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 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.’ Signed …………………………………………… Date …………………………………………… COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968 I retain all proprietary rights, such as patent rights I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only) I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed …………………………………………… Date …………………………………………… AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ 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 i 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 ii 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 iii Table of Contents Abstract……………………………………………………………………… Acknowledgements…………………………………………………………… List of figures…………………………………………………………………… List of tables…………………………………………………………………… Abbreviation…………………………………………………………………… List of publications…………………………………………………………… Introduction…………………………………………………………………… Literature review……………………………………………………………… 2.1 Vetiver grass, Vetiveria zizanioides: a choice plant for phytoremediation of heavy metals and organic wastes……………………………………… 2.1.1 2.1.2 Introduction……………………………………………………… Vetiveria zizanioides and its outstanding characteristics………… 2.1.2.1 Vetiveria zizanioides…………………………………… 2.1.2.2 Outstanding characteristics of Vetiveria zizanioides…… Morphological and genetic characteristics………… Agronomic characteristics………………………… Physiological characteristics ……………………… Microorganism association ………………………… 2.1.2.3 Phytoremediation of heavy metals and organic wastes… Heavy metals………………………………………… Arsenic…………………………………………… Boron…………………………………………… Cadmium………………………………………… Copper…………………………………………… Chromium……………………………………… 137 Cesium and 90Strontium……………………… Lead……………………………………………… Zinc……………………………………………… Multi-heavy metals……………………………… Phytoremediation potential of vetiver versus other plant species……………………………… Organic Wastes……………………………………… Phenol…………………………………………… 2,4,6-Trinitroluene……………………………… Ethidium bromide………………………………… Benzo[a]pyrene………………………………… iv i iii viii x xii xiii 5 7 8 10 10 15 16 16 16 18 18 19 21 22 22 28 30 31 35 35 35 36 37 Petroleum hydrocarbon………………………… Atrazine………………………………………… 2.1.3 Conclusion……………………………………………………… 2.1.4 References………………………………………………………… 2.2 Vetiver essential oil……………………………………………………… 2.2.1 Introduction……………………………………………………… 2.2.2 Production ……………………………………………………… 2.2.3 Properties ……………………………………………………… 2.2.4 Chemical composition ………………………………………… 2.2.5 Applications……………………………………………………… 2.2.5.1 Perfumery……………………………………………… 2.2.5.2 Aromatherapy………………………………………… 2.2.5.3 Insecticides……………………………………………… 2.2.5.4 Herbicides……………………………………………… 2.2.5.5 Antioxidant activity…………………………………… 2.2.5.6 Anticancer activity…………………………………… 2.2.5.7 Antimicrobial activity………………………………… 2.2.5.8 Other uses……………………………………………… 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 57 58 58 58 60 64 64 66 67 67 68 68 69 69 70 71 72 3.1 3.2 79 81 81 81 83 83 Introduction……………………………………………………………… Materials and methods…………………………………………………… 3.2.1 Plant materials…………………………………………………… 3.2.2 Soil treatments…………………………………………………… 3.2.3 Plant cultivation………………………………………………… 3.2.4 Extraction………………………………………………………… v 78 3.2.5 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 Introduction…………………………………………………………… Materials and methods………………………………………………… 4.2.1 Plant materials………………………………………………… 4.2.2 Soil treatments………………………………………………… 4.2.3 Plant cultivation………………………………………………… 4.2.4 Extraction……………………………………………………… 4.2.5 Gas-chromatographic and Gas chromatography-Mass spectrometry analysis…………………………………………… 4.2.6 Statistical analysis……………………………………………… 4.3 Results and discussion………………………………………………… 4.3.1 Soil properties…………………………………………………… 4.3.2 Effect of lime on soil pH………………………………………… 4.3.3 Vetiver growth performance…………………………………… 4.3.4 Heavy metals in roots and shoots of vetiver…………………… 4.3.5 Oil content and oil yield………………………………………… 4.3.6 Chemical components of vetiver essential oil…………………… 4.4 Conclusion……………………………………………………………… 4.5 References………………………………………………………………… Response surface method applied to supercritical CO2 extraction of Vetiveria zizanioides essential oil……………………………………………… 