2400 series 2

Total extractive content of RCW samples was determined by Soxhlet extraction for 6 h with anhydrous toluene-ethanol (0.427:1.000, v/v), followed by hot water extraction. Klason and acid-soluble lignin contents of G. arborea and S. latifolius RCW were determined using standard methods⁶⁴ . Carbohydrates were determined using the protocol of National Renewable Energy Laboratory (NREL, Golden, CO, USA)⁶⁵ . The analysis was conducted by high performance liquid chromatography using a refractive index detector (HPLC-RID), and performed on an Agilent Technologies 1200 series system equipped with a Rezex RHM-Monosaccharide H + (8%) column (300 × 7.8 mm). Ash contents were determined according to the ASTM standard method⁶⁶ . Mineral contents (Ca, Mg, K, P, Mn) of study samples were determined using an optical emission spectrometer with excitation by inductively coupled plasma (ICP) on an Optimum Machine DV 4300 (Perkin Elmer, Norwalk, CT, USA) with a Scott-type of nebulizer. Elemental analysis of RCW samples were performed using a Perkin Elmer 2400 Series II (CHNO analyzer). The contents (%) of C, H and N in RCW sawdust are based on ash-free dry mass, while O content was computed by difference, i.e., O = 100 – (C + H + N). Each result corresponds to the mean of three replicates.

Three sediment cores 1–5 cm long and with 2.5 cm inner diameters were collected at each field site using an Ekman Box-core (Bottom sampler Ekman-Birge, Hydro-Bios) at depths of 20 and 60 m in the middle of bay (MB), and at 20, 30, and 60 m depth Off OL between July and November 2017, respectively. The sediment for DNA and RNA were immediately preserved at 4°C during transport to the laboratory. Subsequently, the uppermost 3 cm of the sediment cores were cut into slices every 1 cm and stored at −20°C for DNA and −80°C for RNA, the latter preserved with RNAlater® solution (Ambion, United States) until the extraction process. Sediment subsamples from each of the same core fraction used in the nucleic acid analyses were used for measuring Cu and Fe concentration, total carbon (TC), nitrogen (TN), Sulfur content (TS) and grain size distribution. Briefly, the content of TC, TN, and TS was measured by analyzing ~3 mg of dry sediment on a LECO CHNS elemental analyzer (2400 series II, Perkin Elmer). Then the analytical procedure was verified with the analysis of sulfanilic acid (C₆H₇NO₃S) as a reference material (Castillo et al., 2019 (link)). At the collection sites, pH, salinity, conductivity, dissolved oxygen (DO), and temperature (°C) of the water column were measured using a CTD-O profiler (SBE 19 plus V2 Sea cat, SeaBird).

¹H NMR, ¹³C and 2D NOESY NMR spectra were obtained on a Bruker Avance-400 spectrometer (¹³С{¹H} – 100 MHz and ¹H and 2D NOESY, ROESY – 400 MHz). The chemical shifts were determined against the signals of residual protons of deuterated solvent (DMSO-d₆, D₂O). The 2D ¹H,¹H-ROESY spectra were recorded using the roesyphpr standard pulse sequence at 298 K. The mixing time was 500 ms. The concentration of sample solutions was 3–5%. The attenuated total internal reflectance IR spectra were recorded with a Spectrum 400 (Perkin Elmer) Fourier spectrometer. Elemental analysis was performed with a Perkin Elmer 2400 Series II instrument. Mass spectra (MALDI-TOF) were recorded on an Ultraflex III mass spectrometer in a 4-nitroaniline matrix. The melting points were determined using a Boetius Block apparatus. Additional control of the purity of compounds and monitoring of the reaction were carried out by thin-layer chromatography using Silica G, 200 µm plates, UV 254. Most chemicals were purchased from Aldrich and used as received without additional purification. The organic solvents were purified in accordance with standard procedures. Pillar[5]arene 1 was synthesized according to the literature procedures [48 (link)].