Determination of a suitable molar absorption coefficient (ε) for lignin analysis of fibrous plants using the CASA method

Antoine Bozek; Fabienne Mathis; Laurent Guillaumat; Jörg Müssig; Nathalie Boizot; Alaa Ismael; John Ralph; Fachuang Lu; Richard Sibout; Johnny Beaugrand

doi:10.1016/j.ijbiomac.2025.142324

Determination of a suitable molar absorption coefficient (ε) for lignin analysis of fibrous plants using the CASA method

Int J Biol Macromol. 2025 May;308(Pt 1):142324. doi: 10.1016/j.ijbiomac.2025.142324. Epub 2025 Mar 19.

Authors

Affiliations

¹ UR1268 BIA, INRAE Nantes, BP 71627, 44316 Nantes Cedex 03, France; HEMP-IT-ADN, 6 rue Louis Lumière, 49250 Beaufort en Anjou, France. Electronic address: antoine.bozek@inrae.fr.
² HEMP-IT-ADN, 6 rue Louis Lumière, 49250 Beaufort en Anjou, France.
³ LAMPA, ENSAM Angers, boulevard du Ronceray, 49100 Angers, France.
⁴ The Biological Materials Group, Biomimetics, HSB - City University of Applied Sciences, Neustadtswall 30, D-28199 Bremen, Germany.
⁵ INRAE, ONF, BioForA, UMR 0588, F-45075 Orleans, France; Phenobois Facility, INRAE, 45075 Orleans, France.
⁶ UR1268 BIA, INRAE Nantes, BP 71627, 44316 Nantes Cedex 03, France.
⁷ DOE Great Lakes Bioenergy Research Center, U. Wisconsin, Madison, WI 53726, USA; Department of Biochemistry, U. Wisconsin, Madison, WI 53706, USA.
⁸ DOE Great Lakes Bioenergy Research Center, U. Wisconsin, Madison, WI 53726, USA.
⁹ UR1268 BIA, INRAE Nantes, BP 71627, 44316 Nantes Cedex 03, France. Electronic address: johnny.beaugrand@inrae.fr.

PMID: 40118417
DOI: 10.1016/j.ijbiomac.2025.142324

Abstract

In this study, the benchmarking and applicability of the CASA method for the analysis of fibrous plants, such as flax, hemp, and jute, is evaluated. Lignin is a phenolic biopolymer present in plant cell walls and is composed of three primary monomeric units, designated G, S, and H. Various factors, such as the genetic variability, influence the relative proportions of these units in plant samples. Recently, the cysteine-assisted sulfuric acid (CASA) method has been introduced as a rapid method for the quantification of lignin in wood samples. The aim of this study is to establish a suitable molar absorption coefficient (ε, L·g^-1·cm^-1) to adapt the CASA method for use with annual plant fibers. This investigation was motivated by the technical advantages of the CASA method, including higher throughput, lower reaction temperatures, and ecological benefits due to the absence of carcinogenic, mutagenic, or reprotoxic (CMR) substances and the need for minimal sample quantities. In this method, lignin solubilization is facilitated by cysteine, which is an amino acid that enhances the reaction kinetics, using a one-hour incubation period. However, as with any spectrophotometric technique, the CASA method depends on a molar absorption coefficient (ε) that varies according to the ratio of the aromatic units within the polymer. To evaluate the suitability of CASA for quantifying lignin in economically significant plant fibers, we investigate the impact of the unit ratios on the accuracy of ε. The results are compared with those of two widely recognized lignin analytical methods: the Klason method, which is a gravimetric reference method, and the acetyl bromide soluble lignin method, which is the most commonly used spectrophotometric approach. The final ε obtained in this study reveals a relative difference in lignin content ranging from -8 % to +9 % based on a comparison between the CASA and Klason methods across different industrial hemp varieties. Using known G:S ratios in annual fibrous plants, ε can be estimated from our results.

Keywords: Absorption spectroscopy; Biomass; Composition.

MeSH terms

Cysteine* / chemistry
Lignin* / analysis
Lignin* / chemistry
Plants* / chemistry
Sulfuric Acids* / chemistry

Substances

Lignin
Sulfuric Acids
Cysteine
sulfuric acid