5.1 Introduction……………………………………………………………… 5.2 Materials and methods………………………………………………… 5.2.1 Plant material preparation……………………………………… 5.2.2 Soxhlet extraction………………………………………………… 5.2.3 Hydro-distillation………………………………………………… vi 84 85 86 86 87 88 89 91 94 98 99 104 105 108 108 108 109 109 110 110 111 111 111 112 114 115 116 120 121 125 126 129 129 129 130 5.2.4 5.2.5 5.2.6 5.2.7 5.2.8 Supercritical CO2 extraction…………………………………… Experimental design…………………………………………… Kinetic study…………………………………………………… Yield calculation……………………………………………… Gas chromatography and gas chromatography-mass spectrometry analysis………………………………………………………… 5.3 Results and discussion………………………………………………… 5.3.1 Optimization of SCF extractions……………………………… 5.3.2 Kinetic study…………………………………………………… 5.3.3 Chemical components of SCE vetiver extract………………… 5.3.3.1 Khusimol……………………………………………… 5.3.3.2 Zizanoic acid………………………………………… 5.3.4 Comparison with conventional extraction methods…………… 5.4 Conclusion……………………………………………………………… 5.5 References……………………………………………………………… 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………………………………………………………………… Conclusions and Recommendations…………………………………………… 7.1 Summary of conclusions………………………………………………… 7.2 Recommendations………………………………………………………… Appendix A………………………………………………………………………… vii 130 131 134 134 135 136 136 143 144 147 149 150 153 154 158 159 162 162 162 164 165 166 166 167 167 168 176 179 180 183 184 187 187 189 190 List of Figures Vetiveria zizanioides grass from left to right: mature plant, thick hedge, deep and extensive root system……………………………………………………………… 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 extraction…………………………………………………………………………… 131 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 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 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 6.1 6.2 viii 164 discussed previously, the operation at low temperature, low pressure and high concentration of added ethanol produced high oil yield Therefore, the yields produced at this condition (100 bar, 40ºC and 15% ethanol) and the optimal conditions were used to compare with the yield obtained by hydrodistillation and SCE (operated at 190 bar and 50C) The optimal condition (Ex 8) of modified SCE produced the highest oil yield as compared to other processes This yield was over three times and nearly double that of hydrodistillation and SCE without modifiers, respectively When SCE with ethanol as a modifier was operated at 100 bar, 40C and 15 vol% ethanol (Ex 5) it produced a yield that was 90% of the highest yield and 142% of pure SCE yield In summary, operation at low temperature and pressure and at high concentration of added ethanol is recommended for the extraction of vetiver oil Results indicate that however high ethanol levels in the extracting medium determine favorable process yields, the combination of ethanol with CO2 is essential to generate a product of commercial interest Table 6.5 Yield of vetiver oil extracted by hydrodistillation, pure SCE and ethanolmodified SCE Operating conditions Methods Time (min) Yields (%) Hydro-distillation 720 Temperature (C) 100 SCE 105 50 190 3.74  0.12 Ex 105 40 100 15 5.31 Ex 105 50 190 15 5.90 Pressure (bar) NA Ethanol (%) NA 1.69  0.07 Ethanol-modified-SCE 6.3.5 Heavy metals contents in SCF extracts In order to be accepted for application in the perfumery and food industries, vetiver extracts must not contain any toxic substances that may cause a health hazard to the consumers However, vetiver plants have great potential in accumulating high 180 concentrations of heavy metals, particularly Pb, Zn and Cu, in their roots This characteristic may cause cross-contamination of vetiver oils extracted from the roots with heavy metals Essential oils of vetiver grown on heavy metal contaminated soil extracted by hydrodistillation were shown to contain no heavy metals (Danh et al., 2010) However, there are no studies reported in the literature about heavy metal contents in the essential oil of vetiver or other plant extracted by supercritical fluids A standard procedure of heavy metal analysis of plant materials requires 0.5 gram of sample However, the oil yields