed to survive and produce higher biomass (Paul et al.,

2005). There are several protocols for testing the tolerance of plants in contaminated soil;

one is to rely on the literature report and other is to assess toxicity tolerance by germination

test and subsequent growth. There are abundant reports in the literature which suggest

alfalfa as tolerant crop to petroleum hydrocarbons both in germination and subsequent

growth and survival (Banks et al., 2000). There are various attributes that make alfalfa

(Medicago sativa L.) a tolerant crop such as being a perennial grass with long, dense and

fibrous root. Therefore, alfalfa has been suggested as an ideal candidate to be used in

rhizoremediation process (Villacieros et al., 2003; Fan et al., 2008). Wiltse et al. (1998)

studied 20 genotypes of alfalfa for their potential to degrade crude oil at concentration of

20 g/kg and observed reduction of 56% in one year. Graj et al. (2013) bioaugmented

alfalfa and other three crops with pre-isolated bacteria instead of indigenous bacteria to

assess the combined impact of plants and consortia of bacteria on degradation of diesel

The recorded plant growth attributes such as germination index, root shoot length and

biomass of the alfalfa was increased significantly as compared to non-inoculated control.

Literature suggests that perennial crops with thick cuticle and less number of stomata are

thought to be more tolerant than annual crops (Hutchinson and Hellebust, 1974). The

waxy cuticle present on the leaf protects the plant from dehydration and disease (Olson,

2003). Less number of stomata and cuticle layer reduces transpiration and traits related to

high water uptake may reduce bioremediation by reducing oxygen diffusion from roots


83

and root surface area available for colonization of microorganisms (Chang and

Corapcioglu, 1998).

Similarly, maize has been used by many workers for phytoremediation or plant

assisted bioremediation of hydrocarbons contaminated soil (Chaineau et al., 2000; Adam

and Duncan, 2002; Sharifi et al., 2007; Zand et al., 2010). As for as canola (Brassica

napus L.) is concerned, it has been extensively studied for reclamations of elemental

contamination, however, for petroleum hydrocarbons contamination a few reports does

exist in literature (Graj et al., 2013; and Asghar et al., 2013). Different factors such as

metrological conditions, contaminant type and concentration, genotypes even of same

crop, interacting microorganism may make the results regarding germination, tolerance,

biomass of root and shoot agreeable with the literature reports and also chances of

opposing the literature. For example, ethylene production in response to environmental or

contaminant stress surely reduces the root biomass and subsequently shoot biomass but if

the interacting microorganisms possess ACC-deaminase enzyme then this reduction in

biomass may be overcome by alleviating the stress hormone ethylene (Arshad et al.,

2007). Hence germination test and recording plant growth attributes such as root shoot

length and biomass of plants during plant assisted bioremediation is of obvious

importance. The aim of this chapter was to assess the tolerance of selected crop to

petroleum contamination, assessment of effect of inoculation of bacterial isolates with

ACC-deaminase activity and bioremediation potential on growth of crops under petroleum

stress and to observed the combined effect of bacterial isolates and plant on degradation of

petroleum hydrocarbons in controlled and ambient conditions of light and temperature.

5.2 Methodology

5.2.1. Seed viability

International Rules for Seed Testing procedure (ISTA, 1985) was applied for testing

the viability of seed. Briefly, 0.5% tetrazolium chloride was used to soak seeds to assess

dehydrogenase activity which is indicator of seed viability. The viable seed turned red and

no stain was occurred for dead seeds.

5.2.2 Germination Test

Coarse textured and 2 mm sieved sand was spiked with crude oil to attain a

concentration of 1%, 2% and 3% (w/w). Ten viable seeds of each crop were grown in

plastic container of diameter 13×6 cm where 13 cm was length of jar and 6 cm was internal

diameter. The germination test was carried out in controlled light and temperature


84

incubation room. Half strength Hoagland (Hoagland and Arnon, 1950) solution was used

for nutrition. Germination was assessed by emergence of seedling. Results were recorded as

percentage of total seeds sown.

5.3. Pot trial on artificially spiked sand

Laboratory experiment on artificially petroleum hydrocarbons spiked sand filled in

pots was conducted. Sequentially screened final 8 bacterial isolates were used alone and in

combination with alfalfa, maize and canola crop. Initially, this plant assisted

phytoremediation trial on artificially spiked sand was conducted in growth room under

controlled light and temperature and finally the same set up of experiment was exposed to

ambient light and temperature.

5.3.1 Soil analysis

5.3.1.1 Maximum water holding capacity

Sand water holding capacity was measured following the protocol of Rayment and

Higginson (1992) which is described briefly as follow. A column of 50 g of experimental

sand was developed in a funnel lined inside by a Whatman No. 1 filter paper (185 mm).

The sand was oven dried to determine the moisture content already contained in soil. A

clipped hose was attached to the lower end of funnel to avoid drainage while pouring

water in the column. Distilled water was used to fully saturate the soil in column. This

saturated sand was allowed to stand for half an hour. The gravitational water was then

allowed to drain by removing the clip from hose. Maximum water holding capacity in

percentage was calculated by measuring the difference between the volume of added

water and volume of gravitational water plus volume of water retained by filter paper and

water already present in sand.

5.3.1.2 Soil pH

A sand/water suspension of 1:5 was prepared using 20 g air-dried 2 mm sieved

sand and 100 mL of deionized water. The suspension was shaken mechanically and

allowed to settle for half an hour (Rayment and Higginson 1992). The suspension was

allowed to settle for 30 min, and pH was determined by calibrated pH meter (Kent Eil

7015).

5.3.1.3 Electrical conductivity

Electrical conductivity (EC) was determined using a 1:5 sand/water suspension

(Rayment and Higginson 1992). The suspension was prepared by weighing 10 g air dried,

sieved (1.5 mm) soil and adding 50 mL deionized water. The suspension was

mechanically shaken at 150 rpm on an orbital shaker for 1 h to dissolve the soluble salts.


85

Electrical conductivity was measured using a calibrated EC meter (Jenway 4510

Conductivity meter, UK).

5.3.1.4 Soil organic carbon

Wet oxidation-redox titration method (Tiessen and Moir, 1993) was used to

determine soil organic carbon content. Briefly, one gram of sieved air-dried sand sample

was digested in 7.5 mL of potassium dichromate digestion mixture. Digestion flasks

along with sand sample and digestion mixture were heated to150°C on hot plate for 45

min. Digestion flasks and its contents were allowed to cool down. After cooling digested

samples were transferred to 250 mL conical flasks. Prior to titration with (NH4)2SO4, 25

mL of water, 2.5 mL 85% phosphoric acid (H3PO4), and two drops of ferroin indicator

solution were added to the same conical flask. Ammonium sulphate (0.2M) was used to

titrate the content of flasks to brown color end point. The analysis was carried out three

times to ensure accuracy of results. For each analysis a sample without sand was run as

blank control. Organic carbon was calculated from the titration of ferrous ammonium

sulfate used in the back titration of unused dichromate. Organic carbon content was

calculated as a percentage carbon (w/w).

5.3.2 Spiking of sand with crude oil

Crude oil containing mixture of different petroleum products such as diesel,

gasoline and kerosene with specific gravity of 0.81% and API 45° was used to spike the

sand. Sieving through 2 mm sieve was carried to remove gravel and debris. Sand

sterilization at 120±1°C for 20 minutes was done thrice to kill the indigenous

microorganism (fungi, bacteria and actinomycetes). After sterilization sand was cooled

under laminar flow hood so that volatilization of the oil should be avoided upon spiking the

sand. Ten kg of sterilized, homogenously spiked with crude oil contamination of 10,000 mg

kg-1 (w/w) was filled in each plastic container. A period of 15 days under axenic conditions

at controlled light and temperature was allocated to the spiked pots for establishment to

avoid volatilization toxicity to seed and emerging seedling.

Seeds were inoculated by dressing with mixture of inoculum, sticky material,

sterilized peat and 10% sugar solution. Prior to inoculation of seeds, seed surface

disinfection was carried out by dipping them 95% ethanol for few seconds and 0.2%

solution of HgCl2 for two minutes followed by several washing with distilled water to

remove disinfectants (Russell et al., 1982). Inoculated seeds were sown at a depth of 2 cm

and this depth was covered with standard sand (sterilized but not contaminated) to ensure

maximum germination of seeds. In case of the un-inoculated control, the seeds were


86

dressed with the same mixture but without bacterial inoculation. Ten seeds per pot were

sown and laterally thinned to 5 plants per pot. Sterilized Hoagland solution was applied

twice a week for provision of mineral nutrition. In remaining days of the week, sterilized

distilled water was applied to maintain moisture level. Data regarding shoot length, root

length, fresh biomass and oven-dried biomass were recorded at harvesting and sand was

analyzed for remaining TPH.

5.3.3 Treatment plan

In treatment plan, control pots were contaminated with crude oil but neither plant

was grown nor bacterial inoculation applied. The control was kept to assess evaporation

losses. Another treatment (shown as Phyto in Figs. 30-41) was kept where only plants were

grown in contaminated sand but there was no inoculation. This treatment was kept to

segregate the effect of plants alone on degradation of petroleum hydrocarbons. This

treatment also fulfills the purpose of assessing plant tolerance to petroleum hydrocarbon

contamination and effect of bacterial inoculation on plant growth under petroleum stress.

Moreover, each plant was grown in non-contaminated and un-inoculated pots to ensure that

there was no environmental factor other than petroleum hydrocarbon suppressing plant

growth (Shown as NC control in Fig. 22 - Fig. 29). This treatment was called as non-

contaminated control. In all other treatments inoculated plants were grown in contaminated

sand. For assessment of potential of bacteria alone to degrade petroleum hydrocarbons,

each bacterium was inoculated in separate pot spiked with same oil concentration.

