Crude lipid
In the present study, lipid yield of the treatment with HCl in the reflux, osmotic shock, enzyme and ultrasonic homogenizer cell disruption methods with Bligh and Dyer and Soxhlet crude lipid methods are shown in the Fig. 1. Bligh and Dyer and Soxhlet lipid extraction methods, which were not applied any disruption processes were accepted as control groups. Lipid ratio of the control groups Bligh Dyer control (BDC) and Soxhlet control (SC) was determined as 18.87 ± 0.4% and 5.54 ± 0.08%, respectively. Lipid ratio of Bligh and Dyer applied groups BDOS, BDE, BDHCl and BDUH was found as 20.92 ± 1.87%, 21.72 ± 0.74%, 19.38 ± 1.20% and 14.52 ± 0.36%, respectively. The lipid yield of Soxhlet method applied groups SOS, SE, SHCl and SUH groups was found as 8.21 ± 1.60%, 11.98 ± 1.41%, 12.26 ± 0.15% and 14.70 ± 0.3%, respectively. The highest lipid value was detected in the BDE group (P< 0.05) and followed by BDOS, the difference between these groups was found to be statistically insignificant (P> 0.05). The lowest value was found in the SC group (P < 0.05). The highest lipid value in the Soxhlet applied groups was found in the SUH group with 14.70 ± 0.30%. It was found statistically significant from other Soxhlet applied groups (P < 0.05). Comparing the BDC and SC control groups showed all cell disruption methods were able to disrupt algal cells, although the lipid although lipid yield showed differences (P< 0.05).
Fig. 1 Lipid yield of different cell disruption methods
Difficulties arise in obtaining lipid due to the cell walls of microalgae. For this reason, in this study, lipid was obtained fromSchizochytrium sp . with the HCl treatment, osmotic shock, enzyme and ultrasonic homogenizer cell disruption methods to increase the lipid yield to be used for feed, food and cosmetic raw material for future studies. In our study, it was observed that the cell disruption methods increased the lipid yield compared to the control groups. The highest lipid yield was detected in the BDE group (21.72 ± 0.74%). Higher results were obtained in the Bligh and Dyer crude lipid method compared to Soxhlet. Lipid ratio of BDOS (20.92 ± 1.87%) group also followed the BDE group lipid ratio and there was no statistically significant difference (P > 0.05). In the Soxhlet groups, the highest lipid value was found in SUH group (14.70 ± 0.3%), followed by the SHCl (12.26 ± 0.15%). The difference between these two groups was statistically significant (P < 0.05). When our results were compared, it was observed that the lipid yield was lower in the Soxhlet applied groups, and the laboratory scale applicability of the Bligh and Dyer method was more appropriate. Araujo et al. (2013) reported that the Soxhlet extraction mechanism is mainly diffusion and the procedure does not involve mechanic disruption to the biomass. Their results showed that simply diffusion of lipids through the cell membrane is a slow process and results in low yield of lipid. Similar with the study of Araujo et al. (2013), low results were obtained in the Soxhlet method compared with the Bligh and Dyer method, in our study. In the literature studies, different applications such as autoclave, bead-beating, microwave, ultrasonication and osmotic shock were applied to increase the lipid amount of microalgae. Positive results were obtained with different mechanical applications using different solvents, however environmentally friendly applications such as use of enzymes is promising. Byreddy et al. (2015) studied cell disruption methods to increase the efficiency of lipid extraction ofSchizochytrium sp. using different solvents in their study. The maximum yield was obtained from the chloroform:methanol (2:1) solvent. Chloroform:methanol (2:1) was used in our study with reference to Byreddy et al. (2015). Liquid nitrogen grinding, osmotic shock, vortexing, water bath and sonication were used in their study to increase the efficiency of lipid extraction. In the osmotic shock application, the highest efficiency of lipid was obtained forSchizochytrium sp. as 48.7%, while lipid yield of the control group was below 10%, in their study. In our study, there was an increase in lipid yield in cell disruption methods compared to the control group.
Araujo et al. (2013) declared the Bligh and Dyer method assisted by ultrasound resulted in the highest extraction of lipid from C. vulgaris (52.5%) and lipid yield with Soxhlet method was obtained quite low (1.8%). Lee and Han (2015) applied hydrodynamic cavitation with sulfuric acid for different durations in Nannochloropsis salina species to increase lipid yield. While sulfuric acid concentrations were 0.1% and 2% lipid contents were determined as 21.7% and 45.4%, respectively. Lipid yield was increased with the increase of acid concentration and time. In our study, the lipid yield of HCl disruption combined with Soxhlet and Bligh and Dyer methods, was obtained respectively as; 12.26 ± 0.15% and 19.9 ± 1.20% and an increase occurred compared to the control group. Although its high efficacy for disrupting microalgal cells, sulfuric acid treatment is not appropriate for use in a microalgal biorefinery because of destroying the activity of valuable cellular components (such as protein and pigment) (Halim et al., 2012). Liang et al. (2012) studied to increase lipid yield of Chlorella vulgaris , Scenedesmus dimorphusand Nannochloropsis sp. by enzyme (cellulase, neutral protease and alkaline protease) and sonication treatment. The highest lipid yield was obtained as 49.82% in the combined sonication-enzyme treatment. Lipid yield of control group increased from 15.11%. A single method of extraction cannot be sufficient to reach the maximum yield. Combination of pretreatment methods with different solvents increase lipid yield of the algae (Mubarak et al., 2015). Studies have also demonstrated that lipid extraction efficiency of enzymatic treatment is higher than the mechanical methods including microwave and ultrasonication. The yield of lipid achieved through enzymatic hydrolysis depends on the type of enzyme used, pH, temperature and microalgal type (Nagappan et al., 2019). In our study, the highest amount was observed in enzyme application and in order to get better results, the enzyme was treated at 55 °C, which is the optimum temperature for the cellulase enzyme. Taher et al. (2014) in their on extracting lipid with enzyme (lysozyme and cellulase) from Scenedesmus sp., declared Soxhlet method, the lipid value was 4% in the control group without any application. The highest value was obtained with lysozyme (16.6%) followed by the cellulase enzyme (15.4%). Algal cell disruption methods to increase lipid extraction from microalgae depends on species, age of the culture and composition of cell wall. Only few mechanical methods such as application of enzymes/chemicals either or alone can be considered for industrial applications. Osmotic shock method can also be applied industrially for lipid extraction, because of reducing energy consumption and production cost (Byreddy et al., 2015). Although acid treatment process is efficient in cellulose degradation, sulfuric acid is toxic and corrosive, so this process is not recommended (Taher et al., 2014). As a result in our study, the best results were obtained in the enzyme applied groups, but also, osmotic shock, ultrasonic homogenizer increased lipid yield compared to the control group, and these methods can be evaluated positively on an industrial scale.