Improving the extraction efficiency of sedimentary carbohydrates by sequential hydrolysis

Highlights

• A sequential hydrolysis protocol is developed to extract sedimentary carbohydrates.

• The protocol greatly reduces lose of labile carbohydrates by strong acid treatment.

• Carbohydrate recoveries are increased by on average ~60% by this protocol.

• This protocol is appropriate for different sediment types.

• It provides more accurate information on carbohydrate inventories and compositions.

Abstract

Carbohydrates serve as structural materials, energy storage compounds, and energy carriers in organisms and provide insights into the dominant sources and the degradation state of organic matter (OM). Several studies have analyzed dissolved and/or particulate carbohydrates in the environment, often with the aim of studying particular carbohydrate groups of interest. These studies have often employed different acids and hydrolytic conditions, making comparisons across studies a challenge. Here, we introduce a sequential acid hydrolysis protocol for the comprehensive extraction, quantification and compositional investigation of carbohydrates in sediment samples. This protocol has four acid hydrolysis steps that sequentially increase in extraction strength, where each subsequent hydrolysis step is performed on the unextracted remains previously hydrolyzed. The four hydrolysis steps consist of 1 M HCl for 2 h at (1) room temperature, (2) 50 °C, and (3) 105 °C, followed by incubation for 8 h using (4) 6 M HCl and 105 °C. Based on tests using diverse carbohydrate standard compounds, the protocol recovers most di- and oligosaccharides and soluble polysaccharides during the initial hydrolysis steps, whereas the recovery of most insoluble polysaccharides is highest during the final extraction step. Applying this protocol to different sediment types shows that recoveries of neutral sugars, amino sugars and sugar alcohols are on average ~60% higher than with a reference one-step extraction method using only hydrolysis with hot 6 M HCl. This sequential extraction protocol thus provides an important new tool for the quantitative and compositional analysis of carbohydrates in aquatic sediments.

Introduction

Carbohydrates are present in Bacteria, Archaea, and Eukarya and are the most abundant group of biomolecules on Earth, accounting for up to ~90% of land plant, 20–40% of phytoplankton, and ~ 20% of total microbial biomass (Knudsen, 1997, Panagiotopoulos and Sempéré, 2005, Lever et al., 2015). Carbohydrates can be grouped into four major size classes, namely monosaccharides (monomers), disaccharides (dimers), oligosaccharides (typically 3–10 units), and polysaccharides (>10 units). They can serve as metabolic intermediates, as energy storage compounds, or as essential structural components in organisms. Mono- and disaccharides are generally considered to be highly biologically labile and are respired or fermented by many organisms to obtain energy for growth and maintenance (Handa and Mizuno, 1973, Pel and Gottschal, 1986, Killops and Killops, 2005). Depending on the chemical configuration, oligo- and polysaccharides are used to store energy within cells (e.g., raffinose, starch and glycogen) or to form essential structural components. The latter include cell walls of phytoplankton and vascular plants (e.g., cellulose and hemicellulose), exoskeletons of arthropods and cell walls of fungi (e.g., chitin and chitosan), cell walls of many Bacteria and Archaea (e.g., peptidoglycan and pseudomurein), and side chains that link and stabilize other biomolecules, such as proteins and lipids (e.g., glycoproteins, glycolipids) (Hecky et al., 1973, Benner and Kaiser, 2003, Barton, 2005; Madigan and Martinko, 2005; Souza et al., 2011, Meyer and Albers, 2014, Gangl and Tenhaken, 2016). Structural carbohydrates account for the largest pools of carbohydrates, with cellulose and chitin representing the two most abundant biopolymers on Earth (Souza et al., 2011).

