ORIGINAL PAPER P. Barghini á F. Montebove á M. Ruzzi á A. Schiesser Optimal conditions for bioconversion of ferulic acid into vanillic acid by Pseudomonas fluorescens BF13 cells Received: 1 July 1997 / Received revision: 28 October 1997 / Accepted: 16 November 1997 Abstract Pseudomonas ¯uorescens BF13 is especially capable of promoting the formation of vanillic acid during ferulic acid degradation. We studied the possi- bility of enhancing the formation of this intermediary metabolite by using suspensions of cells at high density. The bioconversion of ferulic into vanillic acid was af- fected by several parameters, such as the concentration of the biomass, the amount of ferulic acid that was treated, the carbon source on which the biomass was grown. The optimal yield of vanillic acid was obtained with 6 mg/ml cells pre-grown on p-coumaric acid and 2 mg/ml ferulic acid. Under these conditions the bio- conversion rate was 95% in 5 h. Therefore BF13 strain represents a valid biocatalyst for the preparative syn- thesis of vanillic acid. Introduction The use of microbial cells and their enzymes as catalysts in the manufacture of economically important substances is a rapidly developing ®eld of biotechnology. For nearly every type of reaction known in organic chemistry (oxi- dative, reductive, hydrolytic or conjugative) there is a biocatalytic equivalent (Turner 1995). The key advantage of biocatalysis is that enzymes and microorganisms cat- alyse reactions speci®cally under mild conditions, there- by saving energy (Faber 1992; Wong and Whitesides 1994). Moreover, bioprocesses are the only ones legally accepted in Europe and the US for the production of some food additives such as natural ¯avour. At present we are studying the microbial degradation of lignin by isolating several aerobic bacterial strains able to utilise phenylpropenoids as carbon source, particularly ferulic acid. This compound occurs, free or in combined form, in many Monocotyledoneae and Dicotyledoneae (Hartley and Harris 1981; Smith and Hartley 1983) and can be easily extracted from some agriculture by-products. It represents a feedstock for the biocatalytic conversion into other valuable molecules such as styrenes, polymers, epoxides alkylbenzenes, vanillin and vanillic acid derivatives, protochatecuic- acid-related catechols, guaiacol and catechol (Rosazza 1995; Rosazza et al. 1995). This paper describes the results that we have obtained with one of the isolated strains identi®ed as Pseudo- monas ¯uorescens, named BF13, which utilised some phenylpropenoids (ferulic, m- and p-coumaric acids) as sole carbon source. This strain, when degrading ferulic acid, transiently forms in the culture medium a certain amount of vanillic acid. The latter is also important for biotechnological applications since vanillic acid is used as the starting material in the chemical synthesis of ox- ygenated aromatic chemicals, such as vanillin, one of the most important ¯avour molecules (Cheetham 1993; Rosazza 1995). In this work we have studied the possibility of in- creasing the level of vanillic acid that accumulates in the medium during ferulic acid degradation. This was done through bioconversion experiments performed with suspensions of cells at high density. Conditions for the optimum production of vanillic acid were investigated. Materials and methods Bacterial strain and growth conditions Strain BF13 was isolated from soil for its ability to utilise ferulic acid as the sole carbon and energy source. It is a gram-negative, aerobic and motile rod, that is catalase- and oxidase-positive. On the basis of the phenotypic properties, it was identi®ed by using an API 20 NE identi®cation kit (bioMerieux) as a P. ¯uorescens (99.9% identity; Ruzzi et al. 1997). The medium used in all experiments described in this paper was the M9 minimal medium (Sambrook et al. 1989). Phenylpropenoids (ferulic, m- and p-coumaric acids), glucose, glutamate and succinate Appl Microbiol Biotechnol (1998) 49: 309±314 Ó Springer-Verlag 1998 P. Barghini á F. Montebove á M. Ruzzi (&) á A. Schiesser Department of Agrobiology and Agrochemistry, University of Viterbo, via C. de Lellis, blocco B, I-01100 Viterbo, Italy Fax: +39 761 357242