E L S E V I E R Bioresource Technology 67 (1999) 73-82

blOi BOUI ([ TKHI LOQT

Exploitation of the tropical oil seed plant Jatropha curcas L.

G.M. Giibitz a, M. Mittelbach b*, M. Trabi c "Institute of Microbiology, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria

blnstitute of Organic Chemistry, Karl-Franzens University Graz, HeinrichstraJ3e 28, A-8010 Graz, Austria ~Institute of Biotechnology, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria

Received 20 February 1998; accepted 12 March 1998

Abstract

In the last few years the potential of the drought resistant tropical tree Jatropha curcas L. (Euphorbiaceae) for the production of biofuels and industrial products has been assessed by several groups. Various novel methods for the cultivation and genetic improvement of J. curcas have been presented. A trans-esterification process of the seed oil for its use as a biofuel was evaluated on an industrial scale (1500 t/a). Various biologically active substances have been isolated and characterized from all parts of the plant. Their mechanisms of action have been studied in relation to a great number of applications of J. curcas in traditional medicine. Substances such as phorbol esters, responsible for the toxicity of J. curcas to animals and humans, have been isolated and their molluscicidal, insecticidal and fungicidal properties have been demonstrated in lab-scale experiments and field trials. Newly developed biotechnological processes related to the exploitation of J. curcas include the genetic improvement of the plant, biological pest control, enzyme-supported oil extraction, anaerobic fermentation of the press cake and the isolation of anti- inflammatory substances and wound-healing enzymes. © 1998 Elsevier Science Ltd. All rights reserved.

Keywords: Jatrophacurcas; Biofuel; Wound-healing; Molluscicide

1. Introduction

Bioresource technology involves the exploitation of natural substances and/or biotechnological approaches in production processes. The utilization of various parts of Jatropha curcas L., reviewed for the first time in this paper, combines these targets (Fig. 1), thus potentially improving the economic situation of various tropical countries.

J. curcas is a drought-resistant shrub or tree belonging to the genus Euphorbiaceae , which is culti- vated in Central and South America, South-east Asia, India and Africa. From the Caribbean, where this species had been already used by the Mayas (Schmook & Serralta-Peraza, 1997), J. curcas was probably distri- buted by Portuguese seafarers via the Cape Verde Islands and Guinea Bissau to other countries in Africa and Asia (Heller, 1996). J. curcas, which can easily be propagated by cuttings, is widely planted as a hedge to protect fields, as it is not browsed by cattle. Like many other Jatropha species J. curcas is a succulent that

*Corresponding author.

sheds its leaves during the dry season. It is well adapted to arid and semi-arid conditions and often used for erosion control (Heller, 1996).

The first commercial applications of J. curcas were reported from Lisbon, where the oil imported from Cape Verde was used for soap production and for lamps. The press cake was used as a fertilizer for potatoes. Even today, J. curcas is mainly cultivated for the production of oil as a fuel substitute. However, trans-esterification of the oil for use in standard diesel engines is gaining more importance than direct utiliza- tion of the oil in adapted engines. Costs of US $0.2 per liter were calculated for the methyl ester from a Nicar- aguan plant which aimed to produce 1450 tons in 1997 (Foidl & Eder, 1997). New techniques such as the enzyme-supported oil extraction and more efficient trans-esterification processes have been evaluated (Foidl et al., 1996; Winkler et al., 1997).

All parts of J. curcas have been used in traditional medicine and for veterinary purposes for a long time (Duke, 1985). The oil has been used as a purgative, to treat skin diseases and to soothe pain such as that caused by rheumatism. Decoctions of the leaves have been used against coughs or as antiseptics after birth,

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74 G.M. Gabitz et al./Bioresource Technology 67 (1999) 73-82

and the branches as chewing sticks (Heller, 1996). Recently, the substances responsible for wound healing and anti-inflammatory effects have been isolated and characterized (Nath & Dutta, 1991; Staubmann et al.,

1997b). Various extracts from J. curcas seeds and leaves

showed molluscicidal, insecticidal and fungicidal properties (Nwosu & Okafor, 1995; Liu et al., 1997; Solsoloy & Solsoloy, 1997). Phorbol esters have been suggested to be one of the toxic principles.

