Supercritical CO2 extraction of vetiver essential oil 
& 
Economic incentive for use of vetiver grass in 
phytoremediation  

 
by 

 
 

Luu Thai Danh 

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
‘I hereby declare that this submission is my own work and to the best of
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

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.

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.

iii


Table of Contents

1
2

Abstract………………………………………………………………………....
Acknowledgements……………………………………………………………..
List of figures……………………………………………………………………
List of tables…………………………………………………………………….
Abbreviation……………………………………………………………………
List of publications……………………………………………………………..
Introduction…………………………………………………………………….
Literature review……………………………………………………………….

Ethidium bromide…………………………………
Benzo[a]pyrene…………………………………...
iv

i
iii
viii
x
xii
xiii
1
5
5
6
7
7
8
8
10
10
15
16
16
16
18
18
19
21
22
22

2.2.7 References…………………………………………………………
2.3 Supercrtical fluid extraction ………………………………………………
2.3.1 Introduction……………………………………………………….
2.3.2 Characteristics of supercritical fluid extraction…………………..
2.3.3 Selection of the operating conditions…………………………….
2.3.3.1 Pressure………………………………………………….
2.3.3.2 Temperature…………………………………………….
2.3.3.3 CO2 flow rate……………………………………………
2.3.3.4 Particle size……………………………………………...
2.3.3.5 Time…………………………………………………….
2.3.3.6 Modifiers or co-solvents………………………………..
2.3.4 Optimization of operating conditions…………………………….
2.3.5 References…………………………………………………………
Economic incentive for applying vetiver grass to remediate lead, copper
and zinc contaminated soils………………………………………………….....

37
37
38
40
51
51
51
53
53
54
54
55
55
57

3.2.1 Plant materials…………………………………………………….
3.2.2 Soil treatments……………………………………………………
3.2.3 Plant cultivation…………………………………………………...
3.2.4 Extraction…………………………………………………………
v

78


3.2.5

4

Gas-chromatographic and Gas chromatography-Mass
spectrometry analysis……………………………………………
3.2.6 Statistical analysis……………………………………………….
3.3 Results and discussion…………………………………………………...
3.3.1 Soil characteristics………………………………………………
3.3.2 Growth performance…………………………………………….
3.3.3 Content and yield of vetiver oil………………………………….
3.3.4 Heavy metal contents in vetiver roots and shoots……………….
3.3.5 Chemical components of vetiver essential oil…………………...
3.4 Discussion………………………………………………………………..
3.5 Conclusion……………………………………………………………….
3.6 References………………………………………………………………..
Effect of calcium on growth performance and essential oil of vetiver grass
grown on lead contaminated soils……………………………………………
4.1
4.2


84
85
86
86
87
88
89
91
94
98
99
104
105
108
108
108
109
109
110
110
111
111
111
112
114
115
116
120
121
125

Extraction of Vetiver essential oil by ethanol modified supercritical carbon
dioxide……………………………………………………………………………
6.1
6.2

Introduction………………………………………………………………..
Materials and methods…………………………………………………….
6.2.1 Plant material preparation…………………………………………
6.2.2 Extraction………………………………………………………….
6.2.3 Experimental design………………………………………………
6.2.4 Gas chromatography and gas chromatography–mass spectrometry
analysis…………………………………………………………….
6.2.5 Heavy metal analysis……………………………………………...
6.2.6 Statistical analysis…………………………………………………
6.3 Results and discussion…………………………………………………….
6.3.1 Kinetic study………………………………………………………
6.3.2 Effect of operating parameters on oil yield and optimization of
ethanol-modified-SCE…………………………………………....
6.3.3 Chemical components of ethanol-modified SCE extracts………..
6.3.4 Comparison with hydrodistillation and pure SCE………………..
6.3.5 Heavy metals contents in SCF extracts……………………………
6.4 Conclusion…………………………………………………………………
6.5 References…………………………………………………………………
7 Conclusions and Recommendations……………………………………………
7.1 Summary of conclusions………………………………………………….
7.2 Recommendations…………………………………………………………
Appendix A………………………………………………………………………….
vii

130

187
187
189
190


List of Figures
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………………………………………….
Central composite design with three operating conditions of supercritical fluid


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

Response surface plot showing the effect of pressure and temperature on khusimol
content at the extraction time of 50 minutes…………………………………………

149

2.1.

5.2.

5.9.

132

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………………………
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


The effect of pressure and temperature on oil yield at the ethanol concentration of
5%...............................................................................................................................

175

ix


List of Tables
2.1.

Threshold levels of heavy metals to vetiver growth (Truong 1999b) based on single
element experiments………………………………………………………………… 12

2.2.

Concentrations of heavy metals accumulated in VZ roots and shoots (Truong,
1999b)……………………………………………………………………………….

14

2.3.

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

25

2.4.



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
Pb, Cu and Zn………………………………………………………………………..

92

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…..



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



Metal contents of vetiver roots before and after extraction by pure SCE and ethanol
modified SCE……………………………………………………………………….

182

6.5.
6.6.

xi


Abbreviation

VG

Vetiver grass

VZ

Vetiveria zizanioides

SCE

Supercritical carbon dioxide extraction

SFE

Supercritical fluid extraction


Copper

Hg

Mercury

Ni

Nickel

Pb

Lead

Se

Selenium

Zn

Zinc

137

137

90

Cs



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. 6th
International Conference on Phytotechnologies, St. Louis, Missouri, USA, December 1-4,
2009.

xiii 
 


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.

xiv 
 


1. Introduction


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

2



Optimization of ethanol-modified supercritical CO2 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 coextract heavy metals with essential oil from vetiver roots.

4


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

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).

6


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

(Hengchaovanich, 1998) and acquires a total length of 7 meters after 36 months (Lavania,
2003). The features of the root system support the survival of VZ under extreme drought
conditions as it can utilize deep soil moisture. The root system prevents VZ from
dislodgement under high velocity flows (Hengchaovanich, 1999; Hengchaovanich and
Nilaweera, 1998). Stiff and erect stems of VZ, up to 2-m high, can stand also in relatively
deep-water flow (Truong, 2002). The peculiarity of VZ's root system ensures high contact
surfaces with soil particles and, consequently, efficient phytoremediation and possible
prevention of leaching of contaminants into ground waters (Figure 2.1).

8


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