Compounds, Nitrogen

Carbon aerogels (CAGs) based on different types of starch (potato, maize, and rice) were obtained by the carbonization of organic aerogels. Precursors of carbon aerogels were prepared via the sol–gel polycondensation process according to the procedure described in our previous paper [25 (link)]. Briefly, starches of potato, maize, and rice origins (Sigma-Aldrich, Saint Louis, MO, USA) were dispersed in water with appropriate dilution ratio (the concentration of the solutions for potato and rice starches was 10 wt %, for maize starch it was 15 wt %). Suspensions of starches were stirred and heated up to the gelatinization temperature. Subsequently, the solvent exchange by immersing the aqueous gels for 12 days in the ethanol (96%, Avantor Performance Materials - formerly POCH S.A., Gliwice, Poland) was carried out. Afterwards, the alcogels were dried under ambient pressure in air at 50 °C for 1 day. As-obtained organic aerogels (OAGs) based on potato (OAGPS), maize (OAGMS) and rice starch (OAGRS) were analyzed in the context of thermal decomposition and morphology characteristics. Starch aerogels were then pyrolysed under argon flow (purity 99.999%, 50 mL∙min⁻¹, Air Products, Allentown, PA, USA) at 700 °C, 800 °C, and 900 °C for 6 h, which allowed carbon aerogels (so called CAGPS, CAGMS, CAGRS, consequently) to be obtained. At this point, it should be mentioned that the OAGs for carbonization were prepared as coarse powders and CAGs after the heat treatment remained in this form. However, for electrochemical application, the CAGs need to be ground into uniform fine powders, and the samples in this form are presented in the following paper. That is why the grinding of CAGs samples after pyrolysis was performed in an agate mortar for about 30 min for each sample.
The thermal decomposition of organic aerogels was studied by means of thermogravimetric analysis coupled with evolved gas analysis with infrared spectroscopy detection (EGA(FTIR)-TGA/DTA/DTG method). The experiments were carried out using SDT Q600 thermobalance (TA Instruments, New Castle, DE, USA) coupled with a Fourier transform infrared (FTIR) spectrometer (Nicolet 6700 FTIR, Thermo Fisher Scientific, Waltham, MA, USA) by FTIR-TGA interface (Thermo Fisher Scientific, Waltham, MA, USA). The measurements were performed in an inert gas flow (N₂, 20 mL∙min⁻¹) for samples with the weight of 20 mg placed in a corundum crucible, in the temperature range of 20–1000 °C and at a heating rate equal to 5 °C∙min⁻¹. The 2D and 3D FTIR spectral maps of evolved gaseous products were recorded with resolution of 4 cm⁻¹ collecting eight scans for each spectrum. The morphology of the materials was characterized using an FEI Versa 3D (FEG—Field Emission Gun) scanning electron microscope (FEI Company, Hillsboro, OR, USA). The crystal structure of the carbon aerogels was characterized by powder X-ray diffraction (XRD) using BRUKER D2 PHASER (Billerica, MA, USA). The Cu Kα radiation ( $\lambda =0.154184 \mathrm{nm})$ in the range of 10–60° (2θ) with a step of 0.02° was used. To determine the amount (a weight percent) of carbon, hydrogen, and nitrogen elements in the obtained carbon compounds, the elemental analysis (CHN analysis) was conducted using micro analyzer vario MICRO cube coupled with microbalance (Elementar, Langenselbold, Germany). Before the CHN determination, the CAG samples were dried in vacuum oven under 80 mbar for 3 h at 80 °C. The evaluation of chemical composition was performed with an accuracy of 0.3%. The electrical conductivity (EC) studies were carried out using semi-4-probe method with 1 mA alternating current (at a fixed frequency of 33 Hz) within temperature range from −20 to +40 °C by means of state of the art Sigma1 apparatus. The powder samples (with a thickness of about 2.5 mm) were placed in a glass tube between the parallel flat and gold circular electrodes (with 5 mm in diameter) and pressed by an electrode piston until the measured resistance of the samples remained constant and appropriate electrical contact was assured. Porous features of the resulting samples were evaluated from N₂ sorption at −196 °C measured with 3Flex v1.00 automated gas adsorption system (Micromeritics, Norcross, GA, USA). Before the analysis, the samples were degassed under vacuum at 350 °C for 24 h. The specific surface area (S_BET) was determined by the single point surface area at $\frac{p}{p_0}=0.2$ .