Biotechnological processes related to the exploita- tion of J. curcas include genetic improvement of the

plant, biological pest control, enzyme supported oil extraction, anaerobic fermentation of the press cake and the isolation of anti-inflammatory substance and wound healing enzymes (Table 1).

2. Cultivation

J. curcas shows some unusual features among tree crops such as its modular construction. The plant's branching pattern and the position of the inflores- cences conform to Leeuwenberg's model in the classifi-

Ja t ropha c u r c a s

-Erosion control -Fire wood -Hedge plant -Plant protectant

Fruits

l Seeds -Insecticide -Food / fodder

(non toxic varieties)

Leaves -Development of Eri Silkworm -Medicinal uses -Anti-inflammatory substance

Fruit hulls -Combustibles -Green manure -Biogas production

+ + Seed oil Seed, ~ k e -Soap production -Fertilizer -Fuel -Biogas production -Insecticide -Fodder -Medicinal uses (non toxic varieties)

Fig. 1. Exploitation of J. curcas.

Seed shells -Combustibles

Latex -Wound healing Protease

(Curcain) -Medicinal uses

Table 1 Biotechnology processes involved in the utilization of J. curcas

Process/product Principle Reference

Rapid propagation and (genetic) improvement Biological pest control Enzyme supported oil extraction Detoxification of seeds Biogas production Anti-inflammatory substance Wound healing substance

Tissue cultures Entomopathogenous fungi Proteases, (hemi) cellulases Fermentation with R. oryzae Anaerobic fermentation of press cake and fruit shells Isolated from the leaves Protease curcain from latex

(Machado et al., 1997) (Grimm & Guharay, 1997) (Winkler et al., 1997) (Trabi et al., 1997) (Staubmann et al., 1997a) (Staubmann et al., 1997b) (Nath & Dutta, 1991)

G.M. Giibitz et al./Bioresource Technology 67 (1999) 73-82 75

cation of architectural types. Plant growth and reproduction are tightly linked in Jatropha, and some of the consequences of this have been explored in studies carried out in Nicaragua and India (Aker, 1997; Sharma et al., 1997). On the basis of the results obtained from detailed studies on the response of J. curcas to various parameters such as rainfall and nutrient deficiency, these authors discussed recommen- dations for agronomic practice and pest management.

New techniques using cell and tissue cultures have been developed for the propagation and storage of selected genotypes of tropical plants (Engelmann, 1991). Compared to conventional techniques, these protocols provided higher multiplication rates, minimized the risk of infections by microorganisms and insect pests, reduced genetic erosion, space require- ments and expenses in labor costs. The multiplication phase and initiation of aseptic cultures from J. curcas seeds have been optimized for a range of different genotypes from various geographical origins such as Nicaragua, Mexico, Cape Verde, and Madagascar (Machado et al., 1997). Both the addition of different substances (such as cytokinins, the auxin IBA or gibberellic acid) to the culture media and the genotype, influenced the success of micropropagation through axillary branching. The genotype Mexico was not suitable for micropropagation. Promising results for J. curcas genotypes from both India and Nicaragua were also obtained by adventitious shoot formation from l~af discs which formed a base for future genetic ifnprovement of this species (Sujatha & Mukta, 1996; Machado et al., 1997).

Only few insect pests of economic importance have been described for J. curcas due to the toxicity of all parts of the plant and their insecticidal qualities (Grainge & Ahmed, 1988; Sauerwein et aL, 1993; Solsoloy, 1993; Mengual, 1997). However, true bugs have been found to feed on the fruit both in the paleo- tropics (Heller, 1996) and the neotropics (Grimm & Guharay, 1997). The two most common species found in Nicaragua are Pachycoris klugii Burmeister and Leptoglossus zonatus. Both species damage the developing fruit thus causing abortion and malforma- tion of seeds. Yield loss was found to be up to 18.5% of viable seeds at low insect densities (Grimm & Guharay, 1997). The potential of biological control of P klugii and L. zonatus pests using the entomopatho- genous fungi Beauveria bassiana and Metarhizium anisopliae was assessed on a laboratory scale, obtaining up to 99% mortality of L. zonatus and 64% of P. klugii with M. anisopliae (Grimm & Guharay, 1997). Besides the two species mentioned above, 12 further species of true bugs also feed on the nut. Other pests include a stem borer (Lagocheirus undatus), grasshoppers, leaf eating beetles and caterpillars as well as leaf hoppers (Meshram & Joshi, 1994; Grimm & Maes, 1997).

