Cn analyzer

Soil pH was determined by pH meter using 1:1 water:soil suspension (PHS-3C, LIDA, Shanghai, China). EC (µs cm⁻¹), was measured by microprocessor conductivity meter (DDS-12DW, Xiaoshan, China by taking soil and distilled water (w/v) in a 1:5 ratio. Total nitrogen, organic carbon, and C:N of RGS were measured with a CN Analyzer (Vario Max, Elementar, Germany). Soil organic carbon was measured by the oxidation method with 1N K₂Cr₂O₇ and 98% H₂SO₄ solution, followed by titration with 0.2 N FeSO₄ [62 ]. Total nutrients (N, P, K) of RGS were determined colorimetrically by digesting samples in H₂SO₄ and HClO₄, as described by Reference [63 (link)]. Available phosphorous in soil was determined following Olsen et al. [64 ] in a wet digestion through the calorimetric method of molybdenum antimony resistance, and absorbance was taken at 880 nm using a spectrophotometer (UV-VIS spectrophotometer, Model UV-2450, Shimadzu, Kiyoto, Japan). Available K was extracted using a neutral solution of NH₄OAc (1 mol L⁻¹) followed by emission spectroscopy (FP 6410, Shanghai Bante Instrument Co., Ltd., China) [65 ].

The net nitrification rate was determined using a laboratory incubation procedure42 . We collected soil samples in July and analyze the net nitrification rate at the similar temperature as July. Samples (10 g) of field moist soil were incubated in 100 ml polyethylene bottles at 20 °C in the dark for 14 days. The net nitrification rate on a dry mass basis was calculated as the change in NO₃⁻-N between the initial samples and the incubated samples (mg NO₃-N kg⁻¹ soil d⁻¹). Total soil organic carbon (SOC) was determined by a CN Analyzer (Vario Max, Elementar, Germany) using a high temperature combustion method. Soil NH₄⁺-N and NO₃⁻-N were analyzed from filtered 2M KCl-extracts colorimetrically with a continuous flow analyzer (AutoAnalyzer3, BranLuebbe, Germany). Soil pH was measured with a glass electrode in a 1:2.5 soil/water suspension43 .

Lake water pH was measured in triplicate in situ by pH probe V3.0 (Atlas Scientific) at the time of sampling. Concentrations of major cations and anions ( F⁻, Cl⁻, NO₂⁻, SO₄²⁺, Br^-, NO₃⁻, Li⁺, Na⁺, and K⁺) were measured using ion chromatography (Dionex, ICS-2100). Total organic carbon (TOC) and total nitrogen (TN) were measured by CN Analyzer (Vario Max CN, Elementar, Germany). GPS coordinates were recorded at each sampling point, ranged from 28° 58′ 29.53″ N to 31° 49′ 19.45″ N and 85° 44′ 57.78″ N to 91° 06′ 49.54″ N (Additional file 1: Table S1). The mean of lake water temperatures for the month (August 2015) prior to sampling dates was extracted from public data [23 (link)].

During both days in the chamber, 24-hour urine was collected for the analysis of urinary nitrogen excretion as an estimation of protein metabolization (Vario Max, CN-analyzer, Elementar Analysesysteme GmbH). Urine bottles were prepared with 10 mL hydrochloric acid (4 mmol/L) to prevent nitrogen degradation. Nitrogen levels were then multiplied by 6.25 to calculate the daily protein intake (g/day).