Treatments are given below:

1. Contaminated control (no vegetation & no inoculation)

2. Un-inoculated plant (Phyto)

3. Non-contaminated un-inoculated control (NC Control)

4. PM32Y+plant

5. SFD2S2+Plant

6. WZ3S1+Plant

7. JM44+Plant

8. MZT72+Plant

9. SP104Y +Plant

10. SM73 +Plant

11. WZ3S3+Plant

12. Bioremediation (Each bacterial isolate with 3 replicates)


87

5.3.4 TPH by infrared spectroscopy

Infrared spectroscopy measures energy absorbed by a molecule in electromagnetic

spectrum. Horiba-350 oil content analyzer was used to assess remaining TPH in sand

after harvest of trial. TPH oil content analyzer principally measure TPH using infrared

light. Five gram of sand was taken in china dish and five gram of sodium sulfate was

added to absorb moisture in the sand. These were mixed thoroughly and 40 mL of RDH-

CCl4 was added in mixture of sand and sodium sulfate. This mixture was shaken for 30

minutes and filtered using Whatman No.40 (11 cm). During filtration, 100 mesh size

silica was placed in funnel to absorb moisture and biogenic hydrocarbons. Filtrate was

filled in 1.5 cm cell and placed in instrument to get the reading in mg/kg. Dilutions were

made using RDH-CCl4 to bring the concentration into range of the standards and

instrument. Prior to measurement of TPH, the instrument was calibrated using the same

solvent and working standards were prepared using the same crude oil that was used for

the experiments.

5.3.5 Analysis of sand for remaining diesel on GC

After the analysis of TPH the ratio of different components of crude oil remained

in the sand were analyzed using a modified shaking extraction method (Schwab et al.,

1999) followed by GC-FID analysis. A 2 g sample of sand was mixed with 1 g of sodium

sulfate and 4 ml of 50:50 (v/v) hexane: acetone solvent, shaken for 10 min, and then

centrifuged at 1300 rev min-1 for 10 min. Supernatants were transferred to a clean vial

containing 0.5 ml toluene, evaporated without heating to 0.5 ml, transferred to a GC vial,

and brought to a final volume of 1.8 ml with toluene. Samples were analyzed on a GC-

17A Shimadzu with DB-WAX column. Helium was used as carrier gas with flow rate 30

mL/min. Detector and injector temperatures were 310°C and 250°C, respectively.

Column oven temperature was 35°C for 5 min to 310°C at 20°C /min. For calculation of

TPH concentration, internal standard was used. Standard solution was used to make TPH

concentration ranging from 1000 ppm to 4000 ppm (total 8) using internal standard. Peak

areas and peak ratios between TPH and internal standards were calculated. Between TPH

standards and peak area a standard line was built and this standard line was used to

calculate TPH concentration.

5.3.6 Statistical analysis of data.

Data of different trials were analyzed by using standard statistical procedure as described

by Steel and Torrie (1980). Completely randomized design with three replicates was used

in all studies and mean value was obtained. Duncan Multipl Range (DMR) test was used


88

to compare the means for significance. The statistix 9.1 statistical software was used to

analyze the data.

5.4. Results

5.4.1 Plant growth performance in petroleum contaminated soil under controlled


5.4.1.1 Physicochemical properties of sand

Soil used for germination test as well as for plant assisted bioremediation trials

was of coarse texture as it contained 90% sand particles with low organic carbon (0.2%).

The aim of using sand was to provide maximum bioavailability of petroleum

hydrocarbons to plants and bacterial isolates. Organic carbon may also restrict

bioavailability of petroleum hydrocarbons by the process of humification, so sand with

low organic carbon also ensured maximum bioavailability of petroleum hydrocarbons to

microorganism and plants. Drawback of using sand is limited supply of mineral nutrients

which was regulated by using Hoagland solution. The physicochemical properties of the

soil used for experiments are given below in table 15.

5.4.1.2. Germination

Viable seeds of each crop alfalfa, maize and canola were sown in sand spiked with

crude oil concentration of 0%, 0.5%, 1%, and 3% to assess germination potential of each

crop in freshly spiked petroleum contaminated sand. At 0% contamination, there was

100% germination of all three crops indicating that there was no suppression factor other

than petroleum hydrocarbon contamination. Crude oil concentration at level 0.5% did not

severely affect germination of all three crops; however, at 1% and 3% concentration

germination of canola was affected significantly as compared to maize and alfalfa.

Germination of maize and alfalfa was statistically at par with each other at all three

(0.5%, 1% and 3%) concentration. Emergence of alfalfa and maize seedling was

considerable up to 3% concentration of crude oil. Both in alfalfa and maize seeds

germination percentage was 90%. However, germination of canola was severely affected

at 3% crude oil concentration as less than 50% seed germination was observed.


89

Table 16 Physicochemical properties of soil

Parameter Value

Maximum water holding capacity 26±2.00%

Texture Sand

Sand 90±0.5%

Silt 4.5±1.00%

Clay 6.5±1.00%

EC 1.8±0.5 dS m-1

Organic Matter 0.2±0.10 %

Fig. 21 Germination percentage at different concentration of crude oil

40

50

60

70

80

90

100

110

0 0.5 1 3

% G

erm

inat

ion

% Concentration

maize alfalfa canola


90

5.4.1.3 Root length

Fig. 22 displays the increase in root length of alfalfa, maize and canola in TPH

contaminated soil over their corresponding un-inoculated control (un-inoculated plant).

Root length of all three crops was significantly increased by PM32Y as compared to un-

inoculated control. As for as root length of maize and alfalfa is concerned, PM32Y

approximately increased 85% of alfalfa and 75% root length of maize as compared to un-

inoculated control. While inoculation of PM32Y (Bacillus subtilis) caused 61% increase

in root length of canola. Other prominent bacteria in enhancing root elongation was

SFD2S2 (Bacillus Sp.) which increased root length over un-inoculated control (un-

inoculated plant) by 70%, 58% and 39% of alfalfa, maize and canola, respectively.

Bacterium JM44 identified as Bacillus sp. also prominently increased root length of alfalfa and

maize. The increase in root length of alfalfa and maize by this bacterium was 75% and 72%,

respectively. Overall, effect of bacterial inoculation on root elongation of alfalfa and maize

was more pronounced as compared to canola revealing that under controlled light and

temperature the performance of maize and alfalfa was better than canola.


Shoot elongation of alfalfa was greatly improved by inoculation with bacterial

isolate PM32Y which increased maximum shoot height among all the bacterial isolates

(Fig. 23). The PM32Y increased shoot length of alfalfa by 77% followed by an increase

of 76% caused by JM44 inoculation. Other bacterial isolates SFD2S2, WZ3S3, SM73

and WZ3S1 were also prominent and elongated shoot of alfalfa by 65%, 61% , 56% and

53%, respectively, as compared to un-inoculated control. In case of maize, bacterial

isolate PM32Y caused a maximum increase of 74% in shoot elongation. While bacterial

isolates SFD2S2 and WZ3S3 increased shoot length of maize by 43% and 31%,

respectively, as compared to un-inoculated control. Likewise root length, shoot length of

canola was not significantly improved by bacterial inoculation. The most consistent

bacterial isolate with all three crops was PM32Y which only increased shoot length of

canola by 33% as compared to un-inoculated control. Other noticeable increase of 28%

and 22% in shoot length caused by bacterial isolates WZ3S3 and SM73, respectively.


91


In growth room under controlled light and temperature conditions, alfalfa

produced maximum fresh biomass in response to PM32Y inoculation (Fig. 24). This

maximum increase in fresh biomass of alfalfa was 91% as compared to un-inoculated

control. Other efficient bacterial isolates were JM44, SFD2S2 and SM73 with 86%, 72%

and 70% increase in fresh biomass of alfalfa, respectively. Inoculation with bacterial

isolates WZ3S1, MZT72, SP104Y and WZ3S3 also increased fresh biomass significantly

as compared to un-inoculated control. Response of maize growing under TPH

contaminated soil to bacterial inoculation regarding increase in biomass was also

pronounced. The PM32Y was the best among all the bacterial isolates in increasing fresh

biomass of maize as it increased 67% over un-inoculated control (Fig. 24). Bacterial

isolate WZ3S3, SM73, JM44 and SFD2S2 also prominent as these bacterial isolates

caused more than 50% increase in fresh biomass of maize as compared to un-inoculated

control. Bacterial isolate PM32Y maintained its consistancy in plant growth promotion

with all three crops (alfalfa, maize and canola) under stressed conditions of TPH

contamination as it caused maximun increase in fresh biomass of canola like alfalfa and

maize. For canola, the PM32Y inoculation caused 58% increase in fresh biomass

followed by 55% increase resulted from the inoculation of WZ3S3 as second best

bacterial isolate with respect to increase in fresh biomass of canola.


Considerable increase in oven dried biomass of alfalfa was observed in response to

bacterial inoculation. Maximum 62% increase was observed with inoculation of bacterial

isolate PM32Y as compared to un-inoculated control followed by 38% and 32% increase in

response to inoculation of MZT72 and WZ3S3, respectively (Fig. 25). In case of maize,

maximum increase of 71% over control in oven dried biomass was recorded in treatment

receiving PM32Y inoculation which was followed by 43% as second highest increase in

oven dried biomass resulted from the inoculation of WZ3S3. Other noticeable increase in

oven dried biomass of maize under TPH contaminated soil was due to inoculation with

SM73 and MZT72 as they caused 33% and 28% increase, respectively. Canola response to

bacterial inoculation under TPH contaminated soil with respect to increase in oven dried

biomass was comparatively less as compared to maize and alfalfa. The PM32Y inoculation

was able to cause increase in oven dried biomass by 34% as compared to un-inoculated

control.


