## 3 Recent Advances in Biotransformation of Terpenes: Study Case of Limonene and Alpha Pinene
### Biotransformation of \(\alpha\)-pinene  
One of the prominent antimicrobial monoterepenes found in various essential oils is \(\alpha\)-pinene. This natural compound holds significant importance as it is widely utilised in the food and fragrance industries, pharmaceuticals, fine chemicals, and as a renewable fuel source [33]. Currently, industrial production of \(\alpha\)-pinene primarily relies on tree tapping (gum turpentine) or as a byproduct of paper pulping (crude sulfate turpentine, CST). However, extracting \(\alpha\)-pinene from trees is laborious, inefficient, and depletes natural resources due to its low content [34]. Hence, exploring sustainable technologies for the biotransformation of \(\alpha\)-pinene would be a promising avenue (Figure 3). In a study conducted by [35], a cold-adapted fungus, _Chrysosporium panororum_, was identified as a biocatalyst for the oxidation of refining \(\alpha\)-pinene. Gas chromatography-mass spectrometry (GC-MS) analysis of the fermentation process revealed the production of two commercially valuable molecules, verbenol and verbenone, through the biotransformation of \(\alpha\)-pinene. The oxidative activity of _C. panororum_ was observed over a wide temperature range (5-25 \({}^{\circ}\)C), with the optimum temperature being 10 \({}^{\circ}\)C to produce these bioactive compounds. By sequentially adding the substrate over a period of three days, the yield of verbenol and verbenone significantly increased to 722 mg/L and 176 mg/L, respectively, resulting in a twofold enhancement compared to a single supply of \(\alpha\)-pinene. The total concentration of conversion products in the culture medium reached 1.33 g/L. Another study by [36] focused on the biotransformation of \(\alpha\)-pinene to (+)-\(\alpha\)-terpineol using _Candidia tropicalis_ MTCC 230. Under continuous agitation at 30 \({}^{\circ}\)C for 96 h, a concentration of 0.5 g/L of (+)-\(\alpha\)-terpineol was obtained. The strain demonstrated efficient bioconversion capabilities, achieving a conversion rate of 77% and a production of 0.43 g/L under the specified conditions. [PERSON] et al. [37] reported the metabolism of \(\alpha\)-pinene by the yeast _Hormonema_ sp. UOPS Y-0067, isolated from pine tree samples. After 72 h, this yeast produced transverbenol (0.4 g/L) and verbenone (0.3 g/L)  
Figure 2: Biosynthetic pathways in plants starting from limonene.  
through \(\alpha\)-pinene biotransformation. It is worth noting that all these experiments were conducted using pure or refine \(\alpha\)-pinene. To the best of our knowledge, no research has been conducted on the biotransformation of \(\alpha\)-pinene using residual wood, such as tree bark. Further investigations should include GC-MS analysis of different tree species or parts to identify sources with high \(\alpha\)-pinene content. Based on the market value of the molecules obtained by the transformation of \(\alpha\)-pinene, it would be important to study the technological feasibility of the biotransformation of \(\alpha\)-pinene contained in wood residues rich in pinene. The key parameters for a good microbial fermentation in a semi-solid medium are widely studied in the literature. The particle size and specific surface area are key parameters to ensure a good contact surface between the microorganisms and the molecules to be biotransformed. Also, the particle size is an important factor in oxygen transfer during solid or semi-solid fermentation. Other important factors are the moisture content, the inoculum size and the fermentation temperature [38]. Regarding terpen, the volatilisation of molecules and the presence of lignin can be factors slowing down the yield of molecules obtained. The following parts will discuss in detail the current practices to address these challenges.  
Figure 3: Degradation pathways of alpha pinene by Pseudomonas.