Production of Lovastatin, (+)-Geodin and Sulochrin by Aspergillus Terreus ATCC 20542 using Pure and Crude Glycerol

A. terreus ATCC 20542 is a prolific fungi strain known for its ability to produce lovastatin, a potent cholesterol-lowering drug. Lovastatin is synthesised via type I polyketide pathway (PKS), a common pathway used to produce secondary metabolites in microorganisms. This pathway is also responsible for the production of two cometabolites of lovastatin, namely (+)-geodin and sulochrin. This study aimed to characterise the production of lovastatin and its co-metabolites, sulochrin and (+)geodin by A. terreus, and to investigate the relationship between lovastatin, sulochrin and (+)-geodin production using bio-waste crude glycerol (CG) as the substrate. The first part of this study investigated the effects of the major components of culture medium for the production of lovastatin, (+) -geodin and sulochrin, and pellet morphology. This investigation revealed that the types of carbon source have a major influence on lovastatin production, but not (+) -geodin and sulochrin. By contrast, the types of nitrogen source mainly influence (+)-geodin and sulochrin production. Of note, reasonable lovastatin production (25.68 mg/L), with high production of (+)-geodin (9.00 mg/L) and sulochrin (22.35 mg/L) can be achieved using glycerol as the carbon source, and yeast extract as the nitrogen source. Further, culture with glycerol produced pellets with hairy morphology, which are optimal for the production of lovastatin, (+) -geodin and sulochrin. These results provide a basis for optimum culture conditions for subsequent experiments to study the production of metabolites by A. terreus. The second part of the study investigated the potential of crude glycerol (CG), a common bio-waste product from biodiesel industry, as the substrate for A. terreus cultivation and the production of metabolites of interest. At 30 g/L of CG, the production of (+)-geodin (13.14 mg/L) and sulochrin (14.79 mg/L) increased almost 2fold, with a significant inhibition of lovastatin production (~35% reduction) when compared to pure glycerol (PG). The major contaminants from CG were then identified, and their effects on A. terreus’ growth and metabolite production were determined to further explain these observations. These studies show that the presence of contaminants in CG including saturated fatty acids (up to 48% reduction) and soap (up to 90% reduction) could contribute to the inhibitory effect of CG on lovastatin production, with no inhibition was observed on (+)-geodin and sulochrin production. Conversely, some contaminants, including double-bonded fatty acid such as oleic acid, methanol (MeOH) and salt (NaCl) could enhance lovastatin production up to 72%, with varying effects on (+)-geodin and sulochrin production. Partial purification of CG using solvent and activated carbon (AC) resulted in an improved yield of all three metabolites. This investigation revealed that CG can potentially be used to cultivate A. terreus, provided that sufficient purification is conducted on CG. Elicitor refers to a substance that provokes the microorganism’s defense system. The third part of this study investigated the effects of selected ‘elicitors’ on the production of lovastatin, (+)-geodin’s and lovastatin, and to elucidate the relationship between these three metabolites, if any. CG, which was subjected to partial purification by AC, was used for this part of the study, as the product purity was comparable to pure glycerol. In this study, the elicitors of choice were chemical elicitors such as sodium alginate, cholesterol, malonic acid, and physical elicitors such as shear force and viscosity. It was found that chemical elicitor stimulated the production of both lovastatin and sulochrin, with a lesser degree of stimulation on (+)-geodin’s production. On the other hand, (+)-geodin’s production was suppressed in the presence of high viscosity ( 500 mg/L). These observations indicate that lovastatin and sulochrin may play a role in A. terreus’ defense mechanism. Conversely, (+)-geodin may be important for fungal pellet integrity or immediate response to injury, as physical force greatly enhanced its production. In conclusion, CG is a promising alternative substrate for metabolite production by A. terreus, provided that sufficient purification and culture conditions are applied. This study, however, demonstrates no apparent relationship between the production of lovastatin, (+)-geodin and sulochrin by A. terreus using glycerol or CG as the substrate.