# In situ micro-spectroscopic investigation of lignin in poplar cell walls pretreated by maleic acid

## TL;DR

This study uses micro-spectroscopy to investigate how maleic acid pretreatment changes lignin structures in poplar cell walls, which could improve biomass breakdown for biofuel production.

## Contribution

The study introduces a novel micro-spectroscopic method to distinguish and track dense and loosely packed lignin forms during acid pretreatment.

## Key findings

- Maleic acid pretreatment modifies dense lignin into loosely packed lignin at low concentrations.
- High acid concentrations remove both dense and loosely packed lignin, forming lignin–carbohydrate complex droplets.
- Loosely packed lignin removal increases enzyme accessibility for biomass digestion.

## Abstract

In higher plant cells, lignin provides necessary physical support for plant growth and resistance to attack by microorganisms. For the same reason, lignin is considered to be a major impediment to the process of deconstructing biomass to simple sugars by hydrolytic enzymes. The in situ variation of lignin in plant cell walls is important for better understanding of the roles lignin play in biomass recalcitrance.

A micro-spectroscopic approach combining stimulated Raman scattering microscopy and fluorescence lifetime imaging microscopy was employed to probe the physiochemical structure of lignin in poplar tracheid cell walls. Two forms of lignins were identified: loosely packed lignin, which had a long (4 ns) fluorescence lifetime and existed primarily in the secondary wall layers; and dense lignin, which had a short (0.5–1 ns) fluorescence lifetime and was present in all wall layers, including the cell corners, compound middle lamellae, and secondary wall. At low maleic acid concentration (0.025 and 0.05 M) pretreatment conditions, some of the dense lignin was modified to become more loosely packed. High acid concentration removed both dense and loosely packed lignins. These modified lignins reformed to make lignin–carbohydrate complex droplets containing either dense or loosely packed lignin (mostly from secondary walls) and were commonly observed on the cell wall surface.

We have identified dense and loosely packed lignins in plant cell walls. During maleic acid pretreatment, both dense lignin droplets and loosely packed lignin droplets were formed. Maleic acid pretreatment more effectively removes loosely packed lignin in secondary walls which increases enzyme accessibility for digestion.