The matrix can be viewed as the water-insoluble fraction of macroaggregates. Major spectrum bands (Figure 1A) could be assigned: 3000–3600 cm⁻¹ (O-H, N-H stretching band region), 3000–2800 cm⁻¹ (aliphatic groups), 1730 cm⁻¹ (C=O stretching of COOH, ketones or aldehides), 1650–1640 cm⁻¹ (C=O stretching of amide I; C=N stretch; stretching of COO⁻; C=C signals of aromatic ring and/or olefins C=C, water deformational modes), 1430 cm⁻¹ (aliphatic C-H deformation of CH₂ and CH₃ groups; symmetric stretching of COO⁻), 1150–1000 cm⁻¹ (carbohydrates, Si-O stretch). Spectra of surface matrix display also main calcite peaks situated at 2516, 1420–1450, 876 and 713 cm⁻¹ and that of silicates (527–535 and 472 cm⁻¹). The elemental composition of surface and water column samples revealed higher values for surface macroaggregates, averaging 16.4% of C_org., 0.7% of N_tot. and 254 ppm of P_tot., with a mean C:N:P ratio (molar) of 772:26:1. Accordingly, higher contents of total carbohydrates and proteins, averaging 13.5% and 4.7%, respectively, were determined in surface samples compared to deeper seawater layer samples. This statement is supported also by FTIR analyses (Figure 1) showing the presence of carbohydrates, evidenced by bands at ~1150–900 cm⁻¹ and proteins, evidenced by bands at 1654–1635 cm⁻¹, in both samples. The lipid contents, clearly indicated by bands at 2950–2850 cm⁻¹ in FTIR spectra (Figure 1A), are lower, averaging 2.2%.

FTIR spectra of interstitial water colloids (the “water-soluble” fraction) show the presence of both basic constituents, i.e., carbohydrates and proteins (Figure 1B). In FT-IR spectra of all colloidal fractions (not shown), the presence of a band at 2240 cm⁻¹ probably indicates the cyanogenic glycosides [24] represents the greatest part of organic nitrilated compounds [25]. Their existence was previously confirmed also by monosaccharide analyses in bulk macroaggregates [26]. Their source is most probably linked to amino acids [25]. The bands assigned to lipids, i.e., aliphatic components, at 2800–3000 cm⁻¹ are less evident. The important contribution of carbohydrates in all colloid fractions was supported by higher UV absorption at λ = 250 nm, more typical of polysaccharides, compared to λ = 205 nm, being more typical of proteins [27] and humics [28]. The highest carbohydrate content and the lower C/N and C/P ratios (Figure 2) were found in the higher molecular weight (MW) colloidal fraction indicating the important presence of N and P containing [29] carbohydrates as well as their important role in mucilage composition. The δ¹⁵N data exhibiting low values, especially in the >30 kDa fraction [30] and previous reports on non-protein nitrogen compounds [31], also sustain the presence of non-protein N compounds, i.e., amino sugars, chlorophyll and nucleic acids, in the colloidal fraction. In contrast, the lower MW fractions seem mostly composed of oligo- and polysaccharides.

According to macroaggregate enzymatic hydrolysis with α-amylase and β-glucosidase, the α (1–4) reserve polysaccharides, abundantly present in dinofllagelates in surface bulk macroaggregate samples taken in July 2004, are the most susceptible for enzymatic hydrolysis. In contrast, in the water column macroaggregates, containing prevalently diatoms, the impact of α-amylase is lower (Figure 3).

The presence of α- and β-glycosidic linkages in macroaggregates was previously confirmed by ¹H-NMR spectroscopy [32] and a shift from α- to the more refractory β-glycosidic linkage was described in aged macroaggregates dominated by diatoms containing storage β-glucans [19,22]. These findings are in accordance with the observations of Herndl et al. [33] and Zaccone et al. [34] who found that the activities of α- and β-glucosidase in macroaggregates were opposite due to microbial response to the composition of substrates, i.e., macroaggregate components, where β-glycosidic linkage seems more refractory. The compositional difference between “fresher” surface and aged water column macroaggregate samples is also indicated by results of enzymatic hydrolysis (Figure 4). The addition of α-amylase and β-glucosidase produces high carbohydrate release in both samples (Figure 4A and 4B). Protease and proteinase K, the most active ectoenzymes found in particles [35] and macroaggregates [22], produce, in addition to proteins, higher carbohydrate release in the water column sample which is clearly evident from FTIR spectra (Figure 4B) showing a greater decrease in the carbohydrate band at ~1150–1000 cm⁻¹.

FTIR spectra of the water phase from macroaggregate matrix slurries, obtained after hydrolysis with α-amylase + β-glucosidase, indicate the presence of minerals (Figure 5) such as calcite (signals at 2516, 1430, 876 cm⁻¹) and silicates (530 and 472 cm⁻¹). These results confirm the importance of associations between carbohydrates and minerals (inorganic species) for mucilage events. The release of minerals was not observed in the case of the same treatment of more mature water column sample, probably due to more intense interactions between organic and inorganic components; the latter acting as a stabilizing agent.

