## 4 Avenues and Approaches for Improving the Biotransformation of Limonene and Alpha Pinene
### Selection of Microorganisms  
Synthetic limonene or pinenes are often used instead of residues for biotransformation [41]. It would be of interest to use known strains to carry out solid or semi-solid fermentations of residual media containing limonene (citrus like orange, lemon) or pinene (white birch bark). This practice, rather than the use of oils, would minimise the toxicity of pinene or limonene at the start of fermentation. In addition, the use of solvents for juice extraction will be eliminated at the start of the process. Another possible step would be to isolate microbial strains from the residual wood and orange media. Indeed, the natural presence of strains in these residues testifies to their ability to withstand toxic doses of the molecules in question, as well as their capacity to metabolise them. Among strategies, bioprospecting is used by some authors in an attempt to identify new strains of microorganisms for the production of bioaromas [42]. Particularly, soil samples have been frequently used for the prospecting of microorganisms that are potential bio-transformers of terpene compounds. To illustrate this approach, some examples of microorganisms isolated from soil may be cited: Bacillus fusiformis, which was able to convert isoeugenol to vanillin (production of 8.10 g L1 after 72 h) [43]; Bacillus pumilus, which was also able to convert isoeugenol to vanillin (production of 3.75 g L1 after 150 h) [44]; Pseudomonas putida, which was able to convert isoeugenol to vanillin acid (98% of molar conversion after 40 min); Bacillus subtilis, which was able to convert isoeugenol to vanillin (production of 0. 9 g L1 after 48 h) [45] and _Chrysosporium panorum_, which was able to convert a-pinene to verbenone and verbenol. [PERSON] et al. [37] have isolated strains from effluents of citrus industry as well as other samples (soil from the plantation of citrus, citrus fruits, and citrus leaves). Of the 405 strains isolated [37], eight were able to bioconvert limonene and fifteen converted pinene, generating \(\alpha\)-terpineol as a product in both cases. The transformation of limonene to \(\alpha\)-terpineol typically involves the oxidation of limonene. The first step is the hydroxylation of limonene, which can occur at different positions to yield a variety of alcohols. This is typically catalysed by cytochrome P450 monooxygenases and the formation of Limonene-1,2-diol: One of the primary alcohols formed from the hydroxylation of limonene is limonene-1,2-diol. Then the conversion to Terpineol: Limonene-1,2-diol can then be dehydrated to yield \(\alpha\)-terpineol. This step can be enzymatically catalysed bydehydratase enzymes [46]. While the biotransformation of \(\alpha\)-Pinene occurs typically with the oxidation of \(\alpha\)-pinene to yield \(\alpha\)-pinene oxide. This reaction is facilitated by the enzyme cytochrome P450 monooxygenase or similar enzymes present in Pseudomonas or other fungi. The \(\alpha\)-pinene oxide can undergo a rearrangement to form generally verbenol. The verbenol formed can then be hydroxylated to produce terpineol. This hydroxylation can be mediated by specific hydroxylase enzymes present in fungi [46].  
[PERSON] et al. [9] have obtained 248 microorganisms, of which seventy were developed in a medium containing limonene as the sole source of carbon. However, as reported by [PERSON] et al. [30], there are several challenges to be overcome to enable the production of aromas by biotransformation, among them the high toxicity of both the substrate and the product and the low yields obtained. According to these authors, such difficulties can be overcome with the isolation and selection of strains as the studies mentioned above.