With the aim of understanding the contribution of enzymes to the cost of lignocellulosic biofuels, we constructed a techno-economic model for the production of fungal cellulases. We found that the cost of producing enzymes was much higher than that commonly assumed in the literature. For example, the cost contribution of enzymes to ethanol produced by the conversion of corn stover was found to be $0.68/gal if the sugars in the biomass could be converted at maximum theoretical yields, and $1.47/gal if the yields were based on saccharification and fermentation yields that have been previously reported in the scientific literature. We performed a sensitivity analysis to study the effect of feedstock prices and fermentation times on the cost contribution of enzymes to ethanol price. We conclude that a significant effort is still required to lower the contribution of enzymes to biofuel production costs.

/pdf/the-challenge-of-enzyme-cost-in-the-production-of-1bd9pll6gk.pdf

The challenge of enzyme cost in the production of lignocellulosic biofuels

Greater understanding of the mechanisms contributing to chemical and enzymatic solubilization of plant cell walls is critical for enabling cost-effective industrial conversion of cellulosic biomass to biofuels. Here, we report the use of correlative imaging in real time to assess the impact of pretreatment, as well as the resulting nanometer-scale changes in cell wall structure, upon subsequent digestion by two commercially relevant cellulase systems. We demonstrate that the small, noncomplexed fungal cellulases deconstruct cell walls using mechanisms that differ considerably from those of the larger, multienzyme complexes (cellulosomes). Furthermore, high-resolution measurement of the microfibrillar architecture of cell walls suggests that digestion is primarily facilitated by enabling enzyme access to the hydrophobic cellulose face. The data support the conclusion that ideal pretreatments should maximize lignin removal and minimize polysaccharide modification, thereby retaining the essentially native microfibrillar structure.

https://science.sciencemag.org/content/sci/338/6110/1055.full.pdf

How Does Plant Cell Wall Nanoscale Architecture Correlate with Enzymatic Digestibility

In the primary walls of growing plant cells, the glucose polymer cellulose is assembled into long microfibrils a few nanometers in diameter. The rigidity and orientation of these microfibrils control cell expansion; therefore, cellulose synthesis is a key factor in the growth and morphogenesis of plants. Celery (Apium graveolens) collenchyma is a useful model system for the study of primary wall microfibril structure because its microfibrils are oriented with unusual uniformity, facilitating spectroscopic and diffraction experiments. Using a combination of x-ray and neutron scattering methods with vibrational and nuclear magnetic resonance spectroscopy, we show that celery collenchyma microfibrils were 2.9 to 3.0 nm in mean diameter, with a most probable structure containing 24 chains in cross section, arranged in eight hydrogen-bonded sheets of three chains, with extensive disorder in lateral packing, conformation, and hydrogen bonding. A similar 18-chain structure, and 24-chain structures of different shape, fitted the data less well. Conformational disorder was largely restricted to the surface chains, but disorder in chain packing was not. That is, in position and orientation, the surface chains conformed to the disordered lattice constituting the core of each microfibril. There was evidence that adjacent microfibrils were noncovalently aggregated together over part of their length, suggesting that the need to disrupt these aggregates might be a constraining factor in growth and in the hydrolysis of cellulose for biofuel production.

/pdf/structure-of-cellulose-microfibrils-in-primary-cell-walls-2pp7smdyc1.pdf

Structure of Cellulose Microfibrils in Primary Cell Walls from Collenchyma

Thermostable cellulases: Current status and perspectives.

Abstract This article recounts, from my perspective of four decades in this field, evolving paradigms of primary cell wall structure and the mechanism of surface enlargement of growing cell walls. Updates of the structures, physical interactions, and roles of cellulose, xyloglucan, and pectins are presented. This leads to an example of how a conceptual depiction of wall structure can be translated into an explicit quantitative model based on molecular dynamics methods. Comparison of the model’s mechanical behavior with experimental results provides insights into the molecular basis of complex mechanical behaviors of primary cell wall and uncovers the dominant role of cellulose–cellulose interactions in forming a strong yet extensible network.

/pdf/building-an-extensible-cell-wall-2ppnpm1v.pdf

Building an extensible cell wall

The low efficiency of enzymes used in the bioprocessing of biomass for biofuels is one of the primary bottlenecks that must be overcome to make lignocellulosic biofuels cost-competitive. One of the rate-limiting factors is the accessibility of the cellulase enzymes to insoluble cellulolytic substrates, facilitated by surface absorption of the carbohydrate-binding modules (CBMs), a component of most cellulase systems. Despite their importance, reports of direct observation of CBM function and activity using microscopic methods are still uncommon. Here, we examine the site-specific binding of individual CBMs to crystalline cellulose in an aqueous environment, using the single molecule fluorescence method known as Defocused Orientation and Position Imaging (DOPI). Systematic orientations were observed that are consistent with the CBMs binding to the two opposite hydrophobic faces of the cellulose microfibril, with a well-defined orientation relative to the fiber axis. The approach provides in situ physical evidence indicating the CBMs bind with a well-defined orientation on those planes, thus supporting a binding mechanism driven by chemical and structural recognition of the cellulose surface.

In situ imaging of single carbohydrate-binding modules on cellulose microfibrils

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In situ imaging of single carbohydrate-binding modules on cellulose microfibrils