Methods in Molecular Biology (2020) 1980: 181–190
DOI 10.1007/7651_2017_105
© Springer Science+Business Media New York 2017
Published online: 14 November 2017
Complete Acid-Based Hydrolysis Assay for Carbohydrate
Quantification in Seaweed: A Species-Specific Optimized
Approach
Emily T. Kostas, Stuart J. Wilkinson, Daniel A. White, and David J. Cook
Abstract
Accurate quantification of the carbohydrate content of biomass is crucial for many bio-refining processes.
The most commonly followed protocol is typically a modification of the NREL-based assay (specifically
designed for carbohydrate analysis from lignocellulosic biomass). However, this NREL protocol was revealed
to be excessively thermochemically harsh for seaweed biomass. This can result in erroneously low total sugar
quantification as the reaction severity can degrade a proportion of the liberated sugars to decomposition
products such as furans. Here we describe an optimization of the total acid hydrolysis protocol for accurate
quantification of the carbohydrate content of seaweeds. Different species of seaweed can be accurately
evaluated for their carbohydrate contents by following this optimized method.
Keywords Carbohydrate, Composition, Macroalgae, Mass balance, Optimization, Species
1 Introduction
In order to measure the carbohydrate content of seaweed biomass,
modified versions of the NREL two-stage acid hydrolysis protocol [1]
are typically applied [2–4]. The NREL carbohydrate assay is composed
of two distinct stages and was initially developed for the quantification of
total carbohydrate in lignocellulosic biomass (Fig. 1). The first stage of
the assay is a low-temperature (37
C) treatment with concentrated acid
(typically 12 M H 2 SO 4 ) which induces the initial swelling of the biomass
followed by the fragmentation of the larger polysaccharide complexes
found in the biomass. The second stage of the protocol involves using
dilute acid (1 M H 2 SO 4 ) but at a higher temperature (100
C) which
then fully hydrolyzes the larger sugar fragments (oligosaccharides) into
their constituent monomeric units (monosaccharides). These monosaccharide sugars can then be quantified either by high-performance liquid
chromatography or gas chromatography mass spectrometry (HPLC or
GC-MS, respectively) or alternatively via colorimetric methods [5]. Seaweed polysaccharides are distinctly different to those of terrestrial plants
in terms both of the sugar subunits (monomers) which are present and
also the specific linkages between the monomers. In addition seaweed181
DOI 10.1007/7651_2017_105
© Springer Science+Business Media New York 2017
Published online: 14 November 2017
Complete Acid-Based Hydrolysis Assay for Carbohydrate
Quantification in Seaweed: A Species-Specific Optimized
Approach
Emily T. Kostas, Stuart J. Wilkinson, Daniel A. White, and David J. Cook
Abstract
Accurate quantification of the carbohydrate content of biomass is crucial for many bio-refining processes.
The most commonly followed protocol is typically a modification of the NREL-based assay (specifically
designed for carbohydrate analysis from lignocellulosic biomass). However, this NREL protocol was revealed
to be excessively thermochemically harsh for seaweed biomass. This can result in erroneously low total sugar
quantification as the reaction severity can degrade a proportion of the liberated sugars to decomposition
products such as furans. Here we describe an optimization of the total acid hydrolysis protocol for accurate
quantification of the carbohydrate content of seaweeds. Different species of seaweed can be accurately
evaluated for their carbohydrate contents by following this optimized method.
Keywords Carbohydrate, Composition, Macroalgae, Mass balance, Optimization, Species
1 Introduction
In order to measure the carbohydrate content of seaweed biomass,
modified versions of the NREL two-stage acid hydrolysis protocol [1]
are typically applied [2–4]. The NREL carbohydrate assay is composed
of two distinct stages and was initially developed for the quantification of
total carbohydrate in lignocellulosic biomass (Fig. 1). The first stage of
the assay is a low-temperature (37
C) treatment with concentrated acid
(typically 12 M H 2 SO 4 ) which induces the initial swelling of the biomass
followed by the fragmentation of the larger polysaccharide complexes
found in the biomass. The second stage of the protocol involves using
dilute acid (1 M H 2 SO 4 ) but at a higher temperature (100
C) which
then fully hydrolyzes the larger sugar fragments (oligosaccharides) into
their constituent monomeric units (monosaccharides). These monosaccharide sugars can then be quantified either by high-performance liquid
chromatography or gas chromatography mass spectrometry (HPLC or
GC-MS, respectively) or alternatively via colorimetric methods [5]. Seaweed polysaccharides are distinctly different to those of terrestrial plants
in terms both of the sugar subunits (monomers) which are present and
also the specific linkages between the monomers. In addition seaweed181