Aspergillus sclerotiorum PSU-RSPG178 was isolated from a soil sample collected from the Plant Genetic Conservation Project under the Royal Initiation of Her Royal Highness Princess Maha Chakri Sirindhorn at Ratchaprapa Dam in Suratthani Province, Thailand. It was grown on potato dextrose agar (PDA) which was obtained from HiMedia, Mumbai, India at 28 °C for 7 days. Mycelial plugs (0.5 cm×0.5 cm) cut from the periphery of an actively growing colony were used as the inoculum according to the method of Daengrot et al. (15). The mycelial plugs were used since a spore inoculum gave a low yield of lovastatin in a previous study (14).

The agricultural wastes used in this study were corn trunks, rice husks, wild sugarcane (Saccharum spontaneum) and soya bean sludge. The corn trunks, rice husks, and wild sugarcane were obtained from farmers in Khuan Khanun, Phatthalung, Thailand, while fresh soya bean sludge was obtained from a tofu shop in Hat Yai, Songkhla, Thailand. The corn trunks and wild sugarcane were washed with water and cut to a length of 0.5 cm. All the wastes were dried at 60 °C to a constant mass. Wheat flour, molasses and palm oil (Morakot, olein palm oil) were obtained from a local market in Hat Yai, Songkhla, Thailand. All chemicals were of analytical grade, including glucose (d-glucose anhydrous, Kemaus, Cherybrook, NSW, Australia), urea (99.5%, Kemaus), KH₂PO₄ (anhydrous, Loba Chemie, Mumbai, India), MgSO₄·7H₂O, CaCl₂ (dihydrate, FeSO₄·7H₂O and ZnSO₄·7H₂O (Fisher Scientific, Leicestershire, UK), and methanol, hexane, ethyl acetate, Na₂SO₄ and acetonitrile (all RCI Labscan, HPLC grade, Bangkok, Thailand).

For SmF, 5 g (dry mass) samples of waste were placed in 250-mL Erlenmeyer flasks to which 100 mL of distilled water were added. The mixture was autoclaved for 15 min at 103 kPa and 121 °C, then cooled to room temperature. Five mycelial agar plugs (0.5 cm×0.5 cm) of A. sclerotiorum PSU-RSPG178 were added to each Erlenmeyer flask. The flasks were incubated at 25 °C for 14 days. The solid waste that gave the highest yield of lovastatin concentration was selected to be cultured under SSF.

For SSF, 5 g (dry mass) samples of the selected solid waste were placed in 250-mL Erlenmeyer flasks and moistened with distilled water (20 g) to maintain a total moisture content of the solid waste of 80% (5). The flasks were autoclaved for 15 min at 103 kPa and 121 °C, cooled to room temperature and then five mycelial agar plugs of A. sclerotiorum PSU-RSPG178 were added to each flask. The flasks were incubated at 25 °C for 14 days. The obtained lovastatin yield was compared to that of SmF and the condition that gave the highest yield of lovastatin was selected for further experiments with other carbon sources.

After the adjustment of moisture content, 10 mL of the supplements, comprising glucose 20 mg/mL, wheat flour 300 mg/mL, urea 2 mg/mL, molasses 0.03 mL/mL, palm oil and trace elements were added separately to the substrates, then incubated with five mycelial agar plugs of A. sclerotiorum at 25 °C for 14 days. The trace elements consisted of (in mg/mL): KH₂PO₄ 2, MgSO₄ 0.3, CaCl₂ 0.3, FeSO₄ 0.11 and ZnSO₄ 0.3 (11).

The best agricultural waste (soya bean sludge), cultivation process (SSF) and supplementary carbon source (palm oil) were selected from the above experiments to use in the optimization studies. The conditions were varied, including the amount of supplement (10, 20 or 30 mL of palm oil), the number of mycelial agar plugs (10, 20 or 30 plugs) and the cultivation time (14, 18 or 21 days).

Samples were weighed before drying, then placed in a heat-resistant container which was heated at 135 °C for 2 hours, then weighed again to calculate the moisture mass fraction (16). The moisture content was calculated as the mass difference before and after drying and expressed as a mass fraction of the final dry mass of the sample. The analysis of moisture content was conducted in triplicate.

