Materials

Biochar was produced from mixed woody waste materials via slow pyrolysis (700 °C) by Swiss Biochar (Belmont-sur-Lausanne, VD, Switzerland) with a Pyreg reactor37 and was characterized by Eurofins Umwelt Ost GmbH, Halsbrücke-Tuttendorf, Germany, as requested by the European Biochar Certificate1. Results are displayed in Supplementary Tables 1–3. This biochar was part of the COST Action-TD1107 biochar ring trial38 and both pristine/non-co-composted and co-composted aliquots from the same composting experiment were subject to previous studies39.

Biochar was co-composted at the Ithaka Institute at St. Léonard, VS, Switzerland, from August to October 2014. Biochar was mixed into compost feedstock composed of cow, horse and poultry manure and green waste at a rate of 4.3% (dry matter w/w). Compost was managed aerobically as described by Kammann et al.24 resulting in temperatures above 60 °C for more than 2 weeks. Compost feedstock was composted without biochar amendment as a control. After frost-protected storage during winter, biochar was picked manually from the mature compost in spring 2015. For biochar picking, 50 kg of biochar-amended compost were stored for analysis in a plastic bag. For each analytical procedure (extraction, characterization), a subsample of compost was collected as a composite sample from this bag. 10–30 char particles were separated from this individual subsample and then prepared for analysis. The actual number of biochar particles finally involved in analysis varied strongly between the different techniques. E.g. only 3 particles of each treatment could by analyzed by STEM and 2 for STXM, while extraction or powder-based analysis (gas adsorption) where based on at least 20 particles.

The same type of biochar was already purchased from Swiss Biochar in 2012 (separate analysis in Supplementary Tables 1–3). It was soaked in a diluted commercial fertilizer solution overnight (1:1 w/v, 1.25 g N kg−1 biochar) and incorporated at a rate of 60 Mg ha−1 into the upper 15 cm of a Terric Anthrosol (top- and subsoil of a Cambisol mixed by construction activities) at the Tuebingen-Sand field site (lat. 48.5342, long. 9.0711). The plot was cropped with Emmer wheat (Triticum dicoccon) in 2012 and winter vetch (Vicia villosa) in 2013 and green fallow thereafter. Biochar was manually picked from soil samples obtained from the upper 15 cm of three sampling sites in spring 2015.

Serial extraction of nitrate

Nitrate was repeatedly extracted from both pristine and co-composted biochar 1:10 (w/v) with 2 M KCl in 50 ml Falcon tubes (n = 3) on a roller shaker (ROLLER 10 digital, IKA, Staufen, Germany) at 50 r.p.m. at room temperature (22 ± 3 °C). After each extraction step, the extractant was decanted through a 0.5 mm sieve. The volume of the decanted extractant was measured to correct the data for the residual extractant (2 M KCl) that cannot be removed by decanting (residual = water content of biochar + added extractant—decanted extractant) which carries a small quantity of already extracted nitrate from one to the subsequent extraction. Fresh 2 M KCl was added to the biochar at the same volume. Each sample was extracted six times: three times for 1 h, for 18 h, for 48 h and for 96 h, respectively, resulting in a total extraction time of 167 ± 1 h. Nitrate was quantified using a continuous flow analyzer (SEAL Analytical, Germany) after reduction of the nitrate to nitrite with hydrazine, which was prepared according to SEAL’s advice for soil extracts. Nitrite was quantified by UV–vis absorption at 550 nm after reaction with N-1-naphtyl-ethylendiamin. The SEAL system is equipped with a dialysis membrane that removes any extraneous microparticles to prevent side reactions or additional absorbance. Standards were prepared in 2 M KCl to provide the same matrix.

