Bacillus subtilis SPB1 biosurfactant: Production optimization and insecticidal activity against the carob moth Ectomyelois ceratoniae Inès Mnif a, b , Mouna Elleuch a, b , Semia Ellouze Chaabouni a , Dhouha Ghribi a, b, * a Unité « Enzymes et Bioconversion », National School of Engineers of Sfax, Tunisia b Higher Institute of Biotechnology of Sfax, Tunisia article info Article history: Received 4 July 2012 Received in revised form 10 March 2013 Accepted 16 March 2013 Keywords: Bacillus subtilis Lipopeptide biosurfactant Insecticidal activity Ectomyelois ceratoniae Optimization Solid state fermentation abstract Bacillus subtilis SPB1 was shown to produce a lipopeptide biosurfactant. The insecticidal activity of this biosurfactant was evaluated against Ectomyelois ceratoniae Zeller, a moth pest of stored dates in Tunisia. The LC 50 and LC 90 values after six days of contact were 152 mg/g and 641 mg/g, respectively. To promote an economical production of this highly effective bioinsecticide, statistical experimental designs and response surface methodology were employed to optimize the concentrations of agro-industrial residues and humidity, for lipopeptide biosurfactant production by B. subtilis SPB1 under solid state fermentation. The optimal medium leading to a production yield near to 28 mg of crude lipopeptide preparation per g of wet solid material was composed of a mixture of 4.34 g of tuna fish flour and 5.66 g of potato waste flour with a moisture content of 76%. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction In the south of Tunisia, dates are the main agro-resource of oasies and have a major role in the Tunisian agriculture and the development of the national economy. Nevertheless, dates are subjected to many diseases and pests that decrease their yield and deteriorate their quality. The carob moth, Ectomyelois ceratoniae Zeller (Lepidoptera, Pyralidae), is the most important and destructive insect pest attacking dates (Phoenix dactylifera Linn) both in the field and during storage (Jarraya and Vinson, 1980; Dhouibi, 1989; Mediouni et al., 2004; Ben Jemâa et al., 2012). It infests 20% of the harvestable crop annually (Anonymous, 2009), degrades stored dates and causes weight loss and downgrading of the commercial value of the fruit (Al-Azawi et al., 1984; Dhouibi, 2000). Fumigation is among the most current available methods for postharvest control of theses stored pests (Dhouibi, 1989; Beckett, 2011). Nevertheless, their application is being restricted due to concerns about possible health and environmental damage (Ben Jemâa et al., 2012). Therefore, research on effective alternative methods is needed. Recently, the use of essential oils as insecticidal agents have shown some promise (Ben Jemâa et al., 2012; Haouel et al., 2010). Also, insecticidal proteins produced by Bacillus thuringiensis (Berliner) species have been reported to be effective for biological control of Lepidoptera larvae and adults. Surface active compounds produced by Bacillus subtilis (Ehrenberg) species were also reported as exhibiting insecticidal activities against larval and pupal stages of mosquitoes (Geetha and Manonmani, 2010; Manonmani et al., 2011) and against lepidopterean and Spodoptera littoralis (Bois- duval) (Abd El-Salam et al., 2011; Ghribi et al., 2011a) larvae, mainly, Prayes olea (Bernard) (Ghribi et al., 2011b) and Ephestia kuehniella (Zeller) (Ghribi et al., 2012a,b). Biosurfactants are natural active compounds with amphiphilic structure consisting of a hydrophilic head and a hydrophobic tail produced by a large range of microorganisms (Muthusamy et al., 2008). They can decrease surface and interfacial tension in watereoil and oilewater systems. Based on their chemical composition, biosurfactants can be classified into glycolipids, lip- opeptides, lipoproteins, phospholipids, fatty acids and polymeric surfactants (Muthusamy et al., 2008). They are popular in many fields: environment, food industry and biopharmaceutic technol- ogy for their interfacial, emulsifying, foaming, antimicrobial activ- ities and their biodegradable nature, low toxicity and their temperature, pH and ionic strength tolerance (Cameotra and Makkar, 2004; Mulligan, 2005; Nitschke and Costa, 2007; Muthusamy et al., 2008). But, the high cost of production and re- covery limit their use. Hence, to overcome this problem, scientists * Corresponding author. Unité « Enzyme et Bioconversion », ENIS, BP W 3038 Sfax, Tunisia. Tel.: þ1 216 74674364; fax: þ1 216 74675055. E-mail address: dhouhag@yahoo.fr (D. Ghribi). Contents lists available at SciVerse ScienceDirect Crop Protection journal homepage: www.elsevier.com/locate/cropro 0261-2194/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cropro.2013.03.005 Crop Protection 50 (2013) 66e72