a thesis submitted in partial fulfillment for the degree of doctor of philosophy in the school of chemical engineering faculty of engineering

Vetiver grass, Vetiveria zizanioides: a choice plant for phytoremediation of heavy metals and organic wastes……….. 99 4 Effect of calcium on growth performance and essential oil of vetiv

A thesis submitted in partial fulfillment for   the degree of Doctor of Philosophy in the  School of Chemical Engineering  Faculty of Engineering 

March 2010 

ORIGINALITY STATEMENT

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my knowledge it contains no materials previously published or written

by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged

in the thesis I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’

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Signed ………

Date ………

ABSTRACT

Vetiver grass (VG) can be used for soil phytoremediation of various pollutants The plant is of high tolerance for extreme climatic variations and hostile soil conditions and can produce high biomass Vetiver can accumulate high concentrations of heavy metals as well as absorb and promote biodegradation of organic wastes

Essential oil extracted from roots of VG has aromatic and biological properties employed in several applications VG oil and its fractions are extensively used for blending in oriental types of perfumes, cosmetics, foods and aromatherapy, and have applicable potential as pharmaceutics, insecticides and herbicides

The effect of Pb, Zn and Cu on vetiver oil yield and chemical composition was investigated Oil content and yield are not affected at low and moderate concentrations of

Cu and Zn However, Pb has a significant detrimental effect on plant growth, oil yield and composition Vetiver oils extracted by hydrodistillation were free of heavy metals Results show that phytoremediation of Cu and Zn contaminated soils by vetiver can generate revenue from the production of oil extracts

To improve growth, oil yield and quality of VG grown on lead contaminated soils, the addition of CaCO3 was investigated Calcium treatment increased vetiver growth and survival, but did not improve vetiver oil yield and chemical composition

A response surface method was applied to optimize the extraction yields produced

by supercritical CO2 extraction (SCE) Operation at optimal conditions (190 bar, 50ºC and 100 minutes) produced vetiver oil yield about four times higher than that of hydrodistilation Extraction pressure has a major linear effect on oil yield, whilst temperature and time have a lesser impact

The addition of ethanol increased extraction efficiency of SCE At optimal conditions of 190 bar, 50ºC and 15 vol% ethanol, ethanol-modified-SCE produced a yield nearly double that of SCE without modifier operated at 190 bar and 50ºC The operation

at 100 bar, 40ºC and 15 vol% ethanol had nearly the same yield as that of optimal conditions This finding allows extraction operated at low pressure and temperature Metals accumulated in vetiver roots were not co-extracted with essential oils by either ethanol-modified SCE or SCE without modifier

ACKNOWLEDGEMENTS

I would like to express my deep gratitude to my supervisor, Prof Neil Foster, who allowed

me to work in his laboratory with full support I specially appreciate his encouragement, guidance, valuable suggestions and feedbacks during the course of my graduate study

I would also like to thank my co-supervisors, Dr Paul Truong and Dr Raffaella Mammucari, for many lengthy discussions, advices and encouragement throughout the study I am especially indebted to Dr Paul Truong for his hospitality during the period of my stay in Brisbane and his time and energy for field work I am also indebted to Dr Raffaella Mammucari for her feedback on the writing of this thesis

I would like to extend my gratitude to my co-workers, Roderick, Roshan, Adam, Wendy, Jane and Grace for your friendship and valuable discussions I am also grateful to Van Bong Dang, Thanh Ngoc Vo and Peter Valtchev for their technical support I also send my gratitude to my friends, Tran Chau Duc and Dao Hong Quang, for their friendship and support

I would like to thank “cau Ba” and “mo Ba” for their support and hospitality during the time

we stayed with them I am also grateful to “chu Tam” for his support and encouragement

I would like to thank Ministry of Education and Training, Vietnam for financial support during the time I studied abroad The financial support from Gelita, Australia is highly appreciated

Finally, I would like to thank my family for their support and encouragement during the course of study Most of all, I would like to thank my wife, without her support this work would not have been possible

Table of Contents

Abstract……… i

Acknowledgements……… iii

List of figures……… viii

List of tables……… x

Abbreviation……… xii

List of publications……… xiii

1 Introduction……… 1

2 Literature review……… 5

2.1 Vetiver grass, Vetiveria zizanioides: a choice plant for phytoremediation of heavy metals and organic wastes……… 5

2.1.1 Introduction……… 6

2.1.2 Vetiveria zizanioides and its outstanding characteristics………… 7

2.1.2.1 Vetiveria zizanioides……… 7

2.1.2.2 Outstanding characteristics of Vetiveria zizanioides…… 8

Morphological and genetic characteristics………… 8

Agronomic characteristics……… 10

Physiological characteristics ……… 10

Microorganism association ……… 15

2.1.2.3 Phytoremediation of heavy metals and organic wastes… 16 Heavy metals……… 16

Arsenic……… 16

Boron……… 18

Cadmium……… 18

Copper……… 19

Chromium……… 21

137 Cesium and 90 Strontium……… 22

Lead……… 22

Zinc……… 28

Multi-heavy metals……… 30

Phytoremediation potential of vetiver versus other plant species……… 31

Organic Wastes……… 35

Phenol……… 35

2,4,6-Trinitroluene……… 35

Ethidium bromide……… 36

Benzo[a]pyrene……… 37

Petroleum hydrocarbon……… 37

Atrazine……… 37

2.1.3 Conclusion……… 38

2.1.4 References……… 40

2.2 Vetiver essential oil……… 51

2.2.1 Introduction……… 51

2.2.2 Production ……… 51

2.2.3 Properties ……… 53

2.2.4 Chemical composition ……… 53

2.2.5 Applications……… 54

2.2.5.1 Perfumery……… 54

2.2.5.2 Aromatherapy……… 55

2.2.5.3 Insecticides……… 55

2.2.5.4 Herbicides……… 57

2.2.5.5 Antioxidant activity……… 57

2.2.5.6 Anticancer activity……… 58

2.2.5.7 Antimicrobial activity……… 58

2.2.5.8 Other uses……… 58

2.2.7 References……… 60

2.3 Supercrtical fluid extraction ……… 64

2.3.1 Introduction……… 64

2.3.2 Characteristics of supercritical fluid extraction……… 66

2.3.3 Selection of the operating conditions……… 67

2.3.3.1 Pressure……… 67

2.3.3.2 Temperature……… 68

2.3.3.3 CO2 flow rate……… 68

2.3.3.4 Particle size……… 69

2.3.3.5 Time……… 69

2.3.3.6 Modifiers or co-solvents……… 70

2.3.4 Optimization of operating conditions……… 71

2.3.5 References……… 72

3 Economic incentive for applying vetiver grass to remediate lead, copper and zinc contaminated soils……… 78

3.1 Introduction……… 79

3.2 Materials and methods……… 81

3.2.1 Plant materials……… 81

3.2.2 Soil treatments……… 81

3.2.3 Plant cultivation……… 83

3.2.4 Extraction……… 83

3.2.5 Gas-chromatographic and Gas chromatography-Mass

spectrometry analysis……… 84

3.2.6 Statistical analysis……… 85

3.3 Results and discussion……… 86

3.3.1 Soil characteristics……… 86

3.3.2 Growth performance……… 87

3.3.3 Content and yield of vetiver oil……… 88

3.3.4 Heavy metal contents in vetiver roots and shoots……… 89

3.3.5 Chemical components of vetiver essential oil……… 91

3.4 Discussion……… 94

3.5 Conclusion……… 98

3.6 References……… 99

4 Effect of calcium on growth performance and essential oil of vetiver grass grown on lead contaminated soils……… 104

4.1 Introduction……… 105

4.2 Materials and methods……… 108

4.2.1 Plant materials……… 108

4.2.2 Soil treatments……… 108

4.2.3 Plant cultivation……… 109

4.2.4 Extraction……… 109

4.2.5 Gas-chromatographic and Gas chromatography-Mass spectrometry analysis……… 110

4.2.6 Statistical analysis……… 110

4.3 Results and discussion……… 111

4.3.1 Soil properties……… 111

4.3.2 Effect of lime on soil pH……… 111

4.3.3 Vetiver growth performance……… 112

4.3.4 Heavy metals in roots and shoots of vetiver……… 114

4.3.5 Oil content and oil yield……… 115

4.3.6 Chemical components of vetiver essential oil……… 116

4.4 Conclusion……… 120

4.5 References……… 121

5 Response surface method applied to supercritical CO 2 extraction of Vetiveria zizanioides essential oil………. 125

5.1 Introduction……… 126

5.2 Materials and methods……… 129

5.2.1 Plant material preparation……… 129

5.2.2 Soxhlet extraction……… 129

5.2.3 Hydro-distillation……… 130

5.2.4 Supercritical CO2 extraction……… 130

5.2.5 Experimental design……… 131

5.2.6 Kinetic study……… 134

5.2.7 Yield calculation……… 134

5.2.8 Gas chromatography and gas chromatography-mass spectrometry analysis……… 135

5.3 Results and discussion……… 136

5.3.1 Optimization of SCF extractions……… 136

5.3.2 Kinetic study……… 143

5.3.3 Chemical components of SCE vetiver extract……… 144

5.3.3.1 Khusimol……… 147

5.3.3.2 Zizanoic acid……… 149

5.3.4 Comparison with conventional extraction methods……… 150

5.4 Conclusion……… 153

5.5 References……… 154

6 Extraction of Vetiver essential oil by ethanol modified supercritical carbon dioxide……… 158

6.1 Introduction……… 159

6.2 Materials and methods……… 162

6.2.1 Plant material preparation……… 162

6.2.2 Extraction……… 162

6.2.3 Experimental design……… 164

6.2.4 Gas chromatography and gas chromatography–mass spectrometry analysis……… 165

6.2.5 Heavy metal analysis……… 166

6.2.6 Statistical analysis……… 166

6.3 Results and discussion……… 167

6.3.1 Kinetic study……… 167

6.3.2 Effect of operating parameters on oil yield and optimization of ethanol-modified-SCE……… 168

6.3.3 Chemical components of ethanol-modified SCE extracts……… 176

6.3.4 Comparison with hydrodistillation and pure SCE……… 179

6.3.5 Heavy metals contents in SCF extracts……… 180

6.4 Conclusion……… 183

6.5 References……… 184

7 Conclusions and Recommendations……… 187

7.1 Summary of conclusions……… 187

7.2 Recommendations……… 189

Appendix A……… 190

List of Figures

2.1 Vetiveria zizanioides grass from left to right: mature plant, thick hedge, deep and

extensive root system……… 9

2.2 Phase diagram of a pure compound (Smith et al., 1996)……… 65

4.1 Soil pH under effect of different Ca treatments……… 112

5.1 Schematic diagram of SCF extraction V1, V2, V3: stopping valve; F: filter; CV:

check valve, HC: heating coil; E: extraction vessel; CH: circulating heater; PM:

pressure meter; MV: micro-metering valve……… 131

5.2 Central composite design with three operating conditions of supercritical fluid

5.3 Goodness of fit of empirical model……… 138

5.4 Response surface plot showing the effect of pressure and temperature on oil yield at extraction time of 50 minutes……… 139

5.5 Response surface plot showing the effect of temperature and time on oil yield at

fixed pressure of 190 bar……… 140

5.6 Response surface plot showing the effect of pressure and time on oil yield at fixed

temperature of 50°C……… 141

5.7 Scanning electronic microscopy of dry vetiver root: localization of oil glands…… 142

5.8 Yield of vetiver oil extracted by SCE as a function of the total amount of CO2 used

at different operating conditions……… 144

5.9 Response surface plot showing the effect of pressure and temperature on khusimol

content at the extraction time of 50 minutes……… 149

6.1 Schematic diagram of ethanol-modified SCF extraction V1-V3: stopping valve,

MV1-MV2: micro-metering valve, HC: heating coil, E: extractor, SM: static mixer, CV: check valve, CH: circulating heater, PM: pressure meter……… 164

6.2 The cumulative yield of vetiver extracts over time at the fixed operating conditions

of 145 bar, 45C and different amounts of added ethanol (0, 5, 10 and 15%)……… 168

6.3 Goodness of fit between the experimental and predicted yields………. 170

6.4 The effect of pressure and the concentration of ethanol on oil yield at the extraction temperature of 50°C……… 172

6.5 The effect of temperature and the concentration of ethanol on oil yield at the

extraction pressure of 100 bar ……… 174

6.6 The effect of pressure and temperature on oil yield at the ethanol concentration of

5% 175

2.3 Ratio of accumulated Pb in vetiver shoots over roots in different experiments…… 25

2.4 Effect of EDTA on Pb uptake and translocation ratio of VZ……… 26

2.5 The level of Zn in soils and in VZ roots and shoots……… 29

2.6 Phytoremediation ability of Vetiveria zizanioides and other heavy metal

hyperaccumulators……… 33

3.1 Concentrations of nutrients (mg kg-1 dry soil) added into Pb, Cu and Zn treatments 82

3.2 Physical and chemical properties of river sand……… 86

3.3 The dry bimomass of VG grown on soils with different concentrations of lead,

copper and zinc for 7 months……… 87

3.4 Yield of VG oil under effect of lead, zinc and copper……… 89

3.5 Level of Pb, Cu and Zn in VG shoots, roots and hydro-distilled roots……… 90

3.6 Chemical components of vetiver essential oil under effect of different treatments of

4.1 Growth characteristics of vetiver grass grown on lead contaminated soils under

different treatments of calcium……… 113

4.2 Level of heavy metals in leaves of Vetiver grown under different Ca treatments… 115

4.3 The oil content and yield of vetiver grass……… 116

4.4 Chemical composition of vetiver essential oil under different treatment of Ca…… 118

5.1 Size ranges of roots particles……… 129

5.2 Coded and uncoded levels of independent variables……… 133

5.3 Central composite design with coded and uncoded levels of independent variables and experimental yield………. 136

5.4 Regression coefficients and corresponding t and P-values for vetiver oil yield…… 137

5.5 Chemical components of vetiver oils extracted by SCE at different operating

conditions, hydrodistillation and hexane extraction……… 145

5.6 Regression coefficients and corresponding t and P-values for khuismol content 148

5.7 Yields obtained by hydro-distillation, solvent and supercritical fluid extraction of

Vetiver essential oil……… 151

6.1 Coded and uncoded levels of independent variables……… 165

6.2 Central composite design with coded and uncoded levels of independent variables, and

experimental yield………. 169

6.3 Regression coefficients and corresponding t and P-values for vetiver oil yield…… 170

6.4 Chemical compositions of vetiver oil extracted by hydrodistillation, supercritical

CO2 and ethanol modified supercritical CO2……… 177

6.5 Yield of vetiver oil extracted by hydrodistillation, pure SCE and ethanol-modified

6.6 Metal contents of vetiver roots before and after extraction by pure SCE and ethanol

xii   

Abbreviation

VZ Vetiveria zizanioides

SCE Supercritical carbon dioxide extraction

SFE Supercritical fluid extraction

SC-CO2 Supercritical carbon dioxide

RSM Response surface method

List of publications

Published papers

Luu Thai Danh, Raffaella Mammucari, Paul Truong and Neil Foster 2009 Response

surface method applied to supercritical carbon dioxide extraction of Vetiveria zizanioides essential oil Chemical Engineering Journal, 155, 617-626

