Flash ea 1112

The ¹³C isotopic discrimination in the algal samples (δ¹³C_alga) was determined by mass spectrometry using a DELTA V Advantage (Thermo Electron Corporation, USA) Isotope Ratio Mass Spectrometer (IRMS) connected to a Flash EA 1112 CNH analyser, as described by Iñiguez et al. (2016) . The ¹³C isotopic discrimination of the dissolved inorganic carbon found in the medium (δ¹³C_DIC) was measured with the same IRMS connected to a GasBench II (Thermo Electron Corporation) system, using 20 ml FSW collected from each cylinder, previously filtered (Whatman GF/F). The δ¹³C_alga was corrected with the δ¹³C_DIC values from the medium, since the CO₂ source used in the experiment for the CO₂-enriched treatment came from previously fixed CO₂ that had been already discriminated.

Soil horizons of each soil core were divided into Oi and Oe horizons (organic materials), and OA and A horizons. The OA horizon was placed in between organic and mineral A layers, and most A horizons were found below 50 cm depth. We defined here Oi horizon as “top soil,” and Oe, OA, and A horizons as “sub-soil.” Soil texture was determined by the pipette method (Gee and Bauder, 1986 ). Electrical conductivity (EC) and soil pH were measured in a soil-water suspension (1:5 ratio, w/v) using a pH/EC meter (Orion Star A215, Thermo Scientific, Waltham, MA, USA). Water content (WC) was determined by measuring the weight change in soils after oven-drying at 105°C for 48 h. Total carbon (TC) and total nitrogen (TN) contents in soils were measured using an elemental analyzer (FlashEA 1,112 Thermo Electron corporation, Waltham, Massachusetts, USA). For NH₄⁺-N and NO₃⁻-N contents analysis, fresh soil was extracted using 2 M KCl solution and subsequently filtrates were analyzed on an auto-analyzer (QuAAtro; Seal Analytical, Norderstedt, Germany). Fresh soil was mixed with deionized water and then filtered through Whatman filter paper #42 firstly and then 0.45-μm filter to acquire water extractable carbon (WEC) and nitrogen (WEN).

For determination of the organic carbon (OC) content, sediment samples were first freeze-dried or dried at 60°C in an oven overnight, acidified to remove carbonate, and further vacuum-dried. The OC content of the sediment was determined with an elemental analyzer (NA-1500n, Fisons, Rodano-Milan, Italy: for the Japanese sediments and FlashEA 1112, Thermo Electron, Bremen, Germany: for the sediments of other samples).

Harvested sorghum was firstly dried at 50 ℃ for 2 days and then crushed to the size of less than 1 cm before the experiments. The characteristics of the prepared sorghum are shown in Table 1. The organic elements C, H, O, N were analyzed via a fundamental measurement service of National Institute for Environmental Studies by using an element analyzer Flash EA 1112 (Thermo Electron Corporation, US). The TS, VS and COD were analyzed according to the standard methods [20 ]. The cellulose (ADF-ADL), hemi-cellulose (NDF-ADF) and lignin (ADL) contents of the sorghum were analyzed in the fractions of NDF (neutral detergent fiber), ADF (acid detergent fiber) and ADL (acid detergent lignin) were analyzed according to a previous report [21 (link)]. The inoculated sludge was taken from a lab-scale anaerobic CSTR (continuous stirred tank reactor) reactor treating sorghum at mesophilic condition. The total solids (TS) of the reactor was around 30 g/L and the organic loading rate (OLR) of the reactor was 2 g−COD/L/d.Table 1

Characteristics of dried sorghum used in this study (based on raw material weight).

Table 1

Component	Value
C (%wt)	43.59 ± 0.25
H (%wt)	5.66 ± 0.08
O (%wt)	35.76
N (%wt)	0.78 ± 0.09
TS (%)	91.8 ± 0.3
VS (%)	85.8 ± 0.23
Cellulose (%wt)	37.9
Hemi-cellulose (%wt)	23.3
Lignin (%wt)	4.9

TS: total solids.

VS: volatile solids.

C and N content (in % dry weight) of samples from the Alps (n = 272) were analysed using a CHN elemental analyser (Flash EA 1112, Thermo Electron Corporation). A subset of these samples with enough remaining material for further analyses (n = 104) were analysed for P content and an additional set of samples (n = 54) were analysed for both N and P content by colorimetry using a segmented flow analyser after chemical digestion¹⁵ . One set of samples from Fennoscandia (n = 152) were analysed for their C and N content by a CNS elemental analyser (Flash 2000 Organic elemental analyser, Thermo Scientific, UK), one set (n = 59) were analysed for their P content and yet another set of samples (n = 74) were analysed for both N and P content, using the same colorimetric method as for the samples from the Alps. For all chemical analysis the recovery was at least 90% of Certified Reference Material (BCR-129 Institute for Reference Materials and Measurements at the European Commission Joint Research Centre).

For stable C and N isotope analyses, the samples (approximately 0.5 mg: a few individuals of termites and ants from each colony) were placed in folded tin capsules. Stable C and N isotope ratios were measured using a mass spectrometer (Delta^plus XP, Thermo Electron, Erlangen, Germany) coupled with an elemental analyser (Flash EA 1112, Thermo Electron, Erlangen, Germany). The precision of the on-line procedure was better than ± 0.2‰ for both isotope ratios. Natural abundances of ¹³C and ¹⁵N are expressed in per mil (‰) deviation from international standards: δ¹³C or δ¹⁵N = (R_sample/R_standard − 1) × 1000, where R in δ¹³C or δ¹⁵N is ¹³C/¹²C or ¹⁵N/¹⁴N, respectively. Pee Dee Belemnite and atmospheric nitrogen were used as the international standards for carbon and nitrogen, respectively.

Carbon isotope ratio (CIR), calculated as δ¹³C (per mil), can be used to estimate long-term water-use efficiency of leaves in natural vegetation²⁶ . The CIR was measured with an elemental analyzer (Flash EA 1112, Thermo Electron Corporation, Waltham, MA, USA) interfaced to an isotope ratio mass spectrometer (Thermo Finnigan MAT DELTAplusXP, Thermo Electron Corporation, MA, USA) at the Institute of Desertification Studies, Chinese Academy of Forestry (Beijing, China).