3. Application ofJ. c u r c a s in traditional medicine

In India, Africa and also in Latin America various parts of J. curcas have been and are still used in tradi- tional medicine. In Africa, the seeds are used as an anthelmintic and as a purgative, with the leaves as a haemostatic (Watt & Breyer-Brandwijk, 1962). In Mali the leaves are known as a treatment for malaria (Henning, 1997). Leaves, seeds and bark are boiled and the watery extracts are used as a purgative (Mampane et al., 1987). The leaf decoction is applied externally for rheumatism and inflammation. The root decoction is drunk against pneumonia, syphilis, as an abortifacient, vermifuge and purgative (Chhabra et al., 1990). In South Sudan, the seeds as well as the fruit are used as a contraceptive or as an abortifacient (List & Horhammer, 1979). Furthermore, ascites, gout, paralysis and skin diseases are treated with different medicines made from the seeds (Joubert et al., 1984). In Mexico the latex is used for fungal infections in the mouth, bee and wasp stings and digestive troubles of children (Watt & Breyer-Brandwijk, 1962; Schmook & Serralta-Peraza, 1997).

4. Toxicity of the plant

There are reports from Mexico that the White Winged Dove (Zenaida asiatica) usually feeds from the seeds of J. curcas (Rivera-Lorca & Ku-vera, 1997b). In some cases chickens or pigs consume the seeds and in Mexico the boiled or roasted seeds are even used to prepare traditional dishes (Neuwinger, 1994; Rivera- Lorca & Ku-Vera, 1997a; Schmook & Serralta-Peraza, 1997). Furthermore, there are reports of normal weight gain in mice fed with J. curcas seeds from Veracruz, Mexico (Panigrahi et al., 1984). However, the seeds of J. curcas are, in general, toxic to humans and animals. Numerous feeding experiments with different animal species have demonstrated the toxicity of the seeds as well as of the oil and the press cake.

Raw or cooked seeds killed rats within 2-3 days, whereas oil (raw or cooked) or defatted seed meal (raw or cooked) caused death within 6-8 days and roasted and cooked seeds after 14-16 days (Liberalino et al., 1988). The minimal lethal dose of Jatropha seeds for three different animal species has been determined (Table 2) (Felke, 1913; B6hme, 1988). The test animals - - regardless of the species - - showed inappetance, abdominal pain, diarrhoea, respiratory problems and imbalance. Histopathological findings include gastro- intestinal inflammation, necrosis of the liver, heart and kidneys as well as haemorrhages in the liver (Adam, 1974; Adam & Magzoub, 1975; Ahmed & Adam, 1979a; Ahmed & Adam, 1979b; Abdu-Aguye et al., 1986; Liberalino et al., 1988; Becker, 1995).

76 G.M. Giibitz et aL/Bioresource Technology 67 (1999) 73-82

Table 2 Minimal lethal dose of J. curcas seeds

Animal Amount of seeds fed Estimated curcin Death intake (day)

(g kg-~) (g total) (mg total)

Sheep 7.4 67 460 9 Goat 1.5 8 55 12 Calf 3.0 36 248 12

While no signs of intoxication have been found in mice fed with Jatropha oil from Veracruz, Mexico (Cano et al., 1989), other authors have reported severe diarrhoea and inflammation of the rats' intestines when fed with Jatropha oil from an Indian variety. A LDs0 of 6 ml kg -1 body weight was determined (Gandhi et al., 1995).