The lovastatin was extracted from the cells after cultivation according to the method of Daengrot et al. (15) and Phainuphong et al. (13). The fermented material was immersed in methanol for three days, followed by filtration through Whatman paper no. 0.5, then the methanol was evaporated with a rotary evaporator until the solution was viscous. Next, distilled water (50 mL) was added to the solution, and the mixture was extracted with hexane (2×100 mL). The hexane layer was separated and evaporated over anhydrous Na₂SO₄ under reduced pressure to obtain a crude extract in the form of a dark brown gum. The aqueous residue after extraction with hexane was further extracted twice with an equal amount of ethyl acetate (EtOAc). The EtOAc layer was dried over anhydrous Na₂SO₄ under reduced pressure to obtain a crude extract in the form of a dark brown gum.

The crude extracts were transferred to a microcentrifuge tube and dissolved in 1.0 mL of acetonitrile. Lovastatin (98%, Acros Organics, Geel, Belgium) was used to prepare standard solutions in the same way as described for the sample solutions, containing 0, 40, 80, 160, 320 and 640 mg/L. Quantitative HPLC analyses were performed on an Agilent 1200 series DAD HPLC system using an ACE^® Generix 5 C18 column (250 mm×4.6 mm×5 mm i.d.). Acetonitrile was used as mobile phase A, and mobile phase B was aqueous 0.1% H₃PO₄ (Fisher Scientific). The flow rate was 1 mL/min, the injection volume 20 μL and peak detection was at 238 nm. Analysis started with 60% A and 40% B and lasted for 20 min (13).

The total carbon and nitrogen mass fraction (in %) in the fermented material were determined using a combustion method according to the AOAC method 993.13 (17) by a carbon to nitrogen determinator (CN628, LECO Corporation, St. Joseph, MI, USA) at the Central Equipment Division, Faculty of Science, Prince of Songkla University, Songkhla, Thailand.

Statistical analysis was performed by one-way analysis of variance (ANOVA) and the Tukey’s post-hoc test with a significance level of p<0.05, using SPSS v. 17 (18). The data are presented as the mean value±S.D., with p<0.05 considered statistically significant.

Table 1 shows the initial moisture content of the substrates and the lovastatin yields obtained from them using SmF and SSF. Soya bean sludge had the highest moisture content (86%). The SmF system, using corn trunks as a substrate, showed the highest yield of lovastatin (0.004 mg/g) followed by soya bean sludge (0.002 mg/g), while A. sclerotiorum PSU-RSPG178 did not grow on the rice husks or on the wild sugarcane. Thus, corn trunks and soya bean sludge were used as substrates for SSF. In SSF with soya bean sludge and a 14-day cultivation period, the lovastatin yield was (0.040±0.002) mg/g. In the corn trunk medium, there was no growth after 14 days; however, there was growth after 30 days with a lovastatin yield of (0.164±0.001) mg/g. Although lovastatin production using corn trunks as the substrate was higher than that obtained using soya bean sludge, corn trunks require a size reduction step before use. Soya bean sludge has the advantage of being used directly. Additionally, it is plentiful, with about 1.7 million tonnes generated in Thailand during 2019 (19). Therefore, SSF using soya bean sludge was chosen for optimization of the other process conditions for lovastatin production.

Experiments were carried out in SSF with the soya bean sludge medium supplemented with other carbon sources. The soya bean sludge medium supplemented with palm oil gave the highest lovastatin yield (1.0 g/g) (Table 2). This lovastatin yield was almost 27-fold higher than the yield achieved in the control (without supplementation). At the end of the 14-day fermentation, the mycelium cultured on the soya bean sludge medium supplemented with palm oil was denser than on the culture without supplementation (Fig. 1). The mixture of residual solid substrate and fungal cells after lovastatin extraction was dried at 105 °C to a constant mass. The dry mass obtained for the soya bean sludge medium with or without palm oil was 47.5 and 9.7 g, respectively. These results agree with previous reports that vegetable oil improves lovastatin production (20, 21).

