Molecular composition of plant parts and sediment organic matter in a Mediterranean seagrass (Posidonia oceanica) mat

Highlights

• Analytical pyrolysis applied to Posidonia seagrass organs and marine mat deposit.

• Posidonia phenolic composition dominated by p-hydroxybenzoic acids.

• Peat-like mat deposits composed predominantly of root, rhizome and sheath materials.

• We suggest a link between Posidonia chemistry and C accumulation in mats.

Abstract

Posidonia oceanica forms extensive peat-like deposits (mats) in Mediterranean coastal waters, which have a potential as carbon sinks and archives of environmental change. Nonetheless, the organic chemistry of both P. oceanica plant materials, as well as the environmental and diagenetic effects on the composition of its detritus, is poorly understood. We analyzed plant organs of P. oceanica and the coarse organic matter from a mat core spanning 750 yrs using pyrolysis techniques (PY-GC–MS and THM-GC–MS) to improve our understanding of their molecular properties and their preservation upon mat development. It appeared that leaf sheaths, roots and the outer parts of rhizomes were composed predominantly of phenolic constituents based on p-hydroxybenzoic acid (p-HBA), which is atypical for vascular plants, in addition to carbohydrates and lignin. The inner rhizome and leaf blades had a different composition, with predominance of carbohydrates. The seagrass detritus in the mat was composed mainly of p-HBA phenolic material and carbohydrates, confirming earlier studies showing that the coarse detritus in the mat originates largely from Posidonia sheath, root and rhizome debris. The intermolecular arrangement of the p-HBA remains unclear, as they seem to correspond to ester-bound phenols yet their persistence in the mat attributes them a refractory nature. Variations in molecular composition within the mat are probably associated with diagenetic alteration of P. oceanica detritus, such as the decomposition of minor amounts of fatty acids, chlorophyll and syringyl lignin, and selective preservation of p-HBA relative to carbohydrates. This work lays the foundations for a molecular understanding of carbon storage within the mats and the environmental changes recorded therein.

Introduction

Some terrestrial vascular plants living in coastal environments re-adapted to life in the sea more than 100 million years ago, evolving into marine phanerogams (seagrasses). Since then, seagrasses have inhabited shallow (<40–60 m depth) coastal areas worldwide, forming extensive and rich ecosystems along most of the world’s coasts. Among the many services they provide (Orth et al., 2006), two have been recognized recently and are related to the accumulation of biogeochemical elements in the underlying sediments. Firstly, even though seagrasses only occupy 0.1% of the ocean surface, their ecosystems are considered a significant carbon (C) sink (Duarte et al., 2005, Duarte et al., 2010, McLeod et al., 2011). This can be explained by the fact that a few large seagrass species, mostly inhabiting protected and shallow environments, produces substantial accumulation of seagrass detritus buried in marine peat-like deposits. The most extraordinary example is exhibited by the Mediterranean endemic seagrass Posidonia oceanica L. (Delile) (Lavery et al., 2013), which forms up to several meters thick and several thousand years old deposits (Boudouresque et al., 1980, Mateo et al., 1997, Serrano et al., 2012, Serrano et al., 2014) also known as ‘mat’ (or ‘matte’; Boudouresque et al., 1980). The organic C stocks beneath P. oceanica meadows have been estimated to 0.5–12 × 10¹⁵ g C for the Mediterranean Sea (Serrano et al., 2014). The role of P. oceanica in sequestering and storing CO₂ on centennial to millennial scales in the mats is being evaluated as a novel aspect of the global C cycle (Fourqurean et al., 2012, Duarte et al., 2013) and the recent focus on C trading has intensified the interest in quantifying the capacity of these ecosystems to store C (Lavery et al., 2013). Secondly, these mats form a repository of palaeo-environmental information (Mateo et al., 2002, Mateo et al., 2010, López-Sáez et al., 2009, Serrano et al., 2011, Serrano et al., 2012, Serrano et al., 2013, López-Merino et al., 2015), very much like terrestrial peat archives. Although a wealth of technical and scientific literature is available on the role of seagrass sediments as C sinks and palaeoarchives, these fields of research have critical knowledge gaps concerning some fundamental issues: (i) the origin of the accumulated C (i.e. the sources and composition of organic C retained in the sink), and (ii) the post-burial diagenetic alteration of P. oceanica mats (factors that control degradation/preservation dynamics).

Similar to other seagrasses, P. oceanica consists of long strap-like leaves connected to the rhizome through a leaf sheath (Fig. 1). Over the lifetime of an individual plant, leaves fall through abscission (by currents and waves, or during senescence), while the leaf sheaths remain attached to the rhizome. As such, various layers of sheaths can be identified on mature specimens, and confer a “hairy” appearance to the rhizome (Hemminga and Duarte, 2000). The root is often short but strong and grows adventitiously. The detritus of belowground organs (rhizomes, roots and sheaths), which constitute the vast majority of the plant’s dry weight (Alcoverro et al., 2001), are incorporated directly into the seabed, thereby protected against subsurface currents, wave action and herbivores. The aboveground organs (leaves), on the other hand, are largely eroded from the meadows and exported to adjacent systems including supra-littoral environments forming the so-called banquettes and agaepropili (Mateo et al., 2002, Simeone and De falco, 2012). Indeed, no coarse (>1 mm) leaf detritus had been found in seagrass sedimentary debris (Serrano et al., 2012). It is worth mentioning here that this P. oceanica beach material causes annoying flies, odours and beach size reduction in touristic areas along the Mediterranean coast, and associated expenditure on removal (Khiari et al., 2010, Simeone and De falco, 2012, Plis et al., 2014). On the other hand, it is a potential feedstock for biofuel, animal fodder, cellulose and activated charcoal (Torbatinejad et al., 2007, Ncibi et al., 2009, Coletti et al., 2013, Bettaieb et al., 2015), even though the ecological consequences of such usages have not been assessed. Knowledge on the molecular composition of Posidonia raw materials is obviously valuable to these potential future applications.

