Biofuels have a strategic value in reducing our dependence on foreign oil and the ability to increase our energy security. Growing and processing oil-rich biomass into biofuel in the U.S. would contribute to reduce this dependence. The objective of this project is to evaluate the conversion of oilseeds that could be grown in the U.S. and U.S. territories into a substitute of hydrocarbon fuel. One particular crop is Jatropha curcas, a bush belonging to the family Euphorbiaceae. A feasibility study shows that the crop will be economical if plantation by-products can be of commercial value, which would decrease the energy crop costs, hence make it competitive. As a parallel, oil-rich feedstock like soybean is grown in the U.S. not just for its oil, but for the value of its protein-rich meal. As for Hevea, Jatropha curcas stem and other plant parts contain a sap-like extract known as latex that is the primary source of natural rubber. It also contains substances that are toxic and that renders any by-product unsafe for human or animal consumption. Jatropha curcas oil press cake cannot be sold or easily disposed of, making the seeds less attractive economically. Some of these substances may be used as a natural pesticide or could find direct medical applications, e.g. in fighting lice and scabies (Mange). Another notable physiological property of phorbol esters is their capacity to act as tumor promoters in cancer research. The husks of J. curcas fruits can be directly used as a fuel in the oil seed processing facility, which lowers the power consumption. Also, plant mass built under- and above ground could be traded on the growing carbon markets. These studies will be conducted as an integrated part of a larger project focusing on the development of Jatropha curcas as a viable biofuel crop on sub-prime lands. OBJECTIVES: Biofuels have a strategic value in reducing our dependence on foreign oil and the ability to increase our energy security. Growing and processing oil-rich biomass into biofuel in the U.S. would contribute to reduce this dependence. The objective of this project is to evaluate the conversion of oilseeds that could be grown in the U.S. and U.S. territories into a substitute of hydrocarbon fuel. One particular crop is Jatropha curcas, a bush belonging to the family Euphorbiaceae. A feasibility study shows that the crop will be economical if plantation by-products can be of commercial value, which would decrease the energy crop costs, hence make it competitive. As a parallel, oil-rich feedstock like soybean is grown in the U.S. not just for its oil, but for the value of its protein-rich meal. As for Hevea, Jatropha curcas stem and other plant parts contain a sap-like extract known as latex that is the primary source of natural rubber. It also contains lectins, carbohydrate-binding proteins or glycoproteins which have specific coagulation properties. Jatropha curcas lectins are called Curcin. Jatropha curcas also contains phorbol esters which is highly toxic and renders any by-product unsafe for human or animal consumption. Jatropha curcas oil press cake cannot be sold or easily disposed of, making the seeds less attractive economically. A component called Jatropherol-I, a phorbol-type diterpene has found applications as a pesticide. Besides being a natural pesticide, Jatropherol-I could find direct medical applications, e.g. in fighting lice and scabies (Mange). Another notable physiological property of phorbol esters is their capacity to act as tumor promoters in cancer research. The husks of J. curcas fruits amount up to 35% in weight and at harvest, the exocarp is dry and can be directly used as a fuel in the oil seed processing facility. Plant mass build up during the establishment period could be traded as sequestered on the carbon trading markets. AAF will investigate each of these by-products to enhance the attractiveness of J.curcas as an energy crop. The Project objectives are: Determine the natural rubber content of twenty plants (10 from Indian origin and 10 from Madagascar origin) with different forms to ascertain the amount and degree of variation of rubber content between forms and origins. Determine the meal, leaf and stem content of phorbol esters, Jatropherol-I and lectins from twenty plants as described above. Determine the calorific value of different plant parts, e.g. husks as described above. APPROACH: The approaches for each objectives are: Objective 1: Rubber content of stems and leaves and oil extracted meal from twenty Jatropha curcas plants with different forms will be determined using the method of Black et al (1983). Plant material will be harvested from target plants and air dried. After cutting into small pieces they will be ground in a mill and then dried further in a blower oven at 50oC for 3 hours. Two grams of dried powdered sample will be homogenized with 67 mls of acetone in a Polytron homogenizer for 30 seconds at high speed and then centrifuged at 6,000g for 5 minutes. The pellet will be re- extracted with acetone and then the rubber extracted from the pellet by homogenizing with 67 mls of cyclohexane. After centrifugation, the supernatant will be kept and the pellet re-extracted with cyclohexane. The two supernatants will be combined and then evaporated to dryness in a blower oven at 105oC. The resulting rubber will be weighed and expressed as a % of the dry weight of the plant. Objective 2: An agglutination test will be performed on each plant part (stems, leaves, oil extracted meal) using the latex agglutination method of Kaul et al. (1991). Briefly, after de-fatting the ground and dried plant parts to remove lipids, the remaining solid will be extracted with 0.9% saline for 1 hour at room temperature and the supernatant filtered and then clarified by centrifugation at 4oC for 15 minutes at 10,000rpm. Fifteen microliters of the resulting solution (containing curcin) will be mixed with an equal volume of 10% latex beads coated with ovalbumin, containing 20 mM MnCl2 in a 12 well multi-dish and incubated at room temperature for 2 hours and then observed for visible agglutination. Curcin activity will be expressed as the inverse of the minimum amount of sample (mg/ml) in the assay which produces visible agglutination. Phorbol ester content of the each of the plant parts will be determined using the method of Makkar et al. (1997). 2.5 to 5.0 grams of dried sample will be mixed with 20 mls of dichloromethane and homogenized for 1 minute. After filtration, the filtrate will be stored and the retentate re-extracted with dichloromethane three more times. The filtrates will be combined. The retentate will be sonicated for 3 minutes with a further 20 mls of dichloromethane the filtrate again combined with the previous filtrates. The combined filtrates will be dried under vacuum at 40C using a rotary evaporator, re-dissolved in 5 mls of tetrahydrofuran and 20 ul injected into an HPLC equipped with a reversed phase C18 column (LiChrospher 100, endcapped, 5 um, Merck) . A ternary gradient (1.3mls/minute)using phosphoric acid, acetronitrile and tatrahydrofuran will be used to separate compounds of interest and eluted peaks will be detected using absorbance at 280 nm. Following Hirota et al., 1988, four phorbol esters elute between 41 and 48 minutes. Quantitation will be expressed as phorbol-12-myristate 13 acetate (Sigma-Aldrich) which elutes between 52 and 53 minutes. Objective 3: The net calorimetric values of husks and other parts of the Jatropha curcas fruits will be evaluated using a plain jacket calorimeter from Parr
