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

Dimitrios Kapsokalyvas*, Joachim Loos, Ilco A. L. A. Boogers, Maaike M. Appeldoorn, Mirjam A. Kabel, Marc Van Zandvoort

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review


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.

Original languageEnglish
Article numbere23347
Number of pages10
Issue number3
Early online date23 Dec 2019
Publication statusPublished - Mar 2020


  • biomass
  • cellulose
  • enzymatic hydrolysis
  • FLIM
  • pretreated corn stover (pCS)
  • two-photon microscopy

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