In addition to being important components of living organisms, carbohydrates are present in vast quantities as detritus within aquatic sediments. Accordingly, it has been estimated that 1–40% of total organic carbon (TOC) (for review see Burdige, 2007), and 5–85% of dissolved organic carbon (DOC) in recent aquatic sediments is made of carbohydrates (Arnosti and Holmer, 1999, Burdige et al., 2000). A large fraction of these carbohydrates is made up of neutral sugars, including arabinose, fucose, galactose, glucose, mannose, rhamnose, and xylose (Handa and Mizuno, 1973, Burdige et al., 2000, da Cunha et al., 2002, Duan et al., 2017), and amino sugars, including glucosamine, galactosamine, and muramic acid (Niggemann and Schubert, 2006, Carstens et al., 2012).

In recognition of the importance of carbohydrates as building blocks and energy sources to many organisms and as major components of organic carbon pools in sediments, a substantial effort has been made over the last decades to improve the identification and quantification of carbohydrates in the environment (e.g., Cowie and Hedges, 1984a, Pakulski and Benner, 1992, Amelung et al., 1996, Myklestad et al., 1997, Kerhervé et al., 2002, Zhu et al., 2014, Quijada et al., 2015, Nouara et al., 2019). A typical carbohydrate extraction protocol begins with an acid hydrolysis that is followed by neutralization, purification, and final determination of carbohydrate monomers by colorimetric or chromatographic detection (see review by Panagiotopoulos and Sempéré, 2005). The hydrolysis step is crucial to release carbohydrate monomers from biopolymers and/or sample matrices into the extraction solution. Monosaccharide yields after hydrolysis depend on many factors, including type of acid, acid strength, hydrolysis duration, hydrolysis temperature, and types of glycosidic bonds (e.g., α or β anomer) within the carbohydrates present (Panagiotopoulos and Sempéré, 2005). Both mild and strong hydrolysis protocols have been applied to environmental samples. Mild hydrolysis is usually performed using trifluoroacetic acid (TFA), hydrochloric acid (HCl), or sulfuric acid (H₂SO₄) at concentrations of 0.09–2 M (e.g., Mopper, 1977, Uzaki and Ishiwatari, 1983, Walters and Hedges, 1988, Borch and Kirchman, 1997) and is suitable for the release of carbohydrates from biologically labile biopolymers (e.g., fucoidan, laminarin, mannan; Borch and Kirchman, 1997, Pielesz and Paluch, 2011). By contrast, strong acid hydrolysis is often employed for structural polysaccharides, such as alginic acid, chitin, and cellulose, and typically involves hot HCl (e.g., 6 M HCl heated at 105 °C for 8 h; Zhang and Amelung, 1996, Zhu et al., 2014), or concentrated H₂SO₄ (e.g., 12 M H₂SO₄ at ambient for 2 h, followed by diluting to 1.2 M H₂SO₄ and heated at 100 °C for 3 h; Mopper, 1977, Cowie and Hedges, 1984a).

Previous evaluations of hydrolysis methods suggest that hydrolysis efficiencies vary with sample types (seawater or sediment) and depend on carbohydrate source compounds and sugar losses during recovery with each method (Cowie and Hedges, 1984a, Borch and Kirchman, 1997). These sample- and method-inherent biases make it difficult to directly compare environmental carbohydrate data produced by different protocols, or to estimate the contribution of carbohydrates to global organic carbon pools.

The goal of this study is to establish a universal extraction protocol that maximizes the recovery of carbohydrates from a wide range of aquatic sediments. We first compare the monomer recoveries from chemically diverse soluble di-, oligo- and polysaccharides and insoluble polysaccharides based on hydrolysis protocols that involve different acid types (TFA, HCl, H₂SO₄), acid strengths (0.5–12 M), durations (0.5–8 h), and temperatures (room temperature to 105 °C). Based on these tests, we identify four hydrolysis steps that, when applied sequentially, maximize monomer recoveries across the entire suite of compounds tested. We then apply this sequential protocol to diverse marine and lacustrine sediment types, and compare the hydrolysis yields and extraction efficiency to those obtained with a previously published one-step hydrolysis method based on gas chromatography with flame-ionization detection (GC-FID). Finally, we use pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) to estimate the amount of remaining, non-extracted carbohydrates after our sequential extraction protocol.