Synergistic action of enzyme preparations towards recalcitrant corn silage polysaccharides

Highlights

• High degrees of substitution (Ac, Ara and UA) are present in recalcitrant corn xylan.

• Commercial enzymes vary in their activity towards recalcitrant corn polysaccharides.

• Synergy occurs between an A. niger/T. emersonii and commercial enzyme preparations.

• Different mixtures of enzyme preparations are needed for optimal feedstock conversion.

Abstract

Corn silage, its water unextractable solids (WUS) and enzyme recalcitrant solids (ErCS) and an industrial corn silage-based anaerobic fermentation residue (AFR) represent corn substrates with different levels of recalcitrance. Compositional analysis reveals different levels of arabinoxylan substitution for WUS, ErCS and AFR, being most pronounced regarding acetic acid, glucuronic acid- and arabinose content. By screening for enzymatic degradation of WUS, ErCS and AFR, enzyme preparations exhibiting high conversion rates were identified. Furthermore significant synergistic effects were detected by blending Aspergillus niger/Talaromyces emersonii culture filtrates with various enzymes. These findings clearly highlight a necessity for a combinatorial use of enzyme preparations towards substrates with high recalcitrance characteristics to reach high degrees of degradation. Enzyme blends were identified, outperforming the individual commercial preparations. These enzyme preparations provide a basis for new, designed enzyme mixtures for corn polysaccharide degradation as a source of necessary, accessory enzyme activities.

Introduction

Corn silage, a major biogas feedstock [1], provides a source of fermentable sugars. In order to enable efficient utilization of recalcitrant corn residues or wastes for biogas production, enzymatic preparations are necessary that can degrade these substrates.

Multiple enzyme classes are involved in the degradation of cellulose, including β-1,4-endoglucanases, exoglucanases/cellobiohydrolases and β-glucosidases. Hemicellulose requires even more enzyme activities for its complete hydrolysis. β-1,4-endo-xylanases and β-1,4-xylosidases are necessary to degrade the (arabino-)xylan backbone. Important accessory activities are α-arabinofuranosidases, arabinoxylan arabinofuranohydrolases, α-glucuronidases, α-1,4-galactosidases, β-1,4-galactosidases, feruloyl/p-coumaroyl esterases and acetyl xylan esterases [2]. Furthermore, GH61 and CBM33 classified enzymes catalyze oxidative cleavage of polysaccharides [3]. Other non-hydrolytic proteins such as expansins, expansin-like proteins (swollenin), carbohydrate-binding modules or low molecular weight peptides promote disruption of cellulosic material by amorphogenesis and deagglomeration [4].

While a broad range of microorganisms express enzymes that are capable of hydrolyzing (hemi)cellulose, only a few strains secrete a complex of (hemi)cellulolytic enzymes with potential for practical application as in biofuels production [5]. Genera that are commonly commercially exploited for (hemi)cellulase production include Aspergillus, Trichoderma, Humicola (Fungi, Ascomycota), Thermomonospora (Bacteria, Actinobacteria) and Bacillus (Bacteria, Firmicutes). Bioconversion by (hemi)cellulolytic enzyme preparations is limited by the recalcitrance of feedstocks. Recalcitrant cell wall structures, like a high degree of backbone substitution (87%) of corn glucuronoarabinoxylan has been reported [6]. Several substituents, like ferulic acid, diferulates, acetic acid, galactose, arabinose and uronic acid, make corn fiber resistant to enzymatic hydrolysis following mild acid pretreatment [7]. Furthermore, biomass recalcitrance is derived from the architecture of cell walls, influencing the accessibility for enzymes [4].

Different strategies to overcome plant cell wall recalcitrance have been applied. A frequently used strategy is the application of harsh physical and chemical pretreatment conditions, like steam explosion and acid hydrolysis. Such conditions typically cause the hydrolysis of the hemicellulose fraction, while rendering the cellulose fraction more susceptible to enzymatic degradation [8]. Disadvantages of these methods are their high costs, energy needs [9], their contribution to environmental pollution and the formation of inhibitors [10]. Another promising approach is to diminish the necessity of intense pretreatment by improved enzymatic hydrolysis. The latter would allow better utilization of substrates in biogas production where usually no pretreatment is applied. Furthermore a better utilization of C-5 sugars without (intense) pretreatment would be of relevance for 2nd generation ethanol production as C-5 fermenting yeast has been reported as a powerful tool to ferment xylose and arabinose [11], [12].

Here we present fermentable sugar release by enzyme preparations screened on corn silage derived substrates: Water unextractable solids (WUS), enzyme recalcitrant solids (ErCS) and an anaerobic fermentation residue (AFR). WUS and ErCS (laboratory prepared corn silage model and -recalcitrant polysaccharides) and AFR (industrial residue) are substrates with increasing recalcitrance characteristics as determined by compositional analysis.

Substrate conversion by screening individual and blended enzyme preparations is assessed. For the latter a state-of-the-art Aspergillus niger/Talaromyces emersonii enzyme mixture is supplemented with a variety of commercial enzyme preparations resulting in increased conversion rates. Significant synergistic effects towards cellulose and hemicellulose degradation are identified for all substrates tested.