Na 1500

Bacteria in the fermenters were labeled using ¹⁵N. On day 7, 0.3 g/L (NH₄)₂SO₄ in McDougall's buffer was replaced with 0.3 g/L ¹⁵N-enriched (NH₄)₂SO₄ (Sigma Chemical Co., St. Louis, MO, USA; minimum ¹⁵N enrichment 10.01 atom%) until the end of the experiment. On d 13, 14, and 15, daily effluent samples were preserved with 3 mL of a sodium azide solution (20%; wt/vol) and 40 mL were subsampled for isolation of liquid-associated bacteria.
To determine ¹⁵N concentration, effluent liquid samples were centrifuged (20,000 × g, 30 min, 4°C) and the resulting pellets were washed using de-ionized water and centrifuged 3 times (20,000 × g, 30 min, 4°C). The pellets were then re-suspended in distilled water and frozen at −20°C until lyophilized. The FPA bacterial samples collected after stomaching were centrifuged (500 × g, 10 min, 4°C) to remove large feed particles and the supernatant was decanted and centrifuged (20,000 × g, 30 min, 4°C) to isolate a bacterial pellet which was washed 3 times as previously described. The pellet was then resuspended in distilled water and stored at −20°C. Washed feed residues (FPB fraction) were dried at 55°C for 48 h, weighed for DM determination, ball ground (MM 400; Retsch Inc., Newtown, PA, USA), and analyzed for total N and ¹⁵N by combustion analysis using a mass spectrometer (NA 1500, Carlo Erba Instruments).

Forage and concentrate bag residues were dried at 55°C for 48 h and pooled over 3 days (d 9, 10 and 11, and d 13, 14, and 15 for each fermenter) to ensure that there was sufficient sample for chemical analysis. Samples were ground through a 1 mm screen in a Wiley mill (standard model 4; Arthur H. Thomas Co., Philadelphia, PA, USA) before chemical analysis. Substrates were analyzed for DM (method no. 930.15) and ash content (method no. 942.05) according to AOAC (2006 ). The concentration of neutral detergent fiber (aNDF) was assayed with a heat stable amylase and sodium sulphite and expressed inclusive of residual ash (Van Soest et al., 1991 (link)). The concentration of acid detergent fiber (ADF) was determined according to method 973.18 (AOAC, 2006 ). The concentration of total N (method no. 990.03; AOAC, 2006 ) was determined by combustion analysis using a mass spectrometer (NA 1500, Carlo Erba Instruments). Concentrations of VFA and NH₃-N in the liquid effluent were analyzed by gas chromatography (Wang et al., 2001 (link)) and the modified Berthelot method (Rhine et al., 1998 (link)), respectively.

Total organic C (TOC) and total nitrogen (TN) concentrations were determined
using a Carlo Erba NA 1500 elemental analyzer (Carlo Erba Instruments, Milan,
Italy). Soil water content was determined by oven-drying a sub-sample of the
soil at 105 °C to constant weight. Soil pH was measured
in a suspension of 1:2 soil:water (w:v) using a portable pH meter (PCE
Instruments GmbH, Meschede). Soil texture was measured following the hydrometer
method described in Kroetsch and Wang58 .

The quantitative elemental composition of the 401 topsoil samples (0–10 cm) was analyzed by energy dispersive X‐ray fluorescence (ED‐XRF) spectrometry, using a XEPOS spectrometer (Ametek Corporation). First, each sample was air‐dried, sieved (<2 mm), ground finely (1–3 g), and compressed with a plunger (250–260 gm) on a Teflon cup with a Prolene membrane at the bottom. Four‐point ED‐XRF spectra obtained from each cup were averaged and compared with the values obtained by measuring a certified reference material (NBS‐1572; Gaithersburg MD). We considered the total content of S, Fe, Si, and Al obtained by the ED‐XRF spectrometry to assess soil inorganic components (see Table S1). In addition, C and N concentrations (%) were also determined for all samples previously oven‐dried (110°C), sieved (<2 mm), weighed on a Mettler Toledo microbalance (+0.0005 mg), and then combusted in a Carlo Erba NA‐1500 elemental analyzer (Sobral et al., 2017 (link)). The total elemental content of C, N, S, Fe, Si, and Al was centered log‐ratio (CLR) transformed for removing closure effects of analytical chemical data prior to undertaking a Principal Component Analysis (Aitchison, 1984 (link)).

Small punches (3.14cm²) of each quartz fiber filter sample were analyzed for total aerosol carbon content using an elemental analyzer (EA) (Carlo Erba, NA-1500). The filter punch was rounded using a pair of flat tipped tweezers, placed into a tin cup, and then caked into a ball. Before use, the tin cup was washed with acetone under ultrasonication to remove organic and other contaminants. Carbon was converted to CO₂ in the combustion furnace (1020°C). Stable carbon isotopic analyses were conducted using the same EA interfaced (ThermoQuest, ConFlo II) to isotope ratio mass spectrometer (IRMS) (ThermoQuest, Delta Plus). The isotopic compositions (δ13 C) were determined using the standard isotopic conversion: where R is the ratio of 13 C/¹²C. Vienna Pee Dee Belemnite (¹³C) was used as the isotope standard. Samples were analyzed in duplicate and averaged concentrations and isotopic ratios are reported here after the blank correction. Reproducibility of TC measurement was within 2%. Analytical error in the carbon isotope ratios was within 0.2‰.

The aboveground biomass was destructively sampled by randomly cutting four representative plants from each plot. All the plant samples were heated to 105°C, oven dried at 70°C until a constant weight was achieved, and the samples were then weighed. The dry plant material was then ground so that it passed through a 240-mesh screen, and the ground samples were analyzed for total N using a Carlo Erba NA 1500 dry combustion analyzer (Carlo Erba, Milan, Italy)36 . The quantitative analysis of the total soluble sugar content was measured with the anthrone colorimetric method37 .