of pure SCE and ethanol-modified SCE were smaller than the suggest level (Table 6.6) Therefore, an indirect method was employed to determine the amount of metals in oil extracts by comparing metal contents in plant materials before and after extraction (Zheljazkov et al., 2006) Metal contents in the roots of vetiver grown on Pb, Zn and Cu contaminated soils are shown in Table 6.6 All analyzed metals, particularly Pb, Zn and Cu, showed no significant differences in plant materials before and after extraction The finding indicated that all metals were retained in plant materials during extraction As accumulated in roots, metals tend to form metal-organic complexes that can not be dissolved by supercritical CO2 or ethanol-modified-supercritical CO2 It can be concluded that vetiver oils extracted by SCE contained no or negligible amounts of metals However, metal analysis on larger samples of vetiver oils is advisable to further confirm that oil extracted from VG grown on heavy metal contaminated soils using supercritical fluid technology can be acceptable in the market 181 Table 6.6 Metal contents of vetiver roots before and after extraction by pure SCE and ethanol modified SCE Metals (mg kg-1 DW) Before extraction Ca After extraction Pure SCE * Ethanol modified SCE ** 1890.0 ± 31.5 a 1896.7 ± 28.9 a 1904.5 ± 24.1 a Cd 0.3 ± 0.0 a 0.1 ± 0.1 a 0.2 ± 0.2 a Cu 243.0 ± 10.6 a 244.6 ± 5.4 a 245.4 ± 7.0 a Fe 3218.5 ± 38.1 a 3264.4 ± 64.8 a 3277.5 ± 25.1 a K 7205.6 ± 66.4 a 7311.8 ± 45.0 a 7309.7 ± 81.2 a Mg 2861.2 ± 23.8 a 2873.5 ± 1.9 a 2858.6 ± 28.1 a Na 3862.9 ± 69.0 a 3880.1 ± 28.1 a 3898.6 ± 31.5 a Ni 18.7 ± 1.5 a 20.6 ± 0.9 a 19.7 ± 0.6 a Pb 834.7 ± 12.6 a 853.8 ± 15.5 a 851.1 ± 8.5 a Zn 868.2 ± 25.3 a 877.2 ± 19.5 a 880.1 ± 13.3 a Oil yield (%) NA 1.32  0.32 a 2.1  0.24 b Note: the same letter in the same row indicated no significant difference at 5% level of significance * pure SCE was performed at 190 bar and 50C ** ethanol-modified-SCE was operated at 190 bar, 50C and 15% ethanol 182 6.4 Conclusion The application of the response surface method coupled with the central composite design allowed a full investigation of the effect of pressure, temperature and amounts of added ethanol on vetiver extracts by using SCE Based on the statistical and graphic analysis, pressure and amounts of added ethanol were found to have the most significant influence on vetiver oil yield, while temperature and interactive effects of all tested parameters were not significant The SCE with ethanol modified CO2 at 190 bar, 50ºC and 15% ethanol produced the highest oil yield (5.9%) over three times and nearly double that of hydrodistillation and SCE with pure CO2, respectively Interestingly, the increment of pressure at high concentrations of ethanol resulted in a negligible increase in oil yield In addition, the rise in temperature generally caused a slight reduction in oil yield Therefore, the extraction at low temperature, low pressure and high concentration of ethanol (100 bar, 40ºC and 15% ethanol) produced a similar yield (5.3%) compared to that of the optimal conditions The chemical analysis of extracts showed no significant difference in chemical compositions (except for zizanioic acid content) of all SCE extracts Heavy metal analysis of plant materials before and after extraction indicated that in all cases, SCE produced extracts free of metals In short, ethanol-modified-SCE performed at low temperature, low pressure and high concentration of ethanol has great application potential for producing high yields of vetiver oil with low pressure extraction apparatus Vetiver extracts would be suitable for a wide range of applications, such as aromatherapy, food, and perfumery 183 6.5 References Antiochia, R., Campanella, L., Ghezzi, P and Movassaghi K 2007 The use of vetiver for remediation of heavy metal soil contamination Anal Biochem., 388, 947-956 Armando, T.Q., Oro, K., Shunsaku, K and Takashi, M 2006 Recovery of oil components of okara by ethanol-modified supercritical carbon dioxide extraction Bioresour Technol., 97, 1509–1514 Caredda, A., Maringiu, B., Porcedda, S., Soro, C 2002 Supercritical carbon dioxide extraction and