In the year 1854, in Birmingham, more than 30 children were intoxicated by J. curcas seeds (Felke, 1913). In general, ingestion of 3-5 seeds causes marked nausea, gastro-intestinal irritation, abdominal pain, vomiting and sometimes diarrhoea. In severe cases patients were clinically dehydrated, but made a rapid recovery after intravenous fluid replacement (Joubert et al., 1984; Abdu-Aguye, 1986; Mampane et al., 1987).

In Mexico, achiote seeds (Bixa orellana L.) are said to be an antidote for poisoning with J. curcas (Rivera- Lorca & Ku-Vera, 1997), whereas in South Africa, traditional healers use a watery extract of Peltrophorum africanum as an antidote (Mampane et al., 1987).

5. Composition and utilization of various parts of J. curcas

5.1. Leaves and twigs

Chemical components isolated from the leaves and the young twigs of J. curcas include the cyclic triterpenes stigmasterol, stigmast-5-en-3fl,7fl-diol, stigmast-5-en-3fl,%t-diol, cholest-5-en-3fl,7fl-diol, cholest-5-en-3fl,7~t-diol, campesterol, fl-sitosterol, 7-keto-fl-sitosterol as well as the fl-D-glucoside of fl-sitosterol. Furthermore, these parts of the plant contain the flavonoids apigenin, vitexin and isovitexin (Mitra et al., 1970; Khafagy et al., 1977; Hufford & Oguntimein, 1987; Neuwinger, 1994). These authors also report the existence of 1-triacontanol, CH3(CH2)28CH2OH a waxy alcohol, and ~-amyrin. The leaves also contain the dimer of a triterpene alcohol (C63H1t7Og) and two flavonoidal glycosides (Khafagy et al., 1977).

An ethanolic extract of the defatted leaves and twigs has shown both in vivo and in vitro activity against P-388 lymphocytic leukaemia (Hufford & Oguntimein,

1987). Recently, an anti-inflammatory substance has been isolated from the leaves (Staubmann et al., 1997b). Ethyl acetate leaf extracts showed anti-inflam- matory properties in a carrageenin induced rat-paw- edema-test. On the base of this testing procedure two active substances with the stoichiometric formulas C4HaN202 and C4HTNO~ were isolated.

J. curcas leaves were also used as a substrate for eri silkworm Samia cynthia ricini. Although with castor leaves (Ricinus communis L.), several other plants, mainly from the family of Euphorbiaceae, are known as secondary hosts, a survival rate of only 21% was deter- mined with J. curcas leaves from Nicaragua (Grimm et al., 1997). The same authors obtained even worse results with other J. curcas varieties such as from Cape Verde Islands, Madagascar, India and Mexico. In general, the weights of the shells obtained were signifi- cantly lower than those of silkworms fed with castor leaves.

5.2. Aerial parts, stem and root

Various organic acids (o and p-coumaric acid, p-me and p-OH-benzoic acid, protocatechiuc acid, resorsilic acid and t-me-cinnamic acid) as well as iridoids, saponins and tannins have been found in aerial parts of J. curcas from India (Hemalatha & Radhakrishnaiah, 1993). Friedelin, epi-friedelinol, the tetracyclic triter- pene ester jatrocurin and scopoletin methyl ester have been isolated from the stem (Talapatra et al., 1993). The stem bark contains fl-amyrin, fl-sitosterol and taraxerol (Mitra et al., 1970).

The roots contain fl-sitosterol and its fl-D-glucoside, marmesin, propacin, the curculathyranes A and B and the curcusones A-D. Furthermore, the diterpenoids jatrophol and jatropholone A and B, the coumarin tomentin, the coumarino-lignan jatrophin as well as taraxerol have been found (Naengchomnong et al., 1986; Naengchomnong et al., 1994). Recently, it has been demonstrated that accumulation of the macro- cyclic diterpenoid jatrophene, the active principle of many folk medicines, was highest with callus and suspension cultures derived from J. curcas and J. ellip- tica (Pletsch & Charlwood, 1997).

