Cyclotec 1093

Individual spikes were marked at anthesis to monitor grain growth. At seven-day intervals during grain development, from seven days after anthesis until harvest maturity, ten spikes were collected from each subplot. The spikes (a total of seven samples per subplot) were lyophilized using a HALDRUP LT-15 laboratory thresher (HALDRUP GmbH, Ilshofen, Germany). Once the grains were threshed, thousand grain weight (GW) was determined with a Marvin system (GTA Sensorik GmbH, Neubrandenburg, Germany) according to the standard MSZ 6367/4-86 (1986) method. The grains were then milled using a Foss Cyclotec 1093™ (FOSS, Barcelona, Spain) mill equipped with a 0.5 mm screen. Finally, the flour was immediately stored at −20 °C in the dark until analysis.

In order to remove the foliar P fertiliser potentially adhering to the sprayed surfaces, all plant parts were thoroughly washed in a three-step process with deionized water after fresh weight determination. Subsequently, all samples were dried at 65 °C, then weighed and ground to powder (Cyclotec 1093, Foss Tecator, Höganäs, Sweden). For nutrient analysis by inductively coupled plasma-mass spectrometry (ICP-MS; Agilent Technologies 7700 Series, Böblingen, Germany), 200 mg of each replicate was transferred to 10 mL 69% HNO₃ (ROTIPU-RAN Supra for ICP, 69%) and dissolved in an 1800 W microwave-oven (MARS 6 Xpress; CEM, Matthews, MC, USA) for 45 min, as described by Jezek et al. [29 (link)].

The seeds of all the wheat samples were cleaned using the Mini-Petkus seed cleaner and then milled using a laboratory mill equipped with a 1 mm sieve (Cyclotec 1093, FOSS, Hillerod, Denmark) to obtain the whole-grain flour. ATIs were quantitatively extracted from 1 g of whole-grain flour using 5 mL of extraction buffer (10 mM sodium bicarbonate, 500 mM sodium chloride, pH 7.8) with constant spinning at 4 °C overnight. The suspension was centrifuged at 4fSubsequently, the best linear unbiased 600 × g for 30 min, the supernatant collected and the procedure was repeated with an additional 5 mL of extraction buffer. Supernatants were combined and sterile-filtered (0.22 µm). Our prior study showed that this procedure extracted > 90% of ATIs, while maintaining their native protein conformation and solution stability (Sielaff et al. 2021 (link)).

Digestate samples from each reactor were taken weekly and analysed for pH, dry matter (as a TS content) and organic matter (as VS content), following the TS and VS procedure (APHA, 2005 ) respectively. Dissolved VFA were determined weekly using a gas chromatograph (5560-D of APHA, 2005 ) equipped with a flame ionization detector (HP 68050 series Hewlett Packard). Total ammonia nitrogen was also determined weekly from fresh samples using photometric kits (Spectroquant kit, Merk, USA).
At the end of each experimental period, samples from each reactor were taken, dried (48 h at 60°C) and milled using a mill with a 0.8 mm of diameter (Cyclotec 1093, Foss, North America). Fiber fractions (total neutral detergent fiber, acid detergent fiber and lignin), crude fat and TKN were analysed from the dried milled samples. Fiber fractions were determined according to the Van Soest procedure (Van Soest, 1991 (link)) and corrected for ash. Crude fat was determined by measuring extracted lipids with petroleum ether (Soxtec 2050, Foss analytical, Hillerød, Denmark) after hydrolysing with HCl (Stoldt, 1952 ). Total kjeldahl nitrogen was determined according with APHA (2005 ).

Insects of the desired age (eight-days-old adult males and ten-days-old adult females) where freeze-killed and stored at -20 °C. At a later phase, specimens were allowed to defrost for 10 min, the gut of each specimen was removed to avoid contamination due to residuals of plant tissue, and all carcasses were dried for 48 h at 60 °C. Dry insects were individually ground to fine powder with a ball mill (MM400, Retsch, Germany) and total carbon and nitrogen were measured by using an elemental analyser (EA 3000, EuroVector, Italy). Carbon (C) and Nitrogen (N) percentage and the C/N ratio were used as response variables.
Plant matter was dried for 48 h at 60 °C, grounded using a cyclone mill (Cyclotec 1093, Foss A/S, Sweden) and analysed for nitrogen concentration by near-infrared spectroscopy (Spectra Star 2400, Unity Scientific, USA). The concentration was derived after calibration models covering a spectra range from 1250 to 2350 nm^{101 (link)} and the accuracy of the measurements was confirmed with a subset of samples measured with an elemental analyser (EA 3000, EuroVector, Italy).