From plants to bioproducts

Plants are a major source for the production of renewable products, which are an alternative to fossil-based products. More specifically, plant cells are made up of a wall composed of lignocellulose, a network of molecules such as cellulose, hemicellulose, and lignin. This lignocellulose is attracting a lot of attention because the cellulose it contains contains glucose molecules. It is this glucose that is then used as a raw material to produce biomaterials or biofuel. However, to make cellulose (and therefore glucose) accessible, the components of lignocellulose must first be separated, and this process is still complex and costly.

The process of plant cell wall degradation

To “break down” the lignocellulose in the cell wall, enzymes are used. This process of cell wall deconstruction is called enzymatic hydrolysis or saccharification. It is a complex process that is still poorly understood at the cellular level. To optimize the process of transforming plants into bioproducts, it is therefore essential to better understand how enzymes break down plant cell walls at the microscopic level.

A new technique for tracking plant decomposition at the cellular level

Although many imaging techniques exist, accurately tracking the enzymatic breakdown of plant walls remains particularly difficult, especially when degradation is very advanced. Current methods rely on data with favorable properties: acquisition on flat, slightly degraded samples that remain stable over time. However, these requirements are generally not met when enzymatic wall breakdown is significant, limiting or even preventing quantitative analysis at advanced stages of degradation.

This was the focus of the study conducted as part of the FillingGaps project. The researchers developed HydroTrack, a method that allows the degradation of plant cell walls to be studied in three dimensions over time, even with samples that deform, break down, and move, which had previously blocked existing techniques. They tested this method on spruce wood, a species particularly affected by climate change in Europe, and observed its degradation.

The first step is to acquire 3D images of the wood samples every hour for 24 hours using a confocal fluorescence microscope. These images show the evolution of the cell walls, which emit a signal naturally thanks to their autofluorescence. The images are then processed using software called HydroTrack, which makes it possible to:

• Precisely align the images over time, even if the sample deforms and moves considerably;
• Track the same cells throughout the experiment;
• Measure the volume and surface area of cell walls, which gives an idea of the rate of degradation by enzymes.

Identifying key markers to track the degradation process

Using this method, researchers were able to identify critical markers for tracking the degradation process of plant walls:

• The autofluorescence of cell walls is a key marker for tracking the progress of enzymatic hydrolysis.
• The morphological parameters of the cell wall are indicators of the efficiency of cellulose conversion.

Indeed, the natural autofluorescence distribution of the walls changes characteristically under enzymatic action. At the same time, it is possible to quantify the geometric properties of the cells (volume and accessible surface area of the walls) during hydrolysis. This monitoring reveals a decrease in cell volume that precedes that of their surface area, showing that accessibility to enzymes varies over time.

What are the future industrial applications?

By providing a detailed assessment of the effectiveness of enzymes and pretreatments, this approach would therefore make it possible to accurately analyze the effectiveness of enzymes in biomass treatments and improve cellulose conversion processes, in order to support the development of more sustainable solutions for the manufacture of bio-based products.

Thanks to Yassin REFAHI, PhD – Research Fellow, UMR FARE INRAE, and Gabriel Paës, PhD – Research Director, UMR FARE INRAE, for reviewing this article.