5.3. Latex

Curcacycline A, a cyclic octapeptide from the latex of J. curcas, displayed a moderate inhibition of classical pathway activity of human complement and prolifera- tion of human t-cells (Vandenberg et al., 1995). Curca- cycline B, a cyclic nonapeptide, has been shown to enhance the rotamase activity of human cyclophilin B (Auvin et al., 1997). The latex itself has been found to be strongly inhibitory to watermelon mosaic virus (Tewari & Shukla, 1982). The latex has also been used

G.M. Gabitz et al./Bioresource Technology 67 (1999) 73-82 77

to promote healing of wounds, refractory ulcers, septic gums and as a styptic in cuts and bruises. A proteolytic enzyme (curcain) with a molecular weight of 22 kDa and the isoelectric point at pH 5.6-6.0 has been extracted (Nath & Dutta, 1988; Nath & Dutta, 1989; Nath & Dutta, 1991; Dutta, 1997). Histopathological studies on the wound-healing properties of this enzyme revealed that curcain ointments showed better effects in mice than nitrofurazone ointment or propamidine isethionate cream (Nath & Dutta, 1997). Acute toxicity studies demonstrated that curcain is nontoxic on oral administration and its LDs0 on intraperitoneal admini- stration in mice is 0.96 g kg-1 body weight.

5.4. Seeds

The chemical composition of the seed shells is given in Table 3. Besides the main constituents, a positively inotropic cardioactive principle has been found in the seed shells ofJ. curcas (Panigrahi et al., 1984b).

Table 3 also lists the different constituents of J. curcas kernels calculated on the base of dry matter. The existence of dulcitol, sucrose and the fl-D-glucoside of fl-sitosterol in the kernel have been reported (Mitra et al., 1970).

The main toxic principle of the press cake, a hemag- glutinin named curcin, had already been described in 1913 (Felke, 1913). It was found that curcin inhibits protein synthesis in vitro, but does not affect protein synthesis in Ehrlich ascites cells, probably due to its lack of a carrier moiety (Stirpe et al., 1976). Later, a hemagglutinin was obtained from the seeds which showed an exceptionally high molecular weight (660.000), two different subunits and a stable activity up to 60°C. But it could not be proved that this hemag- glutinin was in fact curcin (Cano et al., 1989).

Further toxic and antinutritional components in the kernel and the press cake include phytates, saponins and a trypsine inhibitor, respectively (Table 4) (Aregheore et al., 1997; Makkar & Becker, 1997; Wink et al., 1997). Tannins, amylase inhibitors, glucosinolates and cyanogens were not detected (Makkar et al., 1997).

Various methanolic extracts of the seeds have shown contraceptive and abortive action in rats (Mameesh et al., 1963; Goonasekera et al., 1995).

Depending on the variety, the decorticated seeds contain 43-59% of oil (Mtinch & Kiefer, 1986; Liber- alino et al., 1988; Neuwinger, 1994; Gandhi et al., 1995; Sharma et al., 1997; Wink et al., 1997), which is used for lighting, as a lubricant and for making soap (Rivera-Lorca & Ku-Vera, 1997a). The fatty acid composition of the oil is given in Table 5.

Although there are several reports in the literature on the use of the oil for boiling and cooking, such as in a village of the Rio Hondo zone (Mexico), the oil can, in general, not be utilised in human nutrition due to several toxic components (Schmook & Serralta-Peraza, 1997). A toxic fraction initially called curcanoleic acid has been isolated from the seed oil of J. curcas (Felke, 1913). Later it was demonstrated that the toxic principle did not involve any hydroxylated fatty acid (Miinch & Kiefer, 1986). Like many other plants belonging to the families of Thymeleaceae and Euphor- biaceae, the kernels of J. curcas contain between 0.03 and 3.4% of phorbol esters (depending on the variety), which can be expressed with the oil (Adolf et al., 1984; Horiuchi et al., 1987; Hirota et al., 1988; Sauerwein et al., 1993; Makkar & Becker, 1997; Wink et al., 1997). Phorbol esters are known to activate protein kinase C (PKC), a key enzyme in signal transduction and development processes of most cells and tissues. Many of the PKC target substrates are components of signal transduction pathways and include proteins that regulate ion channels, calcium and calmodulin-binding proteins, growth factor receptors, structural and regula- tory proteins of the cytoskeleton, components of the transcriptional machinery, efflux pumps and many other proteins (Azzi et al., 1992; Kikkawa & Nishizuka, 1986). In contrast to the transitory activation of PKC during normal signal transduction the prolonged inter- action of phorbol esters with PKC is thought to sustain the mitogenic response during tumorgenesis (Roten- berg et al., 1991).