characterization of Laurus nobilis essential oil J Agric Food Chem., 50, 1492–1496 Castillo, M., Fonseca, R and Candia., J.R 2007 Report on the pilot study on the use of Vetiver grass for Cu mine tailings phytostabilisation at Anglo American El Solado mine, Chile Fundacion Chile (report in Spanish) Cochran, H.D., Lee, L.L 1989 Solvation Structure in Supercritical Fluid Mixtures Based on Molecular Distribution Functions ACS Symp Ser., 406, 27 Danh, L.T., Mammucari, R., Truong, P and Foster, N 2009a Response surface method applied to supercritical carbon dioxide extraction of Vetiveria zizanioides essential oil Chem Eng J., 155, 617-626 Danh, L.T., Truong, P., Mammucari, R and Foster, N 2009b Vetiver grass, Vetiveria zizanioides: a choice plant for phytoremediation of heavy metals and organic wastes Int J Phytorem., 11, 664 – 691 Danh, L.T., Truong, P., Mammucari, R and Foster, N 2010 Economic incentive for applying Vetiver grass to remediate lead, copper and zinc contaminated soils Int J Phytorem., Article in Press Debenedetti, P.G 1987 Clustering in Dilute, Binary Supercritical Mixtures: AFluctuation Analysis Chem Eng Sci., 42, 2203 Dobbs, J.M., Wong, J.M and Johnston, K.P 1986 Non-polar co-solvents for solubility enhancement in supercritical fluids J Chem Eng Data., 31, 303 Dobbs, J.M., Wong, J.M and Johnston, K.P 1986 Nonpolar cosolvents for solubility enhancement in supercritical fluids, J Chem Eng Data, 31, 303 Eckert, C.A., Ziger, D.H., Johnston, K.P and Kim, S 1986 Solute Partial Molar Volumes in Supercritical Fluids J Phys Chem., 90, 2738 184 Hasbay-Adil, I., Cetin, H.I., Yener, M.E and Bayındirli, A 2007 Subcritical (carbon dioxide + ethanol) extraction of polyphenols from apple and peach pomaces, and determination of the antioxidant activities of the extracts J Supercrit Fluids, 43, 55–63 Kajimoto, 0., Futakami, M., Kobayashi, T and Yamasaki, K 1988 Charge-Transfer-State Formation in Supercritical Fluids: (N,N-Dimethy1amino) benzonitrile in CF3H J Phys Chem., 92, 1347 Ke, J., Mao, C., Zhong, M., Han, B and Yan, H 1996 Solubilities of salicylic acids in supercritical carbon dioxide with ethanol cosolvent, J Supercrit Fluids, 9, 82 Ke, J., Mao, C., Zhong, M., Han, B and Yan, H 1996 Solubilities of salicylic acids in supercritical carbon dioxide with ethanol cosolvent, J Supercrit Fluids., 9, 82 Kopcak, U and Mohamed, R.S 2005 Caffeine solubility in supercritical carbon dioxide/cosolvent mixtures J of Supercrit Fluids, 34, 209–214 Lim, J.S., Lee, Y.Y and Hai, S.C 1994 Phase equilibria for carbon dioxide-ethanol-water system at elevated pressures J Supercrit Fluids, 7, 219-230 Massardo, D.R., Senatore, F., Alifano, P., Giudice, L.D., Pontieri, P 2006 Vetiver oil production correlates with early root growth Biochem Syst Ecol., 34, 376-382 Myers, R.H and Montogomery, D.C 2002 Response of surface methodology, Process and Product Optimization using Designed Experiments (2nd ed.) (Wiley, New York) Petsche, I.B and Debenedetti, P.G 1989 Solute-Solvent Interactions in Infinitely Dilute Supercritical Mixtures: A Molecular Dynamics Investigation J Phys Chem., 93, 7075 Rozzi, N.L and Singh, R.K 2002 Supercritical fluids and the food industry Comp Rev Food Sci Food Safe., 1, 33–44 Ruckenstein, E and Shulgin I 2002 The solubility of solids in mixtures composed of a supercritical fluid and an entrainer Fluid Phase Equilib., 200, 53–67 Ting, S.S.T., Macnaughton, S.J., Tomasko, D.L and Foster, N.R 1993a Solubility of naproxen in supercritical carbon dioxide with and without cosolvents Ind Eng Chem Res., 32, 1471 Ting, S.S.T., Tomasko, D.L., Macnaughton, S.J and Foster, N.R 1993b Chemical-physical interpretation of cosolvents effects in supercritical fluids, Ind Eng Chem Res., 32, 1482 Xiang, X., Yanxiang, G., Guangmin, L., Qing, W and Jian, Z 2008 Optimization of supercritical carbon dioxide extraction of sea buckthorn (Hippopha thamnoides L.) oil using response surface methodology, LWT, Food Sci Technol., 41, 1223–1231 185 Yonker, C.R and Smith., R.D 1988 Solvatochromic behaviour of binary supercritical fluids: The carbon dioxide/2-Propanol system J Phys Chem., 92, 1261 Yuan, H., Gao, G.T and Zeng, X.C 1997 Effects of the