Cellulosic ethanol production: Progress, challenges and strategies for solutions

The surplus availability of rice straw, its limited usage, and environmental pollution caused by its inefficient burning has fostered research for its valorization to biofuels. This review elucidates the current status of rice straw potential around the globe along with recent advances in revealing the critical factors responsible for its recalcitrance and chemical properties. The role and accumulation of high silica content in rice straw has been elucidated with its impact on enzymatic hydrolysis in a biorefinery environment. The correlation of different pre-treatment approaches in modifying the physiochemical properties of rice straw and improving the enzymatic accessibility has also been discussed. This study highlights new challenges, resolutions, and opportunities for rice straw based biorefineries. © 2017 Society of Chemical Industry and John Wiley & Sons, Ltd

Rice straw as a feedstock for biofuels: Availability, recalcitrance, and chemical properties

Miscanthus is a leading bioenergy crop with enormous lignocellulose production potential for biofuels and chemicals. However, lignocellulose recalcitrance leads to biomass process difficulty for an efficient bioethanol production. Hence, it becomes essential to identify the integrative impact of lignocellulose recalcitrant factors on cellulose accessibility for biomass enzymatic hydrolysis. In this study, we analyzed four typical pairs of Miscanthus accessions that showed distinct cell wall compositions and sorted out three major factors that affected biomass saccharification for maximum bioethanol production. Among the three optimal (i.e., liquid hot water, H2SO4 and NaOH) pretreatments performed, mild alkali pretreatment (4% NaOH at 50 °C) led to almost complete biomass saccharification when 1% Tween-80 was co-supplied into enzymatic hydrolysis in 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. By comparison, three optimized pretreatments distinctively extracted wall polymers and specifically altered polymer features and inter-linkage styles, but the alkali pretreatment caused much increased biomass porosity than that of the other pretreatments. Based on integrative analyses, excellent equations were generated to precisely estimate hexoses and ethanol yields under various 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). Using four pairs of Miscanthus samples with distinct cell wall composition and varied biomass saccharification, this study has determined three main factors of lignocellulose recalcitrance that could be significantly reduced for much-increased biomass porosity upon optimal pretreatments. It has also established a novel standard that should be applicable to judge any types of biomass process technology for high biofuel production in distinct lignocellulose substrates. Hence, this study provides a potential strategy for precise genetic modification of lignocellulose in all bioenergy crops.

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A finalized determinant for complete lignocellulose enzymatic saccharification potential to maximize bioethanol production in bioenergy Miscanthus.

Genetic modification of plant cell walls has been implemented to reduce lignocellulosic recalcitrance for biofuel production. Plant glycoside hydrolase family 9 (GH9) comprises endo-β-1,4-glucanase in plants. Few studies have examined the roles of GH9 in cell wall modification. In this study, we independently overexpressed two genes from GH9B subclasses (OsGH9B1 and OsGH9B3) and examined cell wall features and biomass saccharification in transgenic rice plants. Compared with the wild type (WT, Nipponbare), the OsGH9B1 and OsGH9B3 transgenic rice plants, respectively, contained much higher OsGH9B1 and OsGH9B3 protein levels and both proteins were observed in situ with nonspecific distribution in the plant cells. The transgenic lines exhibited significantly increased cellulase activity in vitro than the WT. The OsGH9B1 and OsGH9B3 transgenic plants showed a slight alteration in three wall polymer compositions (cellulose, hemicelluloses, and lignin), in their stem mechanical strength and biomass yield, but were significantly decreased in the cellulose degree of polymerization (DP) and lignocellulose crystalline index (CrI) by 21–22%. Notably, the crude cellulose substrates of the transgenic lines were more efficiently digested by cellobiohydrolase (CBHI) than those of the WT, indicating the significantly increased amounts of reducing ends of β-1,4-glucans in cellulose microfibrils. Finally, the engineered lines generated high sugar yields after mild alkali pretreatments and subsequent enzymatic hydrolysis, resulting in the high bioethanol yields obtained at 22.5% of dry matter. Overproduction of OsGH9B1/B3 enzymes should have specific activity in the postmodification of cellulose microfibrils. The increased reducing ends of β-1,4-glucan chains for reduced cellulose DP and CrI positively affected biomass enzymatic saccharification. Our results demonstrate a potential strategy for genetic modification of cellulose microfibrils in bioenergy crops.

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Overproduction of native endo-β-1,4-glucanases leads to largely enhanced biomass saccharification and bioethanol production by specific modification of cellulose features in transgenic rice.

Mild chemical pretreatments are sufficient for bioethanol production in transgenic rice straws overproducing glucosidase

Genetic modification of plant cell walls has been posed to reduce lignocellulose recalcitrance for enhancing biomass saccharification. Since cellulose synthase (CESA) gene was first identified, several dozen CESA mutants have been reported, but almost all mutants exhibit the defective phenotypes in plant growth and development. In this study, the rice (Oryza sativa) Osfc16 mutant with substitutions (W481C, P482S) at P-CR conserved site in CESA9 shows a slightly affected plant growth and higher biomass yield by 25%-41% compared with wild type (Nipponbare, a japonica variety). Chemical and ultrastructural analyses indicate that Osfc16 has a significantly reduced cellulose crystallinity (CrI) and thinner secondary cell walls compared with wild type. CESA co-IP detection, together with implementations of a proteasome inhibitor (MG132) and two distinct cellulose inhibitors (Calcofluor, CGA), shows that CESA9 mutation could affect integrity of CESA4/7/9 complexes, which may lead to rapid CESA proteasome degradation for low-DP cellulose biosynthesis. These may reduce cellulose CrI, which improves plant lodging resistance, a major and integrated agronomic trait on plant growth and grain production, and enhances biomass enzymatic saccharification by up to 2.3-fold and ethanol productivity by 34%-42%. This study has for the first time reported a direct modification for the low-DP cellulose production that has broad applications in biomass industries.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.12700

OsCESA9 conserved-site mutation leads to largely enhanced plant lodging resistance and biomass enzymatic saccharification by reducing cellulose DP and crystallinity in rice

Fan Hu

Papers

OsCESA9 conserved-site mutation leads to largely enhanced plant lodging resistance and biomass enzymatic saccharification by reducing cellulose DP and crystallinity in rice