12 Industrial Extraction of Microalgal Pigments
291
liquid permeabilisation) or a combination of two or more pretreatments (HCl hydrolysis followed by ultrasonication) in order to disrupt the thick cell walls of H. pluvialis cysts. This was then followed by a subsequent extraction step where organic
solvent, pressurised solvent or supercritical carbon dioxide is used to extract the
released pigment from the cell debris. For organic solvent extraction or pressurised
solvent extraction, acetone, ethanol, ethyl acetate, hexane and methylene chloride are
frequently used. The cell rupture step (ultrasonication, microwave or HCl treatment)
and the extraction step are often combined and can be carried out simultaneously as
a single step (Dong et al. 2014; Zhao et al. 2009; Zou et al. 2013).
Across the different studies, the integration of a cell rupture step prior to or during
astaxanthin extraction consistently led to an improved extraction efficiency and
overall increase in the pigment yield. In Molino et al. (2018), subjecting H.pluvialis
red cysts to bead milling prior to the extraction step was able to triple the amount
of extracted astaxanthin. Similarly, the use of ultrasonication in Zou et al. (2013)
doubled the astaxanthin yield obtained by both ethyl acetate and ethanol extraction. In Sarada et al. (2006), a higher astaxanthin yield was obtained from acetone
extraction of HCl-treated biomass compared to that from untreated biomass.
For SCCO 2 extraction, the addition of ethanol to the extraction process was shown
to be able to consistently improve astaxanthin yield (Fujii 2012; Jaime et al. 2010;
Machmudah et al. 2006; Reyes et al. 2014). SCCO 2 is a non-polar solvent and the
addition of a polar entrainer such as ethanol to the solvent was able to improve its
affinity (and thus interaction) with astaxanthin molecules.
Free astaxanthin has a stronger antioxidant property and thus a higher market
value than its corresponding mono- or diester forms. Since up to 95% of astaxanthin
in H.pluvialis cells can exist in its ester forms, astaxanthin extract from the biomass
is often subjected to a post-recovery hydrolysis step in order to saponify any ester in
the crude extract to free astaxanthin (Jaime et al. 2010). The hydrolysis step typically
involves reaction of the extracted astaxanthin with a base (such as NaOH or KOH)
or cholesterol esters (Denery et al. 2004; Jacobs et al. 1982; Jaime et al. 2010; Kang
and Sim 2007).
We note that the majority of previous studies on astaxanthin recovery have
used freeze-dried H. pluvialis powder as the starting biomass for their extraction
(Tables 12.2 and 12.3). Since biomass drying or dehydration (whether it is thermal
drying or freeze drying) forcefully removes water molecules from cell cytoplasm, it
inadvertently disrupts the cell walls and can be considered as an extra cell rupture
or pretreatment step. Biomass drying, however, requires a tremendous amount of
energy and is unnecessary if the biomass is later to be subjected to another cell
rupture step (such as high-pressure homogenisation or ultrasonication). We have not
included drying in our discussion in Sect. 12.2 as we do not consider it as a scalable
pretreatment. Recent life-cycle analyses on microalgal biorefineries for both food
and fuel applications have established that biomass drying on a commercial scale
is energetically prohibitive and thus has to be avoided in order for the systems to
achieve a positive energy balance (Dong et al. 2016; Martin 2016). The economic
prospects of biomass dehydration will be further discussed in Sect. 12.6.
291
liquid permeabilisation) or a combination of two or more pretreatments (HCl hydrolysis followed by ultrasonication) in order to disrupt the thick cell walls of H. pluvialis cysts. This was then followed by a subsequent extraction step where organic
solvent, pressurised solvent or supercritical carbon dioxide is used to extract the
released pigment from the cell debris. For organic solvent extraction or pressurised
solvent extraction, acetone, ethanol, ethyl acetate, hexane and methylene chloride are
frequently used. The cell rupture step (ultrasonication, microwave or HCl treatment)
and the extraction step are often combined and can be carried out simultaneously as
a single step (Dong et al. 2014; Zhao et al. 2009; Zou et al. 2013).
Across the different studies, the integration of a cell rupture step prior to or during
astaxanthin extraction consistently led to an improved extraction efficiency and
overall increase in the pigment yield. In Molino et al. (2018), subjecting H.pluvialis
red cysts to bead milling prior to the extraction step was able to triple the amount
of extracted astaxanthin. Similarly, the use of ultrasonication in Zou et al. (2013)
doubled the astaxanthin yield obtained by both ethyl acetate and ethanol extraction. In Sarada et al. (2006), a higher astaxanthin yield was obtained from acetone
extraction of HCl-treated biomass compared to that from untreated biomass.
For SCCO 2 extraction, the addition of ethanol to the extraction process was shown
to be able to consistently improve astaxanthin yield (Fujii 2012; Jaime et al. 2010;
Machmudah et al. 2006; Reyes et al. 2014). SCCO 2 is a non-polar solvent and the
addition of a polar entrainer such as ethanol to the solvent was able to improve its
affinity (and thus interaction) with astaxanthin molecules.
Free astaxanthin has a stronger antioxidant property and thus a higher market
value than its corresponding mono- or diester forms. Since up to 95% of astaxanthin
in H.pluvialis cells can exist in its ester forms, astaxanthin extract from the biomass
is often subjected to a post-recovery hydrolysis step in order to saponify any ester in
the crude extract to free astaxanthin (Jaime et al. 2010). The hydrolysis step typically
involves reaction of the extracted astaxanthin with a base (such as NaOH or KOH)
or cholesterol esters (Denery et al. 2004; Jacobs et al. 1982; Jaime et al. 2010; Kang
and Sim 2007).
We note that the majority of previous studies on astaxanthin recovery have
used freeze-dried H. pluvialis powder as the starting biomass for their extraction
(Tables 12.2 and 12.3). Since biomass drying or dehydration (whether it is thermal
drying or freeze drying) forcefully removes water molecules from cell cytoplasm, it
inadvertently disrupts the cell walls and can be considered as an extra cell rupture
or pretreatment step. Biomass drying, however, requires a tremendous amount of
energy and is unnecessary if the biomass is later to be subjected to another cell
rupture step (such as high-pressure homogenisation or ultrasonication). We have not
included drying in our discussion in Sect. 12.2 as we do not consider it as a scalable
pretreatment. Recent life-cycle analyses on microalgal biorefineries for both food
and fuel applications have established that biomass drying on a commercial scale
is energetically prohibitive and thus has to be avoided in order for the systems to
achieve a positive energy balance (Dong et al. 2016; Martin 2016). The economic
prospects of biomass dehydration will be further discussed in Sect. 12.6.