92

a.

b.

c.

Fig. 22 Effect of bacterial inoculation on root length of alfalfa, maize and canola as

compared to un-inoculated control (Phyto) under controlled conditions of light and

temperature

a

f

bbd ce

bc

e

f

de e

5

15

25

35

45

NC

Control

Phyto PM32 SFD2S2 WZ3S1 JM44 MZT72 SP104Y SM73 WZ3S3

Ro

ot

length

(cm

)

Bacterial Isolates

Alfalfa

a

g

b

cdde

bc

efe

ef ef

5

15

25

35

NC

Control


Ro

ot

length

(cm

)

Bacterial Isolates

Maize

a

g

bcd

def

e ef

c c

5

15

25

35

45

NC

Control


Ro

ot

length

(cm

)

Bacterial isolates

Canola


93

a.

b.

c.

Fig. 23 Effect of bacterial inoculation on shoot length of alfalfa, maize and canola as


temperature

a

e

abad

bd

ac

cd d bd ad

5

15

25

35

45

NC

Control


Sho

ot

length

(cm

)

Bacterial Isolates

Alfalfa

a

g

b

c

df df ef

de d

5

15

25

35

45

55

65

NC ControlPhyto PM32 SFD2S2 WZ3S1 JM44 MZT72 SP104Y SM73 WZ3S3

Sho

ot

length

(cm

)

Bacterial Isolates

Maize

a

g

bde

efde

fgef

cd bc

5

15

25

35

45

55

NC

Control


Sho

ot

length

(cm

)

Bacterial Isolates

Canola


94

a.

b.

c.

Fig. 24 Effect of bacterial inoculation on fresh biomass of alfalfa, maize and canola as


temperature

a

f

bbd

de

bc

ede

bece

0

5

10

15

20

25


Fre

sh b

iom

ass

(g)

Bacterial Isolate

Alfalfa

a

e

bc

cc

d d

c c

5

10

15

20

25

30

35

40

NC

Control


Fre

sh b

iom

ass

(g)

Bacterial Isolates

Maize

a

e

b

c c cd d

bc

b

5

10

15

20

25

30

35

NC

Control


Fre

sh b

iom

ass

(g)

Bacterial Isolates

Canola


95

a.

b.

c.

Fig. 25 Effect of bacterial inoculation on oven dried biomass of alfalfa, maize and canola

as compared to un-inoculated control (Phyto) under controlled conditions of light and

temperature

a

f

b

de efde

c

ef de

cd

0

2

4

6

8

10

12

14

NC Control Phyto PM32 SFD2S2 WZ3S1 JM44 MZT72 SP104Y SM73 WZ3S3

Oven

dri

ed b

iom

ass

(g)

Bacterial Isolates

Alfalfa

a

g

b

deeg

df ce fgcd

c

0

5

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20


Oven

dir

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bio

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s (g

)

Bacterial Isolates

Maize

a

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(g)

Bacterial Isolates

Canola


96

5.4.2 Plant growth performance in petroleum contaminated soil under ambient light

and temperature

The same set of experimental conditions regarding bacterial isolates, TPH

contamination level, crops, mineral nutrition was brought under ambient light and

temperature in wire house to assess growth response of alfalfa, maize and canola as

affected by bacterial inoculation under TPH contamination and TPH removal by bacterial

isolates under natural conditions of light and temperature in combination with alfalfa,

maize and canola. Growth attributes of alfalfa, maize and canola recorded after harvest of

the experiment were as follow.

5.4.2.1 Root length

Bacterial isolate PM32Y maintained its consistency in increasing root length of

alfalfa in wire house (ambient light and temperature) experiment as indicated by maximum

increase of 63% over un-inoculated control. Other noticeable increase in root length of

alfalfa was observed in treatments receiving inoculation of bacterial isolates WZ3S3, SM73

and WZ3S1 which caused root elongation by 53%, 46% and 41%, respectively, as

compared to un-inoculated control. Bacterial isolates SFD2S2, JM44 and MZT72 whose

performance with respect to increase in root length of alfalfa in controlled light and

temperature was considerable, failed to maintain their consistency when exposed to

fluctuating conditions of light and temperature. As for as the root elongation of maize by

the effect of bacterial inoculation in TPH contaminated soil under ambient light and

temperature is concerned, bacterial isolates showed their consistency in improving root

length of maize. Maximum increase of 77% in root length was observed in the treatment

receiving inoculation of PM32Y (Fig. 26). This maximum increase was followed by 74%

increase in root length caused by the isolate JM44. However, increase in root length by

SFD2S2, WZ3S1, MZT72, SM73 and WZ3S3 could not be neglected as these isolates

caused considerable increase of 59%, 50%, 42%, 41% and 38%, respectively, as compared

to un-inoculated control. Similarly, root length of canola was also increased by bacterial

inoculation significantly in wire house experiment. Maximum 66% increase over un-

inoculated control was observed in response to inoculation of bacterial isolate PM32Y

followed by 57% increase caused by bacterial inoculation WZ3S3. Bacterial isolate

SFD2S2 response to increase in root length of canola under ambient light and temperature

was not consistent as its performance was better under controlled conditions of light and

temperature.


97


Bacterial isolates PM32Y, WZ3S3, SM73 and WZ3S1 were prominent with respect

to increase in shoot length of alfalfa under ambient light and temperature. Inoculation with

these bacterial isolates caused 59%, 52%, 41% and 37% increase, respectively. Although

SFD2S2, WZ3S1, JM44 and MZT72 increased shoot length of alfalfa as compared to un-

inoculated control yet not to the extent as it was under controlled conditions of light and

temperature (Fig. 27). Response of maize with respect to increase in shoot length to

bacterial inoculation was significant under ambient light and temperature. The PM32Y was

most efficient as it increased maximum shoot length. Maximum 63% increase in shoot

length caused by inoculation with PM32Y which was followed by 33% increase with

inoculation of SFD2S2 and JM44. In case of canola, maximum 59% increase was observed

in treatment receiving inoculation of PM32Y. Bacterial isolate WZ3S3 increased 51%

shoot length of canola as second highest increase. Interesting thing to note was that the

growth performance of canola in TPH contaminated soil under ambient light and

temperature conditions was better than that under controlled conditions of light and

temperature.


Maximum 67% increase in fresh biomass of alfalfa was noticed in response to

inoculation of bacterial isolate PM32Y as compared to un-inoculated control. Bacterial

isolates WZ3S3, SM73 and WZ3S1 were also prominent and caused 58%, 44% and 39%

increase in fresh biomass of alfalfa as compared to un-inoculated control (Fig. 28).

Bacterial isolate JM44 lost its efficiency in increasing biomass of alfalfa under ambient

light and temperature. Maize also showed positive response to bacterial inoculation as

maximum 64% increase in fresh biomass as compared to un-inoculated control was

observed in response to inoculation of bacterial isolate PM32Y. Among rest of the bacterial

isolates, WZ3S3, JM44, WZ3S1 and SM73 were also found efficient in enhancing biomass

of maize in TPH contaminated soil. Fresh biomass of canola was maximally increased with

inoculation of bacterial isolate PM32Y which caused 66% increase as compared to un-

inoculated control. The bacterial isolate WZ3S3, WZ3S1 and SM73 were also efficient

because they caused 57%, 49% and 44% increase in fresh biomass of canola, respectively,

as compared to un-inoculated control.


Inoculation with PM32Y most significantly improved oven dried biomass of alfalfa

under ambient light and temperature. This increase was 63% over un-inoculated control


98

(Fig. 29). Other bacterial isolate which effectively increased oven dried biomass of alfalfa

were WZ3S3 and SM73 with 51% and 32% increase, respectively. Bacterial isolate

PM32Y increased 93% oven dried biomass as compared to un-inoculated control in case of

maize which was followed by 61% increase as compared to un-inoculated control caused

by inoculation with WZ3S3. Bacterial isolates SM73, MZT72 and SFD2S2 were also

effective in increasing oven dried biomass of maize as they caused 51%, 43% and 42%

increase, respectively, over un-inoculated control. Canola also showed great response to

bacterial inoculation as out of 8 bacterial isolates, 4 bacterial isolates PM32Y, WZ3S3,

SM73 and SFD2S2 significantly increased oven dried biomass of canola as compared to

un-inoculated control. These bacterial isolates caused 85%, 63%, 63% and 43% increase,

respectively.


99

a.

b.

c.

Fig. 26 Effect of bacterial inoculation on root length of alfalfa, maize and canola as

compared to un-inoculated control (Phyto) under ambient conditions of light and

temperature

a

h

b

ede

fg gh

cdc

0

10

20

30

40

50

60

NC

Control


Ro

ot

length

(cm

)

Bacterial Isolates

Alfalfa

a

g

bcd

d

bc

eff

ef ef

5

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15

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NC

Control


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(cm

)

Bacterial Isolates

Maize

a

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c

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10

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30

40

50

60

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NC

Control


Ro

ot

length

(cm

)

Bacterial Isolates

Canola


100

a.

b.

c.

