www.nature.com/scientificreports OPEN Enhanced fatty acid methyl esters recovery through a simple and rapid direct transesterifcation of freshly harvested biomass of Chlorella vulgaris and Messastrum gracile Saw Hong Loh1,2*, Mee Kee Chen1,2, Nur Syazana Fauzi2,3, Ahmad Aziz1,2 & Thye San Cha1,2* Conventional microalgae oil extraction applies physicochemical destruction of dry cell biomass prior to transesterifcation process to produce fatty acid methyl esters (FAMEs). This report presents a simple and rapid direct transesterifcation (DT) method for FAMEs production and fatty acid profling of microalgae using freshly harvested biomass. Results revealed that the FAMEs recovered from Chlorella vulgaris were 50.1 and 68.3 mg with conventional oil-extraction-transesterifcation (OET) and DT method, respectively. While for Messastrum gracile, the FAMEs recovered, were 49.9 and 76.3 mg, respectively with OET and DT methods. This demonstrated that the DT method increased FAMEs recovery by 36.4% and 53.0% from C. vulgaris and M. gracile, respectively, as compared to OET method. Additionally, the DT method recovered a signifcantly higher amount of palmitic (C16:0) and stearic (C18:0) acids from both species, which indicated the important role of these fatty acids in the membranes of cells and organelles. The DT method performed very well using a small volume (5 mL) of fresh biomass coupled with a shorter reaction time (~ 15 min), thus making real-time monitoring of FAMEs and fatty acid accumulation in microalgae culture feasible. Te key processes involved in the fatty acid profling of microalgae are cultivation, biomass harvesting and drying, oil extraction and transesterifcation to produce fatty acid methyl esters (FAMEs). Among these pro- cesses, biomass drying and oil extraction are two costly and tedious processes. Te conventional microalgae oil extraction method require several steps. Te efciency of the oil extraction depends on microalgae species, the cell wall disruption method and solvent system 1, 2. To disrupt microalgae cell walls, various approaches such as grinding, ultra sonication 1, microwave treatments 3, enzymes digestion 4 autoclaving, bead beating and high salt solution5 have been reported. Te major problems faced by the conventional oil extraction-transesterifcation (OET) method are biomass drying and oil extraction processes. Te drying of fresh/wet biomass prior to oil extraction requires high energy that is time-consuming. It is estimated that over 90% processed energy is used during the biomass drying and oil extraction steps 6, 7. Furthermore, the solvent system cannot completely extract the fatty acids, especially the fatty acids which are attached to the membrane lipids such as phospholipids and glycolipids8. Tis leads to lower FAMEs recovery and compromises the fatty acid profling6, 7. In addition, the oil extraction yield is adversely afected when wet biomass is used9. Over the years, direct transesterifcation (DT) of biomass has received enormous attention due to its high ef- ciency in the reduction of energy and time 10. Te DT method enables simultaneous extraction of lipid and FAMEs conversion processes by alcohol in the presence of a catalyst. Te alcohol acts as an extraction solvent and an acyl acceptor for transesterifcation 11–13. Te DT method makes it possible to produce FAMEs in a single step using microalgae fresh/wet biomass while also eliminating both biomass drying and oil extraction processes7, 14. Tere 1Faculty of Science and Marine Environment, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia. 2Satreps-Cosmos Laboratory, Central Laboratory Complex, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia. 3Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia. *email: [email protected]; [email protected] Scientifc Reports | (2021) 11:2720 | https://doi.org/10.1038/s41598-021-81609-6 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1. Te growth curve of C. vulgaris UMT-M1 and M. gracile SE-MC4 cultured under nitrate defcient condition. Te cultures were harvested at stationary growth phase when cell growth became plateau. Data shown as the mean ± SD, n = 3. are plenty of recent research on DT of microalgae biomass along with relatively higher FAMEs yield reported. Tere are DT methods established using dry microalgal biomass15–17 or in combination with novel approaches such as microwave irradiation 18, ultrasound treatment 19 and vortex fuidic device (VFD)-based20, as well as the application of immobilized lipases19, 21, among others. One of the major drawback of the above-mentioned DT methods was the transesterifcation reaction time which was relatively longer and difers greatly depending on the approaches used that may range from less than one hour to several hours15–24. In addition, a large amount of dry/wet