13C solid-state nuclear magnetic resonance spectroscopy

Solid-state NMR spectra were collected using a Bruker (Billerica, USA) Avance III 600 MHz spectrometer equipped with a 4.0 mm magic-angle spinning Efree triple resonance HCN probe. 22–29 mg of each sample were packed into 50 µl 4.0 mm zirconia rotors. 1D-13C-spectra were acquired using standard Bruker cross-polarization (CP) pulse sequence at 10 or 12 kHz sample rotation frequency and at 27.0 °C VT inlet gas temperature. 24,576 scans were signal averaged for each spectrum with 3 s recycle delay.13C polarization was achieved using a ramped-amplitude1H-13C CP pulse for 2.0 ms, and the spectra were acquired for 15 ms under 81 kHz two-phase-modulated (TPPM)1H decoupling.13C chemical shifts were referenced externally to 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt using the adamantane methylene peak at 40.48 ppm40. All data were processed with applying a 30 Hz exponential line broadening using Bruker Topspin 3.5.

Gas adsorption

N 2 and CO 2 adsorption were combined to characterize the SSA and pore volume (PV) of the biochar samples. To this end, the adsorption and desorption isotherms for both gases were measured at 77 K and 273 K, respectively. Before the measurements, the char samples were pretreated to clean the char surface from other adsorbed species (degassing) by heating them in vacuum at 120 °C during 6 h. The samples were also milled to reduce the influence of transport limitations on the measurements, especially in the case of N 2 adsorption, due to the low temperatures. The N 2 isotherms were determined measuring the amount of N 2 adsorbed/desorbed as function of the N 2 relative pressure P/P 0 in the measurement station, where P is the pressure of N 2 in the measurement station and P 0 is the N 2 saturation pressure at the measurement temperature (~77 K). The relative pressure P/P 0 ranged from ~0.005 to ~0.995. In the case of CO 2 the relative pressure ranged from ~0.00005 to ~0.028. For both degassing and adsorption/desorption measurements the gas sorption system Nova 2000 provided by Quantachrome Instruments (Boynton Beach, FL, USA) was used. Several methods were applied to the isotherms in order to derive pore surfaces areas and volumes, which are included in the NovaWin software (Quantachrome Instruments). In the case of N 2 adsorption, the Brunauer, Emmett and Teller (BET)41 method was used to determine the total internal SSA of the samples. The DFT (density functional theory)42 method was applied to the adsorption isotherm to characterize the pore size distribution, in particular, the QSDFT (quenched solid-state functional theory) method, considering slit/cylindrical pores. With this method, micropores and mesopores up to ~34 nm in pore width could be measured. The Barrett, Joyner and Halenda (BJH)43 method was applied to characterize mesopores bigger than ~3 nm and macropores up to the detectable limit of N 2 adsorption. The total pore volume was determined from the volume of N 2 adsorbed at a relative pressure (P/P 0 ) ≈1, assuming that at this point the pores are filled with liquid N 2 . For CO 2 adsorption, the nonlinear DFT (NLDFT) was applied to determine the SSA and volume due to micropores.

X-ray photoelectron spectroscopy

XPS uses X-rays to excite atoms of a sample and analyzes the energy of the emitted photoelectrons, which provides information on both elemental composition and chemical bonds of the outermost 10 nm44. XPS was performed on an ESCALAB250Xi (Thermo Scientific, UK) using monochromated Al K α (1486.68 eV, 150 W) at a spot size of 500 µm under high-vacuum conditions (<2×10−9 mbar) and a photoelectron takeoff angle of 90° was used. It was calibrated with Au (Au 4f7 = 83.96 eV), Ag (Ag 3d5 = 368.21 eV) and Cu (Cu 2p3 = 932.62 eV). Pass energy was 100 eV for survey scans and 20 eV for region scans. Biochar particles were gently washed with DI water three times, dried at 40 °C and were measured both as a particle and as a powder after grinding in an agate mortar.

Scanning electron microscopy

In SEM, a focused beam of accelerated electrons scans over the samples and creates a multitude of characteristic secondary emissions. We used secondary electrons, which provide predominantly topographic information of the sample, and EDS. The energy of the X-rays is characteristic for the atom that was excited by a primary electron (beam electron) and thus provides information about the elemental content of the outermost hundreds of nm to ~2 µm of the sample, depending on the composition of the sample and the energy of the incident electron beam45. SEM micrographs and X-ray spectra were obtained on a Zeiss Sigma SEM with Bruker EDS at a working distance of 8–9 mm and an acceleration voltage of 10–15 kV. Individual biochar particles were mounted on SEM stubs with conductive carbon paint. Sputter coating was not necessary.