Fig. 27 Effect of bacterial inoculation on shoot length of alfalfa, maize and canola as


temperature

a

g

ab

ed

ef ef

cdbc

5

10

15

20

25

30

35

40

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50


Shoot

length

(cm

)

Bacterial Isolates

Alfalfa

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Maize

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Bacterial Isolates

Canola


101

a.

b.

c.



temperature

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dc

de de e

cb

5

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40


Fre

sh b

iom

ass

(g)

Bacterial Isolates

Alfalfa

a

e

b c c c

d dc

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0

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NC

Control


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sh b

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ass

(g)

Bacterial Isolates

Maize

a

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ecd

f eff

dbc

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Fre

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(g)

Bacterial Isolates

Canola


102

a.

b.

c.



temperature

a

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b

de cd bdef f

bcbc

0

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Oven

dri

ed b

iom

ass

(g)

Bacterial Isolates

Alfalfa

a

g

b

deeg

de cffg

cdc

0

5

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25


Oven

dri

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iom

ass

(g)

Bacterial Isolates

Maize

a

f

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Oven

dri

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(g)

Bacterial Isolates

Canola


103

5.4.3 TPH removal by bacteria in association with alfalfa, maize and canola under


5.4.3.1 TPH removal in association with alfalfa

Plant assisted bioremediation of petroleum hydrocarbons by bacterial isolates in

association with alfalfa was assessed in growth room on artificially spiked soil. Results

(Fig. 30) revealed that TPH removal by bacterial isolate PM32Y was maximum (46%) as

compared to control (contaminated un-inoculated non-vegetated) while 32% more than

alfalfa alone (Fig. 30). Alfalfa without any bacterial inoculation reduced TPH

contamination by 19% when compared with control (contaminated un-inoculated non-

vegetated). Other efficient isolates were WZ3S1, JM44, SM73 and WZ3S3 which were

statistically at par with the most efficient strain PM32Y.

5.4.3.2 TPH removal in association with canola

Efficiency of TPH removal by canola alone was negligible as it dissipated only

7% more TPH than control. However, bacterial inoculation and canola synergism was

effective in degradation of TPH, as 37% removal was observed in the experimental unit

receiving PM32Y bacterial inoculation in combination with canola as compared to

control. While compared to canola alone, the combination of canola and PM32Y bacterial

isolate was 33% more efficient in the removal of TPH (Fig. 31). Other efficient bacterial

isolates were WZ3S3, WZ3S1, SM73, JM44 and SP104Y which in combination with

canola degraded petroleum hydrocarbon by 34%, 30%, 29%, 27% and 25% as compared

to control (un-inoculated non-vegetated). While compared to canola alone the increased

removal of TPH by the combined effect of canola and these bacterial isolate were 30%,

25%, 24%, 22% and 20%, respectively.

5.4.3.3 TPH removal in association with maize

Maize facilitated the degradation of petroleum hydrocarbons under controlled

conditions of light and temperature. Maximum 43% TPH removal as compared to control

was observed by combined effect of PM32Y and maize. Efficiency of maize alone for

TPH removal was not impressive as it caused 12% removal as compared to control. This

can also be noticed by comparing with maize alone, and by combined effect of maize and

PM32Y, the later one caused TPH removal 35% more than the former one (Fig. 33). The

bacterial isolate PM32Y was most efficient; however, efficiency of other bacterial isolates

such as WZ3S1 and WZ3S3 cannot be neglected as these bacteria in association with

maize bioremediated petroleum hydrocarbons by 37% and 36% as compared to control

and 28% and 27% as compared to un-inoculated maize.


104

Summarizing the results of plant assisted bioremediation under controlled

conditions of light and temperature; it was observed that among crops alfalfa was the best

with respect to removal of TPH contamination when crops were un-inoculated. Among 8

bacterial isolates, most efficient and consistent with all three crops was PM32Y which

considerably degraded petroleum hydrocarbons. Other consistent bacterial isolates were

WZ3S1, WZ3S3, SM73, as these also degraded petroleum hydrocarbons at meaningful

rate. The bacterial isolates MZT72, SFD2S2 and SP104Y comparatively caused less

degradation of petroleum hydrocarbons, however, the degradation by these bacteria in

association with crops was more than the crops alone. Bacterial isolate JM44 degraded

petroleum hydrocarbons more efficiently in the rhizosphere of alfalfa and canola but in

combination with maize the TPH removal was comparatively less.


105

Fig. 30 TPH removal by bacterial isolates in association with alfalfa under controlled


Fig. 31 TPH removal by bacterial isolates in association with canola under controlled


Fig. 32 TPH removal by bacterial isolates in association with maize under controlled


a

b

e

bdde de

bccd de

de

0

2

4

6

8

10

Control Phyto PM32Y SFD2S2 WZ3S1 JM44 MZT72 SP104Y SM73 WZ3S3

TP

H R

emai

nin

g (

g k

g-1

)

Bacterial Isolates

Alfalfa

a b

d

bccd cd

bccd cd

d

0

2

4

6

8

10


TP

H R

emai

nin

g (

g k

g-1

)

Bacterial Isolates

Canola

a

b

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cdde

bc cd bc

cd de

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TP

H R

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g (

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Bacterial Isolates

Maize


106

5.4.4. TPH removal by bacteria in association with alfalfa, maize and canola under

ambient light and temperature

5.4.4.1 TPH removal in association with alfalfa

Alfalfa when grown under ambient light and temperature, removal of petroleum

hydrocarbons was relatively less as compared to controlled conditions of light and

temperature. Alfalfa alone reduced 14% TPH under ambient light and temperature (Fig.

33) while under controlled conditions of light and temperature the degradation was 19%

as compared to un-inoculated control. The bacterial isolate PM32Y in combination with

alfalfa degraded most efficiently among all the treatments. This maximum degradation

was 45% more as compared to un-inoculated non-vegetated control while 35% as

compared to alfalfa alone. Bacterial isolates WZ3S1 and WZ3S3 also degraded petroleum

hydrocarbons prominently by 38% and 37%, respectively, as compared to un-inoculated

control and 28% and 27% compared to alfalfa alone. Bacterial isolate JM44 which

efficiently degraded petroleum hydrocarbons in the rhizosphere of alfalfa under

controlled conditions of light and temperature was less efficient when exposed to ambient

light and temperature. Minimum reduction of 22% in TPH content was observed in the

treatment receiving bacterial inoculation SP104Y, however, this decrease was 9% more

as compared to alfalfa alone.

5.4.4.2 TPH removal in association with canola

Canola facilitated the degradation of petroleum hydrocarbons under ambient light

and temperature comparatively better as compared to that of under controlled conditions

of light and temperature. Maximum TPH removal as compared to control was observed

by combined effect of PM32Y and canola which resulted into 41% TPH removal (Fig.

34). Efficiency of canola alone for TPH removal was not impressive as it caused only 9%

removal as compared to control. Compared to canola alone, maximum reduction of 41%

in TPH by combined action of PM32Y and canola was observed. Other noticeable

reductions in TPH were 34% and 31% caused by WZ3S3 and WZ3S1 bacterial isolates in

combination with canola.

5.4.4.3 TPH removal in association with maize

Bacterial isolates in the rhizosphere of maize under ambient conditions of light

and temperature degraded petroleum hydrocarbons efficiently as compared to maize

alone. Maize alone only reduced 13% TPH contamination while 42% reduction was

observed by the combined action of PM32Y and maize compared to un-inoculated

control. This reduction was 33% more as compared to maize alone (Fig. 35). Among rest


107

of the bacterial strains, WZ3S1 and WZ3S3 in association with maize were also efficient

as they reduced TPH content by 37% and 36% more as compared to un-inoculated

control. Likewise in the rhizosphere of canola, bacterial isolates SP104Y also reduced

minimum TPH in association with maize. This minimum reduction was 21% more than

un-inoculated control and 9% more than maize alone.

Summarizing the results of plant assisted bioremediation under ambient light and

temperature; it is evident that PM32Y was most efficient bacterial strain in association

with all three crops to degrade petroleum hydrocarbons (Fig .33; 34 and 35). Bacterial

isolates WZ3S1 and WZ3S3 were also consistent with all three crops.


108

Fig. 33 TPH removal by bacterial isolates in association with alfalfa under ambient light

and temperature

Fig. 34 TPH removal by bacterial isolates in association with canola under ambient light

and temperature

Fig. 35 TPH removal by bacterial isolates in association with maize under ambient light

and temperature

a

b

f

cdef

bc cdbc

d de

0

2

4

6

8

10

Control Phyto PM32YSFD2S2 WZ3S1 JM44 MZT72 SP104Y SM73 WZ3S3

TP

H R

emai

nin

g (

g k

g-1

)

Bacterial Isolates

Alfalfa

aab

g

bcef

ce df cddf fg

0

2

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TP

H R

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nin

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Canola

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TP

H R

emain

ing (

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g-1

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Bacterial Isolates

Maize


109

5.4.5.1 TPH removal by bacterial isolates independent of plants association under


Bacterial isolates independent of plant association were also evaluated for

bioremediation of petroleum hydrocarbons both under controlled and natural conditions

of light and temperature. All other agronomic practices were kept same as these were in

biophytoremediation trials (bacterial isolates in convergence with plants). As bacterial

isolates were obtained from soils having previous history of contamination with

petroleum products, therefore bacterial isolates alone also degraded petroleum

hydrocarbons considerably. As indicated in Fig. 36, bacterial isolate PM32Y removed

TPH most efficiently among all the bacterial isolates. Compared to un-inoculated control

bacterial isolate PM32Y caused 33% reduction in TPH followed by 29%, 28% and 27%

caused by WZ3S1, WZ3S3 and SM73, respectively. The least reduction in TPH was

observed in the treatment receiving SFD2S2 bacterial inoculation.

5.4.5.2 TPH removal by bacterial isolates independent of plants association under


The bacterial isolates efficiently degraded petroleum hydrocarbons in growth

room (controlled conditions of light and temperature) were also found consistent upon

exposure to natural conditions of light and temperature. The bacterial isolate PM32Y

caused 31% reduction in TPH while statistically non-significant to PM32Y were WZ3S3,

WZ3S1 and SM73 which caused 30%, 27% and 26% reduction in TPH of contaminated

soil (Fig. 37). The bacterial isolate SFD2S2 was found the least efficient among all

bacterial isolates with respect to degradation of petroleum hydrocarbons. Bacterial isolate

JM44 however failed to maintain its consistency upon exposure to natural conditions of

light and temperature. As bacterium JM44 degraded 20% petroleum hydrocarbons in

growth room under controlled conditions of light and temperature while under natural

conditions of light and temperature 14% TPH reduction was observed.