microalgal biomass was needed for the transesterifcation reaction 15–24. Nevertheless, there is a lack of research reported on small-scale DT using fresh/wet microalgal biomass directly harvested from microalgal cultures for real-time monitoring of oil accumulation and fatty acid composi- tion. Terefore, this study compared the efciency of the conventional OET and DT methods25 for total FAMEs recovery from small amount of microalgae biomass that was freshly and directly harvested from microalgae cultures. In this report, the DT method was optimized using diferent small sample volume sizes of freshly har- vested microalgae biomass. Te variation of fatty acid composition from the two methods was also compared. Results and discussion Cell growth, biomass production and oil content using conventional oil extraction-transester- ifcation (OET) method. Figure 1 shows the growth curve of C. vulgaris and M. gracile. Te cell density of the C. vulgaris culture increased exponentially in the frst 5 days of the culture and attained early stationary growth phase at day 8. No signifcant change in cell density was observed until day 12 where the cell density of 2.26 × 107 cells mL−1 was recorded. On the other hand, the exponential growth of M. gracile lasted for 7 days and attained early stationary growth phase at day 8, where no signifcant change in cell density was observed until day 12 with cell density of 2.08 × 107 cells mL −1. Although an equal initial cell density (5 × 106 cells mL−1) was used for both species, the cell densities recorded everyday were diferent between the two species with C. vulgaris consistently recording higher cell density throughout the growth curve (Fig. 1). All cultures were harvested on day 12 where cell growth reached a plateau, which represents the stationary growth phase. Te logistic growth pattern of microalgae involves lag phase, log phase, stationary phase and senescence phase26. In the lag phase, which was at the beginning of the culture, both species were adapting to a new culture environment. Afer full adaptation to the new culture condition, the log phase occurred in which the population growth was greatly increased until around day 7 (Fig. 1). When the cultures reached the stationary growth phase, the nutrients in the culture medium were expected to be depleted and cell growth turned into a regression straight line with no signifcant change in cell density at the end of the growth curve (Fig. 1). Generally, microalgae grow slowly and attain low biomass production, but with higher lipid 25–27 and carbohydrate28, 29 production when culture is cultivated under low nitrate concentration. Furthermore, station- ary growth phase has been recognized as a period of active oil accumulation phase for microalgae species due to the conversion of carbohydrate to lipids30–32. Terefore, both C. vulgaris and M. gracile cultures were harvested at the stationary growth phase to ensure high oil accumulation for further analysis. As shown in Table 1, the total oil content of C. vulgaris and M. gracile at the stationary growth phase was 47.5% and 40.6% (percent of dry weight), respectively. Conversely, M. gracile recorded higher dry biomass production of 0.42 g L−1 against 0.37 g L−1 produced by C. vulgaris (Table 1). Te higher biomass production was concomitant with lower cell density and total oil content recorded by M. gracile versus C. vulgaris. Te larger cell size of M. gracile could compromise the efciency of biological and physiological processes 33, 34. Scientifc Reports | (2021) 11:2720 | https://doi.org/10.1038/s41598-021-81609-6 2 Vol:.(1234567890) www.nature.com/scientificreports/ Species Cell density (× 10–7 cells mL−1) Total biomass (mg DW) Biomass production (g DW L−1) Oil content (% of DW) C. vulgaris 2.26 ± 0.06 112.1 ± 5.0 0.37 ± 0.02 47.5 ± 2.4 M. gracile 2.08 ± 0.14 126.3 ± 2.1 0.42 ± 0.01 40.6 ± 1.4 Table 1. Cell density, total biomass and oil content of C. vulgaris and M. gracile harvested at stationary growth phase from 0.3 L cultures. Data shown as the mean ± SD (n = 3). DT method (sample volume) OET method 5 mL 10 mL 20 mL 30 mL C. vulgaris Total FAME (mg) 50.1 ± 2.9b 68.3 ± 5.5a 62.3 ± 5.9ab 53.2 ± 1.1b 49.5 ± 4.5b Total FAME (% DW) 44.7 ± 2.6b 60.9 ± 4.9a 55.6 ± 5.3ab 47.4 ± 1.0b 44.1 ± 4.0b M. gracile Total FAME (mg) 49.9 ± 2.8b 76.3 ± 9.2a − − − Total FAME (% DW) 39.5 ± 2.2b 60.4 ± 7.3a − − − Table 2. Total FAME obtained from C. vulgaris and M. gracile using oil-extraction transesterifcation (OET) and direct transesterifcation (DT) methods from 0.3 L culture. For DT method, fresh cells were harvested from 5, 10, 20, and 30 mL of stationary phase microalgal cultures. Te values are in mean ± SD (n = 3). Diferent letters in the same row indicate values are signifcantly diferent according to one-way ANOVA at p < 0.05.