Acetyl Bromide Soluble Lignin (ABSL) Assay for Total Lignin Quantification from Plant Biomass

Abstract

Lignin is the second most abundant biopolymer on Earth, providing plants with mechanical support in secondary cell walls and defense against abiotic and biotic stresses. However, lignin also acts as a barrier to biomass saccharification for biofuel generation (Carroll and Somerville, 2009; Zhao and Dixon, 2011; Wang et al., 2013 ). For these reasons, studying the properties of lignin is of great interest to researchers in agriculture and bioenergy fields. This protocol describes the acetyl bromide method of total lignin extraction and quantification, which is favored among other methods for its high recovery, consistency, and insensitivity to different tissue types ( Johnson et al., 1961 ; Chang et al., 2008 ; Moreira- Vilar et al., 2014 ; Kapp et al., 2015 ). In brief, acetyl bromide digestion causes the formation of acetyl derivatives on free hydroxyl groups and bromide substitution of α-carbon hydroxyl groups on the lignin backbone to cause a complete solubilization of lignin, which can be quantified using known extinction coefficients and absorbance at 280 nm (Moreira- Vilar et al., 2014 ).

Keywords: Acetyl bromide; Biochemical measurement; Lignin; Plant biomass; Plant cell walls.

Figure 1.. Representation of insufficiently and sufficiently ground dry plant material

Figure 2.. Image of non-destarched and destarched AIR following staining with Lugol’s Iodine solution

Figure 3.. Representative absorbance spectrum of ABSL reading measured against a blank.

A²⁸⁰ absorbance is due to the presence of ABSL, and A⁶⁰⁰ is depicted to show that there is minimal residual AIR particulate matter present in the sample during absorbance reading.