110

Fig. 36 TPH removal by bacterial isolates independent of plants association under


Fig. 37 TPH removal by bacterial isolates independent of plants association under


a

g

b

fd

ce

ff

0

2

4

6

8

10

Control PM32Y SFD2S2 WZ3S1 JM44 MZT72 SP104Y SM73 WZ3S3

TP

H R

emai

nin

g (

g k

g-1

)

Bacterial Isolates

Growth room

a

e

b

de

bc bccd

dee

0

2

4

6

8

10

Control PM32Y SFD2S2 WZ3S1 JM44 MZT72 SP104Y SM73 WZ3S3

TP

H R

emai

nin

g (

g k

g-1

)

Bacterial Isolates

Wire house


111

5.4.6 Plant assisted bioremediation of diesel under controlled conditions of light and

temperature

Crude oil used for the study comprised of 36% diesel, 34% gasoline and 20% heavy

naphtha (boiling point between 90°C and 200°C with carbon rang C6 to C12). So, the

main constituents of crude oil were diesel, gasoline and naphtha. Gasoline and naphtha

being short chain hydrocarbons and containing mono-aromatics were supposed to be

degraded completely and easily by the bacterial isolates as bacteria were isolated from

petroleum contaminated soils and isolated through selective salt mineral media with

petroleum hydrocarbons as sole source of carbon. However, diesel range hydrocarbons

being long chain and containing 30-40% PAHs are considered recalcitrant. That is why

for more accurate assessment, GC analysis for assessing the degradation of diesel range

hydrocarbons (C10-C16) were carried out. Being complex mixture of hydrocarbons, TPH

measurement is used as analytical technique in most of the environmental assessment

(Adam and Duncan, 1999). Mechanical shaking extraction instead of Soxhlet extraction

proposed by Schwab et al. (1999) was followed for the extraction of oil from soil. For

calculation of TPH concentration, internal standard was used. Standard solution was used

to make TPH concentration ranging from 1000 ppm to 4000 ppm (total 8) using internal

standard (Supelco, Bellefonte, PA). Peak areas and peak ratios between TPH and internal

standards were calculated. Between TPH standards and peak area a standard line was

built and this standard line was used to calculate TPH concentration.

5.4.6.1 Bioremediation of diesel in association with alfalfa

Alfalfa facilitated bioremediation by bacterial isolates most significantly among

the crops. The most efficient bacterial isolate PM32Y degraded diesel range hydrocarbon

71% in association with alfalfa. This degradation was 69% more than that caused by

alfalfa alone (Phyto control). Bacterial isolates WZ3S3, SM73 and WZ3S1 were also

efficient in degrading diesel range hydrocarbons in association with alfalfa under

controlled conditions of light and temperature. These bacterial isolates degraded 58%,

53% and 48%, respectively in association with alfalfa as compared to un-inoculated

control (Fig. 38).

5.4.6.2 Bioremediation of diesel in association with canola

Maximum degradation of diesel range hydrocarbons degradation in association

with canola was observed by the bacterial isolate PM32Y which caused reduction of 57%

as compared to un-inoculated control and 55% more than the canola alone (Phyto

control). This maximum decrease was followed by 48% and 42% resulted by the


112

combined action of canola and bacterial isolates WZ3S3 and WZ3S,1 respectively (Fig.

39). Bacterial isolate SM73 with 38% reduction was also prominent among rest of the

bacterial isolates.

5.4.6.3 Bioremediation of diesel in association with maize

Among three crops maize was the most efficient in degradation of diesel range

hydrocarbons as maize degraded 7% while alfalfa and canola reduced 6% and 5%,

respectively as compared to control (un-inoculated and non-vegetated). Bacterial isolates

however degraded efficiently in combination with all three crops. Likewise in

combination with alfalfa and canola, bacterial isolate PM32Y in combination with maize

also degraded diesel range hydrocarbons maximally as compared to other combinations

and maize alone. This maximum increase was 66% more compared to un-inoculated

control and to maize alone it was 64% more (Fig. 40). Other prominent increase in

dissipation of diesel range hydrocarbons was noticed in the treatments receiving

inoculation of WZ3S3, SM73 and WZ3S1 which caused 52%, 50% and 46% increased

degradation as compared to un-inoculated control.

Summarizing the results, it was observed that the bacterial isolates PM32Y,

WZ3S3, WZ3S1 and SM73 degraded proficiently in association with all three crops

alfalfa, canola and maize. Bacterial isolate JM44 performed better in the rhizosphere of

alfalfa and maize, however, its efficiency in degrading diesel range hydrocarbons was

relatively less when combined with canola.


113

Fig. 38 Bioremediation of diesel in association with alfalfa under controlled conditions

of light and temperature

Fig. 39 Bioremediation of diesel in association with canola under controlled conditions of


Fig. 40 Bioremediation of diesel in association with maize under controlled conditions of


AB

H

C

FE

DC

FGG

0

500

1000

1500

2000

2500

3000

3500

4000


Die

sel re

mai

nin

g (

mg k

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)

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D

EG

0

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1000

1500

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114

5.4.7 Plant assisted bioremediation of diesel under ambient conditions of light and

temperature

5.4.7.1 Bioremediation of diesel in association with alfalfa

Under ambient light and temperature degradation of diesel range hydrocarbons

slightly reduced as compared to that under controlled conditions of light and temperature.

The most effective bacterial isolate PM32Y increased degradation of diesel range

hydrocarbon by 66% in association with alfalfa over un-inoculated control. This

degradation was 60% more than that caused by alfalfa alone (Fig. 41). Bacterial isolates

WZ3S1, WZ3S3 and SM73 maintained their efficiency of bioremediation of long chain

molecules of diesel range hydrocarbon like under controlled conditions of light and

temperature. These bacterial isolates degraded 58%, 57% and 44%, respectively, as

compared to un-inoculated control.

5.4.7.2 Bioremediation of diesel in association with canola

In the rhizosphere of canola under natural conditions of light and temperature,

maximum degradation of diesel range hydrocarbons was observed by the bacterial isolate

PM32Y which caused 60% reduction as compared to un-inoculated control and 57%

more than the canola alone. This maximum decrease was followed by 50%, 47%, 37%

and 36% resulted by the combined action of canola and bacterial isolates WZ3S3, WZ3S,

SM73 and SP104Y, respectively (Fig. 42).

5.4.7.3 Bioremediation of diesel in association with maize

Under natural conditions of light and temperature maize plant alone was able to

remove diesel range hydrocarbons by 8% as compared to un-inoculated control (Fig. 43).

Bacterial isolate PM32Y in combination with maize degraded diesel range hydrocarbons

maximally as compared to un-inoculated control (63%) and maize alone (60%). Other

prominent increase in dissipation of diesel range hydrocarbons was noticed in the

treatments receiving inoculation of WZ3S3 and WZ3S1 which caused 53% and 52%

degradation as compared to un-inoculated control.

Overall, similar response of bacterial isolates was observed as under controlled conditions

of light and temperature. Bacterial isolates PM32Y, WZ3S1, WZ3S3 and SM73 were

found effective in combination with all three crops both under controlled and natural

conditions of light and temperature.


115

Fig. 41 Bioremediation of diesel in association with alfalfa under ambient conditions of


Fig. 42 Bioremediation of diesel in association with canola under ambient conditions of


Fig. 43 Bioremediation of diesel in association with maize under ambient conditions of


AB

G

C

F

DCD

D

E

F

0

500

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3000

3500

4000


Die

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)

A B

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F

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116

5.4.8. Effect of root biomass in enhancing bioremediation of petroleum

hydrocarbons

Plant roots play an important role in degrading petroleum hydrocarbon because

these provide oxygen to bacteria. Principally, the reductions of petroleum hydrocarbons

occur by terminal oxidation of alkane chains by monooxygenase and aromatics by

dioxygenase enzyme. In both cases, the provision of molecular oxygen is necessary and

plant roots are the factory of oxygen. Moreover, plant roots provide mineral nutrition

through sloughed off cells and containment of petroleum hydrocarbons through lipid

content of roots as petroleum hydrocarbons are highly lipophilic compounds (Banks, 2000).

Correlation between root dry biomass of alfalfa, maize and canola under both controlled

and natural conditions of light and temperature and reduction in content of TPH was

observed. Amazingly, a strong correlation of r= 0.87 and r= 0.83 under controlled

conditions (growth room) and natural conditions (wire house), respectively was observed

between root dry biomass of alfalfa and reduction of TPH (Fig. 44). In case of maize, also

strong correlation was observed between degradation of petroleum hydrocarbons and root

dry biomass of maize (Fig. 45) and for canola similar trend was observed (Fig. 46).

Efficiency of crop in facilitating the bioremediation of petroleum hydrocarbons can also be

correlated with the root dry biomass. It was observed that the crop which produced more

root biomass facilitated more biodegradation as compared to the crop which relatively

produced less root biomass as indicated in Figs. 44; 45 and 46. Alfalfa produced highest

biomass among the crops and thereby minimum TPH remaining was observed in alfalfa

treated experimental units, whether alone or in combination with bacterial isolates.

Comparing crops efficiency in controlled conditions of light and temperature and in natural

conditions of light and temperature, it was observed that alfalfa and maize produced more

root biomass under controlled conditions and consequently more biodegradation of TPH

was observed while canola produced more root biomass under natural conditions of light

and temperature and hence the more degradations of TPH was observed as compared to

controlled conditions.