Fig. 1

Experimental protocol over the 2 generations

Twenty-eight pregnant New-Zealand white female rabbits (INRA1077 line, 1-year old) (F0) were exposed by nose-only inhalation in custom made plexiglas tubes to either diluted DE (1 mg/m³) (exposed group) or clean air (control group) for 2 h/day, 5 days/week, from the 3rd to the 27th day post-conception (dpc) (i.e., 20 days altogether over a 31-day gestation) (Fig. 1). DE exposure was performed with the Mobile Ambient Particle Concentrator Exposure Laboratory [18 (link)] connected to a 25KVA Loxam engine, with a 500 nm particle filter (Additional file 1: Figure S1).
DE is a complex mixture of hundreds of constituents in either a gas or particle form. Gaseous components of DE include carbon dioxide, oxygen, nitrogen, water vapour, carbon monoxide, nitrogen compounds, sulphur compounds, and numerous low-molecular-weight hydrocarbons (some of them individually known to be toxic, such as aldehydes, benzene, 1,3-butadiene, polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHS). The particles present in DE are known to be composed of center core of elemental carbon with absorbed organic compounds and small amounts of sulphate, nitrate, metals, and other trace elements [19 ]. The measured components of the exposure mixture in the present experiment are shown in Additional file 2: Table S1.

The ammonium concentration was estimated by Nessler’s assay carried out in a total volume of 25 mL using 200 μL of each culture and 0.5 mL of Nessler’s reagent. Ammonium rates were measured spectrophotometrically at a wavelength of 410 nm.
Nitrites and nitrate concentrations were determined by using a Dionex ICS-1100 (ThermoFisher Scientific, Waltham, MA, USA) ion chromatography system equipped with a DRS 600 suppressor and a conductivity detector. Anions were separated by a Dionex IonPac AS23 column and a Dionex IonPac AG23 guard column with a flow rate of 1 mL/min of a 0.45 M Na₂CO_3/0.08 M NaHCO₃ eluent. The determinations were conducted according to the APAT CNR IRSA 4020 Man. 29/2003 method, published by the Italian Environmental Protection Agency. The analytical procedure was conducted under UNI CEI EN ISO/IEC 17025:2018 standards; therefore, extensive method validation and the expanded uncertainty were available. Detection limits for nitrites (as NO₂⁻) were 0.09 mg/L and 3.4 mg/L for nitrates (as NO₃⁻).
Total nitrogen was determined by using the small-scale sealed-tube kit (LCK 138, Hach company, Loveland, CO, U.S.A.). Nitrogen compounds present in the samples were oxidized to nitrate according to the method EN ISO 11905-1:1998 which uses peroxodisulfate and a high temperature (120 °C for 30 min) for the digestion. Next, a solution of 2,6-Dimethylphenol was added to the sample, which reacts with the nitrates to form 2,6-Dimethyl-4-nitrophenol. The formed nitrophenol was then determined spectrophotometrically at a wavelength of 345 nm.
COD was determined by using the sealed-tube test method (ISO 15705:2002), while the BOD was determined by using a small-scale sealed-tube kit (LCK 555, Hach company, Loveland, CO, USA).
Chlorides were determined by titration with silver nitrate (APAT CNR IRSA 4090 Man 29:2003) and metals were determined after microwave-assisted aqua regia digestion (UNI EN ISO 15587-1:2002) with an ICP-MS (UNI EN ISO 17294-2:2016).
All the analytical procedures described were performed at the Eco Control Laboratorio Ascolano s.r.l. (Italy), (Certification UNI CEI EN ISO/IEC 17025:2018).

Total protein (nitrogenous compounds content) was determined by the conventional micro-Kjeldahl method [57 ]. A sample of 0.3 g powder of Moringa leaves was weighed into a digestion flask. Ten mL sulfuric acid and 0.5 g digestion mixture (1:4 K₂SO₄:CuSO₄) were added. The sample was then incinerated, the digested sample was quantitatively transferred into the Markham micro-Kjeldahl with the least amount of ammonia-free distilled water, and then 20 mL of 50% NaOH solution was added. A strong current of steam was then passed and the ammonia was distilled into 10 mL of 2.5% boric acid. The ammonia was then titrated against HCl using a mixed indicator (0.4 methylene blue and 0.1 methyl red) until a faint red endpoint was obtained. After correction for the reagent blanks, the titration figures were converted into percentage protein using the following equation: $$Total crude protein\%=\frac{Read\times N of HCl\times 0.014\times Conversion Factor\times 100}{Sample weight}$$
The conversion factor for Moringa = 5.25.