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Significant improvements in pretreatments and a deeper understanding of changes in the physical structure and chemical composition of pretreated lignocellulosic materials are required to guarantee the technical and economic sustainability of second-generation ethanol technologies. In present work, several biophysical techniques were applied to characterize sugarcane bagasse samples that have been subjected to autocatalytic, phosphoric acid and sulfuric acid catalyzed steam explosion under equivalent combined severity factors (CSF). Confocal laser scanning microscopy and fluorescence lifetime imaging studies demonstrated unequal changes in lignin distribution in the plant cell wall that could be nicely correlated with substrate susceptibility to enzymatic hydrolysis. Furthermore, solid state nuclear magnetic resonance corroborated these observations by showing the differences in biomass composition primarily associated with hemicellulose removal, whereas X-ray diffraction analysis revealed changes in crystallinity indexes and average crystallite sizes. Thus, the impact of pretreatment temperature on the physical structure and chemical composition of CSF-equivalent steam-exploded sugarcane bagasse was identified as the key factor for developing substrate accessibility. This calls for caution in using combined severity factor as a chief parameter for comparison of different pretreatment conditions.

The enzymatic mixture containing Novozyme Cellic CTec2 spiked with xylanase derived from Thermomyces lanuginosus (1000 U xylanase per g of substrate) resulted in enhanced saccharification of native, hydrothermally and autohydrolytically pretreated sugarcane bagasse when compared to the benchmark Cellic CTec2. Hydrothermally pretreated bagasse upon hydrolysis with the enzymatic mixture yielded considerably higher levels of sugars as compared to benchmark Cellic CTec2, resulting in saccharification efficiencies of 85.7, 77.8 and 78.9% at 10, 15 and 20% (weight per volume) substrate loadings, respectively. Autohydrolysed and native sugarcane bagasse samples had lower saccharification yields, although xylanase addition to the benchmark cocktail still was beneficial. Chemical compositional analysis and detailed morphological examination of the substrates after pretreatments as well as the enzymatic hydrolysis reactions employing confocal laser scanning microscopy (CLSM), field emission scanning electron microscopy (FESEM), X-Ray diffraction (XRD) and solid state nuclear magnetic resonance (ssNMR) provided fundamental insights into the structural changes during deconstruction and shed light on how such changes affected the biomass enzyme hydrolysis.

Economical sustainability of cellulosic ethanol technology still requires considerable improvements in efficacies of both pretreatment and enzymatic hydrolysis steps. In this work a number of physical techniques were applied to characterize sugarcane bagasse samples that underwent hydrothermal and/or organosolv pretreatments under variable conditions and to correlated the observed changes with the efficiency of enzymatic hydrolysis. Confocal and field emission scanning electron microscopy studies revealed morphological changes in lignin distribution in the plant cell wall. The hydrothermal pretreatment caused a disorder in the arrangement of the lignin, whereas organosolv pretreatment partially removed lignin from bagasse and fraction of it redeposited at the surfaces of cellulose fibers. The delignification process was also analyzed by both chemical composition analysis and nuclear magnetic resonance. Pretreatment conditions leading to a significant increase of the efficiency of enzymatic hydrolysis were identified. Our studies open avenues for further biophysical investigations of pretreated lignocellulosic biomass, which could lead to its improved enzymatic hydrolysis.

Pretreatment is an important technological step for the lignocellulose conversion into fuels and biochemicals. Steam explosion (STEX) pretreatment is a fast method for affecting of plant cell wall architecture by a sharp pressure change at high temperatures. Here, eight different STEX conditions were applied to sugarcane bagasse (SCB) samples using auto-catalyzed and sulfuric acid catalyzed processes under a range of combined severity factors (CSFs; from 0.37 to 1.34). Our results showed that STEX of auto-catalyzed and sulfuric acid-catalyzed SCB led to the removal of 70–80 % and 82–92 % of xylose, respectively. Enzymatic hydrolysis of auto-catalyzed and sulfuric acid catalyzed bagasse was performed of washed and un-washed pretreated material to access the impact of soluble inhibitors on overall cellulosic sugars recovery. Auto-catalyzed bagasse after washing showed significantly higher glucan conversion (96%) than washed bagasse after pretreatment (62%). On the other hand, sulfuric acid-catalyzed bagasse after washing had 94% glucan conversion as opposed to unwashed material which again had significantly smaller conversion levels (73%). Physical properties and chemical composition were characterized using high performance liquid chromatography, X-ray diffraction, 13C solid-state nuclear magnetic resonance and confocal laser scanning microscopy. The obtained results contribute to understanding of an influence of the morphological changes resulting from pretreatments on the efficiency of enzymatic hydrolysis. We show that the changes in physical structure and chemical composition of the pretreated biomass might not be determined solely by combined severity factor of the pretreatments, but by the whole complexity of chemical modifications that different conditions of pretreatments introduce in the plant biomass samples.