International Biodeterioration & Biodegradation 55 (2005) 261–269 Optimisation of physical factors on the production of active metabolites by Bacillus subtilis 355 against wood surface contaminant fungi Ce´lia Moita a , Sonia Savluchinske Feio a,Ã , Lina Nunes b , Maria Joa˜o Marcelo Curto a , Jose´ Carlos Roseiro a a Departamento de Tecnologia de Indu´strias Quı´micas, Instituto Nacional de Engenharia e Tecnologia Industrial, Estrada ao Pac - o do Lumiar, 1699-038 Lisboa, Portugal b Laborato´rio Nacional de Engenharia Civil, Av. do Brasil, 101, 1700-066 Lisboa, Portugal Abstract A Surface Response Model was used to study the effect of pH, temperature and agitation on growth, sporulation and production of antifungal metabolites by Bacillus subtilis CCMI 355. Strong agitation, temperature between 27 and 34 1C and pH 6 favoured cell growth. Alkaline pH, strong agitation and temperature between 28 and 34 1C favoured spore formation. No relationship was found between sporulation and the production of antifungal metabolites. According to the model, pH 8, 37 1C and the absence of agitation were the optimal conditions for the production of broad-spectrum antifungal metabolites against Botrytis cinerea, Penicillium expansum, Trichoderma sp, Trichoderma harzianum, Trichoderma koningii and Trichoderma virgatum. In situ assays using green wood impregnated with Bacillus subtilis CCMI 355 inoculated in Yeast Extract Glucose Broth medium in the conditions above, displayed an efficient protection against wood surface contaminant fungi. r 2005 Elsevier Ltd. All rights reserved. Keywords: Antifungal metabolites; Bacillus subtilis; Surface response model; Wood protection 1. Introduction The growing demand for healthy agricultural and forest products is in line with the increased utilisation of chemical agents to ensure an efficient and rigorous control of their deterioration. Synthetic pesticides and genetically modified plants are now a more current practice (Emmert and Handelsman, 1999). Unfortu- nately, these solutions have proven to induce resistance in the pathogenic populations, to show non-target effects and to be outside the increasing strict safety regulations (Chen and Michailides, 2001; Emmert and Handelsman, 1999). In addition, the growing consumer awareness and/or demand for healthy products in- creased the quest for more efficient methods with minimum health and environmental impact for the control of diseases (Emmert and Handelsman, 1999). Decay of stored wood represents a serious economic problem in the pulp and paper industry. The attack of wood by microorganisms, particularly fungi, may range from minor attack to extractives and deep staining to total decomposition of all the components (Bernier et al., 1986). The decomposition is controlled by syner- gism/antagonism relationships among wood colonising microorganisms. Nutrient competition, mechanical bar- riers or pH changes are some of the antagonistic effects. Both fungi and bacteria are able to synthesise a wide range of metabolites with fungicidal and bacterial ARTICLE IN PRESS www.elsevier.com/locate/ibiod 0964-8305/$-see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2005.02.003 Ã Corresponding author. Tel.: +351217162712; fax: +3511217166966. E-mail address: sonia.feio@ineti.pt (S.S. Feio).