Luu Thai Danh, Paul Truong, Raffaella Mammucari, Tam Tran and Neil Foster 2009

Vetiver grass, Vetiveria zizanioides: A choice plant for phytoremediation of heavy metals and organic wastes International Journal of Phytoremediation 11, 664- 691

Luu Thai Danh, Paul Truong, Raffaella Mammucari and Neil Foster 2010 Economic incentive for applying vetiver grass to remediate lead, copper and zinc contaminated soils

International Journal of Phytoremediation Article in Press

Luu Thai Danh, Paul Truong, Raffaella Mammucari and Neil Foster Effect of calcium on growth performance and essential oil of vetiver grass grown on lead contaminated soils

International Journal of Phytoremediation Article in Press

Submitted papers

Luu Thai Danh, Raffaella Mammucari, Paul Truong and Neil Foster Extraction of Vetiver

essential oil by ethanol modified supercritical carbon dioxide Chemical Engineering

Journal

Conferences

Luu Thai Danh, Paul Truong, Raffaella Mammucari and Neil Foster Effect of calcium on growth performance and essential oil of vetiver grass grown on lead contaminated soils 6thInternational Conference on Phytotechnologies, St Louis, Missouri, USA, December 1-4,

2009

Luu Thai Danh, Raffaella Mammucari, Paul Truong and Neil Foster Extraction of Vetiveria

zizanioides esential oil by supercritical carbon dioxide 11th European Meeting on Supercritical Fluids, Barcelona, Spain, May 4-7, 2008

Luu Thai Danh, Raffaella Mammucari, Paul Truong and Neil Foster Optimization of

essential oil extraction from Vetiveria zizanioides using supercritical CO2 American Institute of Chemical Engineers Annual Meeting, Salt Lake City, Utah, USA, November 4-9,

2007

1 Introduction

An increasingly industrialized global economy and rapid rise in world population over the last century have led to dramatically elevated releases of toxic wastes into the soil environment These pollutants can be readily passed onto human beings through the food chain due to soil-to-plant transfer Consequently, there is a growing global awareness about human health and ecological threats caused by soil pollution

Soil pollution is often treated by conventional methods, such as physical, chemical and biological methods Physical treatments involve removal from contaminated sites (soil excavation), deep burial (land filling) and capping Chemical methods use strong acids and chelators to wash polluted soils Biological methods employ micro-organisms for pollutant removal These approaches are expensive, impractical and at times impossible to carry out,

as the volume of contaminated materials is very large Furthermore, they irreversibly affect soil properties, destroy biodiversity and may render the soil useless as a medium for plant growth

An alternative method called phytoremediation has been developed recently by using

a diverse collection of plant-based technologies from either naturally occurring or genetically engineered plants to clean contaminated soils It represents an environmentally friendly and cost effective technology and attracts the attention of publics and scientists worldwide However, the application of this technology is limited due to several reasons First of all, this method is a time consuming process due to the selection of low biomass plants Secondly, plants survival under the hostile conditions of contaminated soils can be unsatisfactory Finally, plants the accumulation of high concentrations of pollutants in the biomass is not always possible

Among the plants used in phytoremediation, vetiver grass (Chrysopogon zizanioides (L.) Roberty, syn Vetiveria zizanioides (L.) Nash) satisfies nearly all criteria required for an

effective phytoremediation Vetiver grass (VG) is a fast growing plant and produces high levels of biomass It can tolerate well a wide range of hostile conditions at contaminated sites Importantly, it can accumulate high concentrations of heavy metals, particularly lead

and zinc, in the roots The special characteristics that make Vetiveria zizanioides a very

promising candidate for phytoremediation are discussed in Chapter 2, part 2.1

Essential oil extracted from the roots of VG has aromatic and biological properties that can be employed in a wide range of applications VG oil and its fractions are extensively used for blending in oriental types of perfumes, cosmetics and aromatherapy Recently, the discovery of biological properties of vetiver essential oil, such as antifungal, antibacterial, anticancer, anti-inflammatory and antioxidant activities, opens the way to application in the pharmaceutical industry VG extracts can also be used in food and beverages as aromatizing agents Insecticidal activities of VG oil against cockroaches, flies and Formosan subterranean termite have been discovered Production, properties, chemical compositions and uses of VG oil are revised in Chapter 2, part 2.2

Vetiver essential oil is traditionally extracted by hydro-distillation, steam distillation and solvent extraction However, hydro and steam distillation have several disadvantages, such as incomplete extraction of essential oils from plant materials, high operating temperatures with the consequent breakdown of thermally labile components, the promotion

of hydration reactions of chemical constituents, and requirement for a post-extraction process to remove water Solvent extraction overcomes the drawbacks of distillation, but has the major disadvantage of solvent residue in the extracts Supercritical fluid extraction (SFE), a novel and environmentally benign separation technology, represents a green and valuable alternative to the conventional extraction methods for the production of natural extracts Advantages, selection of operating conditions and optimization of SFE process are revised in Chapter 2, part 2.3

The objectives of this study are to investigate the potential of promoting the application of vetiver grass for phytoremediation of heavy metal contaminated soils, specifically lead, zinc and copper, via cash return from essential oil production, and to study

the use of supercritical CO2 for extraction of vetiver essential oil To obtain these goals, the following work was performed:

Investigation of the effect of heavy metals on vetiver essential oil yield and composition (Chapter 3):

The application of VG for phytoremediation or revegetation of soils contaminated by heavy metals can be strongly promoted by economic return from VG extracts However concerns may rise about contamination of oil extracts by heavy metals Lead, zinc and copper are the most common pollutants of the soil environment and they are accumulated in roots of vetiver grass at high concentrations The study was carried out to determine whether

or not high concentrations of these metals accumulated in roots can affect yield, chemical compositions as well as contaminate vetiver essential oil extracted by hydrodistillation

Investigation of the effect of calcium on growth performance and essential oil of vetiver grass grown on lead contaminated soils (Chapter 4):

The study in Chapter 3 showed that the survival rate, and both yield and chemical composition of oil from VG were adversely affected by high levels of lead in soils The toxic effect of heavy metals on plant growth can be reduced by the addition of calcium Therefore, the objective of this study was to investigate the effect of calcium (added as CaCO3) on VG growth and survival rate as well as its effect on yield and chemical composition of oil from VG grown on highly lead contaminated soils

Optimizion of supercritical CO 2 extraction (Chapter 5):

Efficiency of supercritical CO2 extraction (SCE) depends on several variables, including extraction temperature, pressure and time To investigate the effect of these parameters on vetiver oil yield and optimize them for a high yield, the Response Surface Method using a Central Composite Design (RMS-CCD) was employed The yield and chemical compositions obtained from SCE were compared to those obtained from conventional extraction methods (hydro distillation and solvent extraction)

Optimization of ethanol-modified supercritical CO 2 extraction (Chapter 6):