Scanning transmission X-ray microscopy

Ultra-thin-sections for STXM were prepared by cutting pieces of biochar (no pretreatment, no drying) under cryo-conditions in an ultramicrotome Leica UCT, gluing the sample with a droplet of 2.3 M saccharose and catching the ultra-thin-sections with saccharose, too. They were placed on formvar-coated 200 mesh copper grids. Grids were carefully rinsed with water to remove the saccharose and mounted on a STXM sample plate. Despite the rinsing, saccharose was still present on all samples. Therefore, a reference spectrum of pure saccharose was used to quantitatively map saccharose by spectral decomposition in the samples. Ultra-thin-sections were obtained from both center and the edge of pristine biochar particles as well as the edge of co-composted biochar particles. Measurements were conducted at the Canadian Light Source beamline 10ID-1.

Analytical scanning transmission electron microscopy

Pieces of biochar were covered in gold all-around in the Leica EM ACE 600 (Leica Microsystems, Vienna, Austria; 45 mA, 8,0 × 10−3 mbar, working distance 50 mm) using a rotating device to enable 3D coating. This resulted in a minimum of 25-nm-thick gold layer on the biochar. These nuggets were embedded in Spezifix40 (Struers, Willich, Germany) and after hardening carefully trimmed with a trim 90 blade (Diatome, Switzerland). The preparation of 50 nm slices was done at room temperature with the ultra-sonic-knife (Ultra Sonic, Diatome) for reducing compression and allowing best structure preservation in the ultramicrotome Leica EM UC6 (Leica Microsystems). The slices were transferred from the water with a perfect loop (Diatome) on a 200 mesh grid. STEM investigations were performed on a monochromated probe corrected FEI Titan G2 60–300 (STEM) microscope with an X-FEG Schottky field-emission electron source operated at 60 kV. The microscope is equipped with a FEI Super-X detector (Chemi-STEM technology), consisting of four separate silicon drift detectors46 (120 mm acquisition area) and a Dual EELS – Gatan Imaging Filter (GIF) Quantum47. The microscope was run in monochromated mode with a spatial resolution of 0.1 nm and an energy resolution at full-width at half-maximum of 0.17 eV. Two different detectors: high angular annular dark field (HAADF) and annular dark field (ADF), a beam current of approximate 120 pA and acquisition times ranging from 20 to 30 μs/pixel were used for micrographs acquisition. Analytical investigations involved electron energy loss (EELS) and X-ray (EDX) spectroscopy in STEM mode by line scans and spectrum imaging, which, however, were acquired with different acquisition times and pixel sizes depending on the investigated area. Energy loss near edge structure (ELNES) of carbon-, nitrogen- and oxygen- K ionization edges were analyzed considering the specific finger prints and energy shifts of different types of bonding which might be present in the sample, with graphite as a reference. Elemental quantification has been performed by using the k-factor method45, 48. Sample thickness in different areas was determined by EELS48. However, for the mean free path of the sample with the major constituent carbon a calculated value of 76.6 nm (Malis formula48) was used. The images and spectra were recorded by a Gatan Digiscan unit and Digital Micrograph software and were corrected for dark current and gain variations.