Table 3 Chemical composition of kernel, shell and meal*

Kernel Shell Meal

Dry matter (%) 94.2-96.9 89.8-90.4 100 Constituents (% in DM) Crude protein 22.2-27.2 4 .3 -4 .5 56.4-63.8 Lipid 56.8-58.4 0.5-1.4 1.0-1.5 Ash 3.6-4.3 2.8-6.1 9.6-10.4 Neutral detergent fibre 3.5-3.8 83.9-89.4 8.1-9.1 Acid detergent fibre 2.4-3.0 74.6-78.3 5.7-7.0 Acid detergent lignin 0.0-0.2 45.1-47.5 0.1-0.4 Gross energy (MJ kg -j) 30.5-31.1 19.3-19.5 18.0-18.3

*According to Trabi (1998).

6. Insecticidal and molluscicidai properties ofJ. c u r c a s

seed extracts

Ground seeds showed molluscicidal activity against the host of liver fluke (Lymnaea auricularia rubiginosa), a disease which is widely distributed in the Philippines and also against the hosts of Fasciola gigantea in Senegal (Heller, 1996). J. curcas oil and oil extracts have also been successfully applied against golden snails (Pomacea sp.) and vector snails of human schis- tosomes (Heller, 1996; Heller, 1997; Liu et al., 1997). Schistosomiasis (Bilharziasis) is a human disease caused by parasitic blood flukes of the genus Schisto-

78 G.M. Giibitz et al./Bioresource Technology 67 (1999) 73-82

Table 4 Toxic and antinutritional components in the degreased meal of three different varieties of J. curcas (Makkar & Becker, 1997)

Cape Verde N i c a r a g u a Non-toxic Mexico

Lectin mg meal/ml assay which produced hemagglutination 102 102 51 Trypsin inhibitor mg g-~ trypsin inhibited 21.3 21.1 26.5 Phytates % 9.4 10.1 8.9 Saponins % diosgenin equivalent 2.6 2.0 3.4

soma. Schistosomes are endemic in more than 70 tropical and subtropical countries and infect more than 200 million people. The larvae of the parasites, which are infectious for humans, are released from their aquatic vector snails.

J. curcas oil had molluscicidal properties towards Biomphalaria glabrata and Oncomelania hupensis, the vector snails of Schistosoma mansoni and S. japonicum, respectively. The methanol extract of the crude oil was even more active with LDs0 values of 0.004% for B. glabrata and 0.00025% for O. hupensis (Liu et al., 1997; Rug et al., 1997). Further experiments with phorbol esters isolated from J. curcas oil suggested derivatives of 12-deoxy-16-hydroxyphorbol as being one of the toxic principles (Adolf et al., 1984). However, since the aqueous extracts were also toxic to both B. glabrata and O. hupensis, molluscicidal activities of saponins were discussed as well (Hostettmann et al., 1982; Rug et al., 1997).

The crude oil from J. curcas formulated as an emulsifiable concentrate had contact toxicity to corn weevil Callosubruchus chinensis and bean weevil Sitophilus zeamays and deterred their oviposition on corn and sprayed mungbean. The respective LDs0 values were determined to be 0.91% and 1.92% (Solsoloy & Solsoloy, 1997). This formulated product from J. curcas also showed chronic toxicity to the common housefly M. domestica. Although M. domestica maggots were not sensitive, the pupae that later developed from the treated maggots were smaller than normal ones which led to diminutive adults or adults

Table 5 Fatty acid composition ofJ. curcas oil*

Fatty acid (%)

Myristic acid 14:0 0-0.1 Palmitic acid 16:0 14.1-15.3 Stearic acid 18:0 3.7-9.8 Arachidic acid 20:0 0-0.3 Behenic acid 22:0 0-0.2 Palmitoleic acid 16:1 0-1.3 Oleic acid 18:1 34.3-45.8 Linoleic acid 18:2 29.0-44.2 Linoleic acid 18:3 0-0.3

*Adapted from Martin & Mayeux, 1984; Raina & Gaikwad, 1987; Neuwinger, 1994; Gandhi et al., 1995; Bhakare et al., 1996; Sharma et al., 1997; Solsoloy & Solsoloy, 1997; Wink et al., 1997.

with wrinkled wings, resulting in a total population reduction of about 40-60% (Solsoloy & Solsoloy, 1997).