Supercritical CO2 is a non-polar solvent that can not solubilise compounds without

or with weak polarity, resulting in low extraction efficiency The addition of ethanol to supercritical CO2 forms a solvent with higher polarity leading to potential improvements in extraction efficiencies The objectives of this study were to investigate the effect of three operating conditions of ethanol modified CO2 extraction, namely pressure, temperature and amount of added ethanol, on yield and chemical composition of vetiver essential oil, and to optimize these conditions for a high oil yield by using the RMS-CCD method In addition, the study also addressed the risk of heavy metal contamination of VG extracts The study also investigated whether pure SCE (without co-solvent) and ethanol-modified SCE co-extract heavy metals with essential oil from vetiver roots

2 Literature review

2.1 Vetiver grass, Vetiveria zizanioides: a choice plant for

phytoremediation of heavy metals and organic wastes

Abstract

Glasshouse and field studies showed that Vetiver grass (VG) can produce high biomass (>100 tone ha-1year-1) and tolerate extreme climatic variation such as prolonged drought, flood, submergence and temperatures (-15° to 55°C), soils high in acidity and alkalinity (pH 3.3-9.5), high levels of Al (85% saturation percentage), Mn (578 mg kg-1), soil salinity (ECse 47.5 dS m-1), sodicity (ESP 48%), and a wide range of heavy metals (As, Cd, Cr, Cu,

Hg, Ni, Pb, Se, and Zn) Vetiver can accumulate heavy metals, particularly lead (shoot 0.4 wt% and root 1 wt% on dry basis) and zinc (shoot and root 1 wt% on dry basis) The majority of heavy metals is accumulated in roots thus VG is suitable for phytostabilization, and for phytoextraction with addition of chelating agents Vetiver can also absorb and promote biodegradation of organic wastes (2,4,6-trinitroluene, phenol, ethidium bromide, benzo[a]pyrene, atrazine) Although VG is not as effective as some other species in heavy metal accumulation, very few plants have such a wide range of tolerance to extremely adverse conditions of climate and growing medium (soil, sand, and tailings) All these special characteristics make VG a plant of choice for phytoremediation of heavy metals and organic wastes

Note: the content of section 2.1 was published in

Luu Thai Danh, Paul Truong, Raffaella Mammucari, Tam Tran and Neil Foster 2009

Vetiver grass, Vetiveria zizanioides: A choice plant for phytoremediation of heavy metals and organic wastes Inter J Phyt 11, 664- 691

2.1.1 Introduction

The rapid increase in population coupled with fast industrialization and intensive agricultural practices causes serious environmental problems, including the production and release of considerable amounts of toxic wastes into the soil environment (Xuliang et al., 2007) According to a report of the US Environmental Protection Agency (EPA, 2007), there are more than 40,000 contaminated sites in the Unites States alone Soil pollutants can

be classified into two main groups: inorganic and organic The major components of inorganic pollutants are heavy metals, such as lead, arsenic, cadmium, copper, zinc, nickel, and mercury, which are continuously added into the environment via disposal of urban sewage sludge and industrial wastes in agricultural soils and via agrochemical usage (Khan, 2005) These pollutants, together with radionuclides released through various human activities (mining and milling of nuclear fuel, testing of nuclear weapons, occasional nuclear disasters), accumulate in the soils, and can be readily passed onto human beings through the food chain due to soil-to-plant transfer of metals and radionuclides (Khan, 2005; Shaw and Bell, 1991) In addition, vast areas of soils around the world are contaminated with organic pollutants, mainly pesticides, petroleum hydrocarbons, polycyclic aromatic hydrocarbons, and explosives

In recent years, public concerns relating to human health and ecological threats caused by soil pollution have led intensive research of new economical remediation technologies Conventional methods used for treatment of contaminated soils, namely chemical, physical, and microbiological methods, are costly to install and operate A new green technology has been developed, called phytoremediation, which utilizes plants to decontaminate soil, water and air environment (Prasad, 2003; Salt et al., 1998; Chaney et al., 1997), and it is growing of importance in controlling soil pollution Phytoremediation is clean, simple, cost effective, non-environmentally disruptive (Wei and Zhou, 2004; Zhou and Song, 2004), and most importantly, its by-products can find a range of other uses (Truong, 2003)

The application of phytoremediation for pollution control, however, has several limitations that require further research on plant and site-specific soil conditions Phytoremediation is mainly confined to the area occupied by the root systems, and as the consequence, small plants with low yields and reduced root systems do not support efficient phytoremediation and most likely do not prevent the leaching of contaminants into ground water In addition, non-perennial plants, particularly those with slow growth and low biomass production require a long-term commitment for remediation Environmental conditions also determine the efficiency of phytoremediation as the survival and growth of plants are adversely affected by extreme environmental conditions, toxicity, and the general conditions of soil in contaminated lands (Northwestern University, 2007) Finally, different types of pollutants in soils and water require different types of plants for phytoremediation, which adds to the complexity of the technology and requires a wide range of research activities prior to the release of specific plants for commercialization

Vetiveria zizanioides (L.) Nash, is one of the very few plants, if not the only one that

has the potential to meet all the criteria required for phytoremediation (Truong, 2003) Therefore, it has received a great interest from scientists and the wider public for the removal of a wide range of contaminants from soil in recent years In this review, the special

characteristics and potentials that make Vetiveria zizanioides a very promising candidate for

phytoremediation are discussed

2.1.2 Vetiveria zizanioides and its outstanding characteristics

2.1.2.1 Vetiveria zizanioides

Vetiver grass, Vetiveria zizanioides (L.) Nash, syn Chrysopogon zizanioides (L.)

Roberty, belongs to the Poaceae family, subfamily of Panicoideae, tribe Andropogonae and subtribe Sorghina, and the genus includes ten species (Bertea and Camusso, 2002) Vetiver grass originated in the Indian sub-continent is common to flood plains and stream banks, but

can also be found throughout the tropical and subtropical regions of Africa, Asia, America, Australia, and Mediterranean Europe (Maffei, 2002)

Among the Vetiver species, Vetiveria zizanioides (VZ) is the most valuable in term

of economics In fact, essential oil extracted from its roots has been used for a long time in perfumers, medicine, and other areas of applications The use of VZ for land protection purposes has long been practiced in tropical and subtropical countries, but its advantages, namely low cost, effectiveness and ease of soil and water conservation, only emerged in the late 1980s (Truong, 2002) Through the understanding of the extraordinary physiological and morphological characteristics of VZ and their roles in soil and water conservation, researchers found the distinctive attributes, which make VZ particularly suitable for environmental protection purposes (Truong, 2000)

2.1.2.2 Outstanding Characteristics of Vetiveria zizanioides

Figure 2.1 Vetiveria zizanioides grass from left to right: mature plant, thick hedge, deep

and extensive root system

An important aspect of plant use for bioengineering or environmental protection in non-native environments is the prevention of introducing hard-to-control weeds VZ has flowers but setting no seeds, so the potential to become a weed is very low Despite the fact that VZ was introduced from India into Fiji more than 100 years ago, and has been widely used for water and soil conservation over the past 60 years, there are no reports indicating that it is a weed in this new environment (Truong and Creighton, 1994) In addition, a study conducted in Australia for eight years showed that VZ is sterile under various growing conditions (Truong, 2002) In 2007, USDA carried out a new risk assessment of non fertile

VZ cultivars from south India through the Pacific Island Ecosystem at Risk (PIER), typified

by Sunshine (US) and Monto (Australia) genotypes A rating system was adopted based on the Australian/New Zealand weed risk assessment protocol, modified for Hawaii (http://www.vetiver.org/USA_PIER.htm) The protocol is very strict and thorough and considers a score as high as two being acceptable for safe introduction According to such a protocol, VZ was rated minus eight