117

Fig. 44 Effect of root dry biomass of alfalfa on bioremediation of petroleum

hydrocarbons under controlled and natural conditions of light and temperature

6.56.05.55.04.54.0

7.5

7.0

6.5

6.0

5.5

5.0

Root Biomass (g)

TP

H R

emai

ning

(g/

kg)

WZ3S3

SM73

SP104Y

MZT72

JM44

WZ3S1

SFD2S2

PM32Y

Phyto

Growth Room

11109876

8.0

7.5

7.0

6.5

6.0

5.5

5.0

Root Biomass (g)

TP

H R

emai

ning

(g/

kg)

WZ3S3

SM73

SP104Y

MZT72

JM44

WZ3S1

SFD2S2

PM32Y

Phyto

Wire House

r= 0.83

r= 0.87


118

Fig. 45 Effect of root dry biomass of maize on bioremediation of petroleum hydrocarbons

under controlled and natural conditions of light and temperature

3.253.002.752.502.252.00

8.0

7.5

7.0

6.5

6.0

5.5

5.0

Root Biomass (g)

TP

H R

emai

ning

(g/

kg)

WZ3S3

SM73

SP104Y

MZT72

JM44

WZ3S1

SFD2S2

PM32Y

Phyto

Growth Room

7.06.56.05.55.0

8.5

8.0

7.5

7.0

6.5

6.0

5.5

Root Biomass (g)

TP

H R

em

ain

ing (

g/k

g)

WZ3S3

SM73

SP104Y

MZT72

JM44

WZ3S1

SFD2S2

PM32Y

Phyto

Wire House

r= 0.89

r= 0.80


119

Fig. 46 Effect of root dry biomass of canola on bioremediation of petroleum

hydrocarbons under controlled and natural conditions of light and temperature

2.752.502.252.001.751.50

8.5

8.0

7.5

7.0

6.5

6.0

Root Biomass (g)

TP

H R

em

ain

ing (

g/k

g)

WZ3S3

SM73

SP104Y

MZT72

JM44WZ3S1

SFD2S2

PM32Y

Phyto

Growth Room

6.56.05.55.04.5

8.5

8.0

7.5

7.0

6.5

6.0

5.5

Root Biomass (g)

TP

H R

em

ain

ing (

g/k

g)

WZ3S3

SM73

SP104Y

MZT72

JM44

WZ3S1

SFD2S2

PM32Y

Phyto

Wire House

r= 0.91

r= 0.81


120

5.5 Discussion

Plant assisted bioremediation of petroleum hydrocarbons has become the most

attractive technology because of its cost effectiveness and eco-friendliness (Kukla et al.,

2014). This technique can be most effective only if associated microorganisms have

positive activity with plants (Li et al., 2012). Bacterial isolates with significant growth

promoting activity shown under stress free axenic conditions, high bioremediation

capability and high ACC-deaminase activity were used alone and in combination with

alfalfa, maize and canola for removal of TPH. Plant growth was significantly improved as

compared to un-inoculated control of each plant revealing that the petroleum

contamination suppressed plant growth both under natural and controlled conditions of

light and temperature. This suppression in plant growth may be overcome in inoculated

plants due to alleviation or reducing of stress ethylene. Hong et al. (2011) inoculated

maize (Zea mays L.) with bacteria possessing ACC-deaminase activity and observed

significant improvement in growth attributes of maize such as stem, number of leaves,

fresh biomass and root biomass as compared to un-inoculated control. Authors concluded

that improved plant growth was due to the effect of inoculation with bacteria containing

ACC-deaminase activity and resultantly increased biodegradation of diesel. Similarly,

Bisht et al. (2014) studied the plant assisted bioremediation of PAHs by Bacillus sp.

SBER3 possessing IAA, siderophore production and ACC-deaminase activity and

observed 83% reduction of PAHs and authors found increased shoot length, root length

and biomass of inoculated plants. Another reason for improved plant growth may be that

bacterial isolates may have degraded petroleum hydrocarbons and thereby reduced

concentration of growth suppressing factor (petroleum hydrocarbons) as compared to un-

inoculated plant. Graj et al. (2014) observed correlation of germination index and plant

growth of alfalfa and Brassica napus with increasing degree of diesel concentration.

Increased degradation of petroleum hydrocarbons by combined effect of bacterial isolates

and plants may be due to increased biomass of plants especially of roots as roots facilitate

microorganisms in many ways. Petroleum hydrocarbon reduction by plants is principally

facilitated by creating suitable environmental conditions for thriving of microbial

population near roots (Adam and Duncan, 1999). A study conducted by Gunther et al.

(2003) revealed 50% hydrocarbon reduction in experimental unit receiving inorganic

nutrients and no vegetation as compared to treatment where vegetation was present. This

indicates that nutrients were limited in absence of vegetation and thus confirmed the fact


121

that amendment without plants may not be sufficient to reduce petroleum hydrocarbons at

considerable rate. Although plants have potential to remediation organic pollutants by

excreting some enzymes, face toxicity when pollutants are above certain level and

consequently low remediation occurs (Peng et al., 2009; Germaine et al., 2009). The

degradation of contaminants can be enhanced by the increase in plant biomass which can

be possible by the application of plant growth promoting bacteria. Plant biomass under

stressed condition is enhanced by bacteria through detoxification of contaminants via

direct mineralization of organic compounds (Escalante-Espinosa et al., 2005) and

reducing plant stress hormones (Weyens et al., 2009; Glick, 2010). Organic pollutants

may be degraded in the rhizosphere by root-released plant enzymes or through

phytostimulation of microbial degradation. Traditionally, direct plant growth promotion

by plant growth promoting bacteria is attributed to production of phytohormone auxin but

since last few decades it has been known the a number of bacteria possess ACC-

deaminase activity which improve plant growth under stress conditions by reducing level

of stress hormone ethylene (Shaharoona et al., 2006). The present study is also in

agreement with the literature that ACC-deaminase containing bacteria help plants to

produce more biomass (Fig. 44-46) under stressed conditions and consequently increased

reduction of petroleum hydrocarbons.

Chapter 6 General Discussion

122

Chapter 6

General Discussion

Many approaches have been adopted to remediate petroleum hydrocarbon

contamination. Chemical and physical strategies are not encouraged due to many reasons

especially cost effectiveness. So, alternate is biological approach which has been tried by

using microorganisms such as bacteria and fungi (Pizarro et al., 2014). Eukaryotic green

plants have also been used for remediation of petroleum hydrocarbon contamination.

Both plants and microorganisms have ability to degrade petroleum hydrocarbons directly

or indirectly. However, some chemical properties of petroleum hydrocarbons such as high

molecular weight, low or no solubility in water and hydrophobicity make it difficult for

plants to uptake and translocate petroleum hydrocarbons (Hutchinson, 2003). Many

microorganisms have been reported to have metabolic pathway for the degradation of

petroleum hydrocarbons, however, bacteria also face some impedances for complete

mineralization. Since last few decades, convergence of plants and microorganism has

attracted attention due to its success in remediating at meaningful rate (Gerhardt et al.,

2009). Soils polluted with petroleum hydrocarbons have microorganisms that are

acclimated to petroleum contamination and are approximately 10% of the bacterial

population present in contaminated soil (Atlas, 1995). Similar observations have been

made by Leahy and Colwell (1990) that soils with inherent history of contaminations

have bacterial population acclimated to that specific contaminant, however, this

population may be dormant due to low bioavailability of contaminant or scarcity of

nutrients. The challenging task is to isolate these bacteria and to screen out the most

efficient among these isolated bacteria (Gargouri et al., 2014). We have isolated over

three hundred bacterial isolates from soil samples contaminated with different petroleum

products such as gasoline, kerosene, diesel and petroleum sludge collected from different

areas of Punjab, Pakistan. The isolated bacteria were assayed for their ability to remediate

PAH and the results revealed that 189 isolates out of 301 were capable of bioremediation.

The reason for such higher number of microbial isolates may be that the bacterial isolates

were present whether active or dormant because the sites from where samples have been

collected have history of petroleum contamination. Upon growing on mineral salt media

with petroleum hydrocarbon as sole source of carbon, they may have broken their

dormancy and became active. The ability to oxidize PAHs as shown in BRA may be

attributed to their acclamation to aged petroleum hydrocarbons. For example, the most


123

efficient bacterial isolates PM32Y has been isolated from soil samples contaminated with

petroleum sludge, which has been reported to contain PAHs in higher proportion and are

ubiquitous and carcinogenic pollutants (Elektorowicz and Habibi, 2005). According to

suggestions made by Glick (2010) for the enhancement of degradation of organic

contaminants in soil that the bacteria inoculated for remediation of organic contaminants

must possess twin nature of plant growth promotion as well as degrader of soil

contaminant. One indirect mechanism of plant growth promotion adopted by PGPB for

plants growing under stressed conditions is to help the plants in getting rid of stress-

induced ethylene (Shaharoona et al., 2006). Bacteria equipped with ACC-deaminase

enzyme have the ability to reduce stress-induced ethylene (Arshad et al., 2007). Bacterial

colonization around and on roots occurs due to the presence of appreciable amount of

organic molecules in the form of root exudates. These organic molecules stimulate

associated bacteria to synthesize and release indole-3-acetic acid (IAA) phytohormone.

