Cellulose powder

All reagents were purchased from Sigma-Aldrich and TCI Chemicals. All solvents were pro analysis grade from Merck, Carlo Erba Reagents and Scharlab.
Thin layer chromatography (TLC) was performed on precoated silica gel 60 F254 acquired from Merck with layer thickness of 0.2 mm. Reaction control was monitored using ethyl acetate and/or ethyl acetate:methanol (9:1) and spots were visualized under UV detection at 254 and 366 nm. Following the extraction step, the organic layers were dried over anhydrous sodium sulfate. Flash column chromatography was carried out with silica gel 60 0.040–0.063 mm acquired from Carlo-Erba Reactifs. Cellulose flash column chromatography was carried out with cellulose powder 0.01–0.10 mm acquired from Sigma-Aldrich. The elution systems used for flash chromatography were specified for each compound. Solvents were evaporated using a Büchi Rotavapor.

Following previous tyrosine administration studies [6 (link),9 (link)], participants were administered a dosage of 2 g tyrosine (supplied by The Hut.com Ltd.) or placebo (2 g cellulose powder (Sigma-Aldrich Co.), solved in 200 ml orange juice (Solevita, Lidl). According to Tam et al. (1990) [64 (link)], tyrosine administration enhances tyrosine hydroxylation and causes plasma tyrosine levels to peak approximately 60–120 minutes following intake and remain significantly boosted for up to 8h [65 (link)]. Consistent with this, the delay between tyrosine/placebo administration and start of the cognitive assessment was exactly 75 minutes. To test whether the participants were blind to the supplementation condition, we asked them to guess their treatment assignment (placebo—tyrosine / tyrosine—placebo) subsequent to completing the second session.

The biochemical methane potential (BMP) of the rice straw and rice husks was determined using the commercial system AMPTS II (Bioprocess Control, Sweden) and all samples were analyzed in triplicate. Inoculum from all reactors collected from a biogas plant in Uppsala, Sweden, operating with mixed sewage sludge at wastewater treatment plant (WWTP). The inoculum:substrate ratio was set to 3:1 (VS basis) and the organic load was 3 g VS/L. The inoculum from WWTP had a total solids (TS) content of 3.1% of wet weight and a VS content of 2.0%. The TS and VS content was analyzed by drying according to APHA [50 ]. For the rice straw and rice husk powder, TS content were assumed to be the same as the dry weight. Dilution was made with tap water to final volume of 400 mL (flask volume 500 mL). Incubation temperature was set to 37 °C. To monitor background gas production from inoculum alone, three bottles were initiated by adding the same amount of inoculum and water to reach the same final liquid volume, but with no substrate. To confirm the activity of the inoculum, control bottles were also initiated with cellulose powder (Sigma-Aldrich) as substrate. The experiment ended when daily methane production fell below 1% of the accumulated methane production on a volume basis. The gas volumes produced were normalized to 1.01325 bar and temperature 273.2 K.

Cellulose powder (cotton linters, medium fiber) and MTS (purity > 99%) were purchased from Sigma-Aldrich Co. (Seoul, Korea) Lithium hydroxide (LiOH, purity = 99%), urea (purity = 98%), and ethyl alcohol (purity = 95%) were purchased from Duksan Pure Chemicals Company. DIW was purified using an EXL3 pure and ultrapure water system (VIVAGEN CO., LTD., Seongnam, Korea). All the chemicals were used without further purification.

An inoculum from an overnight grown culture in log phase was added to 100 ml NFMM containing cellulose powder (Sigma-Aldrich) as a carbon source in 250-ml Erlenmeyer flasks. After incubation for 48 h, at 37^°C, under shaking condition of 150 rpm, the culture was harvested and growth was measured at OD600 with UV-Vis spectrophotometer (Optima SP-3000 Plus, Slovakia). The cultures were centrifuged at 10,000 rpm for 10 min at 4^°C. After centrifugation, the supernatant (cell-free extract) was used as crude preparation to measure cellulase activity.
For enzyme assay, Cellulose and Sodium CMC were used as the substrates. Enzyme activity was determined by measuring the release of reducing sugars during the enzyme-substrate reaction using dinitrosalicylic acid method [14 (link)]. The values were determined from glucose standard curve. One unit (IU) of activity was defined as the amount of enzyme required to liberate 1μmol of glucose per minute under given assay conditions.