In aged macroaggregates, the carbohydrates can be linked to proteins, probably in the form of glycoproteins, and thus the organic N can be preserved in these probably more crosslinked samples [36]. The rapid hydrolysis of organophosphorus in the macroaggregate matrix by phosphatase, a nearly 300% increase of phosphate concentrations in the first ten minutes, suggests faster cycling of P with respect to N. Phosphatase can also be involved in the hydrolysis and release of organic constituents [37].

The hydrolysis of surface (fresher) macroaggregates with lipase showed primarily the degradation of lipids and polysaccharides, suggesting an association of lipids with polysaccharides possibly in the form of glycolipids. Using lipase (Figure 6) the macroagregate degradation is slower, ranging from days to weeks, compared to proteinase K and protease, ranging from hours to days. In the three week long degradation experiment, the relative intensities of absorptions indicating the lipidic component (2800–3000 cm⁻¹, 1730–1740 cm⁻¹, 1430–1450 cm⁻¹), revealed a large decrease in lipids, only at the end of the experiment due to their lower degradability.

The previous studies [18,36] of temporal compositional changes of the northern Adriatic bulk macroaggregates in the summer stratified seawater column, using ¹H- and ¹³C-NMR and FTIR spectroscopy, also revealed the preferential degradation of carbohydrates in comparison to the aliphatic components—lipids. Similar results were obtained in the spectroscopic ¹H-NMR study of the bulk macroaggregates, showing much faster degradation of carbohydrates compared to lipids during the mucilage event [31]. All these findings seem to contradict the reported observations of the high hydrolytic activity of lipases, one of the most active ectoenzymes in aquatic systems [38] and in macroaggregates [39].

Regardless of bacterial enzymatic activity, the macroaggregate hydrogel structure can slow microbial degradation [40]. The gel microhabitat can represent an important site of organic matter degradation (microbial functioning) but, on the other hand, the gel polymers can be preserved due to their sterical hindrance [40] and the presence of organic-inorganic associations.

The enzymatic hydrolysis of all colloidal fractions using α-amylase and β-glucosidase indicates the presence of α- and β-gycosidic linkages in nearly equimolar proportions. Similar kinetics of hydrolysis appears in the >30 kDa and 10–30 kDa fractions. In the 5–10 kDa fraction, the increase of carbohydrate content after seven days is probably due to the prevalently polysaccharidic nature of this fraction making it more suitable for enzymatic hydrolisis. Contents of C_org., N_tot.and P_tot. and the results of HPSEC analyses [41] from the »natural« degradation of macroaggregate colloidal fractions, indicate faster degradation in the >30 kDa and 30–10 kDa fractions compared to the 10–5 kDa fraction. This could be due to the higher contents of N-containing polysaccharides in the higher MW fractions. The microbial degradation of the carbohydrate component of glycoproteins is reported to proceed up to 3-fold faster compared to the proteinaceous components [42,43] explaining the preservation of organic nitrogen and the decreasing C/N ratio in aged degraded mature macroaggregates.

Macroaggregate samples were collected on 1 July, 2004, in the southern part of the Gulf of Trieste at a fixed sampling site (45° 31.46′ N, 13° 33.72′ E) at the sea surface and at a depth of 10 m above the pycnocline. The sea water temperature was 25 °C. Both macroaggregate samples were in macrogel form; hence it was possible to collect them by hand by SCUBA divers, using polyethylene bottles with a minimal amount of surrounding water. The macroaggregate interstitial water was isolated by filtration through a 50 μm mesh size plankton net and centrifuged at 4000 g. The supernatant was successively filtered through preignited 0.7 μm pore size Whatman GF/F filters. The sediment (water-insoluble fraction) experiments and filtered supernatant (water-soluble fraction) were freeze-dried and used for subsequent degradation and fractionation, and degradation experiments, respectively.

C_org. and N_tot. in freeze-dried samples were analyzed using a Carlo Erba mod. EA 1108 C, H, N, S analyzer and P_tot. colorimetrically [44] after sample digestion with K₂S₂O₈. Total proteins, carbohydrates and lipids were assayed colorimetrically using the Coomassie Brilliant Blue method of Setchell et al. [45], the phenol-sulphuric acid method of Dubois et al. [46] and the Folch [47] method, respectively. Standards comprised solutions of d-glucose (Sigma) for carbohydrates and BSA (Sigma) for proteins in Milli-Q water. All colorimetric analyses were performed in triplicate. FTIR spectra were obtained from homogenized samples using a Perkin-Elmer Spectrum One spectrometer with a diffuse reflectance sampling accessory. The micro-cup of the accessory was filled with the sample diluted by anhydrous KBr to give up to a 5% mixture. Spectra were collected at room temperature with a resolution of 4 cm⁻¹ and 4–10 scans were accumulated for each spectrum in a frequency range of 4000–450 cm⁻¹.