Biofuels from Lignocellulosic Biomass - Sustainable Biotechnology: Sources of Renewable Energy - page 39

lignocellulose situation. Some non-cellulolytic enzymes (e.g. ferulic acid esterases
and various xylanases) have been studied as pretreatment agents and showed
promising results in increasing glucose yield from lignocellulose [51].
Since enzyme cost is a large contributor to the total production cost for lignocel-
lulosic ethanol [30, 44], considerable research has been undertaken in attempts to
increase the efficiency and reduce the cost of enzymes. Addition of protein (bovine
albumen) and other additives (Tween 20 or 80, polyethylene glycerol, etc.) that
reduce the affinity between cellulases and lignin all improve the efficiency of cel-
lulose hydrolysis [27]. A recycling process using an ultrafiltration membrane to
separate hydrolyzed glucose showed that cellulases could be re-used up to 3 times
for pretreated low lignin biomass, or until ∼ 50% of the cellulases were bound on

accumulated lignin [48].
To help lower enzyme costs and possibly improve effectiveness, a research
strategy has been developed to genetically-engineer biomass to express transgenic
endocellulases. Microbial cellulose transgenes have been expressed in several crops:
tobacco, potato, tomato, alfalfa, rice, maize, and barley [52-54]. Endoglucanase 1
(E1) concentration in some transgenic experiments has reached 1% (corn stover)
[55] to 5% (rice straw) [54] of total soluble proteins. In some cases, both treated
and non-treated E1 engineered biomass showed higher digestibility than biomass
of their wild counterparts. Whether transgenic expression of appropriate enzymes
is a viable long-term strategy when used for large-scale production remains under
investigation.

6.4 Fermentation (Including SSF and C5 and C6)

For large-scale, economically viable use of lignocellulose there will be two input
streams of sugars, one from hydrolysis of pretreated cellulose (C6 sugars such
as glucose) and one from the hydrolysis of pretreated hemicellulose (C5 sugars
such as xylose) since the common fermentation yeast ( Saccharomyces cerevisiae )
can only utilize C6 sugars, an additional technology is required for lignocellulose
compared to starch or sucrose based ethanol production. The fermenting process
for lignocellulosic ethanol production will include either two fermentation pro-
cesses ( S. cerevisiae for glucose and bacteria or other yeast for pentoses) or one
C5 and C6 co-fermentation process (e.g. genetically-engineered microorganisms
with specifically-designed metabolic pathways). To-date, several microbial species
have been engineered to ferment both glucose and pentoses, including E. coli ,
Zymomonas mobilis , Pichia stipitis , Thermoanaerobacterium saccharolyticum and
S. cerevisiae [56-58]. While these metabolically-engineered microbes show C6 and
C5 fermentation, the ethanol yields have been too low for commercial applica-
tions [57]. In addition, many engineered organisms are susceptible to inhibitory
compounds generated during pretreatment, and are not as tolerant to high ethanol
concentration as the typical S. cerevisiae strains. Research continues to explore the
possibilities for economic fermentation of both C6 and C5 sugars.