One of the peculiar characteristics of VZ is its ability to form a thick hedge when planted close together (Figure 2.1) A VZ hedge can stand up to water flow of 0.6 m depth, forming a living barrier, which impedes and spreads run-off water The VZ hedge also acts

as a very effective filter for trapping sediments and agricultural chemicals (Truong, 2002) before they reach water courses

Agronomic characteristics

The rate of phytoremediation is directly proportional to the plant growth rate Fast growing and high biomass are favorite criteria used to select plants for phytoremediation (Roger et al., 2000) VZ has a C4 pathway of photosynthesis overcoming the limits of photorespiration found in C3 plants C4 plants show higher rates of photosynthesis at high light intensities and high temperatures due to the increased efficiency of photosynthetic carbon reduction cycle (Hatch, 1987) In favorable environments, C4 plants outperform C3 plants, making them the most productive crops or the worst weeds (Bertea and Camusso, 2002) VZ demonstrates the high growth rates of a C4 grass, as indicated by a radiation use efficiency (RUE) of 18 kg ha-1 per MJ m-1 (Vieritz et al., 2003) The RUE of VZ is

comparable with the one of other C4 grasses such as maize (Zea mays L.) and sugarcane (Saccharum officinarum) which present 16 and 18 kg ha-1 per MJ m-1, respectively (Muchow, Sinclair, and Bennett, 1990; Inman-Bamber, 1974) and much higher than the

RUE of C3 grasses such as coastal couch grass (Cynodon dactylon): 5.3 kg ha-1 per MJ m-1(Burton and Hanna, 1985) According to Truong (2003), under tropical hot and wet conditions, VZ grows very fast, its biomass is extremely high (more than 100 tons of dry matter ha-1 year-1) VZ retains high activity of the key enzymes involved in photosynthesis (NADP-MDH and NADP-MET) even when cultivated in temperate climates (Bertea and Camusso, 2002), which makes this plant an ideal candidate for phytoremediation in different parts of the world

Physiological characteristics

In the last 10 years, extensive research on physiological aspects of VZ discovered special characteristics, which makes VZ a good candidate for a wide range of phytoreme diation purposes

VZ is highly adaptable to extreme environmental conditions, such as up to 6 months drought (Truong, 1999a) The study of Xia et al (2003) conducted over three years showed that VZ could tolerate a time of submergence (more than 120 days) much higher than the

other plants included in the study: bahia grass: 60-70 days, carpet grass: 32-40 days, sour Paspalum: 25-32 days, St Augustine: 18-32 days, centipede grass: only 7-10 days A trial conducted in 2007, to stabilise the Mekong river bank in Cambodia, showed that VZ can survive more than 3 months under muddy water (Toun Van, pers.com.)

Furthermore, although VZ is a tropical grass, it can survive and thrive under extremely cold conditions and can tolerate extreme temperatures ranging from -15°C to 55°C (Xia et al., 1999; Xu and Zhang, 1999) Under frosty weather, VZ top growth was killed but its underground growing points survived In Australia, VZ growth was not affected by severe frost at -11°C and it survived for a short period at -22°C in northern China Recent research showed that 25°C was the optimal soil temperature for root growth, but VZ roots continued to grow at 13°C When the temperature of the soil was in the 15°C (day)-13°C (night) range, very little shoot growth occurred, however root growth continued

at the rate of 126 mm day-1, indicating that VZ was not dormant Extrapolations suggested that root dormancy occurs at about 5°C (Wang, 2000)

One special characteristic of VZ is the high tolerance of a range of extreme soil conditions, especially heavy metal contamination Glasshouse and field experiments have shown that VZ can thrive under a wide range of pH; it is highly tolerant to saline and sodic soil conditions, and the presence of Al and Mn (Truong and Baker, 1998; Truong and Baker 1997) VZ can flourish between pH 3.3 and 9.5 with adequate supply of N and P fertilizers Under extremely acidic soil conditions, with adequate supply of N, P and moisture, VZ can grow with levels of Al and Mn as high as 85% saturation percentage and 578 mg kg-1 soil, respectively (Truong, 1999b) It can survive in saline soil with electrical conductivity (ECse)

up to 47.5 dS m-1, its salinity threshold is at ECse = 8 dS m-1 and soil ECse values of 10 and

20 dS m-1 reduce yield by 10 and 50%, respectively Based on these results, VZ belongs to a group of highly salt tolerant crop and pasture species grown in Australia, such as Rhodes

grass (Chloris guyana) and barley (Hordeum vulgare) (Greenfield, 2002) According to

Northcote and Skene (1972), soil with an exchangeable sodium percentage (ESP) higher than 15 is considered strongly sodic; the growth of VZ was not adversely affected on bentonite tailings with ESP up to 48% (Bevan, Truong, and Wilson, 2000) In addition, a

series of glasshouse experiments proved that VZ has high tolerance to a wide range of heavy metals in soils due to its high threshold levels of these metals in soils (Table 2.1) While most vascular plants are highly sensitive to heavy metal toxicity and most plants were also reported to have very low threshold levels for As, Cd, Cu, Cr, and Ni in the soils In summary, studies showed that VZ can be established under highly hostile environments and indicate that it is highly suitable for phytoremediation of mining and industrial waste sites (Greenfield, 2002)

Table 2.1 Threshold levels of heavy metals to vetiver growth (Truong 1999b) based on

single element experiments

(Bowen, 1979)

Soil level (Baker & Eldershaw, 1993)

Soil level Shoot level

NA

NA 0.5-2.0

NA

NA

2.0 1.5

NA

NA

NA

NA 7-10 2-14

NA

100-250 20-60 50-100 200-600

Note: NA not available

VZ has great potential in absorbing dissolved nutrients such as nitrogen and phosphorous It has been observed that VZ can be used to reduce N and P concentrations in polluted river water (Zheng et al., 1997) The initial concentrations of total N and P in polluted water was 9.1 and 0.3 mg L-1 After 4 weeks of experiment, total N and P was reduced by 71 and 98%, respectively In a field study, VZ could remove up to 740 kg N ha-1and 110 kg P ha-1 over 3 months at a nutrient-rich site, and 1020 kg N ha-1 and 85 kg P ha-1over 10 months at a lower nutrient site (Vieritz et al., 2003) In a hydroponic flow through

system with an effluent flow rate of 20 L min-1 through VZ roots, one square meter of VZ can treat 30,000 mg of N and 3575 mg of P in eight days (Hart, Cody and Truong, 2003) In this application, VZ over-performed other crops and pasture plants such as: Rhodes grass, Kikuyu grass, Green Panic, Forage Sorghum, Rye grass, and Eucalyptus trees (Truong, 2003) In an experiment to determine the upper tolerance limit of VZ to N and P applications, Wagner et al (2003) showed that VZ growth increased with the level of N supply up to 6000 kg ha-1 year-1 However, very little growth response occurred at rates higher than 6000 kg ha-1 year-1; although rates up to 10,000 kg N ha-1 of N did not adversely affect VZ growth Similarly, no growth response occurred at P rates higher than 250 kg ha-1year-1 However, its growth was not adversely affected at P application rates up to 1000 kg

ha-1 year-1

A series of trials demonstrated that VZ could accumulate high amounts of heavy metals in roots and shoots Data from Table 2.2 show that only small amounts of arsenic, cadmium, chromium, and mercury were translocated to the shoots (1-6%) For copper and nickel, a moderate proportion was translocated to the shoots (16-30%) Lead, selenium, and zinc were almost evenly distributed between shoot and root (44-47%)

