Abstract
BackgroundRecent advances in the development of enzyme cocktails for degradation of lignocellulosic biomass, especially the discovery of lytic polysaccharide monooxygenases (LPMOs), have opened new perspectives for process design and optimization. Softwood biomass is an abundant resource in many parts of the world, including Scandinavia, but efficient pretreatment and subsequent enzymatic hydrolysis of softwoods are challenging. Sulfite pulping-based pretreatments, such as in the BALI™ process, yield substrates that are relatively easy to degrade. We have assessed how process conditions affect the efficiency of modern cellulase preparations in processing of such substrates.ResultsWe show that efficient degradation of sulfite-pulped softwoods with modern, LPMO-containing cellulase preparations requires the use of conditions that promote LPMO activity, notably the presence of molecular oxygen and sufficient reducing power. Under LPMO activity-promoting conditions, glucan conversion after 48-h incubation with Cellic® CTec3 reached 73.7 and 84.3% for Norway spruce and loblolly pine, respectively, at an enzyme loading of 8 mg/g of glucan. The presence of free sulfite ions had a negative effect on hydrolysis efficiency. Lignosulfonates, produced from lignin during sulfite pretreatment, showed a potential to activate LPMOs. Spiking of Celluclast®, a cellulase cocktail with low LPMO activity, with monocomponent cellulases or an LPMO showed that the addition of the LPMO was clearly more beneficial than the addition of any classical cellulase. Addition of the LPMO in reactions with spruce increased the saccharification yield from approximately 60% to the levels obtained with Cellic® CTec3.ConclusionsIn this study, we have demonstrated the importance of LPMOs for efficient enzymatic degradation of sulfite-pulped softwood. We have also shown that to exploit the full potential of LPMO-rich cellulase preparations, conditions promoting LPMO activity, in particular the presence of oxygen and reducing equivalents are necessary, as is removal of residual sulfite from the pretreatment step. The use of lignosulfonates as reductants may reduce the costs related to the addition of small molecule reductants in sulfite pretreatment-based biorefineries.
Highlights
Recent advances in the development of enzyme cocktails for degradation of lignocellulosic bio‐ mass, especially the discovery of lytic polysaccharide monooxygenases (LPMOs), have opened new perspectives for process design and optimization
Saccharification of a model cellulosic substrate The model cellulosic substrate Avicel PH-101 was incubated for 48 h with an older generation, LPMO-poor, cellulase mixture (Celluclast® 1.5L supplemented with Novozym 188; 5:1, w/w), as well as a modern, LPMOcontaining, cellulase preparation, C ellic® CTec3, under either aerobic or anaerobic conditions, in the presence or absence of an external electron donor
Saccharification of Avicel with the C elluclast® 1.5L:Novozym 188 mixture was hardly affected by the presence of oxygen or the addition of ascorbic acid (Fig. 1c), and under LPMO-promoting conditions, only low product levels were observed (Fig. 1d). Celluclast® 1.5L is a product based on the T. reesei secretome, in which three AA9 family LPMOs have been identified, which are expressed at low levels [13, 32]
Summary
Recent advances in the development of enzyme cocktails for degradation of lignocellulosic bio‐ mass, especially the discovery of lytic polysaccharide monooxygenases (LPMOs), have opened new perspectives for process design and optimization. Softwood biomass is an abundant resource in many parts of the world, including Scandinavia, but efficient pretreatment and subsequent enzymatic hydrolysis of softwoods are challenging. Sulfite pulping-based pretreatments, such as in the BALITM process, yield substrates that are relatively easy to degrade. Efficient pretreatment and enzymatic hydrolysis of softwoods has been demonstrated for organosolv- and SPORL-pretreated biomasses [4, 5]. The BALITM process is an alternative sulfite-based pretreatment that yields cellulose-rich substrates containing little lignin and hemicellulose relative to, e.g., the SPORL pretreatment, whose enzymatic conversion to glucose is relatively easy [6]. The most studied enzymes are cellulose-degrading glycoside hydrolases, known as cellulases. In natural biomass-converting enzyme systems, the cellulases are accompanied by a wide range of hemicellulose- and lignin-degrading enzymes working in concert to fully decompose lignocellulose
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