Preservation of exopolymeric substances in estuarine sediments

Abstract

The surface of intertidal estuarine sediments is covered with diatom biofilms excreting exopolymeric substances (EPSs) through photosynthesis. These EPSs are highly reactive and increase sediment cohesiveness notably through organo-mineral interactions. In most sedimentary environments, EPSs are partly to fully degraded by heterotrophic bacteria in the uppermost millimeters of the sediment and so they are thought to be virtually absent deeper in the sedimentary column. Here, we present the first evidence of the preservation of EPSs and EPS-mineral aggregates in a 6-m-long sedimentary core obtained from an estuarine point bar in the Gironde Estuary. EPSs were extracted from 18 depth intervals along the core, and their physicochemical properties were characterized by (i) wet chemical assays to measure the concentrations of polysaccharides and proteins, and EPS deprotonation of functional groups, (ii) acid-base titrations, and (iii) Fourier transform infrared spectroscopy. EPS-sediment complexes were also imaged using cryo-scanning electron microscopy. EPS results were analyzed in the context of sediment properties including facies, grain size, and total organic carbon, and of metabolic and enzymatic activities. Our results showed a predictable decrease in EPS concentrations (proteins and polysaccharides) and reactivity from the surface biofilm to a depth of 0.5 m, possibly linked to heterotrophic degradation. Concentrations remained relatively low down to ca. 4.3 m deep. Surprisingly, at that depth EPSs abundance was comparable to the surface and showed a downward decrease to 6.08 m. cryo-scanning electron microscopy (Cryo-SEM) showed that the EPS complexes with sediment were abundant at all studied depth and potentially protected EPSs from degradation. EPS composition did not change substantially from the surface to the bottom of the core. EPS concentrations and acidity were anti-correlated with metabolic activity, but showed no statistical correlation with grain size, TOC, depth or enzymatic activity. Maximum EPS concentrations were found at the top of tide-dominated sedimentary sequences, and very low concentrations were found in river flood-dominated sedimentary sequences. Based on this observation, we propose a scenario where biofilm development and EPS production are maximal when (i) the point bar and the intertidal areas were the most extensive, i.e., tide-dominated sequences and (ii) the tide-dominated deposit were succeeded by rapid burial beneath sediments, potentially decreasing the probability of encounter between bacterial cells and EPSs.

Keywords: EPS-sediment aggregates; FTIR – spectroscopy; cryo-SEM; diatom biofilms; estuarine sediments; exopolymeric substances; preservation; sedimentary core.

Figure 1

(A) Map of the Gironde estuary showing the location of the Bordeaux North point bar, and satellite image of the point bar at low tide (Google Earth Pro) showing the location of the studied core (BXN Long Core) and surface samples. Three sedimentary domains are visible in the intertidal zone of the point bar: the mud flat, the chute channel, and the sand dunes. (B) Photographs and X-Ray image (SCOPIX) of the principal facies found in the BXN long core.

Figure 2

Evolution of exopolymeric substances (EPSs) and sediment properties with depth along the 6 m-long BXN Long Core (see location Figure 1). First column to the left: sedimentary description. (1) X-Ray image (SCOPIX); (2) core photographs; (3) sketch; (4) facies; (5) sedimentary log, and (6) sedimentary sequences. Sample depths are written in red. Second column: median grain size (D₅₀; yellow dots). Third column: particle size class percentage: clay (brown), silt (gray), and sand (yellow). Fourth column: Sugar and protein concentrations: phenol-sulfuric assay (yellow) and protein assay (green). Fifth column: EPS acidic site density measured with the Alcian Blue assay (blue). Sixth column: total organic carbon (TOC) in the sediment (black). Seventh column: rate of metabolic activity using TTC reduction as a proxy (red). Height column: rate of enzymatic activity using hydrolyzation of FDA as a proxy (yellow).

