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

Abstract

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.

Fluorescence Microscopy Methods for the Analysis and Characterization of Lignin

“…In general, the range goes from 400 to 640 nm. However, some methods, such as excitation, use UV ranges from 300 nm of excitation, while some lasers reach up to 820 nm of excitation [ 55 ]. For each technique, the power of the lasers or lamps and the type of filter cube that discriminates the wavelength that is emitted or detected vary.…”

“…Based on the intensity and wavelength in which the compounds are detected, whether in the blue, green, or red channel, some of the structural components present in the samples can be identified. In the case of lignin, the range in which fluorophores emit fluorescence is wide, so their presence can be detected in different channels [ 40 ] depending on the wavelength in which fluorophores are excited and detected in addition to the treatments or dyes that the sample receives, so the detection value can vary based on the method used [ 55 ].…”

“…This type of microscopy has been used to analyze the structural morphology of sugarcane bagasse before and after a hydrolysis and bleaching treatment because when penetrating the sample, better-quality details of the structure are obtained compared to confocal microscopy [ 93 ]. Similarly, the recalcitrant structures of lignin were identified in samples treated with enzymatic hydrolysis [ 55 ]; therefore, TPM is an efficient method to calculate the saccharification rate of lignocellulosic biomass by detecting changes in fluorescence emission [ 43 ]. On the other hand, TPM allows to identify structural changes in the wood coming from musical instruments of different ages [ 101 ] and serves to structurally analyze chitosan—lignin composite films [ 102 ].…”