The use of J. curcas oil for the control of cotton insect pests seemed to be a promising alternative to hazardous chemicals (Solsoloy, 1993). The effect of J. curcas oil extracts on cotton bollworm Helicowerpa armigera and on the cotton flowerweevil Amorphoidea lata has been studied by these authors. In contrast to spraying with chemicals, treatment with J. curcas oil extract did not affect the population of beneficial arthropods. J. curcas also showed potential in control of the sorghum pests (stem borers) Sesamia calamistis and Busseola fusca, the crude oil being more efficient than the methanolic extract (Mengual, 1997).

The potential of certain J. curcas extracts in the treatment of subcutaneous phycomycosis in humans and animals was assessed (Nwosu & Okafor, 1995). While the growth of Basidiobolus haptosporus and B. ranarum was totally inhibited, the extract did not show any effect on the growth of Aspergillus fumigatus, Geotrichum candidum and Candida albicans.

7. Jatropha curcas oil and transesterified oil as fuel substitute

Various methods for recovering the oil from J. curcas seeds, including extraction with organic solvents and water, have been investigated. Compared to hexane extraction (98%) the oil extraction using water yielded only 38%. The enzyme-supported aqueous extraction offers a nontoxic alternative to common extraction methods using organic solvents. Comparing the effect of several cell-wall-degrading enzymes during aqueous extraction a maximum yield of 86% was obtained under optimized reaction conditions with an alkaline protease (Winkler et al., 1997). This is in good agreement with data reported for coconuts (78%) (Buenrostro & Lopezmunguia, 1986). Hemicellulases and cellulases were less effective in enhancing J. curcas oil extraction (73%) than the alkaline protease (Winkler et al., 1997).

Vegetable oils like rape seed, sunflower and soybean oil have proved to be excellent sources of fuel. Two strategies have been performed and evaluated:

1. adaptation of the engine to the fuel;

G.M. Giibitz et al./Bioresource Technology 67 (1999) 73-82 79

2. adaptation of the fuel to the engine.

The first strategy can be accomplished by using specially developed engines like the Elsbett engine. This strategy makes sense only when using neat vegetable oil in stationary engines, as the price of the engine is too costly due to low production numbers. Therefore, even in developing countries the second strategy seems to be more practicable. The production of fatty acid methyl esters from vegetable oils and their use as Diesel fuel has been well tested and evaluated in several European countries such as Austria, France and Italy. The simple technology specially developed for this chemical process can also be performed in less industrialised countries (Mittelbach et al., 1983; Conne- mann, 1994). In a previous study 364 different plant seed oils were surveyed, as pure oil as well as fatty acid methyl esters, for their potential use as diesel fuels (Kalayasiri et al., 1996).

The fact that the oil of J. curcas cannot be used for nutritional purposes without detoxification makes its use as an energy source for fuel production very attrac- tive. Besides the production of soaps, the oil was in former times also used as lamp oil (Martin & Mayeux, 1984). In Madagascar, Cape Verde and Benin the seed oil of Jatropha was used as a diesel fuel substitute during the Second World War. Blends of the oil with mineral fuel have been proposed (Mensier & Loury, 1950; Bhasabutra & Sutiponpeibun, 1982; Banerji et al., Table 6

