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.

Plant cellulose microfibrils are increasingly employed to produce functional nanofibers and nanocrystals for biomaterials, but their catalytic formation and conversion mechanisms remain elusive. Here, we characterize length-reduced cellulose nanofibers assembly in situ accounting for the high density of amorphous cellulose regions in the natural rice fragile culm 16 (Osfc16) mutant defective in cellulose biosynthesis using both classic and advanced atomic force microscopy (AFM) techniques equipped with a single-molecular recognition system. By employing individual types of cellulases, we observe efficient enzymatic catalysis modes in the mutant, due to amorphous and inner-broken cellulose chains elevated as breakpoints for initiating and completing cellulose hydrolyses into higher-yield fermentable sugars. Furthermore, effective chemical catalysis mode is examined in vitro for cellulose nanofibers conversion into nanocrystals with reduced dimensions. Our study addresses how plant cellulose substrates are digestible and convertible, revealing a strategy for precise engineering of cellulose substrates toward cost-effective biofuels and high-quality bioproducts.

Rice is a major food crop containing large amounts of lignocellulose residues usable for biofuels. In this study, we collected transgenic rice plants that over-produced Trichoderma reesei β-1,4-D-glucosidase (BGL I) into the cell walls in the mature straws. Without any pretreatment, the transgenic rice straws showed a consistently higher biomass enzymatic saccharification than the wild-type (WT) cultivar, in particular when 1% Tween-80 or 0.5% PEG-4000 was co-supplied into the enzymatic hydrolysis. Notably, under mild alkali pretreatment (1% NaOH at 50 °C for 2 h), the desirable transgenic line exhibited complete biomass enzymatic hydrolysis, resulting in the highest bioethanol yield of 21% (% dry matter) when compared with the rice and other bioenergy crops subjected to stronger pretreatment conditions reported in previous studies. Meanwhile, despite relatively low hexose yields obtained under 1% H₂SO₄ pretreatment, the transgenic rice straw also showed high bioethanol production at 18% due to an almost complete sugar–ethanol conversion rate. Chemical analyses indicated that the transgenic rice straw had significantly increased biomass porosity and reduced cellulose features (CrI, DP), which contributed to the largely enhanced biomass enzymatic hydrolysis. In addition, the raised arabinose level in hemicellulose and the lignin H-monomer proportion may also positively affect the biomass enzymatic saccharification in the transgenic rice straw. Hence, this study demonstrated a cost-effective and green lignocellulose conversion technology for high bioethanol production in the transgenic rice straw. It also provided a strategy for the potential genetic modification of plant cell walls in bioenergy crops.

Although cellulose represents the most abundant biomass resource on Earth, its natural recalcitrance hinders its application for low-cost biofuels and high-value bioproducts on a large scale. In this study, we generated less recalcitrant cellulose nanofibril substrates by site-specific mutations and knockouts of three OsCESA4,7,9 isoforms that are essential as cellulose synthase complexes for the cellulose biosynthesis of secondary cell walls in rice. As a comparison with the wild type form, three genetically-engineered CESAs isoforms independently generated cellulose nanofibrils with the average lengths reduced by 63%, which accounts for the high density of the amorphous cellulose chains as the initial breakpoints for the consistently enhanced biomass enzymatic hydrolysis into fermentable sugars. The genetically modified cellulose nanofibrils efficiently produce ideal nanocrystals with the most reduced dimension to-date in three knockout lines, which are applicable as optimal intermediates for highly valuable bioproducts. The improved cellulose nanofibrils are also effective in inducing the secretion of lignocellulose-degradation enzymes from fungi (T. reesei) with the two cellulase (exoglucanases, β-glucosidases) activities elevated by 100% and 138%, and the total protein level increased by 44%. Our data thus reveal a novel green strategy for achieving high-quality diverse bioproduction by integrating the precise genetic modification of lignocellulose substrates with efficient biomass process technology.

The genetic modification of plant cell walls has been considered to reduce lignocellulose recalcitrance in bioenergy crops. As a result, it is important to develop a precise and rapid assay for the major wall polymer features that affect biomass saccharification in a large population of transgenic plants. In this study, we collected a total of 246 transgenic rice plants that, respectively, over-expressed and RNAi silenced 12 genes of the OsGH9 and OsGH10 family that are closely associated with cellulose and hemicellulose modification. We examined the wall polymer features and biomass saccharification among 246 transgenic plants and one wild-type plant. The samples presented a normal distribution applicable for statistical analysis and NIRS modeling. Among the 246 transgenic rice plants, we determined largely varied wall polymer features and the biomass enzymatic saccharification after alkali pretreatment in rice straws, particularly for the fermentable hexoses, ranging from 52.8 to 95.9%. Correlation analysis indicated that crystalline cellulose and lignin levels negatively affected the hexose and total sugar yields released from pretreatment and enzymatic hydrolysis in the transgenic rice plants, whereas the arabinose levels and arabinose substitution degree (reverse xylose/arabinose ratio) exhibited positive impacts on the hexose and total sugars yields. Notably, near-infrared spectroscopy (NIRS) was applied to obtain ten equations for predicting biomass enzymatic saccharification and seven equations for distinguishing major wall polymer features. Most of the equations exhibited high R 2/R 2 cv/R 2 ev and RPD values for a perfect prediction capacity. Due to large generated populations of transgenic rice lines, this study has not only examined the key wall polymer features that distinctively affect biomass enzymatic saccharification in rice but has also established optimal NIRS models for a rapid and precise screening of major wall polymer features and lignocellulose saccharification in biomass samples. Importantly, this study has briefly explored the potential roles of a total of 12 OsGH9 and OsGH10 genes in cellulose and hemicellulose modification and cell wall remodeling in transgenic rice lines. Hence, it provides a strategy for genetic modification of plant cell walls by expressing the desired OsGH9 and OsGH10 genes that could greatly improve biomass enzymatic digestibility in rice.

An integrated strategy for complete lignocellulose saccharification to maximize bioethanol yield under a cost-effective and green-like biomass process in rapeseed stalk.