Desorption atmospheric pressure photoionization FT-ICR MS

A modified ThermoFisher LCQ atmospheric pressure photoionization (APPI) source (ThermoFisher Corp., Bremen, Germany) was used for all Fourier-transformed ion cyclotron resonance mass spectrometry (FT-ICR MS) experiments49. Pieces of pristine and co-composted biochar were held by tweezers ∼ 1 mm from the exit of the heated ceramic nebulization tube and ~10 mm from the FT-ICR MS inlet. Gas-phase neutrals were produced through a combination of thermal and chemical desorption50, and ionized by dopant-assisted APPI51. Nitrogen was used as nebulizer gas at 100 psi with toluene as dopant at a flow rate of 50 μl min−1. The temperature of the heated nebulizer gas/solvent plume ranged from 100 to 500 °C depending upon the sample. Mass spectra were acquired by a custom-built FT-ICR MS based on a passively shielded 9.4 T horizontal 200 mm bore diameter superconducting solenoid magnet (Oxford Corp., Oxford Mead, UK) operated at room temperature52. A modular ICR data station (Predator) facilitated instrument control, data acquisition, and data analysis53. Positive ions were accumulated in an external quadrupole for 50–500 ms, and passed through an rf-only quadrupole into an octopole equipped with tilted wire extraction electrodes for improved ion extraction and transmission54. Helium gas was introduced into the octopole to collisionally cool the ions before transfer through rf-only quadrupoles (total length 127 cm) into a seven-segment open cylindrical ICR cell with capacitively-coupled excitation electrodes based on the Tolmachev configuration55. Approximately 25–50 time-domain acquisitions were co-added, Hanning-apodized, and zero-filled once prior to fast Fourier transform and magnitude calculation. ICR frequencies were converted to m/z values by the quadrupolar electric trapping potential approximation56. Spectra were internally calibrated from abundant homologous alkylation series (compounds that differ in elemental composition by integer multiples of CH 2 ). Mass spectral peaks with signal magnitude greater than six times the baseline root-mean-square (r.m.s.) noise level were assigned elemental composition values (C 0–100 H 0–200 N 0–5 O 0–15 S 0–2 ) with PetroOrg software (Y.E. Corilo; Florida State University; all rights reserved), subject to the following constraints for chemical feasibility: mass error < 1 p.p.m.; the computed ring and double bond equivalent must be an integer value ≥ 0; neutral formulas containing an odd number of nitrogen atoms must have an odd nominal mass, and neutral formulas containing zero or an even number of nitrogen atoms must have an even nominal mass (nitrogen rule); 2 ≤ H ≤ (2 C + 2); 0.33 ≤ H/C ≤2.25; 0 ≤ O ≤ (C + 2); O/C <1.2; N/C <0.5; S/C <0.257.

DAPPI FT-ICR MS was performed both on original biochar samples and on washed biochar as described below. However, data on the washed samples was more comprehensive and better calibrated, i.e., more detected masses could be assigned to formulas. In the original co-composted biochar, 6,063 detected masses were not assigned formulas, while only 4,350 could not be assigned after washing.

Biochar washing for liquid extraction of the organic coating

Biochar particles were washed in four steps to remove any compost induced surface alterations and, at the same time, to obtain a suspension of the co-composting derived material that forms the coating of the biochar as identified with STEM. 15 mL DI water were added to 2.5 g moist biochar in a Falcon tube (step 1). After 5 min on a roller shaker at 50 rpm (Roller 10 digital, IKA, Staufen, Germany), water was decanted through a 0.5 mm mesh sieve and 15 ml of DI water were added to repeat this procedure (step 2). After decanting, 15 ml of 0.05 M NaOH solution were added as suggested by Tsechansky and Graber28 and Falcon tubes were placed on the shaker for 90 min (step 3). After decanting, 15 ml of DI water were added for 20 min to remove residual NaOH (step 4). Separate washing procedures were conducted for fresh and co-composted biochar. Afterwards, the biochar was dried at 40 °C for 72 h for DAPPI FT-ICR MS, SEM and STEM as described above. After washing, co-composted biochar was almost black again (Supplementary Fig. 7). Washed biochar was analyzed using SEM and STEM to verify the removal of the coating. SEM showed that the original biochar surface structure was widely exposed again, while few regions still showed surface alterations (Supplementary Fig. 8). In STEM we could not identify a coating after washing, but granular aggregations of Ca and O were present on the biochar surface (Supplementary Fig. 9).