This bacterial induced IAA undergoes different fates such as uptake by plant for growth

promotion and can transcript ACC synthase enzyme within plants and resultantly the

formation of ACC in plants (Glick, 2014). The plant synthesized ACC exuded out

through roots (Penrose et al., 2001) and bacterial isolates with ACC-deaminase activity

attached to roots play a role of sink for plant exuded ACC which was produced

endogenously in plants or due to stimulation of IAA. In a nut shell, the possibility of

ethylene production in plants and consequently the reduction of plant growth due to

abiotic and/or biotic stress reduced due to ACC-deaminase containing bacteria (Glick,

2014). Therefore, bacterial isolates were assayed for presence of ACC-deaminase

enzyme. Out of 104 ACC-deaminase containing rhizobacteria, 29 bacterial isolates

showed high bacterial ACC-deaminase activity. ACC deaminase activity possessed by

wide variety of bacterial isolates present in rhizosphere, phyllospere and in plant tissues

as endophytes. Different levels of ACC deaminase activity have been found from one

organism to another organism in the nature. Bacteria with high level of ACC deaminase

activity are found bounded non-specifically to different kind of plants (Shaharoona et al.,

2006). This non-specificity of bacteria to plants allows using bacteria for inoculation of

different crops. Therefore, bacteria which showed high ACC deaminase activity and

medium to high bioremediation activity (27 isolates) were assessed for their compatibility

with alfalfa, canola and maize under axenic conditions. All bacterial isolates showed

increase in plant growth, however, some bacterial isolates were statistically non-

significant to un-inoculated control. However, finally screened 8 bacterial isolates


124

significantly increased growth attributes of alfalfa, maize and canola. As the growth

conditions were ideal such as no pathogen or competitors due to axenic conditions and no

nutritional or light and temperature stress. It can be postulated that the plant growth

promotion by bacterial isolates may be through direct mechanism and most probably

through the production of phytohormones such as auxin, cytokinin and gibberellin or by

regulating ethylene. The indirect plant growth mechanisms by plant growth bacteria

include bio-control by producing antibiotics and antifungal metabolites and inducing

systemic resistance in plants (Bhatacharyya and Jha., 2012). Direct mechanisms of plant

growth promotion by plant growth promoting bacteria include phosphorus solublization

(Baig et al., 2012), iron availability by siderophore production (Bishnori, 2015),

phytohormone production such as auxin, cytokinin and gibberellins (Glick, 2010) and

more important and plausible mechanism of plant growth promotion in context of

phytoremediation is regulation of stress induced ethylene (Arshad et al., 2007). Lifshtiz et

al. (1987) found growth promotion of canola (rape seed) under gnotobiotic conditions by

Psuedomonos putida and similary Wang et al. (2000) also observed increase in root

length of canola by Pseduomonas flourescens that possessed ACC-deaminase activity.

Shaharoona et al. (2006) found strong positive correlation between root elongations of

maize grown under axenic conditions and bacterial ACC deaminase activity. All three

crops were tested for their ability to germinate under petroleum contamination stress at

three different concentrations (0.5%, 1% and 3%) of crude oil. Results revealed that

germination of all three crops at concentration of 0.5% crude oil was not severely

affected. However, at 3% concentration of crude oil germination of the canola was

severely affected as more than 50% seeds were unable to germinate although the sown

seeds were proved viable in seed viability test. However, germination of maize and alfalfa

was considerable even at 3% concentration of crude oil. This inhibition in germination of

seed may be attributed to presence of oil in growth medium as volatile fraction of oil has

the ability to penetrate seed coat and consequently cause the death of embryo (Merkl et

al., 2004b). Another reason for poor seed germination may be the failure of seed

imbibition due to coating of oil around seed which hindered the supply of water and

oxygen to seed. Results of the present study regarding ability of alfalfa to germinate

successfully at higher concentration of petroleum hydrocarbons agreed with the previous

studies such as Wiltse et al. (1998) observed successful germination of alfalfa at a

concentration of 5% (w/w) crude oil and Merkl et al. (2004b) agreed that leguminous

crops were easier to grow on petroleum contaminated soil.


125

Finally screened 8 bacterial isolates with dual nature of plant growth promotion

and bioremediation potential were applied to artificially spiked sand with petroleum

hydrocarbons in association with alfalfa, canola and maize under controlled conditions of

light and temperature. All bacterial isolates significantly promoted plant growth attributes

such as root length shoot length and biomass of plants. The most efficient bacterial isolate

PM32Y (Bacillus subtilis) caused increase in root length of alfalfa, maize and canola by

85, 75 and 61%, respectively as compared to un-inoculated control while under natural

conditions of light and temperature the same bacterial isolate increased root length by 63,

77 and 66% of alfalfa, maize and canola, respectively. This increase in root length of

plants may be attributed to the presence of bacterial ACC-deaminase which might

relieved the plants from ethylene stress and consequently increased root elongation.

Ghosh et al. (2003) disclosed the presence of ACC-deaminase activity in three Bacillus

sp. for the first time and observed up to 80% increase in root length of canola by these

bacterial isolates as compared to un-inoculated control. This increase in root length may

also be due to successive degradation of petroleum hydrocarbons by microorganism as

the inoculated bacteria not only had ACC-deaminase activity but also had potential to

degrade petroleum hydrocarbons. It is evident from the results yielded by inoculation of

bacterial isolates WZ3S3 and SM73 which increased growth attributes of alfalfa, maize

and canola significantly though the ACC-deaminase activity was comparatively less as

compared to the most efficient bacterial isolate PM32Y. Similar observation has been

recorded by Escalante-Espinosa et al. (2005) that microorganisms capable of mineralizing

organic compounds help plants to get adapted to organic contaminants. Development of

biomass by plants under petroleum contamination stress reflects their tolerance and

adaptation to petroleum contamination and also their capacity of photosynthesis under

contamination stress. Photosynthesis surely facilitates enhanced degradation not only by

helping plants to uptake and metabolizes low molecular weight compounds but also

through secretion of root exudate as approximately 40% of photosynthates are secreted as

root exudates (Kumar et al., 2006). These root exudates facilitate the microbial

degradation of contaminant as many root exudates are structural analogue of compounds

found in petroleum hydrocarbons (Siciliano and Germida, 1998). Therefore biomass is of

key importance when assessing plant tolerance. Bacterial inoculation significantly

improved biomass of alfalfa, maize and canola both under controlled and natural

conditions of light and temperature. Bacterial isolate PM32Y under controlled conditions

of light and temperature increased fresh biomass by 91%, 67% and 58% of alfalfa, maize


126

and canola respectively and under natural light and temperature increase in fresh biomass

of alfalfa, maize and canola by the same bacterial isolate was 66%, 67% and 64%,

respectively. Efficient bacterial isolates other than PM32Y were WZ3S3, WZ3S1 and

SM73, as they considerabley increased fresh biomass as compared to un-inoculated

control. The results are in line with work done by Afzal (2010) who observed that the

bacterial isolates capable of degrading alkanes and having ACC deaminase activity

improved fresh biomass by 12% to 44% as compared to un-inoculated control. Effect of

bacterial inoculation on fresh biomass of alfalfa, maize and canola was compared,

maximum increase in fresh biomass of alfalfa was observed and minimum increase in

fresh biomass of canola revealing that canola was most severely affected by petroleum

contamination. Being a good hyper accumulator, canola has been successfully and

extensively applied for phytoremediation of inorganic pollutants. But our study revealed

that canola was not so impressive for use in remediation program of organic contaminants

such as petroleum hydrocarbons as germination, root length, shoot length and fresh

biomass was most severely affected by petroleum contamination as compared to alfalfa

and maize. Study conducted by Besalatpour et al. (2008) on various plants to assess their

ability to tolerate and phytoremediate petroleum hydrocarbons revealed that the canola

was most sensitive regarding gain of biomass. The retardation in plant growth may be

attributed to the inherent toxicity which results in biomass reduction (Bossert and Bartha,

1985; Chaıneau et al., 1997 and Ogbo, 2009). Furthermore, noticeable observation made

regarding behavior of canola in petroleum contaminated soil was that unlike alfalfa and

maize, canola showed poor growth under controlled conditions of light and temperature

as compared to growth in natural conditions of light and temperature. Other possible

reason for reduction in canola biomass may be volatile toxicity of low molecular weight

compounds as these molecules can easily pass through cell membranes thus causing

phototoxic effect (Adam and Duncan, 2002) and in the close environment (growth room)

these volatile compounds may have affected stomatal functions and thereby

photosynthesis of canola. In the present study alfalfa showed maximum tolerance and

growth followed by maize, both under controlled and natural conditions of light and

temperature. Alfalfa has been extensively used in remediation of organic contaminants

alone or in combination with rhizospheric bacteria. The ideality of alfalfa for use in

enhanced bioremediation/rhizoremediation lies upon many characteristics such as it

supports large number of bacteria due to its highly branched root system (Kuiper et al.,

2004), high root lipid contents which help alfalfa to contain lipophilic organic


127

contaminants such as naphthalene (Schwab et al., 1998), presence of P450 as determined

by genomic DNA sequences in many plants including alfalfa and maize (Schuler, 1996),

being a leguminous crop which fix atmospheric nitrogen that is limiting factor in

remediation of petroleum hydrocarbons (Fan et al., 2008), advantage of being narrow

leaved crops make alfalfa less sensitive to contamination (Smith et al., 2006). Moreover,

higher plants such as trees are sometimes considered unsuitable for use in remediation

plan of petroleum hydrocarbons contamination especially when using in convergence

with bacteria as for long time survival of bacteria in the environment it is essential that

there should be vegetation cover on soil (Mishra et al., 2003; Gaskin, 2008). This

vegetation cover or development of plant shoot gives not only esthetical acceptance, but

also benefits degrading bacteria. Using petroleum degrading bacterial inoculants, which

have positive relationship with plants may compensate decline in plant growth due to

contamination stress (Huang et al., 2004). The bacterial isolate PM32Y (Bacillus subtilis)

not only promoted the survival of canola and alfalfa but also increased hydrocarbon

degradation. This increase in plant growth and survival of plants under contamination

stress may be related to lowering the contamination stress by degradation and also

relieving of the plants from contamination induced stress ethylene. Furthermore, Bacillus

subtilis is reported to produce volatile organic compounds (VOCs) that improve plant

growth due to regulation of auxin homeostasis (Zhang et al., 2008). The results are

consistent with Huang et al. (2004) that the combination of inoculants and plants in

contaminated systems not only improved plant survival, but also resulted in an increased

degradation.

