Quantification of morphochemical changes during in situ enzymatic hydrolysis of individual biomass particles based on autofluorescence imaging.

Enzymatic hydrolysis of biomass is an established method for producing biofuels. Lignocellulosic biomass such as corn stover is very inhomogeneous material with big variation on conversion rates between individual particles therefore leading to variable recalcitrance results. In this study, we used noninvasive optical microscopy techniques, such as two-photon microscopy and fluorescence lifetime imaging microscopy, to visualize and analyze morphological and chemical changes of individual corn stover particles pretreated with sulfuric acid during hydrolysis. Morphochemical changes were interpreted based on the fluorescence properties of isolated building blocks of plant cell wall, such as cellulose, hemicellulose, and lignin. Enzymatic hydrolysis resulted in particle size reduction, side wall collapse, decrease of second harmonic signal from cellulose, redshifting of autofluorescence emission, and lifetime decrease attributed to the relative increase of lignin. Based on these observations, tracking compositional change after hydrolysis of individual particles was accomplished. The methodologies developed offer a paradigm for imaging and analyzing enzymatic hydrolysis in vitro and in situ, which could be used for screening enzymes cocktails targeting specific recalcitrant structures or investigating locally enzyme anti-inhibitory agents.

Biomass Pretreatment and Enzymatic Hydrolysis Dynamics Analysis Based on Particle Size Imaging.

Parameters such as pretreatment method, enzyme type and concentration, determine the conversion efficiency of biomass' cellulose and hemicellulose to glucose and mainly xylose in biomass-based fuel production. Chemical quantification of these processes offers no information on the effect of enzymatic hydrolysis (EH) on particle morphology. We report on the development of a microscopy method for imaging pretreated biomass particles at different EH stages. The method was based on acquiring large field of view images, typically 20×10 mm2 containing thousands of particles. Morphology of particles with lengths between 2 µm and 5 mm could be visualized and analyzed. The particle length distribution of corn stover samples, pretreated with increasing amounts of sulfuric acid at different EH stages, was measured. Particle size was shown to be dependent on pretreatment severity and EH time. The methodology developed could offer an alternative method for characterization of EH of biomass for second generation biofuels and visualization of recalcitrant structures.

Study on decomposition products of norbixin during bleaching with hydrogen peroxide and a peroxidase by means of UPLC-UV and mass spectrometry.

The decomposition products of norbixin, a component of the natural colouring agent annatto, have been studied under bleaching conditions in water and in a whey matrix. In water, several unsaturated aldehydes and ketones of carboxylic acids were identified with UPLC-UV/MS and high resolution mass spectrometry techniques. Based on these products a reaction scheme for the decomposition of norbixin is proposed. In whey, the norbixin is also degraded during bleaching, but no decomposition products are detected. Most likely these products react with endogenous compounds from the whey matrix. For one of these compounds, i.e. cysteine, the formation of a reaction product with 3-acetylacrylic acid (decomposition product of norbixin) was shown.