Optimization of R-(+)-α-terpineol production by the biotransformation of R-(+)-limonene

Abstract

R-(+)-limonene is an abundant and non-expensive by-product of the citrus industry and is, therefore, a suitable starting material for the production of natural flavor and fragrance compounds. The biotransformation of R-(+)-limonene to R-(+)-alpha-terpineol by Fusarium oxysporum 152b has already been reported, although the influence of the main process parameters on the production has not yet been evaluated. In this paper, a Plackett-Burman screening design was used to define the effects of the medium composition (glucose, peptone, yeast extract, malt extract and pH), the presence of a co-substrate (biosurfactant), the cultivation conditions (temperature, agitation), the substrate concentration and the inoculum/culture medium ratio on the absolute amount of R-(+)-alpha-terpineol resulting from this biotransformation. The process conditions were further optimized applying response surface methodology (RSM). The volatiles were extracted using a SPME device and were subsequently quantified by GC-FID and identified by GC-MS. The best results were obtained using 0.5% (v/m) R-(+)-limonene in pure distilled water as the culture medium with an inoculum/culture medium ratio of 0.25 (m/m) and 72 h cultivation at 26 degrees C/240 rpm. Under these conditions the concentration of R-(+)-alpha-terpineol in the culture medium reached 2.4 g L(-1), a production almost six times greater than in earlier trials. The presence of a biosurfactant (0-500 mg L(-1)) did not significantly increase the yield.

Improved monoterpene biotransformation with Penicillium sp. by use of a closed gas loop bioreactor

“…Whereas all previously published quantitative data on -terpineol formation with P. digitatum have been derived from assay or shake Xask scales [22,36,37,39], the novel process design enabled us to also report an -terpineol production in the g L ¡1 range on a bioreactor scale for the Wrst time. Very recently, a newly isolated fungus, Fusarium oxysporum 152b, was shown to produce up to 2.4 g L ¡1 -terpineol from (+)-limonene after medium optimization in shake Xasks [40]. Despite the obviously pronounced product tolerance of this fungus, the high toxicity and high volatility of the precursor were claimed to strongly impede further increased product concentrations.…”

Isolation and Screening of Microorganisms for R-(+)-Limonene and (−)-β-Pinene Biotransformation

“…One of the strains tested, F. oxysporum 152B, converted R-(+)-limonene to R-(+)-α-terpineol, yielding nearly 450 mg/L after 3 days of reaction. Bicas et al [26] optimized the process conditions for the biotransformation of R-(+)-limonene to R-(+)-α-terpineol by F. oxysporum 152b using response surface methodology. Best results were obtained using 0.5% (v/m) of R-(+)-limonene in pure distilled water as culture medium with an inoculum/culture medium ratio of 0.25 (m/m) and 72-h cultivation at 26°C/240 rpm.…”

Optimization of α-Terpineol Production by the Biotransformation of R-(+)-Limonene and (−)-β-Pinene

“…A central composite rotatable design (CCRD) 2 3 was carried out to each tested microorganism, and the studied variables were: substrate concentration (0.01 to 3.85 wt%), ratio between substrate and ethanol (0 to 1:2.7 v/v), and inoculum (0.64 to 7.36 g for filamentous fungi and 0.32 to 3.68 g for yeasts). Results were analyzed by the software Statistica® 6.0.…”

“…Terpenes are secondary metabolites of plants that are produced, in part, as a defense against microorganisms and insects, in addition to their pollinator-attractive properties [3].…”