Figure 3

Field and microscope observations of the surface biofilm. Exopolymeric substances (blue arrow), diatoms (green arrow), and clay-EPS complexes (brown arrow). (A) Field picture of the diatom biofilm at the surface of the sediment. The biofilm forms a millimeter-thick golden lamina at the surface of the sediment. (B) Transmitted light microscopic image of the biofilm colored with Alcian Blue. The blue coloration shows acidic EPS fibers surrounding diatoms. Three diatom genera were identified: Nitzschia sp., Pleurosigma sp., and Navicula sp. (C) Cryo-SEM picture of the surface biofilm showing two diatoms surrounded by a mixture of clay and EPSs. EPS fibers form an organo-mineral complex with clay platelets. (D) Cryo-SEM picture of a diatom. Cryofixation preserves the 3D structure of EPSs and allows the observation of very thin EPS fibers. The clay-EPS complex displays a typical alveolar structure.

Figure 4

Cryo-SEM images of subsurface sediments from the BXN Long Core at three depths. Exopolymeric substances (blue arrow), quartz grain (yellow arrow), mica (red arrow), and clay-exopolymeric substance (EPS) complexes (brown arrow). (A,B) Depth of 0.06 m. Alveolar EPSs cover a large part of a quartz grain. EPSs are locally complexed to clay particles, forming a detrital clay coat. (C,D) Depth of 4.33 m. A clay-quartz-EPS complex covers a large part of a mica (C). A sand quartz grain is fully covered by a clay-rich detrital coat (D). (E,F) Depth of 6.08 m. EPS fibers are partially covered with clay platelets and coat both quartz and mica grains (E). The surface of a quartz sand grain is partially coated by a dense clay-EPS complex (F).

Figure 5

Fourier transform infrared spectroscopy spectra of four exopolymeric substances samples recovered from different depths along the BXN Long Core. Colored lines highlight the EPS absorption bands, while black dotted lines indicate clay mineral absorption bands. The red arrows show the position of the protonated carboxylic acid group stretches, which attenuate or disappear at a certain depth. Green arrows show the shift of the amide II stretches.

Figure 6

Acid–base titration curves from exopolymeric substances (EPSs) extracted from the surface biofilm and the bottom of the BXN Long Core (depth = 6.08 m). The titration curves obtained were analyzed using PROTOFIT 2.1 software, and both show three buffering zones. The first zone located around pH 2 is attributed to carboxyl or sulfate groups, the second zone located near pH 5 is attributed to carboxyl groups, and the third buffering zone near pH 9.5 is attributed to amino groups. Control titration was performed using the same volume of deionized water adjusted to the same initial pH with HCl 1 M.

Figure 7

Middle, Principal Components Analysis circle plot of correlation between the samples from the BXN Long Core. (A) Correlation matrix of Pearson’s r coefficients between EPS properties, sedimentological factors and depth for the 16 samples. Green boxes indicate value of p ≤ 0.05. (B) Principal Components Analysis circle plot of variables for BXN-Long Core.

Figure 8

Multiscale scenario explaining the role of the hydro-sedimentary context and organo-mineral interactions in the production and preservation of exopolymeric substances (EPSs) in estuaries. (A) Sketch of the Bordeaux North point bar during a tide-dominated period (A.1; brown) and a river flood-dominated period (A.2; blue). Biofilm development and EPS production are optimal during tide-dominated intervals, when the point bar is stable and the muddy intertidal domain well developed. During river flood-dominated periods, the migration of the point bar coupled to the erosion of the intertidal zone results in lower EPS production. (B) Formation of clay-EPS complexes: clay-EPS aggregates and detrital coats in surface sediments. (C) The preservation of clay-EPS complexes and EPSs during burial depends on the sedimentation rate: (C.1) rapid burial following, e.g., a river flood protects EPSs from heterotrophic degradation; and (C.2) normal sedimentation rate leads to increased EPS degradation. (D) Vertical trends of EPS concentrations depend on production and sedimentation. Tide-dominated periods followed by rapid burial create the optimum conditions for EPS preservation, while river flood-dominated periods followed by tide-dominated periods are associated with low EPS preservation.