Samples of dry soya bean sludge were maintained at an initial moisture content of 80% and then fermented for 14-21 days. The following parameters were varied: (i) the ratio of soya bean sludge mass (g) to palm oil (mL) from 1:2 to 1:6, and (ii) the ratio of soya bean sludge mass (g) to the number of mycelial agar plugs from 1:2 to 1:6. At a ratio of 1:2 soya bean sludge mass to mycelial agar plugs, where the ratio of soya bean sludge to palm oil was also 1:2, the highest lovastatin yield ((11±1) mg/g) was obtained after 18 days of cultivation (Fig. 2, a). At a ratio of soya bean sludge to mycelial agar plugs of 1:4 and to palm oil of 1:2, the highest lovastatin yield ((20±2) mg/g) was also obtained after 18 days of cultivation (Fig. 2b). At a ratio of soya bean sludge to mycelial agar plugs of 1:6 and to palm oil of 1:2, the highest lovastatin yield ((20±1) mg/g) was obtained after 21 days of cultivation (Fig. 2c). However, these last two were not significantly different (p>0.05).

The optimum conditions for lovastatin production were, therefore, a 1:2 ratio of soya bean sludge to palm oil, a 1:4 ratio of soya bean sludge to mycelial agar plugs and an 18-day cultivation period.The optimum ratio of soya bean sludge to palm oil of 1:2 corresponds to a C:N ratio of 42:1 (Table 3), which is similar to the optimum C:N ratio for lovastatin production of 40:1, found by López et al. (22) for the growth of Aspergillus terreus ATCC 20542 in SmF with lactose, in combination with either soybean meal or yeast extract under nitrogen-limited conditions.

The HPLC chromatograms of the lovastatin obtained from this experiment compared with a lovastatin standard are shown in supplementary material (Fig. S1). The mass of the crude culture extracts obtained with ethyl acetate and hexane was 55.07 and 29.06 mg/g, respectively, and the lovastatin yields from ethyl acetate and hexane extract were 18 and 2 mg/g, respectively. Lovastatin is, therefore, more soluble in ethyl acetate than in hexane. The crude culture extracts obtained with ethyl acetate were further purified by column chromatography over silica gel using 2% MeOH/CH₂Cl₂ as eluent to obtain fourteen fractions. Fractions 5, 7 and 12 were lovastatin (23), penicillic acid (24) and monacolin S (25) respectively. These compounds were determined by comparison of the ¹H NMR data with those previously reported.

The obtained optimum condition was then tested at various moisture contents (50, 60, 80 and 86% after 18 days, with 86% moisture being that of fresh soya bean sludge. The mean values for lovastatin production were compared using Duncan’s new multiple range test (α=0.05). The lovastatin yield with a moisture mass fraction in soya bean sludge of 50% was significantly lower than when soya bean sludge with the higher moisture mass fractions was used (p<0.05) (Fig. 3). The lovastatin yields at 60, 80 and 86% of moisture in soya bean sludge were not significantly different (p>0.05). Therefore, fresh soya bean sludge can be directly used as a medium for lovastatin production without the need for drying and adjustment of moisture.

The lovastatin yields obtained from different fungal species grown on different media are quite difficult to compare and it is not a simple matter to determine which species is the best producer. In this study, A. sclerotiorum PSU-RSPG 178 produced (20±2) mg/g of lovastatin using soya bean sludge and palm oil as a medium, which is comparable with the maximal yield of lovastatin (20 mg/g) produced by Monascus sanguineus (Table 4 (9, 12, 23, 26)). Although M. sanguineus produced lovastatin in a shorter time than A. sclerotiorum PSU-RSPG 178, it required a more complicated medium than A. sclerotiorum PSU-RSPG 178. An advantage of A. sclerotiorum PSU-RSPG 178 is that it also produces monacolin S, which has been found in red rice produced by Monascus spp., and has a more potent cholesterol-reducing effect than lovastatin (unpublished). Therefore, A. sclerotiorum PSU-RSPG 178 has a potential to produce a fermented food that has the same properties as red rice.