One of the possible reasons for increased degradation of contaminant may be the

competency and acclimation to the petroleum hydrocarbon contamination as these

bacterial inoculants were isolated from petroleum contaminated soils. It agrees with the

conclusion by Petersen et al. (1996) that for successful bacterial inoculation it is

necessary that bacteria must survive in soil and have positive interaction with plant.

Alfalfa and canola were already examined in petroleum contaminated soil (Shahriari,

2007; Besalatpour, 2008), especially alfalfa has been studied extensively for

phytoremediation of petroleum or PAHs contaminated soils. Some bacterial inoculants

such as SFD2S2, JM44 performed well in improving plant growth and petroleum

degradation under controlled light and temperature but their response in ambient light and

temperature was consistent. This inconsistency in performance may be due to fluctuation

in temperature, as temperature is one the main environmental factor that affects


128

bioremediation and phytoremediation. Temperature affects microbial activity in the

environment and thereby all other processes that are carried out by micro-organisms such

as biodegradation and plant growth promoting activity. As the microorganism used in this

study were isolated from different agro-ecological zones having different range of annual

mean temperature ranging from 25○C to 45○C. Under natural conditions of light and

temperature, both factors were fluctuating and thereby affecting the activity of micro-

organisms. For instance, JM44 and SFD2S2 performed well in improving plant growth

under controlled and consistent temperature in growth chamber but failed to keep their

performance in ambient light and temperature. Temperature may affect microbial activity

indirectly by affecting the chemical and physical property of oil. According to Cerniglia

(1992), temperature affects the viscosity of oil and increases water solubility in water. In

this context, it may be one of the reasons of failure of the microorganism under natural

conditions of temperature that performed well in controlled temperature conditions. Here

interesting to note that efficiency of PM32Y was not affected by increasing or decreasing

viscosity of oil. This might be due to ability of microbe to produce biosurfactant in

addition to bioremediation activity, which helped the microorganism to cope with the

fluctuating viscosity of oil. DNA sequencing showed that PM32Y is Bacillus subtilis

strain which has been already reported in different studies as a candidate for

bioremediation (Kukla et al., 2014; Khan et al., 2013). For instance, Das and Mukherjee,

(2007) isolated and evaluated the efficiency of Bacillus subtilis to degrade petroleum

hydrocarbons, concluded that this bacteria had ability to degrade petroleum hydrocarbons.

In addition to that Bacillus species are reported to promote plant growth by producing

plant hormones such as indole acetic acid (precursor of auxin) and phosphorus

solublization (Zaidi et al. 2006). Results of this study revealed that vegetated

experimental units have less residues of oil than non-vegetated control and vegetation

alone (non-inoculated control). It could be hypothesized that reduction of petroleum

contamination was due to improved plant growth. Due to improved plant growth bacteria

might use the plant exudates to co-metabolize petroleum hydrocarbons. Radwan et al.

(1995b) found microorganisms of various genera associated with various plants grown in

soil polluted with 10% crude oil by weight. Rhodococcus, Pseudomonas, and Bacillus

genera predominated in the contaminated bulk soil suggesting that bacteria of these

genera used plant exudates to proliferate and co-metabolize petroleum contamination.

Surely, one factor is related to other one, for example, in petroleum polluted soils there is

deficiency of oxygen to work as electron acceptor. Plant roots provide oxygen in its


129

rhizospheric soil. One of reasons for enhanced degradation of petroleum contamination in

planted pots as compared to unplanted ones may be the availability of oxygen in planted

pots as the numbers of bacteria are directly proportional to the presence of oxygen.

Though increased number of bacteria may not always the indicator of the biodegradation,

however, number of bacteria over baseline levels would be strong indicator of

biodegradation. Factory of oxygen in rhizosphere are roots of plants which are severely

suppressed due to petroleum contamination. The difference in the degradation rates of

various microorganisms may be due to their different capability to hydrolyze ACC and to

prevent production of stress ethylene and their association with plants. The best

performance showed by Bacillus subtilis which had high ACC deaminase activity as

compared to other bacteria revealing that biodegradation may be indirectly linked with

this property. As the measured ACC deaminase activity and auxin production was high by

Bacillus subtilis so direct correlation with plant growth promotion was observed and may

indirectly enhanced bioremediation of petroleum hydrocarbons. With high ability to

hydrolyze ACC, Bacillus subtilis improved plant growth especially the roots of plants

which not only served as a source of easily available food for microorganism to

proliferate and co-metabolize high molecular weight compounds such as diesel but also

improved aeration/oxygen in the rhizosphere. From the study it can be concluded that

microorganism with ACC-deaminase activity and plant growth promotion ability can

facilitate bioremediation process of petroleum hydrocarbons.

Chapter 7 Summary

130

Chapter 7

Summary

Bioremediation studies generally rely on seeking ideal microorganisms equipped

with multi-traits. In this study, bacteria with different traits have been isolated from

petroleum contaminated soil samples. A total of 301 bacterial isolates have been assayed

for their bioremediation capability, ACC-deaminase activity and plant growth promotion

and finally screened 8 bacterial isolates. These bacterial isolates were applied alone and in

combination with alfalfa, maize and canola for bioremediation of petroleum hydrocarbons

both under controlled and ambient condition of light and temperature. Moreover, effect of

bacterial inoculation on plant growth in petroleum contaminated soil was also assessed.

Under controlled conditions of light and temperature, bacterial inoculation effect

on growth parameters such as root elongation, plant height and biomass of alfalfa and

maize was more by all strains as compared to canola revealing that the growth

performance of maize and alfalfa grown in petroleum contaminated soil was better than

canola. Results revealed that bacterial inoculation significantly improved root elongation

of all three crops compared to un-inoculated control. The most efficient bacterial isolate

was Bacillus subtilis which increased 85% of alfalfa, 75% of maize and 61% as compared

to un-inoculated control. While under natural conditions of light and temperature increase

in root length of alfalfa, maize and canola by the same bacterial isolate (Bacillus subtilis)

was 63%, 77% and 66%, respectively, revealing that performance with respect to increase

in root length of maize and canola was better as compared to controlled conditions of light

and temperature.

Similarly, shoot elongation of all three crops was significantly improved by the

effect of bacterial inoculation both in controlled and natural conditions of light and

temperature as it was evident from the increase of 77%, 74% and 33% in shoot length of

alfalfa, maize and canola with inoculation of Bacillus subtilis. The same bacterial isolate

under natural conditions of light and temperature improved shoot elongation of alfalfa,

maize and canola by 59%, 63% and 59% respectively as compared to un-inoculated

control.

Bacterial inoculation also improved fresh biomass significantly both under

controlled and natural conditions of light and temperature. Bacillus subtilis under

controlled conditions of light and temperature increased fresh biomass by 91%, 67% and

58% of alfalfa, maize and canola, respectively and under natural light and temperature

Chapter 7 Summary

131

increase in fresh biomass of alfalfa, maize and canola by the same bacterial isolate was

66%, 67% and 64% respectively. Bacillus Sp. (WZ3S3), Bacillus cereus (WZ3S1),

Bacillus Sp. (SM73) also considerabley increased growth attributes of all three crops both

under controlled and natural conditions of light and temperature.

Bioremediation of petroleum hydrocarbons was significant by the bacterial

isolates in combination with alfalfa, maize and canola both under controlled and natural

conditions of light and temperature. Degradation of 46%, 37% and 43% observed was by

Bacillus subtilis in combinatin with alfalfa, canola and maize, respectively, under

controlled conditions of light and temperature as compared to un-inoculated control.

Under natural conditions of light and temperature petroleum hydrocarbons degradation by

Bacillus subtilis was equal with alfalfa, canola and maize. It was 45%, 42% and 41% as

compared to un-inoculated control. In addition to Bacillus subtilis, Bacillus Sp. (WZ3S3),

Bacillus cereus (WZ3S1), Bacillus Sp. (SM73) considerabley degraded petroleum

hydrocarbons in association with all three crops both under controlled and natural

conditions of light and temperature. It was observed that with increasing root biomass the

degradation of petroleum hydrocarbons by bacterial isolates was increased revealing that

roots play critical role in degradation of petroleum hydrocarbon contaminated soil.

Bacterial isolates when applied alone to assess their potential to degrade petroleum

hydrocarbons, it was observed that Bacillus subtilis (PM32Y), Bacillus Sp. (WZ3S3),

Bacillus cereus (WZ3S1), Bacillus Sp. (SM73) were efficient and consistent as compared

to rest of the bacterial isolates.

Conclusion

It can be concluded from the study that there are opportunities to find out bacterial

isolates with high capability of bioremediation of petroleum hydrocarbons. Convergence

of bacteria possessing ACC deaminase and plant growth promoting activity with suitable

plants can be successfully applied for enhanced degradation of petroleum hydrocarbons.

Association of plants with bacteria is far better than application of bacteria alone for

bioremediation of petroleum hydrocarbon contaminated soils. Alfalfa (Medicago sativa

L.) is more suitable crop than maize and canola to include in plant assisted

bioremediation program of organic pollutants such as petroleum hydrocarbons; however,

it totally depends upon the compatibility of plants with applied bacteria.

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