Environmental Engineering Reference
In-Depth Information
pretreated biomass also allows flexibility in terms of the biomass feedstock because any cellulosic
material (including dried materials such as wood chips, corn stover, rice straw, agricultural wastes,
etc.) can be used as the source of biofuels. For this application, the enzymes can be produced in
a wide variety of plant hosts, not necessarily a crop with favorable characteristics as a bioenergy
crop, in stably transformed (e.g., transgenic) plants or using transient expression. Enzymes can be
individually produced in different plants/plant tissues, partially purified, and then mixed to gener-
ate appropriate enzyme cocktails. The primary considerations for this application are achieving
high expression levels of active enzymes [g enzyme/kg fresh weight (FW) plant tissue or % total
soluble protein (TSP)]; ease of recovery; solubilization and formulation of active and stable enzyme
preparations; and minimal influence of endogenous plant proteins and metabolites on enzyme activ-
ity, stability, and subsequent fermentation. If stable transgenic feedstocks are used it is important
that the production of the enzymes does not have a deleterious effect on plant growth. This can be
achieved using several approaches:
• Producing enzymes that are not active under plant growth conditions (e.g., thermostable
enzymes or lacking activity at cellular pH level) or that require further processing (post-
growth) to generate a functional form of the enzyme,
• Sequestering the enzymes in a subcellular space/organelle so that the cell wall matrix is
not exposed to the enzymes during plant growth,
• Using inducible expression systems so that enzymes are produced just before or after
harvest, or
• Choosing a host that does not have a particular substrate as a major component of its
cell wall.
In planta
production of enzymes or proteins that lead to cell wall loosening or modifications
that enhance the accessibility and/or effectiveness of exogenously added enzymes may reduce
the stringency (e.g., temperature, time required, acid/basic conditions, and size reduction) of
biomass pretreatment steps, thereby reducing the pretreatment costs and environmental impact.
For example, expansins and swollinins are thought to disrupt hydrogen bonding between cell wall
polysaccharides without hydrolyzing them. For this application, the plant host should have favor-
able characteristics as a bioenergy crop and, to avoid detrimental influence on plant growth and
viability, the approaches listed above could be used for stable transformants. Alternatively, tran-
sient expression of cell-wall-targeted enzymes could be implemented in intact plants after they
have reached maturity or in harvested leaves. For the self-deconstructing biomass application,
production and use of enzymes in living plant tissue is required. Because the enzymes can be
produced within the plant cell, secreted, and localized in the cell wall matrix, higher local enzyme
concentrations might be achievable at the substrate site. This could lead to higher efficiency
hydrolysis than can be achieved with exogenous application of fermenter-produced enzymes in
which nonspecific binding and external and intraparticle mass transfer effects may dominate.
However, this application is more challenging because it requires temporal separation of the pro-
cesses of biomass growth, enzyme production, and enzyme activation to avoid any detrimental
effects by the enzymes on biomass yield and/or enzyme production and to properly target the
enzymes and minimize nonspecific binding. Additionally, the relative concentrations of each of
the enzymes (endoglucanase, exoglucanase, beta-glucosidase, and hemicellulases) must be con-
trolled to achieve optimal hydrolysis. Some form of biomass pretreatment may also be required
before enzyme activation, so the robustness of the enzymes used for each given pretreatment
method would need to be assessed. For example, Teymouri et al. (2004) have shown that there
is a 65% loss in endoglucanse activity after transgenic tobacco leaves expressing thermostable
Acidothermus cellulolyticus
E1 endoglucanse catalytic domain undergo ammonia fiber explosion
(AFEX) pretreatment. However, even if the plant-made enzymes are not able to cause complete