cosolvent energy parameter and dipolar strength on solute residual chemical potential, Fluid Phase Equilib., 138, 61 Zheljazkov, V.D., Craker, L.E and Xing, B 2006 Effects of Cd, Pb, and Cu on growth and essential oil contents in dill, peppermint, and basil Environ Exp Bot., 58, 9–16 186 Conclusion and Recommendations 7.1 Summary of conclusions Vetiver grass is an excellent candidate for the revegetation of heavy metal mine tailings in order to stop surface soil erosion and off-site spreading of contaminants This is due to the fact that VG is a perennial tropical grass with dense and massive root system and produces high biomass It has high tolerance and adaptability to a wide range of climatic and environmental conditions including high salinity, sodicity, high levels of heavy metals, and agrochemicals VG has great potential for phyto-stabilization of heavy metal contaminated soils in order to stop the spreading of contaminants into surrounding areas; in fact VG can accumulate high concentration of heavy metals in its roots, particularly lead and zinc VG can be used for phytoextraction with the addition of chelating agents Furthermore, VZ also has the ability to take up and promote biodegradation of organic wastes, so it can be used for phytoremediation of these contaminants The use of VG for phytoremediation of Zn or Cu contaminated soils can be enhanced by a cash return from production of vetiver essential oil At low and moderate concentrations, Zn and Cu did not have significant impact on plant height, biomass production and essential oil yield that, however, were adversely affected at high concentrations Regardless of its concentration, Pb had a detrimental effect on plant growth, essential oil production and chemical compositions At high concentration of Pb in soil plants could not survive Heavy metals were retained in plant materials during hydrodistillation The application of N reduced the oil yield, indicating a great applicative potential as grower not need to apply artificial N fertilizers Soil amelioration with Ca dramatically improved VG survival on soils with high levels of Pb contamination The application of Ca at about half of Pb concentration value in 187 soils is sufficient to improve plant survival and growth helping the accumulation of high concentrations of Pb in vetiver roots However, Ca amendment did not mitigate the deleterious effects of Pb on vetiver essential oil yield and chemical composition The effect of pressure, temperature and time of supercritical CO2 extraction (SCE) were investigated and optimized by the response surface method Only pressure had a significant effect on vetiver oil yields; temperature had moderate quadratic effect and interaction effect with pressure on oil yield Optimal operating conditions were identified as 190 bar, 50ºC and 100 minutes and produced the oil yield of 1.38 % on a dry root basis The optimum yield obtained by SCE was about four times higher than that of hydrodistillation The content of khusimol, a desired and main component of vetiver oil, was significantly affected by pressure and by the interaction of pressure and temperature SCE operating conditions producing vetiver oil with high content of khusimol and low content of zizanoic acid (ideal for use in perfumery) corresponded to low yields The addition of ethanol in supercritical CO2 improved the efficiency of SCE The analysis of the response surface method showed that only pressure and ethanol concentration had a significant influence on vetiver oil yield: oil yield increased with both these parameters The SCE with ethanol as a modifier produced the highest oil yield at 190 bar, 50ºC and 15% ethanol The yield obtained was nearly double that obtained under the same operating pressure and temperature in absence of the modifier Interestingly, extraction at lower temperature and pressure (100 bar, 40ºC and 15% ethanol) produced a yield similar to the optimal conditions The chemical analysis of extracts showed no significant difference in chemical compositions within all the SCE experiments, except for zizanioic acid content Heavy metals were not co-extracted by any of the extraction methods In