Transpiration is a key process in the application of phytoremediation to soil or groundwater pollutants, as vegetation must transpire enough water from the soil or groundwater to control or take up the contaminants efficiently (Vose et al., 2003) When growing under wetland conditions, VZ had the highest water use rate compared with other

wetland plants such as Iris pseudacorus, Typha spp., Schoenoplectus validus, Phragmites

australis At the average consumption rate of 600 ml day-1 pot-1 over a period of 60 days,

VZ used 7.5 times more water than Typha (Cull et al., 2000) A correlation between water

use (soil moisture at field capacity) and dry weight (DW) yield of VZ has been observed It has been estimated that for 1 kg of dry shoot biomass, VZ would use 6.86 L day-1 of water

If the dry matter yield of 12-week-old VZ, at the peak of its growth cycle, was 40.7 t ha-1, a hectare of VZ would potentially use 279 KL ha-1 day-1 (Truong and Smeal, 2003)

Table 2.2 Concentrations of heavy metals accumulated in VZ roots and shoots (Truong,

1999b)

Heavy metals Soil

(mg kg -1 )

Shoot (mg kg -1 DW)

Root (mg kg -1 DW)

Shoot/root (%)

Shoot/Total (%) Arsenic (As)

50

600

730 6.17

300 74.3

750

11.2 0.31

13

18 78.2 0.12

448 11.3

880

268 14.20

68

1750 87.8 10.8

1040 24.8

1030

4.2 2.2

L-1 and diuron >0.03 μg L-1 (ANZECC & ARMCANZ, 1999) Under a simulated wetland environment, a study by Cull et al (2000) showed that the growth of VZ was not adversely affected by application of atrazine or diuron at rates up to 2000 μg L-1, whilst the growth of Phragmites australis was significantly reduced by both herbicides when applied at the same rate

A typical attribute of VZ that is relevant to phytoremediation or rehabilitation is its long life span VZ can live up to 50 years (National Resource Council, 1995), so it has an advantage over other annual plants that require replanting over such a long period Once VZ has been established at sites requiring remediation, it will grow and develop under adequate maintenance for a long period until phytoremediation or rehabilitation is completed without requiring revegetation Finally, VZ can be eliminated easily either by spraying with

glycophosate herbicide or uprooting and drying out by hand or using farm machinery (Truong, 2002)

Microorganism association

Soil microorganisms in the rhizosphere of plants growing in contaminated soils play

a significant role in phytoremediation (Khan, 2005) Commonly, soil conditions at contaminated sites are very hostile and low in nutrient contents, the presence of rhizobial microorganisms helps promoting the plant growth They generally function in three different ways (Glick, 2001, 1995): synthesizing compounds necessary to the plants, facilitating the uptake of nutrients from the environment (Çakmakçi et al., 2006; Lucas García et al., 2004a, b), and lessening or preventing plant diseases (Guo et al., 2004; Raj et al., 2003; Jetiyanon and Kloepper, 2002)

In many tropical countries, VZ can grow and survive without application of nitrogen and phosphorous fertilizers, especially in infertile soils (Siripin, 2000) VZ establishes a strong symbiotic association with a wide range of soil microbes in the rhizosphere that provide nutrients (nitrogen fixing bacteria, phosphate solubilizing bacteria and fungi, mycorrhizal and cellulolytic fungi) and phytohormones (plant growth regulator bacteria) for plant development The study of Sunanthaposuk (2000) on soil microbial biodiversity in the rhizosphere of VZ revealed that the total soil microorganisms and cellulolytic microbes were

in the range of 106 to 108 cells g-1, non-symbiotic nitrogen fixing bacteria and solubilizing microorganisms varied from 101-104 cells g1 soil and the endomycorrhiza were 2.5-25.5 spores per 100 g of soil Siripin et al (2000) isolated 35 N2-fixing bacteria strains from a VZ root system that differed in physiology and morphology of the colonies and the cells, and had different N2-fixing abilities Experiments on VZ inoculated with mixed strains

phosphate-of N2-fixing bacteria by using 15N isotope dilution method for measurement of N2-fixing ability showed that up to 40% of N in VZ was derived from the atmosphere (Siripin et al., 2000) Improved growth and development were observed in VZ when roots were inoculated

by N2-fixing bacteria that produced plant growth regulators promoting development of

lateral roots and total biomass Patiyuth et al (2000) demonstrated that the N2-fixing

bacteria Azospirillum grew well outside and inside of the VZ roots and produced the plant

growth hormone idole-3-acetic acid at concentration of 30-40 μg ml-1 in the broth media The inoculation of vesicular-arbuscular mycorrhiza into VZ significantly increased plant biomass and the nutrient uptake (Techapinyawat et al., 2000)

2.1.2.3 Phytoremediation of heavy metals and organic wastes

to evaluate the ability of VZ to remove heavy metals such as arsenic, lead, copper, zinc, cadmium, mercury, and so on, from contaminated soils

The potential of VZ in absorbing heavy metals has been studied through a wide range of experiments involving individual heavy metals or their combinations both in field and glasshouse conditions Naturally, contaminated soils were mainly used to investigate the ability of VZ to deal with combinations of several heavy metals, while artificially contaminated soils were used to investigate the removal of single heavy metals

Arsenic

VZ is tolerant to high concentrations of arsenic (As5+) in the soil In a study by Truong (1999), VZ survived in soils containing 414-959 mg As kg-1 In subsequent studies,

VZ survived as planted in soil amended with 450 mg As kg-1 (Sharma et al., 2006) and 500

mg As kg-1 for six months (Singh et al., 2007a)

Arsenic distribution to the various parts of VZ is quite low as it accumulates mostly

in the roots (Srisatit et al., 2003) When VZ was cultivated in soil contaminated with 125 mg

As kg-1, the concentration of As in the root system after 45 days of experiment was 9.78 mg

kg-1 dry weight (DW) compared to 0.53 mg kg-1 DW in shoots The findings were consistent with the results obtained by Truong and Baker (1998) The accumulation rate of arsenic is lower than the growth rate of VZ as a consequence the accumulation level of the heavy metal decreases with plant growth (Srisatit et al., 2003) When VZ was cultivated in soils containing 100 mg As kg-1 dry soils for 3 months, about 3 mg As kg-1 DW accumulated in shoots, whilst roots contained about 13 mg As kg-1 DW (Chiu et al., 2005)

The potential arsenic uptake of VZ can be significantly improved by using organic amendments or chelating agents Singh et al (2007a) reported that VZ grew well in soil with high dairy waste and up to 500 mg kg-1 of NaH2AsO4, when Mycorrhizae and Azotobacter

were added to the growing medium After six months the total arsenic accumulated in VZ reached 286 mg kg-1 DW in which the amount of arsenic accumulated in the roots (185.4 mg

kg1 DW) was higher than that in leaves (100.6 mg kg1 DW) Overall, VZ removed up to 62.1% arsenic from the contaminated soils The high concentration of As accumulated in VZ was due to the addition of organic compounds to promote the development of microbial population in soils that, in turn, may play an important role in arsenic hyperaccumulation

Cao et al (2003) reported that Chinese braken fern (Pteris vittata), an arsenic

hyperaccumulator, with compost amendments only removed 8.15% arsenic from artificially contaminated soil (125 mg As in form of NaH2AsO4 per kg soil) after three months of cultivation Obviously, the arsenic removal efficiency of VZ obtained from the study of Singh et al (2007a) was higher than the one reported by Cao et al (2003)

