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.