summary, SCE with ethanol as a modifier operated at relatively low temperature and pressure can produce vetiver oil extracts with high yields and is of applicative interest In addition, the extracts from plants grown on heavily contaminated soils were free from heavy metal contaminants thus safe for various applications, such as aromatherapy, food and perfumery 188 7.2 Recommendations Vetiver grass can be used for soil phytoremediation of a wide range of heavy metals and organic wastes However, in this work only three heavy metals (Pb, Cu and Zn) were tested for their effect on vetiver oil yield and quality Further investigation of other heavy metals and organic wastes is necessary to promote the application of vetiver grass for phytoremediation of these pollutants in soils The addition of organic matters improves growth and survival of plants cultivated on heavy metal contaminated soils It is worth to study the effect of the addition of organic matter to contaminated soils on yield and quality of vetiver oil Vetiver extracts from this work were only analysed for chemical composition Further tests of SCE extracts, such as specific gravity, optical rotation, antioxidant and other biological activities are required to evaluate the extracts applicability to perfumery, pharmaceutical and food industies, and for insect and weed control A direct spiking of ethanol on vetiver roots materials can represent an economical and simpler way of performing SCE with modifiers compared to the technique used in this work Direct spiking could reduce the operation cost of SCE process, however, it would present drawbacks such as concentration gradients within the matrix and inconsistent results, therefore an accurate design and evaluation of the process are recommended (Modey et al., 1996; Pourmortazavi and Hajimirsadeghi, 2007; Marsili and Callahan, 1993) Sonication could improve process yields and dynamic through the rapid attainment of high concentrations of solutes in the extracting media The effect of sonication may be investigated to improve the efficiency of SCE of vetiver oil 189 Appendix A: Chemical components of Vetiver essential oil No 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 KI Compounds Beta-Pinene Phenylacetaldehyde o-Guaiacol Verbenone trans-Pinocarvone Myrtenol Decanal p-Vinyl guaicol 1-nonanal nonanoic acid 1-decanal Alpha-Cubebene Alpha-Cyperene cycloisosativene Longicyclene Alpha-Ylangene Alpha-copaene 9,10-dehydro-2-norzizaene Alpha-Duprezianene Beta-Elemene (Z)-Isoeugenol Acoradiene II Alpha-Funebrene Trans-2-nor-zizaene Beta-cubebene Cyperene Alpha-Cedrene Beta-funebrene Cascarilladiene (Eudesma-5,7-diene) Beta-caryophyllene Acora-2,4-diene (Epimer A) Beta-cedrene Beta-copaene Beta-gurjunene 10-epi-Acora-2,4-diene (Epimer B) 11,12-13-tri-nor-Eremophil-1(10)-en-7-one Alpha-guaiene guaia-6,9-diene (6,9-Guaiadiene) Acora-3,9-diene gamma-elemene (E)-Isoeugenol Isoeugenol Aromadendrene Preziza-7(15)-ene prezizaene Khusimene (Ziza-6(13)-ene) Zizaene Selina-4(15)7-diene Dimethyl-6,7-bicyclo-[4,4,0]-deca-10-en-4-one Selina-4,7-diene Beta-acoradiene Gamma-gurjunene 975 1040 1087 1201 1309 1142 1160 1196 1203 1294 1345 1352 1351 1349 1350 1371 1372 1375 1379 1405 1388 1390 1407 1410 1387 1390 1402 1406 1417 1430 1415 1416 1419 1414 1413 1436 1425 1424 1430 1434 1420 1442 1440 1442 1442 1442 1451 1449 1449 1451 1445 1452 1453 1455 1457 1465 1469 1471 1472 190 1459 1462 1375 1468 1450 1453 1449 1452 1452 1475 1453 54 55 56 57 59 60 61 Amorpha-4,7(11)-diene Alpha-Amorphene ar-Curcumene 4,7-Epoxy-spirovetiva-2,11-diene 12,13-Di-nor-6(7 >8)-abeo-eremophil-1(10)-en-7-one Alpha-vetispirene cis-Eudesma-6,11-diene 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 107 108 109 110 111 112 113 114 116 Beta-selinene Beta-Vetispirene ( Spirovetiva-1,7(11),10(14)-triene) Delta-Selinene (Eudesma-4,6-diene) 8α-Methyl-11,12,13-tri-nor-eremophil-1(10)-en-7-one 5,11-Epoxy-eudesmane (Dihydroagarofuran) Gamma-amorphene Eudesm-2,4(11),11-triene Valencene Alpha-Muurolene Delta-Amorphene Beta-Bisabolene 7,10-Epoxy-eremophila-1,11-diene Sesquicineole (4,7-epoxy-bisabol-10-ene) 13-nor-Opposite-4(15)-en-11-one 1-Dodecanamide, N,N-dimethyl- a