23
for production of cellulase, and because the costs of enzymes are often reported in
terms of price per unit quantity of ethanol, rather than the unit cost of enzyme
(Marcuschamer et al. 2012; Liu et al. 2016). While techno-economic studies have
indicated that rather than just lowering enzyme production costs, feasibility for biomass to ethanol conversion needs a comprehensive and broader addressing of multiple steps in biorefinery operation including better pretreatment, reduction of lignin
and phenolics, use of more stable enzymes and reduction of residence times, lower
enzyme loadings, and onsite enzyme production (Marcuschamer et al. 2012), the
contribution of enzyme production cost is still significant. The following discussions will therefore address the common strategies for production and the recent
developments aimed at reducing the cost of enzymes through multifaceted
approaches.
1.5.1 Microbes Used for Industrial Production of Cellulases
Filamentous fungi, especially the ascomycetes fungi Trichoderma reesei and
Aspergillus niger or their derivative strains, are used by enzyme companies like
Novozymes, Genencor (DuPont), and Iogen for commercial production of cellulases, with the exception of Dyadic, which uses engineered strains of Chrysoporium
lucknowense (Zhang and Zhang 2013). Most of the Trichoderma reesei strains currently in use for production of cellulases have been derived from the original isolate
Trichoderma sp. strain QM6a isolated by the US Army Research Laboratories at
Natick over 70 years ago. The strain was improved through random mutagenesis
experiments at the Natick laboratory and Rutgers University, which resulted in the
strain RUT-C30 with over 20-fold improvement in cellulase activity. This strain is
still the prototype cellulase hyperproducer available in the public domain (Bischof
et al. 2016). The titers of extracellular cellulase on cellulase-inducing carbon
source – lactose – can reach 30 g/L as demonstrated by Durand et al. (1988). A
direct comparison of the levels of cellulase production by different organisms is
complicated by the different strategies of production employed, by the different
substrates and inducers employed, and in some cases by the different units used for
expressing their activity. Several such comparisons have been made, which have
mostly concentrated on the published information, and information on the cellulase
yields from studies in the public domain may be obtained from these reports
(Sukumaran et al. 2005; Mathew et al. 2008; Chandel et al. 2012; Hansen et al.
2015; Cunha et al. 2016). The highest cellulase activities (as filter paper units,
FPUs) have been recorded at about 10–15 FPUs/g substrate under solid-state fermentation (SSF) and about 13 FPUs/ml for submerged fermentation (Hansen et al.
2015). Lignocellulose hydrolysis requires the concerted action of several enzymes
including the endoglucanases, cellobiohydrolases, β glucosidases, xylanases,
β-xylosidases, and several other accessory enzymes; and no single organism can
optimally produce all these enzymes. Hence it is a common practice to produce different enzymes using different organisms and the enzymes are blended to form
cocktails with better efficiency for biomass hydrolysis. Typically for T. reesei
1 Enzymes for Bioenergy
for production of cellulase, and because the costs of enzymes are often reported in
terms of price per unit quantity of ethanol, rather than the unit cost of enzyme
(Marcuschamer et al. 2012; Liu et al. 2016). While techno-economic studies have
indicated that rather than just lowering enzyme production costs, feasibility for biomass to ethanol conversion needs a comprehensive and broader addressing of multiple steps in biorefinery operation including better pretreatment, reduction of lignin
and phenolics, use of more stable enzymes and reduction of residence times, lower
enzyme loadings, and onsite enzyme production (Marcuschamer et al. 2012), the
contribution of enzyme production cost is still significant. The following discussions will therefore address the common strategies for production and the recent
developments aimed at reducing the cost of enzymes through multifaceted
approaches.
1.5.1 Microbes Used for Industrial Production of Cellulases
Filamentous fungi, especially the ascomycetes fungi Trichoderma reesei and
Aspergillus niger or their derivative strains, are used by enzyme companies like
Novozymes, Genencor (DuPont), and Iogen for commercial production of cellulases, with the exception of Dyadic, which uses engineered strains of Chrysoporium
lucknowense (Zhang and Zhang 2013). Most of the Trichoderma reesei strains currently in use for production of cellulases have been derived from the original isolate
Trichoderma sp. strain QM6a isolated by the US Army Research Laboratories at
Natick over 70 years ago. The strain was improved through random mutagenesis
experiments at the Natick laboratory and Rutgers University, which resulted in the
strain RUT-C30 with over 20-fold improvement in cellulase activity. This strain is
still the prototype cellulase hyperproducer available in the public domain (Bischof
et al. 2016). The titers of extracellular cellulase on cellulase-inducing carbon
source – lactose – can reach 30 g/L as demonstrated by Durand et al. (1988). A
direct comparison of the levels of cellulase production by different organisms is
complicated by the different strategies of production employed, by the different
substrates and inducers employed, and in some cases by the different units used for
expressing their activity. Several such comparisons have been made, which have
mostly concentrated on the published information, and information on the cellulase
yields from studies in the public domain may be obtained from these reports
(Sukumaran et al. 2005; Mathew et al. 2008; Chandel et al. 2012; Hansen et al.
2015; Cunha et al. 2016). The highest cellulase activities (as filter paper units,
FPUs) have been recorded at about 10–15 FPUs/g substrate under solid-state fermentation (SSF) and about 13 FPUs/ml for submerged fermentation (Hansen et al.
2015). Lignocellulose hydrolysis requires the concerted action of several enzymes
including the endoglucanases, cellobiohydrolases, β glucosidases, xylanases,
β-xylosidases, and several other accessory enzymes; and no single organism can
optimally produce all these enzymes. Hence it is a common practice to produce different enzymes using different organisms and the enzymes are blended to form
cocktails with better efficiency for biomass hydrolysis. Typically for T. reesei
1 Enzymes for Bioenergy