Sisal/Agave - Industrial Crops: Breeding for BioEnergy and Bioproducts - page 339

It is likely that separate reaction vessels will be required for conversion of the
pi˜a and the cellulosic material from bagasse and leaves to sugars. The pi˜ais
primarily comprised of fructans that are more readily converted to reducing sugars
than celluloses. However, a different enzyme mix for hydrolysis is required. A
recent study found that Fructozyme (Novozymes; Bagsvaerd, Denmark) achieves
hydrolytic efficiency of 99.5 % after 3-4 h at 60 C in a 1:1 biomass and water
mixture [ 40 ]. The optimum enzyme concentration for hydrolysis of both solid
particles and aqueous agave extract is 0.17 % solution. With 100 % hydrolysis,
there is a yield of approximately 97.5 g l 1 from aqueous extracts and 110 g l 1
from agave particles [ 40 ]. If a fiber Agave such as sisal or henequen was the
feedstock, providing a continual supply of leaves rather than a harvest of leaves
and pi˜a, then this step would be removed (unless the leaves also prove to have high
concentrations of fructans).
There is a loss of efficiency when employing simultaneous extraction and
hydrolysis in a continuous diffuser. Because the extraction conditions can deacti-
vate enzymes, the hydrolysis efficiency is 50-62 % or 70-89 g l 1 of fructose
[ 40 ]. This, however, would avoid the downtime that is unavoidable with current
batch processing. Further study of the conditions that promote enzyme activity may
improve reaction efficiency at the commercial scale.
Fermentation
Agave extracts from the pi˜a are typically stored for 12-24 h before fermentation
commences. Fermentation for tequila is initiated with a heat treatment of 100 C
that imparts a desired color and flavor to the final product, and then fermentation
continues at a lower temperature of 30-35 C. Fermentation of pi ˜ a extracts can be
accomplished without heat treatment, but the ethanol production is delayed ~14 h
and there is a reduction of yeast cells [ 40 ]. Low heat fermentation may be a more
energy efficient method for biofuel production, but has not been studied in detail for
tequila manufacturing because it negatively affects the flavor and consequently
lowers the value in the beverage market. This would not be a concern for the fuel
market.
The yeast Saccharomyces cerevisiae is used to ferment reducing sugars from
Agave to ethanol because of its high alcohol tolerance, but low conversion efficien-
cies are often observed in commercially scaled tequila manufacturing. Fermenta-
tion efficiency of pi˜a-derived sugars to ethanol is usually at least 80 % [ 41 , 42 ], but
in a fermentation test of Agave bagasse, metzal and metzontete yielded a maximum
of only 33 % conversion efficiency of reducing sugars to ethanol [ 37 ]. Assuming
sufficient nitrogen is available, ethanol yield increases with increasing sugar con-
centrations, with 94 % efficiency achieved in some cases [ 19 ].
Guti´rrez-Lom´li et al. [ 42 ] tested the effect of overexpressing genes that
regulate hexose transport (HXT) and alcohol dehydrogenase (ADH). In solution
with 115 g reducing sugar l 1 at 33 C, they discovered that fermentation efficiency
increased to 94 % with recombinant S. cerevisiae from 89 % in the wild type