Washing solution of step 3 (0.05 M NaOH) was analyzed by FTIR,1H NMR, excitation–emission matrix (EEM) fluorescence spectroscopy, ion chromatography, and for dissolved organic and inorganic carbon (DOC/DIC) and electron accepting and donating capacity (EAC/EDC). Before analysis, resulting washing solutions were filtered through a 0.45 µm syringe filter. For FTIR, washing solutions and original 0.05 M NaOH solution were freeze-dried. For1H NMR, washing procedure was repeated and 0.05 M NaOH was prepared with D 2 O instead of H 2 O. Major anions and cations were quantified by ion chromatography (IC, DX-120, Dionex, Sunnyvale, CA, USA). DOC + DIC was quantified on a Vario cube (Elementar, Hanau, Germany), DOC was quantified on the same device after acidifying the samples to pH 1 with HCl. DIC was calculated as [DOC + DIC]—[DOC].

Excitation–emission matrix

EEM fluorescence spectra of washing solution were analyzed using a Fluoromax4 (Horiba, Jobin-Yvon) spectrofluorometer. EEM fluorescence spectra were recorded over a range of excitation wavelength (300–500 nm) and emission wavelength (400–600 nm) relevant for natural organic matter58.

Electron accepting and donating capacity

The redox properties, i.e., electron accepting and donating capacities (EAC/EDC) of biochar washing solutions were determined by mediated electrochemical reduction (MER) and oxidation (MEO) following established procedures6, 59. In brief, the electrochemical system consists of a glassy carbon cell (Sigradur G, HTW, Germany) as working electrode, a Ag/AgCl as reference electrode (Bioanalytical systems Inc., USA) and a platinum wire (0.5 mm, 99.9 %, Sigma-Aldrich Co., USA) attached to a platinum gauze (52 mesh, 99.9 %, Sigma-Aldrich Co., USA) as auxiliary electrode. The applied potential was measured against Ag/AgCl electrode but is reported against standard hydrogen electrode (EH = −0.49 V in MER and EH = + 0.61 V in MEO). Electron transfer mediators were used during the measurements to ensure a fast electron transfer between the electrodes and the analyte59; diquat-dibromide monohydrate (DQ, Sigma-Aldrich Co., USA) was used for MER and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, Sigma-Aldrich Co., USA) for MEO. EAC and EDC are calculated from the measured reductive and oxidative currents considering normalization to carbon content (µmol e-/mmol C)6. The total EEC is the sum of the individual accepting and donating capacities (EEC = EAC + EDC). As an additional reference, dissolved organic matter was extracted from non-biochar-amended control compost in analogy to the biochar washing procedure (0.05 M NaOH, 1.5 h, same shaker).

Fourier-transformed infrared spectroscopy

Fourier-transformed infrared (FTIR) Spectroscopy provides information on bonding modes in organic molecules by absorption of infrared radiation, which depends the vibrational response of the functional groups60. FTIR absorbance spectra of KBr pellets prepared with 0.2% biochar, 0.4% freeze-dried washing solutions or 0.4% freeze-dried control compost were measured with a Vertex 80 v (Bruker) with 128 scans. A KBr pellet without sample was used for background measurements.

1H solution nuclear magnetic resonance spectroscopy

2 ml of the pristine and the co-composted biochar washing solutions were lyophilized (freeze-dried) overnight. The dry samples were rehydrated up to 500 µl using 100% D 2 O. NMR spectra were collected using a Bruker Avance III 600 MHz spectrometer equipped with a Bruker 5 mm SmartProbeTM.1H 1D spectra were acquired for 2 s with a 20 s recycle delay and 1024 scans using standard Bruker pulse sequence with water suppression. All spectra were processed with applying a 0.30 Hz exponential line broadening using Bruker Topspin 3.5.

