Acta Tropica 140 (2014) 19–25 Contents lists available at ScienceDirect Acta Tropica jo ur nal home p age: www.elsevier.com/locate/actatropica Efficacy of olive mill wastewater for protecting Bacillus thuringiensis formulation from UV radiations Wafa Jallouli ∗ , Sameh Sellami, Maissa Sellami, Slim Tounsi Biopesticides Team (LPAP), Centre of Biotechnology of Sfax, Sfax University, P.O. Box: “1177”, Sfax 3018, Tunisia a r t i c l e i n f o Article history: Received 20 May 2014 Received in revised form 24 July 2014 Accepted 25 July 2014 Available online 2 August 2014 Keywords: Bacillus thuringiensis Biopesticides UV protectants Toxicity Ephestia kuehniella a b s t r a c t The effectiveness of 10 low-cost UV-absorbers in protecting Bacillus thuringiensis subsp. kurstaki BLB1 toxins against inactivation by UV-A and UV-B irradiation was evaluated in this study. Among them, two by-products, molasses and olive mill wastewater (OMW) were selected for further studies. They were tested at different concentrations of 0.05, 0.1, 0.15 and 0.2% using the para-aminobenzoic acid (PABA) as a common UV protectant. Interestingly, addition of PABA and OMW to BLB1 formulations was found to be most effective in protecting BLB1 spores at 90.8 and 76.4% respectively and in preserving delta- endotoxin concentration at a level of 81.7 and 72.2%, respectively when used at a concentration of 0.2%. The lowest preserved spores (46.3%) and delta-endotoxin level (12.4%) was found using molasses. In contrast, spore count and delta-endotoxin concentration were completely reduced after an exposure of unprotected Bt strain BLB1 to UV radiations up to 96 h. SDS-PAGE analysis of protected and unprotected samples revealed that delta-endotoxin bands (130, 65–70 kDa) were conserved until 96 h of UV exposure in presence of PABA or OMW compared with their disappearance in presence of molasses after 72 h of exposure and their dramatically decline from 8 h of exposure in unprotected mixture. A complete loss of larvicidal toxicity against Ephestia kuehniella was found after 24 h of exposure in absence of any UV- absorber. Addition of OMW or PABA offered the highest levels of insecticidal activity with 63.2 and 74.7% of residual toxicity, respectively. Whereas, molasses addition, as UV protectant retained only 26.3% of residual activity after 96 h of exposure. Therefore, addition of OMW by-product to Bt formulation may be a suitable alternative to others synthetic chemical compounds. OMW may also provided added value, be environmentally friendly and less hazardous, when used at low concentration. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Application of microorganisms for biological control is draw- ing increasing attention to environmentally hazardous chemical pesticides. Bacillus thuringiensis (Bt) serotypes are the most stud- ied and markedly used bacterium as bioinsecticide (Schnepf et al., 1998). They are Gram positive soil bacteria characterized by their ability to produce crystalline inclusions containing delta-endotoxin proteins such as Bt kurstaki which express the 27–130 kDa pro- teins (Kim et al., 2013). Many formulations of Bt kurstaki are available and applied under field conditions, however the major drawback of using Bt protoxins and spores as active ingredients in biopesticides against Lepidopteran pest species is their lack of persistence due to UV radiations. Generation of free radicals fol- lowing oxidation of amino acids and destruction of tryptophan ∗ Corresponding author. Tel.: +216 74 874 446; fax: +216 74 874 446. E-mail address: jallouliwafa25@gmail.com (W. Jallouli). and histidine residues were the major mechanisms that have been suggested for the inactivation of Bt protoxins (Ignoffo and Garcia, 1978; Pozsgay et al., 1987; Becker et al., 1992). This disad- vantage led to the search for protective measures to reduce the damaging effect of UV radiations on Bt toxins. Photostability of Bt biopesticide, could be achieved by encapsulation with sodium alginate, gelatin, starch, carboxymethylcellulose and carrageenan (Kuppusamy et al., 1989; Elcin, 1995; Elcin et al., 1995; Prabakaran and Hoti, 2008). It could be accomplished also by using differ- ent synthetic organic compounds such as congo red, uric acid, para-aminobenzoic acid (PABA), benzaldehyde, malachite green and melanin which have been evaluated as suitable UV protec- tants for various entomopathogens (Krieg et al., 1980; Dunkle and Shasha, 1989; Cokmus et al., 2000; Maldonado-Blanco et al., 2002; Lee et al., 2006; Arthurs et al., 2006). However, these compounds were considered as expensive products and are not recommended to be disseminated in the environment. Therefore, in our labora- tory, attempts have been made to look for cheaper alternatives such as the use of molasses and olive mill waste water (OMW). http://dx.doi.org/10.1016/j.actatropica.2014.07.016 0001-706X/© 2014 Elsevier B.V. All rights reserved.