Flash 2000 chns o elemental analyzer

For each defined depth, samples of roots and soil were homogenized, freeze-dried (except PLFA samples that were stored at − 20 °C), and ground in a ball-mill for the determination of total C and N, ¹³C, ¹⁵N, and ¹⁴C activity. Total C, N, ¹³C, and ¹⁵N were measured with a FLASH 2000 CHNS/O Elemental Analyzer (Thermo Fisher Scientific, Cambridge, UK) combined to a Delta V Advantage isotope ratio mass spectrometer via a ConFlo III interface (Thermo Fisher Scientific, Bremen, Germany) at the Centre for Stable Isotope Research and Analysis (Georg August University Göttingen, Göttingen, Germany).
All δ¹³C values are standardized to the Vienna PeeDee Belemnite international isotope standard and δ¹⁵N values standardized to the δ¹⁵N values of atmospheric N₂. ¹³C and ¹⁵N enrichment is expressed as atom% excess as calculated by the atom% difference between the respective labeled and unlabeled samples. The ¹⁴C activity was determined by combustion in a Hidex 600 OX Oxidizer (Hidex, Turku, Finland) and counted on a liquid scintillation counter (Tri-Carb 3180TR/SL, PerkinElmer, Waltham, MA, USA). ¹⁴C enrichment is determined by the difference in the ¹⁴C activity (Bq g⁻¹) between the respective labeled and unlabeled samples.

The enzymatic hydrolysis of Tenebrio molitor meal was conducted in a jacketed reactor connected to an automatic titrator (718 Stat Titrino, Metrohm, Herisau, Switzerland). Briefly, the T. molitor hydrolysis was conducted at 50 °C and pH 8. Thirty g/L protein was dissolved in distilled water and Alcalase 2.4 L (EC 3.4.21.62) was added at the beginning of the reaction at a 3% enzyme-to-substrate (protein) ratio. The reaction continued until the degree of hydrolysis (DH), measured by the pH-stat method [52 ], was 20%. The resulting hydrolysate was then deactivated by heating the solution at 100 °C for 15 min, centrifuged at 5300× g for 15 min, and vacuum-filtered through an 8 µm cellulose filter. The supernatant was lyophilized (LyoMicron, Coolvacuum Technologies S.L., Barcelona, Spain) and the powdered product was stored at −20 °C. The nitrogen content of the obtained hydrolysate powder was determined in triplicate according to the Dumas method using a Flash 2000 CHNS/O elemental analyzer (Thermo Scientific, Waltham, MA, USA). Protein content was calculated assuming a nitrogen-to-protein factor of 5.6 [53 (link)], resulting in 68.47 ± 0.39 wt.%.