Gamma-Cadinene 7Beta, 10b-epoxy-4beta-eremophila-1,11(12)-diene Nootkatene Isocalamenene Spirovetiva-1(10),7(11)-diene Eremophila-1(10),7(11)-diene Delta-Cadinene 11,12-13-tri-nor-cis-Eudesm-5-en-7-one 3,10-Epoxy-muurol-4-ene (lentideus ether) Gamma-Vetivenene (eremophila-1(10),6,11-triene) 6-epi-Shyobunol (elema-1,3-diene-6α-ol) Omega-cadinene Alpha-calacorene Selina-4(15),7(11)-diene 13-nor-cis-Eudesm-6-en-8-one 11,12-13-tri-nor-trans-Eudesm-5-en-7-one 10,11-Epoxy-eremophil-1-ene Furopelargone A Kessane (10,11-epoxy-trans-guaiane) Eudesma-4(15),7(11)-diene 7-epi-cis-Dracunculifoliol Eudesma-3,7(11)-diene (Selina-3,7(11)-diene) Beta-Elemol Alpha-Agarofuran (5,11-epoxy-eudesm-3-ene) 11,12-13-tri-nor-cis-Eudesm-5,8-dien-7-one 15-nor-Prezizaan-7-one 13-nor-Eudesm-5-en-11-one (Epimer A) Elemol 6,12-Epoxy-elema-1,3-diene 13-nor-Eudesm-5-en-11-one (Epimer B) Beta-Vetivenene (eremophila-1,7(11),9-triene) 13-nor-eremophil-1(10),6-dien-11-one Beta-Calacorene Eudesma-5,7-dien-4-ol (cascarilladienol) 12-nor-2,3-Epoxy-ziza-6(13)-ene Elema-1,11-dien-15-al (Epimer A) Elema-1,11-dien-15-al (Epimer B) 1473 1473 1478 1478 1484 1484 1484 1484 1493 1476 1477 1481 1490 1474 1468 1471 1491 1490 1498 1486 1486 1493 1506 1484 1495 1492 1495 1496 1497 1497 1497 1507 1507 1499 1512 1507 1510 1512 1517 1523 1525 1484 1495 1503 1519 1512 1496 1500 1512 1510 1502 1504 1517 1517 1517 1517 1517 1531 1508 1525 1518 1526 1527 1534 1528 1528 1528 1528 1528 1532 1532 1540 1540 1540 1540 1540 1540 1547 1547 1547 1551 1551 1551 1551 1562 191 1508 1520 1540 1533 1552 1538 1547 1571 1550 1603 1552 1561 1566 1548 1574 1566 1554 1542 1548 1554 117 118 119 120 121 122 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 Gamma-Guaiene Cyclocopacaphan-12-al (2 epimers) 15-nor-Funebran-3-one 12-nor-Preziza-7(15)-en-2-one (tentatively) 1,10-Epoxy-amorph-4-ene cis-Eudesm-6-en-11-ol Khusimone (12-nor-Ziza-6(13)-en-2-one) Salvial-4(14)-en-1-one 6,12-Epoxy-spiroax-4-ene cis-Guai-6-en-10-ol Selina-6-en-4-ol 12-nor-ziza-6(13)-en-2β-ol trans-Dracunculifoliol 13-nor-cis-Eudesm-6-en-11-one cis-Eudesm-6-en-12-al (2 epimers) 13-nor-eremophila-1(10)-en-11-one cis-eudesm-6,11-dien-3β-ol Sesquiterpene alcohol (M222) Funebran-15-al 13-nor-Eremophil-1(10)-en-11-one Amorph-4-en-10-ol (Epimer A) 10-epi-Cubenol-12-nor-Ziza-6(13)-en-2β-ol (see M 2) Junenol (trans-eudesm-4(15)-en-6β-ol) Eudesm-4(15)-en-5β-ol 12-nor-Ziza-6(13)-en-2α-ol see (see M 3) Eudesm-6-en-4β-ol 10-epi-γ-Eudesmol 1-epi-Cubenol Methyl cyclocopacamphanoate 13-nor-Eremophila-1(10),6-dien-11-one Khusian-2-one (helifolan-2-one) Alpha-Funebren-15-al (2,6-di-epi-cedren-15-al) Eremophila-1(10),6-dien-12-al (2 epimers) Cedrene-15-al Prezizaan-7β-ol (allo-khusiol) 7,15-Epoxy-prezizaane Eremophila-1(10),4(15)-dien-2α-ol (see M 31) Beta-Eudesmol Valerianol Eudesm-4(15),7-dien-3β-ol (see M 7) Alpha-cadinol Cyclocopacamphan-12-ol (Epimer A see M4) Bisabola-3(15),10-dien-7-ol Alpha-Eudesmol 7,10-Epoxy-10-hydroxysalvialane (see M 8) Cyclocopacamphan-12-ol (Epimer B see M6) 13-nor-trans-Eudesma-4(15),7-dien-11-one Amorph-4-en-10-ol (Epimer B) 10-Hydroxy-calamenene (Epimer A) Khusinol Ziza-6(13)-en-3-one (3β-methyl group) 2-epi-ziza-6(13)-en-3α-ol Ziza-6(13)-en-12-al Ziza-6(13)-en-12-yl methyl ether 10-epi-Acor-3-en-4-one Guai-11-en-10-ol (pogostol) Eudesm-11-en-4-ol (intermedeol) 10-Hydroxy-calamenene (Epimer B) 13-nor-7,8-Epoxy-eremophil-1(10)-en-11-one 1575 1562 1562 1566 1566 1566 1577 1577 1577 1582 1584 1584 1599 1593 1574 1565 1604 1604 1601 1596 1607 1610 1579 1598 1575 1616 1604 1612 1602 1578 1618 1584 1621 1598 1598 1598 1603 1603 1603 1607 1610 1610 1614 1617 1617 1625 1625 1628 1628 1638 1638 1638 1643 1643 1643 1643 1645 1645 1645 1645 1649 1649 1649 1653 1653 1653 1653 192 1618 1617 1605 1604 1632 1619 1736 1646 1648 1648 1705 1651 1644 1638 1663 1663 1653 1645 1655 1666 1680 1682 1683 1697 1698 1660 1699 1665 1653 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 209 210 211 212 213 214 215 216 217 218 219 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 epi-Zizanone Tetradecanol Beta-Bisabolol Porosa-4,6-dien-7-one 2-epi-Ziza-6(13)-en-3-one (2α-methyl group) 13-nor-7,8-Epoxy-eudesm-4(15)-en-11-one