1985; Nasir et al., 1988). Early engine tests with J. curcas oil were done in Thailand showing satisfactory engine performace (Takeda, 1982). A 50 h continuous test and starting experiments were conducted using transesterified J. curcas oil, no. 2 diesel fuel, and their blends in two small pre-combustion-chamber-type diesel engines (Ishii & Takeuchi, 1987). In Mali, pre-combustion-chamber diesel engines of Indian origin have been running with pure Jatropha oil, giving even better results than gas oil at maximum load condi- tions. The oil can also be successfully used as lubricant in these engines (Henning, 1997). Different vegetable oils including J. curcas oil were evaluated in direct and indirect injection diesel engines; performance, fuel conversion efficiency, specific consumption as well as exhaust gas emissions were compared (Vaitilingom & Liennard, 1997). The lowest exhaust gas emissions were obtained with coprah and Jatropha. Also a long- term durability test was conducted with J. curcas oil as fuel in a water pump driven by a modified direct injec- tion diesel engine. This 1000 h test indicated a good behavior as fuel and no wear in the engine. At the same power output, specific fuel consumption was lower and efficiency was higher than those of diesel fuel.

For using Jatropha oil as fuel for transportation, the oil has to be transesterified with methanol or ethanol. Especially for countries with large agricultural areas, the use of ethanol is favoured. For African countries,

Chemical, physical and fuel parameters ofJ. curcas oil and methyl (ethyl) esters

Parameter Unit J. curcas oil Methyl esters Ethyl esters O-NORM for of J.c. oil of J.c. oil FAME (1994)

Density at 15°C [g cm-3] 0.920 0.879 0.886 0.87-0.89 Viscosity at 30°C [cSt] 52 4.84 5.54 3.5-5.0 [20°C] Flash point [°C] 240 191 190 > 100 Neutralization number [mg KOH g-~] 0.92 0.24 0.08 _< 0.80 Sulfated ash [% m/m] - 0.014 0.010 _< 0.02 Cetane number* - - 51 59 _> 48 Conradson carbon residuet [% m/m] - 0.025 0.018 _< 0.05 Methyl (ethyl) ester content [% m/m] - 99.6 99.3 - Monoglycerides [% m/m] Not detected 0.24 0.55 - Diglycerides [% m/m] 2.7 0.07 0.19 - Triglycerides [% m/m] 97.3 Not detected Not detected - Methanol [% m/m] - 0.06 0.05 _< 0.20 Water:~ [% m/m] 0.07 0.16 0.16 Free glycerol [% m/m] - 0.015 Not detected _<0.02 Total glycerol§ [% m/m] - 0,088 0.17 _< 0.24 Phosphorus [rag kg-~] 290 17.5 17.5 _< 20 Calcium [rag kg ~] 56 6,1 4,4 - Magnesium [rag kg-1] 103 1.4 0.8 - Iron [mg kg-~] 2.4 0.9 0.3 -

*ISO 5165. i'The Conradson carbon residue (CCR) was determined using 10.0 g of the original sample. ~The water content is limited by the definition: free from separated water. §Sum of free and bonded glycerol. - No value available.

80 G.M. Gi~bitz et aL/Bioresource Technology 67 (1999) 73-82

the feasibility of the production of fatty acid ethyl esters from Jatropha oil was studied (Eisa, 1997). The fuel properties of the oil of J. curcas as well as methyl esters and ethyl esters were compared (Foidl et al., 1996) (Table 6).

Due to the fact that today, even in developing countries, methanol is cheaper than ethanol, in the next years mainly fatty acid methyl esters will be used as diesel fuel substitute. A development aid project sponsored by the Austrian government, in cooperation with the 'Direccion de Investigacion y Orientacion Tecnologia' (DINOT), a department of the 'Univer- sidad Nacional de Ingenieria' (UNI) in Nicaragua was started in 1989 to cultivate J. curcas and to evaluate the production of biodiesel fuel from the oil. In 1995 an area of 1000 ha was grown with J. curcas and a semi- industrial-scale pilot plant for the production of methyl esters from the seed oil with a capacity of 2.000 Mt methyl ester per year was put into operation in 1997. A two-step trans-esterification process was developed using potassium hydroxide as a catalyst (Zamora et al.,

1997). The economic evaluation has shown that the biodiesel production from J. curcas is very profitable provided the by-products of the biodiesel production can be sold as valuable products (Foidl & Eder, 1997).

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