Pre-treatment not only liberates fermentable monomers but
also weakens hydrogen bonding within and between glucan
chains, which enhances the rate of polysaccharide breakdown into
fermentable sugars in the presence of enzymes. Saccharification
tests were performed on non-treated AIR, acid/autoclaved and
water/autoclaved grape marc based on identical cellulose loadings
following pre-treatment. The liberation of glucose from cellulose
was monitored over 48 h of enzymatic digestion using a cellobiase
preparation (cellobiase activity P250 units/g) from A. niger and a
commercial cellulase cocktail (endoglucanase activity
P700 units/g) isolated from T. reesei.
The acid/autoclave-treated grape marc exhibited the highest
conversion of cellulose to glucose and for all pre-treatments, the
amount of glucose released was higher for white marc than for
red marc at all time points (Fig. 3). The greatest amount of glucose
liberated was observed for acid/autoclave-treated Sauvignon Blanc
samples, with 28% of cellulose hydrolyzed. Thermal pre-treatment
did not increase the rate of glucose liberation from white marc
compared with non-treated samples, with only 18% glucose present
in the hydrolyzate (Fig. 3A). Similar findings have been
recorded for sweet sorghum bagasse, in which water did not significantly
improve the rate of cellulose hydrolysis yield (11.8%) over
96 h compared with the control (12.6%) (Cao et al., 2012).
However, for red marc, the amount of glucose released was correlated
to the severity of the pre-treatment, and both
pre-treatments resulted in higher saccharification rates than
untreated samples. The maximum glucose liberated from
Cabernet Sauvignon was 17% in acid-treated samples, 15% in
thermal-treated samples and 13% in non-treated samples over 48 h (Fig. 3B). The slower and lower rate of glucose release
observed in Cabernet Sauvignon samples compared to Sauvignon
Blanc may be attributed to a higher ratio of fucosylated xyloglucan
oligomers (XXFG; 1393 and XLFG; 1555) present in this tissue. The
presence of fucosylated xyloglucans has been proposed to facilitate
binding to cellulose, thus reinforcing the recalcitrant nature of the
cell walls and making the cellulose polymers less accessible to
enzymatic hydrolysis (Levy et al., 1997).
Overall, the acid pre-treatments were more efficient at degrading
grape marc polysaccharides (Table 4) as well as modifying or
weakening the hydrogen bonding of the crystalline cellulose polymers,
making them more accessible to enzyme attack.
Pre-treatments that increase the number of enzyme binding sites
on cellulose microfibrils may increase biofuel yields, although
studies have shown that saccharification may be a limiting factor
when converting from small to large scale production
(Klein-Marcuschamer et al., 2012). As a result, it may be more economically
favorable to identify and capitalize on the WSC present
in dedicated sources of biomass for biofuel production, such as
grape marc.