Mac 50 1 nh

Bark from P. pinaster (approximate age 15 years) was collected in Ponte de Lima, Portugal (April 2016). Firstly, the bark was washed with distilled water and dried at 40 °C for 48 h and subsequently milled in a cutting mill (Retsch SM 2000) to a granulometry of 0.1–0.45 mm for general chemical composition and 1–1.6 mm for extraction process.
Chemical summative analyses were determined in accordance to the National Renewable Energy Laboratory (NREL) official protocols, and included ethanol extractives (NREL/TP-510-42619), structural carbohydrates (namely cellulose and hemicellulose), klason and acid soluble lignin (NREL/TP-510-42618) and ash content (NREL/TP-510-42622). The mineral content was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES), after PB digestion with HNO₃. Fat content was determined according to the official AOAC method (nº 920.39). Total proteins content estimated by using the N×6.25 conversion factor, was performed using a Kjeldahl distillator (Kjeltec 8400 Analyzer, FOSS, Hilleroed, Denmark) by quantification of Nitrogen after PB digestion. PB moisture was determined gravimetrically using a moisture analyzer (MAC 50/1/NH, RADWAG, Radom, Poland). All experiments were performed in triplicate.

Measurements of optical density in oil‐free cultures were performed at a wavelength of 600 nm using a spectrophotometer (Ultrospec 10, Biochrom). Cell counting was performed using disposable C‐Chip Neubauer Improved chambers (NanoEntek) and a light microscope (ICC 50, Leica Microsystems). CDW was calculated by subtracting the weight of empty filters, which were incubated overnight at 120°C, from the weight of filters used for culture filtration that were also incubated overnight at 120°C. Glass fiber filters with 0.4 μm diameter were used (GF‐5, Macherey Nagel). Weight was measured using a moisture analyzer (MAC 50/1/NH, RADWAG).

OP waste was supplied by Indulleida S.A. (Alguaire, Lleida, Spain). As soon as it was received, OP was placed in an oven and dried for 48 h at 60 °C. Subsequently, it was milled and sieved to obtain a material with a particle size between 0.45 to 0.9 mm and 7.41 ± 0.08% (w/w) of moisture determined through a moisture analyzer MAC 50/1/NH (Radwag, Radom, Poland). Finally, OP was kept at room temperature in a dark bottle until evaluated. All the analytical grade chemicals were acquired from Fisher Scientific (Leicestershire, UK) and Sigma-Aldrich (Steinheim, Germany).