The oil extraction step was the next target for optimization. The
centrifugation step to separate cells from the fermented broth was
eliminated and the broth was directly subjected to homogenization
prior to oil extraction for lysis of cells. Homogenization was
inevitable due to the inherently tough yeast cell walls that are
not easily destroyed, thereby making it difficult to efficiently extract
the intracellular lipids with solvents.
Conventional process of oil extraction of using methanol: chloroform/
dichloromethane was avoided as both chloroform and
dichloromethane are expensive as well as environmentally unsafe.
A cheaper solvent such as n-hexane was chosen for oil extraction
from P. kudriavzevii. However, hexane alone was found to be
unsuitable due to its high hydrophobicity resulting in improper
mixing with the cells and lower oil yield (only 7% (w/w) oil recovered).
Using a modified method described by Markham et al.oil extraction of homogenized P. kudriavzevii was carried out using
n-hexane and IPA. The oil yield improved more than 2.5-fold and
up to 18.6% oil was obtained on dry cell weight basis by this protocol.
Another advantage was that over 95% of n-hexane used for
oil extraction could be recovered from this process and was recycled
in subsequent batches. IPA in the aqueous layer was also partially
recovered (60%) and could be recycled back in the extraction
process. The deoiled cell debris was recycled and used as an alternative
for baker’s yeast autolysate thereby bringing down the cost
of raw material. Thus, the entire oil extraction process was made
least price-intensive as possible.