Gas chromatograph

The short-chain fatty acid concentration was determined by using a gas chromatograph (Thermo Fisher Scientific, Milano, Italy) with a DB-FFAP capillary column (DB-FFAP, 30 m × 0.32 mm × 0.25 µm, Agilent Technologies Co., Ltd., Santa Clara, CA, USA) using the method described by Li et al. [13 (link)]. The feed samples were dried at 65 °C in a forced-air oven for 72 h and air-equilibrated for 12 h, then ground using a 1 mm screen. The feed samples were analyzed for DM (dried at 135 °C for 3 h in a forced-air oven); CP (AOAC 2000, Kjeldahl method 988.05); starch (using a commercial assay kit; Jiancheng Bioengineering Institute, Nanjing, China); and NDF and ADF using heat-stable alpha-amylase and sodium sulfite, following the methods of Van Soest et al. [14 (link)].

The photocatalytic
experiments were performed in a Pyrex reactor
of 250 mL. In a typical experiment, 0.1 g of the photocatalyst was
dispersed in 200 mL of deionized water. Before each experiment, the
reactor was purged with N₂ for 30 min and irradiated with
a wide range UV–vis xenon lamp (simulated solar light). The
photocatalyst was stimulated with irradiation between 400 and 900
nm at 100 mW/cm² in demineralized water. The oxygen and
hydrogen products were analyzed using a gas chromatograph (Thermo
Scientific) coupled with a thermal conductivity detector. No buffer
or electrolyzer was added during the reaction, and the starting pH
was 7. No external potential was applied during photocatalytic experiments.
The solar to hydrogen conversion efficiency (STH) was estimated
from eq 1,^{29 (link)} using the H₂ production, the Gibbs
free energy for the reaction, the incident power of the solar simulator
(100 mW/cm² AM1.5G), and the area of irradiation.
The quantum efficiency
(QE) was calculated with eq 2,^{29 (link)} at
420 nm, where N_H2 is the number of H₂ molecules produced in seconds and N_hv is the photon flux.

The chemical composition of the EOs was determined by GC-MS. The samples analyzed on a Thermo Fisher gas chromatograph were coupled to the mass spectrometry system (model GC ULTRA S/N 210729). The column used was a 5% phenylmethyl silicone HP-5 capillary column (30 m × 0.25 mm × thickness 0.25 μm). The temperature was programmed from 50 °C and, after 5 min of initial holding, to 200 °C at 4 °C/min. The carrier gas was N2 (1.8 mL/min); split mode was used with a flow rate of 72.1 mL/min and a ratio of 1/50; the injector and detector temperatures were 250 °C; and the final hold time was 48 min. 1 μL of essential oil was diluted in hexane and injected manually.

On 30 and 60 days of trial, individual and bulk milk samples were collected. Samples of 50 mL each, collected in triplicate were partly immediately analyzed for composition and partly stored at −20°C for FA assessment. Chemical composition of milk (fat, protein, casein, lactose, urea) was determined by MilkoScan FT 6000 (Foss, Hillerød, Denmark), and somatic cells content (SCC) was determined using a Fossomatic TM FC (Foss, Denmark). Milk lipid fraction was extracted according to the AOAC official method [7 ] and the extracted fat was analyzed for FA composition. Separation of fatty acid methyl esters (FAME) was performed by GC using a gas chromatograph (Thermo Scientific, Waltham, MA, USA) equipped with a capillary column (Restek Rt-2560 Column fused silica 100 m×0.25 mm highly polar phase; Restek Corporation, Bellefonte, PA, USA) and a flame ionization detector (FID). Hydrogen was used as carrier gas. The initial holding temperature was 55°C for 1 min; then it was increased to 170°C at a rate of 10°C/min and held for 30 min. The final temperature of 215°C was reached at a rate of 2°C/min and was held for 4 min. Peak areas were quantified using ChromeCard software, and the relative value of each individual FA was expressed as a percentage of the total FA.

Bacterial biomass was measured by confocal laser scanning microscopy and automatic image analysis41 . Fungal biomass and the percentage of active hyphae were determined by epifluorescence microscopy after staining of soil smears with Differential Fluorescent Stain (DFS)42 . DFS stains active hyphae red because of a higher content of nucleic acids. Bacterial growth rate was determined by incorporation of ¹⁴C-leucine into proteins during a short (1 h) incubation43 . Potential C and N mineralization rates were measured by incubation of soil samples for 6 weeks at 20 °C44 (link). Results of the first week were not used as these reflected initial disturbances of transfer to the microcosm. Oxygen and CO₂ were measured weekly using a gas chromatograph (Thermo Fisher Scientific Inc., Waltham, MA, USA). Nitrogen mineralization rates were calculated from the increase in mineral N (nitrate and ammonium) between week 2 and week 6, which served as a proxy for net mineralization of plant-available N. We evaluated the mineralization rates for only the later two time points (69 d and 132 d).

The GC-MS analysis of the roots and shoots of P. nepalensis was conducted using a Thermo Fisher Scientific Gas Chromatograph equipped with a Tri Plus RSH Autosampler, GC trace-1300, and MS-TSQ Duo. The Thermo Fisher Scientific TG-5MS column was utilized, which measured 40 m in length, 0.15 mm in film, and 0.15 m in internal diameter. The method involved setting the first oven temperature to 80 °C, with a temperature increase of 8 °C/min and a 1-min hold period, followed by increasing the temperature to 150 °C, with a rate of 10 °C/min and a 6-min hold period. The total run time was 32 min, with a 1 µL sample volume injected using helium at a flow rate of 0.7 mL/min as the carrier gas. The MS was operated within the electron ionization (EI) mode, scanning within a 40–450 amu range with a mass spectrometer source temperature and transfer line temperature set at 230 °C and 250 °C, respectively, and an electron multiplier voltage of 1 kV. Mass spectra were interpreted using the NIST/EPA/NIH Mass Spectral Library Version 2.2, 2014, and fragmentation patterns were compared with the instrument database data for all constituents detected.