Addition of nitrilotriacetic acid (NTA) to contaminated soils enhanced As accumulation in both shoots and roots of VZ (Chiu et al., 2005) The As concentrations in plants increased with the amount of NTA With the application of 20 mmol kg-1 NTA, the level of arsenic in VZ shoots (10 mg kg-1 DW) and roots (45 mg kg-1 DW) was three times higher than that of the control treatment supplemented with 100 mg kg1 As Results from the study of Lou et al (2007) showed that among three tested chelating agents (EDTA, HEDTA

and oxalic acid) oxalic acid had the most significant effect on As accumulation in the aboveground parts of VZ with a threefold increase compared with the control Although, VZ

is not an As hyperaccumulator, it can be used to remove heavy metals from contaminated sites and can be disposed off safely elsewhere, thus gradually reducing the contaminant levels (Truong, 1999b)

Boron

The pioneering study on the ability of VZ to remove boron was carried out by Angin

et al (2008) VZ was grown in a series of pots that were artificially contaminated with B

(0-180 mg B kg-1) Boron addition did not impact dry matter yield After 90 days of experiments, plants were harvested for chemical analysis The concentration of B accumulated in roots and shoots increased with the level of B in soils The level of B in VZ roots was greater than in shoots: the treatment of 180 mg B kg-1 resulted in about 28 mg B

kg-1 DW in roots whilst shoots contained about 17 mg B kg-1 DW

Cadmium

Cadmium is very toxic to plant growth The general threshold level of Cd in soil to plant growth is about 1.5 mg kg-1 (Baker and Eldershaw, 1993) However, VZ is tolerant to high Cd concentrations in soil, with a threshold level of cadmium to VZ growth up to 60 mg

kg-1 (Truong, 1999b) VZ survived in mine tailing soils containing 32 mg kg-1 Cd (Yang et al., 2003) Moreover, VZ grown in soils spiked with 10, 20, and 40 mg kg-1 Cd showed satisfactory development with 100% survival (Vo, 2007)

The accumulation of Cd in VZ roots and shoots increased with increase in the concentration of Cd in the soil and exposure time (Vo, 2007) At 70 days exposure time, Cd

in the roots and shoots of VZ increased from 0.1417-0.2252 mg kg-1 and 0.1114-0.1522 mg

kg-1, respectively as Cd concentration in soils increased from 10-40 mg kg-1 The same

pattern was found at exposure times of 30 and 50 days The results also indicated that longer cultivation times on Cd contaminated soils corresponded to higher concentrations of Cd in

VZ roots and leaves The Cd level in roots and shoots of VZ grown in soils amended with 40

mg kg-1 Cd was 0.2252 and 0.1522 mg kg-1, respectively at 70 days of treatment compared

to 0.0909 mg kg-1 in roots and 0.0753 mg kg-1 in shoots at 30 days of treatment

The accumulation of Cd in VZ roots was much higher than in shoots VZ grown in soils contaminated with 0.58-1.66 mg kg-1 accumulated 7.77-14.2 mg kg-1 Cd in roots and very little Cd in leaves (0.13-0.58 mg kg-1) (Truong, 1999b) In addition, Cd was not detected in the shoots of VZ grown on mine tailing soils contaminated with 32 mg kg-1whilst roots had 4.98 mg kg-1 Cd after a growth period of 20 weeks (Yang et al., 2003) In other studies conducted on shale oil tailings (Xia, 2004) and contaminated farm lands (Zhuang et al., 2005), the ratio between the Cd content in VZ roots and in VZ shoots varied between 2 and 3, respectively The highest concentration of Cd in VZ shoots cited in the literature is nearly 25 mg kg-1 DW (Lai and Chen, 2004)

Similarly to Zn, the application of EDTA did not effectively increase the concentration of Cd accumulated in roots and shoots of VZ (Zhuang et al., 2005; Lai and Chen, 2004) The Cd content of VZ roots (8.1 mg kg-1) and shoots (3.9 mg kg1) under treatment of 6 mmol kg-1 was nearly equal to value obtained in roots (10 mg kg-1) and shoots (3.7 mg kg-1) under treatment without addition of EDTA (Zhuang et al., 2005) The application of EDTA was not able to enhance Cd and Zn hyperaccumulation by Thlaspi caerulescens (McGrath et al., 2006), and did not increase Cd uptake in roots and shoots of

Rumex K-1 (Rumex patientia x R timschmicus) (Zhuang et al., 2005) Unlike the cases of Zn

and Pb, the addition of organic matter did not decrease Cd uptake in VZ (Yang et al., 2003)

Copper

VZ tolerates high concentrations of Cu in soils as it survived in soils contaminated with up to 1762 mg Cu kg-1 (Wilde, Brigmon, and Dunn, 2005) In addition, VZ survived

and grew on Cu mine tailing soils that contained concentration of Cu as high as 1084 mg kg

-1 (Chiu et al., 2006) and under daily irrigation with 250 ml of 190 mg kg-1 Cu solution (CuCl2) for 30 days (Antiochia et al., 2007) In Chile, at La Africana mine VZ could grow

on copper tailings with total Cu level at 3921 mg kg-1 (Fonseca pers.com.) and at the Anglo American mine (altitude 2000m) with 2600 mg kg-1 of total Cu in the tailings (Castillo et al., 2007)

The accumulation of Cu in VZ roots and shoots is quite low VZ grown on Pb/Zn mine tailing soils containing 35 mg kg-1 Cu showed Cu uptakes in roots and shoots equal to 58.7 and 3.7 mg kg-1, respectively, after 20 weeks of cultivation (Yang et al., 2003) After nine months of growth on firing range soils, VZ accumulated 820.6 and 39.3 mg kg-1 DW of

Cu in roots and shoots, respectively (Wilde, Brigmon, and Dunn, 2005) VZ grown on Cu mine tailing soils accumulated 330 mg kg-1 Cu in roots and 10 mg kg-1 in shoots (Chiu, Ye, and Wong, 2006) after 4 months of cultivation Results at the Anglo American El Solado mine showed that 4-month-old VZ plants had 69 mg kg-1 in shoot and 371 kg-1 in roots; and 10-month-old VZ plants had 65 mg kg-1 in the shoot and 953 kg-1 in the roots indicating that older plant retained more Cu in the roots (Castillo et al., 2007) VZ irrigated daily with 250

ml of 190 ppm Cu solution for 30 days absorbed about 900 and 750 mg kg-1 Cu in roots and shoots, respectively (Antiochia et al., 2007)

The translocation ratio (TR) of Cu from roots to shoots (the percentage of shoot Cu concentration versus the percentage root concentration) in VZ is generally less than 10% (Chiu et al., 2006; Wilde, Brigmon, and Dunn, 2005; Yang et al., 2003) On the other hand,

a TR value of 19% was recorded by Truong (1999b), and up to 83% by Antiochia et al (2007) In the latter study, VZ was irrigated daily with Cu solutions containing high levels of bioavailable Cu Therefore, Cu uptake rate may have been higher than the Cu fixation rate in roots therefore a large amount of Cu escaped fixation in roots and was available to be translocated into shoots leading to high TR value Truong (1999b) considered TR values less than 5% is low, between 5 and 30% is moderate and higher than 30% is high

