Isolation and Screening of D-limonene-resistant Microorganisms

This study reports the isolation of microorganisms that are resistant to environment containing limonene, the most important residue in the citrus industry. For the isolation, samples collected from strategic places of a citrus processing plant (yellow water, entrance and exit of the bagasse tank, effluent and deteriorated fruits found in bins, machine straps, fruit washers and plant floor), some citrus fruit and mint from local market were used. The samples were incubated in rotary shaker at 30oC/150rpm for 48h or 7 days in YM medium containing 0.1% limonene. Great part of the 112 strains recovered after 48h and the 126 strains recovered after 7 days were identified as Gram positive bacilli, followed by Gram negative bacilli, yeasts and Gram positive cocci, besides five fungi. About half of the Gram positive and Gram negative bacilli and yeasts and some Gram positive Cocci were resistant to limonene concentrations up to 2% in the medium broth. Amongst them seventy were able to grow in mineral medium containing limonene as sole carbon source. The research described in this paper is the initial step for the exploration of flavor compounds production via biotransformation of limonene, a nonexpensive by-product of citrus industry. Key-words: Limonene, industrial by-products, flavor, biotechnology. *Corresponding Author. Mailing address: Laboratório de Bioaromas Departamento Ciência de Alimentos Faculdade de Engenharia de Alimentos UNICAMP Rua Monteiro Lobato, 80 CP 6121 cep 13083-862 Campinas, SP Brasil. Tel.: (19) 3521-3887. E-mail: [email protected] INTRODUCTION D-limonene is the most abundant monocyclic monoterpene in nature and represents more than 90% of orange peel oil (2). Its chemical structure is similar to many oxygenated monoterpenoids with pleasant fragrance and, therefore, dlimonene may be used in flavor industry as a precursor in the synthesis of those compounds (20). Beyond their desirable fragrances, some of these d-limonene oxygenated derivates, e.g. perillyl alcohol, carveol, carvone, geraniol and menthol, have shown biological activity in vivo against certain types of tumors, not only preventing the formation or progression of cancer, but also regressing the existing malignant tumors (8). These characteristics greatly enhance industrial interest in such compounds. In this context, biotransformation process emerges as an attractive alternative for the d-limonene transformation since, comparing to the traditional chemical methods, they proceed under mild conditions, have an elevated regio and enantioselectivety, do not generate toxic wastes and the products obtained might be labeled as “natural” (13,17). In the scientific literature, the biotransformation of limonene by microorganisms has been well documented (12). However, a commercial process, which could be applicable and possess high productivity, is yet to be developed. The main problems involved in monoterpene biotransformation processes are (i) the chemical instability of both precursor and product, (ii) the low solubility of the precursor, (iii) the high volatility of both precursor and product, (iv) the high cytotoxicity of both precursor and product and (v) the low transformation rates (18). The three first topics are very difficult to be overcome as they are related to physicochemical characteristics inherent to the system, but a good limoneneresistant strain screening might surpass the citotoxicity and the low transformation rates. The interest in solvent-tolerant microorganism screening is increasing in the last years. The isolation of microorganisms