Production of natural vanillin from cymbopogon citratus using phanerochaete chrysosporium

Abstract

Malaysia as one of the important agricultural countries in the world producing 70 million tonnes of lignocellulosic biomass. One of the abundant agricultural wastes which has high lignocellulose content is lemongrass leaves (Cymbopogon citratus). A total of 8,000 tonnes of dry leaves are produced annually from 1,150 hectares of C. citratus plantation in Malaysia. Parts of the leave wastes are burned for electric generation while the rest are left in the fields to decompose naturally. The use of lemongrass leaves as ruminant feedstock is, however, not favoured due to animal rejection against its aroma. C. citratus leaves have 58% hemicellulose and lignin content and are also rich in ferulic acid. Ferulic acid is the precursor for vanillin production. The C. citratus leave wastes that contain ferulic acid could be potentially used for vanillin production via microbial approach, which is currently less investigated. The main purpose of this research was to investigate the potential of a one-step natural vanillin production from C. citratus leaves hydrolysate by Phanerochaete chrysosporium, the white-rot fungi of basidiomycetes. The research work focused on the recovery of ferulic acid as well as the optimization of natural vanillin production from C. citratus leaves. Leaves within size of 125-249 µm appeared to be suitable for ferulic acid extraction, with 1.12g/L total recovery of ferulic acid by 55 minutes of boiling. A total of 27 strains of fungi were screened for natural vanillin and vanillic acid production. Among these fungi, Phanerochaete chrysosporium was chosen due to its high natural vanillin productivity. The effect of different nitrogen source in vanillin production was investigated using the General Factorial Design. A combination of ammonium chloride with yeast extract (ratio: 75:4) increased the vanillin production to 15-fold. To further optimize the process using 2-Level Factorial Design, the aforementioned nitrogen sources and 5 others independent variables (incubation temperature, pH, incubation time, agitation and inoculum size) were studied. The ammonium chloride concentration, temperature and pH were found to be the key factors for natural vanillin production and these factors were further examined using the Central Composite Design (CCD). The maximum vanillin production was observed with media composition of 1.0 g/L ferulic acid from hydrolysate of C. citratus leaves, 4.43 g/L ammonium chloride (inorganic nitrogen), 0.25 g/L yeast extract (organic nitrogen), 0.2 g/L KH2PO4, 0.013 g/L CaCl22.H2O, 0.5 g/L MgSO4.7H2O and 0.0025 g/L thiamin hydrochloride, at 36.3 °C and pH 6.84 under shaking condition at 150 rpm with inoculum size of 7 % (w/v). The generated model fitted well to the data set with R2 of 0.9059. The actual optimum conditions yielded 0.20 g/L vanillin (17-fold higher than non-optimised condition). The actual experimental data showed 2-fold higher yield as compared to previous reported studies. Artificial Neural Network model were also found to provide accurate validation and prediction result based on feed-forward back propagation as similar to Response Surface Methodology (RSM). A network with one hidden layer and 25 neurons were found to be the most optimum model with the lowest root mean squared error (RMSE; 0.0002415), absolute average deviation (AAD; 0.0004358) and the highest R2 (0.9024). In this study, it was confirmed that C. citratus leaves could serve as a promising alternative source for vanillin production with potential to scale-up for commercial production in Malaysia.