Cu uptake in VZ roots and shoots can be improved by application of chelating agents The influence of different chelating agents (CDTA, EDTA, EGTA, citric acid, malic acid, HEDTA, HEIDA, NTA and DTPA) on the aqueous solubility of Cu in the soils has been studied (Lou et al., 2007; Chiu et al., 2005) The most effective additive was HEIDA, which provided Cu solubility 2.5 to 34 times higher than the other chelating agents when applied at a rate of 20 nmol kg-1 The addition of HEIDA increased the concentration of Cu

in both roots and shoots up to 4 times compared to controls EDTA was also effective in increasing the content of Cu in roots 2-3 times compared to controls (Wilde et al., 2005) Among the three chelating agents HEIDA, EDTA and oxalic acid, HEIDA had the highest effect on increasing Cu content in VZ shoots (Lou et al., 2007) However, the addition of organic matter such as manure compost and sewage sludge significantly decreased the concentration of Cu in roots and shoots of VZ grown on Pb/Zn tailings (Chiu et al., 2006)

Chromium

VZ is tolerant to high concentrations of Cr up to 600 mg kg-1 in contaminated soils Truong (1999b) In the study of Hoang et al (2007), VZ was shown to survive on canal sludge containing 2290 mg Cr kg-1 that is the highest level of Cr reported in literature for Cr tolerance of VZ It survived and grew well under daily irrigation with 250 ml of water containing 623 mg kg-1 Cr for 30 days (Antiochia et al., 2007)

The majority of Cr accumulation in VZ is located in the root system VZ grown in soils containing 50, 200, and 600 mg kg-1 accumulated 404, 1170, and 1750 mg kg-1 in roots, respectively; whilst the concentration of Cr in the shoots was very low: 4, 5, and 18 mg kg-1, respectively (Truong, 1999b)

The time dependency of Cr accumulation in roots and shoots varies It was observed that whilst Cr level in VZ roots had a threefold increase over 30 days, Cr uptake in leaves was constant over time (Antiochia et al., 2007)

Cesium and 90 Strontium

Human activities—such as mining and milling of nuclear fuel, nuclear weapon testing and occasional nuclear disasters (Chernobyl accident)—release radionuclides, such

as 137Cs and 90Sr, in the soils and water Radionuclides from soils and water can enter the food chain and be transferred to humans (Shaw and Bell, 1991) The ability of VZ to remove

137Cs and 90Sr from solutions spiked with individual radionuclide (5 103 k Bq L-1) was investigated by Singh et al (2008) After 168 hours of treatment, 61% of 137Cs and 94% of

90Sr could be removed from solutions As both 137Cs and 90Sr were supplemented together in the solution, 59% of 137Cs and 91% of 90Sr were removed in the same time frame The locations of 137Cs and 90Sr accumulated in VZ are different as 137Cs accumulated principally

in the roots, whilst 90Sr mostly in shoots The addition of potassium ion (K+) and calcium ion (Ca2+) decreased the removal of 137Cs and 90Sr in the presence of VZ, respectively 90Sr

is an analogue of Ca2+ in living organisms (Kabata and Pendias, 1989), while K+ ion is a member of the same homologous series to which Cs+ belongs (Zhu et al., 2000) When grown in low level nuclear waste solution (7.5 104 Bq L-1), VZ could efficiently remove radionuclides to below detection levels within 15 days (Singh et al., 2008) The results indicated that VZ is a potential candidate plant for the phytoremediation of 137Cs and 90Sr

mg kg-1 Similar results were obtained when VZ was cultivated in firing range-soils contaminated with 3281.6 mg Pb kg-1 (Wilde et al., 2005) and mining tailing soils with 4164

mg Pb kg-1 (Yang et al., 2003)

The growth of VZ planted in highly Pb contaminated soils can be significantly improved by the addition of organic matter and/or inorganic fertilizers and arbuscular mycorrhizal fungi The application of organic matter (pig manure) was very effective in improving the biomass of VZ grown in lead mine soils, whilst the use of inorganic fertilizer was not effective (Rotkittikhun et al., 2007) Furthermore, VZ grown in Pb/Zn tailings under treatment of organic matter (sewage sludge) had biomass over two times higher than in the control treatment In particular, the combination of sewage sludge and inorganic fertilizers increased biomass nearly six fold compared to the control (Chiu et al., 2006) The study of Wong et al (2007) showed that the inoculation of VZ with arbuscular mycorrhizal fungi

(Glomus mosseae and G intraradices) significantly increased growth performance of VZ on

Pb contaminated soils Mycorrhizal colonization increased Pb uptake by plants cultivated in soils with low metal concentrations (at 0 and 10 mg Pb kg-1) However, at higher heavy metal concentrations (100 and 1000 mg Pb kg-1) it decreased Pb uptake

The addition of organic matter into Pb contaminated soils reduced the total and extractable lead in soil (Rotkittikhun et al., 2007) VZ showed a decrease in Pb uptake when grown in soil modified with organic matter, such as pig manure, sewage sludge, manure compost and domestic refuse (Rotkittikhun et al., 2007; Chiu et al., 2006; Yang et al., 2003) Organic matter was shown to be very effective in reducing Pb bioavailability, thus lowering

Pb uptake of plants (Ye et al., 1999; Wong and Lau, 1985; Scialdone, Scogamiglio, and Ramunni, 1980) On the other hand, the application of inorganic fertilizers (N:P:K = 15:15:15) at a high rate (150 mg kg-1) significantly increased lead uptake in VZ (Rotkittikhun et al., 2007)

Lead concentration in the shoots and roots of VZ increased significantly with increasing Pb concentration in the soils (Rotkittikhun et al., 2007; Wong et al., 2007; Chen, Shena, and Lib, 2004) The level of Pb in shoots increased from 0.82 to 43.0 mg kg-1 DW, and in the root from 60 to 556 mg kg-1 DW as VZ was grown in the 500, 2500 and 5000 mg

kg-1 Pb amended soils (Chen et al., 2004) The concentration of Pb in roots presented a similar trend, increasing from 19.4 to 449 mg kg-1 DW as the level of Pb in soil was increased from 0 to 1000 mg (Wong et al., 2007) In the same study, it was observed that Pb

in VZ shoots varied between 6.98 and 17.4 mg kg-1 DW with increasing soil contamination level The concentration of Pb increased from 12.5 to 375 mg kg-1 DW in shoots and from 18.7 to 4940 mg kg-1 DW in roots as VZ was grown in soils spiked with 0, 100, 1000 and

10000 mg Pb kg-1 (Rotkittikhun et al., 2007)

Experiments have proven that VZ is a lead hyperaccumulator According to Shah and Nongkynrih (2007), a plant is a Pb hyperaccumulator if it can accumulate Pb at least 0.1% of DW equivalent to 1000 mg Pb kg-1 DW VZ grown in soils amended with 10000

mg Pb kg-1 accumulated up to 4940 and 359 mg Pb kg-1 DW in roots and shoots respectively with root/shoot ratio about 0.7 (Rotkittikhun et al., 2007) From this information, it can be estimated that VZ accumulated Pb over 0.22% of its total dry matter Moreover, VZ grown

in the experiment of Antiochia et al (2007) accumulated 0.4% and 1% Pb in shoots and roots, respectively after 30 days of experiment The higher Pb uptake rate of VZ in this study compared to others was due to the fact that Pb was supplied daily with 250 ml of 621 ppm Pb in the form of PbCl2 solution making Pb readily available for plant absorption Lead concentrations in VZ tissue samples up to 1390-1450 mg kg-1 DW were detected after nine months of cultivation on firing range soils (Wilde et al., 2005)