Degradation of phorbol esters by Pseudomonas aeruginosa PseA during solid-state fermentation of deoiled Jatropha curcas seed cake Chetna Joshi, Priyanka Mathur, S.K. Khare ⇑ Enzyme and Microbial Biochemistry Lab, Department of Chemistry, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India article info Article history: Received 26 October 2010 Received in revised form 13 January 2011 Accepted 14 January 2011 Available online 22 January 2011 Keywords: Jatropha curcas Pseudomonas aeruginosa PseA Phorbol esters Solid-state fermentation Deoiled seed cake abstract Large amount of seed cake is generated as by-product during biodiesel production from Jatropha seeds. Presence of toxic phorbol esters restricts its utilization as livestock feed. Safe disposal or meaningful uti- lization of this major by-product necessitates the degradation of these phorbol esters. The present study describes the complete degradation of phorbol esters by Pseudomonas aeruginosa PseA strain during solid state fermentation (SSF) of deoiled Jatropha curcas seed cake. Phorbol esters were completely degraded in nine days under the optimized SSF conditions viz. deoiled cake 5.0 g; moistened with 5.0 ml distilled water; inoculum 1.5 ml of overnight grown P. aeruginosa; incubation at temperature 30 °C, pH 7.0 and RH 65%. SSF of deoiled cake seems a potentially viable approach towards the complete degradation of the toxic phorbol esters. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Jatropha curcas, also known as physic nut or purging nut, is an important industrial crop belonging to Euphorbiaceae family. Its seeds are used as a major source of biodiesel fuel currently being used in India, Thailand, and other Southeast Asian countries (Saetae and Suntornsuk, 2010). The seed material comprises of 41% shell and 59% kernel. The kernel consists of 40–50% of oil (Singh et al., 2008). Biodiesel production from Jatropha seeds generate large quantum of residual deoiled seed cake with an aver- age rate of 500 g cake per kg of seed used (Zanzi et al., 2008). While the oil is an excellent biodiesel feedstock, potential utilization or safe disposal of huge amounts of seed cake by-product needs to be addressed (Liang et al., 2010). The average chemical composition of deoiled seed cake is protein, 60%; fat, 0.6%; ash, 9%; fibre, 4% and carbohydrates, 26% (Rakshit et al., 2008). The deoiled J. curcas seed cake cannot be used as cattle feed, unlike other oilseeds mainly due to the presence of toxic phorbol esters in it. Phorbol esters have been identified as main toxicants in cake which could not be destroyed even by heat- ing at 160 °C for 30 min (Makkar and Becker, 1997; Makkar et al., 1997). Phorbol esters are 20 carbon tetracyclic diterpenoids made up of four isoprene units (Ito et al., 1983). Tiglaine is the fundamental alcohol moiety in the phorbol esters. Hydroxylation of this basic tiglaine structure at different positions and then ester bonding to various acid moieties results in the formation of large varieties of phorbol ester compounds mainly responsible for toxicity in Jatropha (Goel et al., 2007). Toxicity to snails (Amin et al., 1972), goats (Adam and Magzoub, 1975), pigs (Chivandi et al., 2006), rats (Rakshit et al., 2008), humans (Rai and Lakhanpal, 2008), and mice (Li et al., 2010) has been reported consequent to the consumption of Jatropha seeds or seed cake. In vertebrates and humans, toxicity of phorbol esters is observed to cause burning/pain in the mouth and throat, vomiting, delirium, muscle shock, decrease of visual capacity, high pulse rate and other associated symptoms (Blumberg, 1988). All these restrict the feed uses of the seed cake. However, if left to decay as such, deoiled J. curcas seed cake will add to existing environmental problems. Therefore removal of phorbol esters is currently an important issue to be addressed. Various chemical and physical methods have been employed for the removal/inactivation of phorbol esters. Complete degradation of phorbol esters by stripping/deodorization at 260 °C with 3 mbar pressure and 1% steam injection has been reported by Makkar et al. (2009). Rakshit et al. (2008) observed about 89% reduction while subjecting to alkali and heat treatment [1:1 (w/v) alkali, autoclav- ing at 121 °C]. Extraction with methanol followed by treatment with 0.07% NaHCO 3 in seeds reduced phorbol esters by 97% (Martı ´ nez-Herrera et al., 2006). Washing of heat treated (auto- claved at 121 °C) meal with methanol degrades 95% of the phorbol esters, a reduction from 1.78 mg/g seed meal to a tolerable level of 0.09 mg/g (Aregheore et al., 2003). Aregheore et al. (2003) have reported that heat treatment alone did not decrease the concentra- tion of phorbol esters and chemical treatment in addition to heat is necessary to bring down the concentration of phorbol ester 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.01.039 ⇑ Corresponding author. Tel.: +91 11 2659 6533; fax: +91 11 2658 1102. E-mail addresses: skkhare@chemistry.iitd.ac.in, skhare@rocketmail.com (S.K. Khare). Bioresource Technology 102 (2011) 4815–4819 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech