Characterization Of Biomass Porosity And Wall Polymer Features That Distinctively Affect Lignocellulose Enzymatic Saccharification And Bioethanol Production Under Various Pretreatments In Miscanthus

Miscanthus is a leading bioenergy crop with enormous lignocellulose residues potential for biofuels and biochemicals.Plant cell wall is mainly composed of cellulose,hemicelluloses and lignin,and their high degree of structural organization and complexity play critical roles in cell wall integrity.Cellulosic ethanol is now well established as an excellent additive to gasoline with reduced carbon release.In principle,cellulosic ethanol conversion involves three major steps:initial physical and chemical pretreatment to deconstruct plant cell walls,sequential enzymatic hydrolysis to release soluble sugars and final yeast fermentation to produce bioethanol.However,the intrinsic recalcitrance of lignocellulose to resist biotic attacks and abiotic stress currently leads to process difficulties for a cost-effective and environmental friendly bioethanol production.Lignocellulose recalcitrance is basically determined by diverse cell wall compositions,specialized wall polymer features and complicated wall-network structures.It has been defined that biomass porosity is the general and integrated factor accounting for biomass enzymatic hydrolysis,but much is yet unknown about how biomass porosity is characteristically determined by wall polymer features and wall network styles.Furthermore,due to diverse and heterogeneous wall structures,different pretreatments distinctively extract wall polymers and typically alter wall polymer features and inter-linkages,leading to difficulties in sorting out a universal standard to judge optimal pretreatment for high biomass enzymatic saccharification and bioethanol production.Initially,this study attempted to use Direct yellow 11?DY 11?and Direct Blue 15?DB15?as substitutes for Direct Orange 15?DO 15?and Direct Blue 1?DB 1?for Simons staining technique.Then,adsorption behavior of alternative Simons?stain was evaluated for the stability and potential of the assay to measure overall accessible surface area of lignocellulosic substrates.The optimized Simons?stain was applied to examine biomass porosity reflecting cellulose accessibility to cellulase enzymes for enzymatic saccharification in Miscanthus.Using four typical pairs of Miscanthus accessions that showed distinct cell wall compositions,this study then performed liquid hot water?LHW?and chemical?i.e.,H₂SO₄,NaOH?pretreatments under various conditions to enhance biomass enzymatic hydrolysis.As a comparison,mild alkali pretreatment?4%NaOH at 50oC?led to complete biomass saccharification with hexoses yield of 100%?%cellulose?while 1%Tween-80 was co-supplied into enzymatic hydrolysis stage for the desirable Miscanthus accessions.Consequently,the highest bioethanol yields were obtained at 19%?%dry matter?from yeast fermentation,with much higher sugar-ethanol conversion rates by 94?98%,compared to the other Miscanthus species subjected to stronger pretreatments as reported in previous studies.Furthermore,three optimized pretreatments distinctively extracted wall polymers and specifically altered major polymer features and inter-linkage styles,but the alkali pretreatment could largely extract lignin with effective removal of hemicellulose-lignin complex linkages,leading to significantly-increased biomass porosity than those of the LHW and H₂SO₄ pretreatments.Based on a series of statistic calculations,excellent equations were generated to precisely estimate hexoses and ethanol yields under three optimal pretreatments,and a hypothetical model was proposed to outline an integrative impact on biomass saccharification and bioethanol production subjective to a predominate factor?CR stain?of biomass porosity and four additional minor factors?DY stain,cellulose DP,hemicellulose X/A,lignin G-monomer?,which interpreted why the mild alkali pretreatment could lead to a complete biomass saccharification with the highest bioethanol yield achieved in the desirable Miscanthus accessions.Therefore,this study has established a novel standard to judge optimal biomass pretreatment technology accounting for a cost-effective biofuel production for Miscanthus.Our study also provides insights into a powerful biotechnology for precise lignocellulose modification in bioenergy Miscanthus and beyond.