Fast field cycling nuclear magnetic resonance relaxometry

1 g of each biochar sample was suspended in 3 g of MilliQ grade water (resistivity of 18.2 MΩ cm at 298 K). Milli-Q water was produced by a Milli-Q Advantage A10 Ultrapure Water Purification System (Millipore Corporation, Massachusetts, USA). The suspensions were allowed to sediment overnight prior to the relaxometry investigations. The samples were put in the probe of a Stelar SpinMaster FFC-2000 fast field cycling relaxometer (Stelar s.r.l., Mede, PV−Italy) and analyzed at 25 °C. The basic theory about FFC NMR relaxometry and the sequence applied for the experiments reported in this study have been already summarized by Conte and Alonzo31. In brief, non-polarized (NP) and polarized (PP) sequences were applied. In the NP sequence, a relaxation field (B RLX ) was changed between proton Larmor frequency (ω L ) values ranging in the interval 0.01–30 MHz. Each B RLX was applied for a period τ arrayed with 32 logarithmic spaced time sets, each of them adjusted at every relaxation field in order to optimize the sampling of the decay/recovery curves. At the end of each B RLX field a1H 90° pulse was applied into an acquisition magnetic field (B ACQ ) held for a fixed time at the ω L value of 7.2 MHz. The 1H 90° pulse was needed to make magnetization observable and the free induction decay acquirable. A time domain of 100 µs sampled with 1000 points was applied. Field-switching time was 3 ms, while spectrometer dead time was 15 µs. For all of the experiments a recycle delay of 20 s was used. In the PP sequence, a polarization field (B POL ) set at the ω L of 9 MHz was applied prior to each B RLX field. The period of time during which B POL was applied (referred to as polarization time or T POL ) corresponded to five times the T 1 estimated at this frequency.

The crossover field between NP and PP sequences was approximately retrieved when the relaxation field intensity was half of that of the polarization field31.

FFC NMR relaxometry data elaboration

The longitudinal relaxation time (T 1 ) values of the observed nuclei were obtained for each B RLX by changing the τ values as reported above. The relationship between signal intensity and τ is modeled by Eq. 1:

$$I\left( \tau \right) = {I_0}\exp {\left[ { - \left( {\tau {\rm{/}}{T_1}} \right)} \right]^k}.$$ (1)

Here, I(τ) is the 1H signal intensity at each fixed B RLX , I 0 is the 1H signal intensity at the thermal equilibrium, T 1 is the average proton spin-lattice relaxation time and k is a heterogeneity parameter related to the stretching of the decay process. This function, which accounts for the large sample heterogeneity resulting in a multi-exponential behavior of the decay/recovery curves31, can be considered as a superposition of exponential contributions, thereby describing the likely physical picture of some distribution in T 1 . Eq. 1 has the advantage that it is able to handle a wide range of behaviors within a single model. For this reason, assumptions about the number of exponentials to be used in modeling nuclear magnetic dispersion (NMRD) data are not necessary. The NMRD profiles resulting from the elaboration of the decay/recovery curves are reported in Supplementary Fig. 13. The data acquired at the relaxation field of 0.01 MHz were transformed by applying the Uniform PENalty regularization (UPEN) algorithm31 which allowed the achievement of the distribution of the longitudinal relaxation times (also referred to as relaxograms).

Liquid chromatography organic carbon detection

1 g of pristine, co-composted and soil-aged biochar was added to 10 ml of distilled water at 50 °C for 24 h and regularly stirred, and then centrifuged and filtered (10,170 g, 5 min, 0.45 µm) to separate the solid and liquid phases. The supernatant solutions were analyzed by Liquid Chromatography – Organic Carbon Detection LC-OCD model 8 (DOC Labor, Germany). Details of the measurement procedure have been described in full by Huber et al61. In this study, a Toyopearl TSK HW50S LC column was used with a phosphate buffered mobile phase of pH 6.4 at a flow rate of 1.1 ml min-1. Injection volumes were 1,000 µl. The supernatant solutions are also analyzed to determine total organic nitrogen and carbon and total inorganic carbon using a thermocatalytic high-temperature oxidation process (Multi N/C 2100, Analytic Jena, Germany).

The chromatographic eluent subdivides into six sub-fractions, biopolymers, humics, building blocks, low molecular-weight neutrals and hydrophobic organic carbon (Supplementary Table 5), as suggested by Huber et al.61

Data availability

The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.