Eudesm-7(11)-en-4-ol Nootkatol 4,7-Epoxy-spirovetiv-2-en-11-ol (see M 9) Guaia-1(5),11-dien-3α-ol (see M 11) Helifol-1-en-14-ol (see M 12) Ziza-6(13)-en-3α-ol (see M 19) Prezizaan-15-al 2-epi-Ziza-6(13)-en-12-al 13-nor-4,5-Epoxy-eudesm-6-en-11-one Beta-Funebren-14-ol (see M 13) Preziza-7(15)-en-3α-ol (see M 14) Zizanal Alpha-Bisabolol 6-epi-α-Bisabolol Khusian-2-ol (helifolan-2-ol see M 10) Eremophila-1(10),7(11)-dien-2β-ol 1,7-Cyclogermacra-1(10),4-dien-15-al Eremophil-7(11)-en-10β-ol Eudesm-7(11)-en-4α-ol (juniper camphor) E-Opposita-4(15),7(11)-dien-12-al Eudesma-4(15),7-dien-2β-ol (see M 18) Ziza-6(13)-en-3β-ol (see M 21) E-Opposita-4(15),7(11)-dien-12-ol cadina-1(10),6,8-triene 13-nor-Eudesma-4,6-dien-11-one Ziza-5-en-12-ol 2-epi-ziza-6(13)-en-3β-ol Methyl ziza-6(13)-en-12-oate trans-Eudesma-4(15),7-dien-12-ol 5,6-seco-6,7-Furoeudesman-5-one Eudesma-3,5-dien-1α-ol (see M 26) Preziza-7(15)-en-12-ol (see M 29) Khusimol (Ziza-6(13)-en-12-ol or Zinanol) 10-epi-Acora-3,11-dien-15-al Methyl 2-epi ziza-6(13)-en-12-oate 7,11-Epoxy-eremophila-1,9-dien-8α-ol 6,12;7,11-Di-epoxy-eudesm-4-ene (epimer A) Eudesma-4,6-dien-3-one (β-cyperone) trans-Eudesma-4(15),7-dien-12-yl formate 13-Hydroxy valencene 6,12;7,11-Di-epoxy-eudesm-4-ene (epimer B) 7,11;8,12-Di-epoxy-eremophil-9-ene (Epimer A) Ziza-6(13)-en-12-yl formate Alpha-costol Isovalencenol or (E)-isovalencenol Isokhusenic acid Sesquiterpene alcohol (M220) 9,10-dehydro-isolongifolene Isonootkatool or Vetiselinenol E-Eremophila-1(10),7(11)-dien-2α-ol (see M 41) Spirovetiva-3,7(11)-dien-12-ol (see M 42) Z-Eremophila-1(10),7(11)-dien-12-al (Z-isovalencenal) 14-Hydroxy-d-cadinene 1669 1671 1657 1657 1657 1657 1672 1669 1671 1700 1715 1663 1663 1663 1663 1683 1689 1715 1660 1671 1668 1668 1668 1679 1679 1679 1679 1694 1736 1717 1700 1707 1687 1687 1705 1740 1692 1712 1713 1714 1396 1704 1704 1708 1712 1726 1726 1726 1726 1740 1745 1745 1697 1701 1687 1715 1689 1651 1690 1694 1729 1761 1743 1762 1774 1742 1735 1742 1767 1753 1753 1762 1782 1786 1813 1793 1783 1773 1793 1795 1798 1771 1771 1771 1771 1793 1730 1812 1803 193 1730 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 Nootkatone (Eremophila-1(10),11-dien-2-one) Beta-Vetivone 7,11;8,12-Di-epoxy-eremophil-9-ene (Epimer B) E-Eremophila-1(10),7(11)-dien-12-al (Epimer B) Sesquiterpene ketone (M218) E-isovalencenal E-Eremophila-1(10),7(11)-dien-12-yl formate Alpha-Vetivone Ziza-6(13)-en-12-yl acetate Preziza-7(15)-en-12-yl acetate Khusenic acid E-Eremophila-1(10),7(11)-dien-12-yl acetate Prekhusenic acid zizanoic acid Cyclohexadecanolide Hexadecanoic acid Manool 1781 1788 1807 1823 1804 1819 1829 1806 1823 1816 1819 1806 1823 1845 1842 1794 1830 1800 1800 1800 1813 1828 1833 1869 1828 1836 1843 1855 1851 1842 1871 1874 1884 1837 1860 1960 2057 1900 1933 1960 Note: Weyerstahl, P., Marschall, H., Splittgerber, U., Wolf, D 2000 Analysis of the polar fraction of Haitian vetiver oil Flavour Fragrance J 15, 153-173 Champagnat, P., Figueredo, G., Chalchat, J C., Carnat, A P., Bessibre, J M 2006 A study on the composition of commercial Vetiveria zizanioldes oils from different geographical origins J Essent Oil Res 18, 416-422 Pripdeevech, P., Wongpornchai, S., Promsiri, A 2006 Highly volatile constituents of Vetiveria zizanioides roots grown under different cultivation conditions Molecules, 11, 817826 Martinez, J., Rosa, P T V., Menut, C., Leydet, A., Brat, P., Pallet, D., Meireles, M A A 2004 Valorization of Brazilian Vetiver (Vetiveria zizanioides (L.) Nash ex Small) Oil J Agric Food Chem 52, 6578-6584 Adams, R P., Habte, M., Park, S., Daffornd, M R 2004 Preliminary comparison of vetiver root essential oils from cleansed (bacteria- and fungus-free) versus non-cleansed (normal) vetiver plants Biochem Syst Ecol 32, 137-1144 Massardo, D R., Senatore, F., Alifano, P., Giudice, L D., Pontieri, P 2006 Vetiver oil production correlates with early root growth Biochem Syst Ecol 34, 376-382 Adams, R P., Nguyen, S., Johnston, D A., Park, S., Provin, T L., Habte, M 2008 Comparison of vetiver root essential oils from cleansed (bacteria- and fungus-free) vs noncleansed (normal) vetiver plants Biochem Syst Ecol 36, 177-182 194