Knowledge on the in situ decomposition of P. oceanica detritus within the mats is limited. It involves an initial phase of abiotic leaching of fast-cycling components, consisting of low molecular weight (LMW) phenolic and carbohydrate compounds, followed by microbial and detritivorous breakdown of more refractory materials (Kenworthy and Thayer, 1984, Harrison, 1989, Peduzzi and Herndl, 1991, Enríquez et al., 1993, Opsahl and Benner, 1993, Mateo and Romero, 1996). The second (biotic) decomposition phase is a much slower process (Romero et al., 1992), and allows the more refractory tissues to accumulate in mats. The mechanism behind this slow biotic decomposition after P. oceanica detritus enters the mat is largely unknown, but the likely candidates are anoxic conditions and biochemical stability of the organic matter (Mateo et al., 1997, Pedersen et al., 2011).

Most knowledge on the chemical composition of P. oceanica corresponds to foliar tissue. The leaves consist primarily of polysaccharides (roughly 60%, two thirds of which corresponds to holocellulose), and the remaining material is largely composed of various phenolic constituents, more specifically lignin, tannin and free and ester-bound phenolic acids (Zapata and McMillan, 1979, Agostini et al., 1998, Arnold and Targett, 2002, Torbatinejad et al., 2007). Reported lignin contents of P. oceanica leaves and agaepropili are in the range of 25–30% of their dry weight (Ncibi et al., 2009, Khiari et al., 2010, Bettaieb et al., 2015). However, this is operationally defined Klason lignin (acid-insoluble residues), only a small portion of which corresponds to “true” polyphenolic lignin (Klap et al., 2000). Tannins stored in tannin cells account for ca. 5–10% in P. oceanica leaves (Pergent et al., 2008). Roughly 2% of the dry weight of P. oceanica leaves corresponds to free or ester bound (water- or aqueous/organic solvent mixtures-extractable) phenols (Agostini et al., 1998), the molecular composition of which has been examined with relative scrutiny, as reviewed by Heglmeier and Zidorn (2010). The nature of the “remaining” phenols (total phenols minus lignin, tannin and extractable phenols), which account for an estimated 50% of the total phenols in P. oceanica leaves, is poorly understood. Other organic components in P. oceanica leaves include chlorophyll pigments, cutin in the cuticles and wax lipids. Molecular examinations of the composition of the rhizomes and particularly the roots are less frequent. Klap et al. (2000) demonstrated that the rhizome and roots have higher lignin contents than the leaves.

The molecular composition of seagrass materials can be determined using techniques such as nuclear magnetic resonance and infrared spectroscopy of bulk samples, and gas and liquid chromatography of extractable phenols. Other approaches rely on the chemical breakdown, usually by CuO oxidation (Hedges and Mann, 1979, Opsahl and Benner, 1993), or thermal breakdown, such as pyrolysis (Sáiz-Jiménez, 1988), of the organic matter followed by gas chromatography and mass spectrometry (GC–MS) for identification of the products. Here we will focus on analytical pyrolysis techniques, i.e. conventional analytical pyrolysis (PY-GC–MS) and Thermally assisted Hydrolysis and Methylation (THM-GC–MS), the latter of which uses a derivatization agent simultaneously to thermal treatment to enhance the structural information obtained for polar aliphatic and phenolic materials (Challinor, 2001, Shadkami and Helleur, 2010). Only Gadel and Bruchet (1987) and Klap et al. (2000) studied Posidonia oceanica materials by PY-GC–MS, but they analyzed specific (and unquantified) fractions of humic/fulvic fractions and milled-wood-lignin preparations, respectively. As far as we know, THM-GC–MS has not been applied to P. oceanica materials yet. Kristensen et al. (2009) analyzed an undefined seagrass, while Maie et al. (2006) analyzed leachates from turtle grass (i.e. Thalassia testudinum) by this technique. Hence, contrary to the water- or aqueous/organic solvent mixtures-extractable fraction, detailed molecular information on the inextricable biocomponents, which constitute the vast majority of the plants’ dry weight, is very limited.

We analyzed different organs of Posidonia oceanica and their debris found along a 750-yr sedimentary record from the Mediterranean Sea by PY-GC–MS and THM-GC–MS. From the mat sediments, we analyzed the coarse (>1 mm) fraction of the organic matter (COM), which mainly consisted of sheath, root and rhizome debris (Serrano et al., 2012), to avoid, or at least reduce to a minimum, the contribution from allochthonous sources (e.g. macroalgae and terrestrial detritus; Holmer et al., 2004) to the results obtained. The specific objectives were to determine the molecular composition of different organs of P. oceanica, compare that to the composition of the detritus in the mat core, and, ultimately start unraveling the processes involved in mat composition and diagenesis.