Environmental Engineering Reference
In-Depth Information
The scale required for production of enzymes to meet the needs of the cellulosic biofuel industry
will likely be unprecedented in the industrial enzyme industry. For example, to meet the require-
ments for annual production of 21 billion gal of cellulosic biofuels using current cellulase enzyme
loadings for steam-pretreated corn stover [~1 kg enzyme per dry ton biomass (K. McCall, personal
comm)] and assuming 72 gal of ethanol produced per dry ton of corn stover, it would require an
annual production of close to 300,000 t of cellulase enzyme per year, assuming that the cellulase
enzymes are not reused/recycled. If this amount of enzyme was produced using traditional fungal
fermentation at a relatively high titer of 100 g enzyme/L with a 200 h batch turnaround time, it
would require a total fermenter capacity of over 74 million L. The current worldwide industrial
enzyme fermenter capacity is estimated at approximately 20 million L. It is of course expected that
protein engineering will result in significant reductions in enzyme loadings and increased stabil-
ity enabling reuse of enzymes and thereby reducing the required fermenter capacity; however, the
capital investment for this will still be substantial.
Further, the amount of energy consumption, CO
2
generation, and organic carbon (cellulosic)
nutrient source consumption for a fungal fermentation-based cellulase enzyme production is
often neglected in cellulosic biofuel life-cycle analyses. Saez and coworkers showed that for
cellulase production in an aerobic, submerged bioreactor containing
T. reesei
growing on an
insoluble cellulosic carbon source (5% w/v Solka floc), for each gram of cellulase enzyme pro-
duced, 1.6 g of carbon dioxide (CO
2
) are emitted and 4.3 g of cellulose are consumed (Saez et al.
2002). Although the enzyme cost and activity are often important considerations in assessing
the feasibility of the overall cellulosic bioethanol production process, there are very few detailed
analyses of the energy requirements, CO
2
gas emissions, and diversion of cellulosic carbon
associated with the enzyme production process. Although the agricultural energy inputs (e.g.,
energy associated with the production of fertilizers, herbicides, farm machinery and biomass
transportation) have been carefully delineated, in many net energy analyses (EBAMM 2007;
Schmer et al. 2008) for cellulosic ethanol production the energy requirements for the biorefin-
ery plant (including the pretreatment, hydrolysis, fermentation, and biofuel recovery steps) are
assumed to be satisfied by burning/gasification of the biomass residue. The energy associated
with the production of the large quantities of enzymes that will be needed for the process are
neglected. Although it is expected that enzyme loading will be dramatically reduced because
of improvements in specific activity, long-term stability and reuse, pretreatment methods, and
enzyme cocktail formulation, enzymes will still need to be produced on a massive scale. Thus
there is an urgent need to develop new enzyme production technologies that will minimize
energy and carbon nutrient consumption, reduce GHG emissions, and lower capital costs and
total production costs. These types of “green” biomanufacturing technologies will not only ben-
efit the biofuels/biorefinery industries but will also have broader effects on the industrial enzyme
industry in general.
One approach to this problem is plant-based production of recombinant cellulase and hemi-
cellulase enzymes or plant cell wall modifying proteins in the plant biomass that is intended for
biofuel production itself (e.g., to initiate self-deconstruction and/or modify the plant cell wall to
facilitate the accessibility of exogenous enzymes). Plants may also serve as an alternative host
for production of cellulase and hemicellulase enzymes to be used as additives to other pretreated
biomass, or a combination of the two, because the spent biomass that produces the enzymes can
also serve as a source of cellulose. This general approach has been referred to as endogenous,
endoplant, or
in planta
enzyme production and has been used in plant-made additive enzymes, cell
wall loosening modifications, and self-processing/deconstruction biomass.
Production of the enzymes in plants has several advantages over microbial fermentation in
large-scale stainless steel bioreactors. These advantages include use of sunlight/photosynthesis and
consumption of CO
2
as the energy and carbon source for host cell growth, lower capital and pro-
duction costs, easier scale-up, and the ability to glycosylate enzymes that may be important for the
activity and/or stability for some cellulases. Use of plant-made enzyme preparations as additives to
