The location of our study was a cropland agroforestry field and a nearby forest which are situated nearby Georg August University, Goettingen from September-November, 2022. The type of soil was middle shell limestone which was weathered from Triassic rocks and Quaternary sediments. The annual mean temperature is of 9°C, yearly mean precipitation 628 mm (World Weather Online; browsed on 12th December 2023). Alley cropping constituting poplar (Populus nigra, Populus maximowiczii) and willow (Salix viminalis, Salix schwerinii, Salix viminalis) with a 6m width for tree rows and 24m width for crop rows was adjusted for the site. Forest sites were comprised of poplar on the upper slope, and willow on the lower slope. Five (5) samples were collected from each sample observation site. Agroforestry field was established in 2011 with respect to growing the trees of similar age from forest sites.
In order to determine the physical properties (soil moisture, water-filled pore space, and bulk density), biochemical characteristics (soil pH, inorganic N, microbial biomass C, microbial biomass N, total N), and biogeochemical properties (Nitrogen fixation, greenhouse gas fluxes), soil cores were collected from the upper 15 cm of the A horizon from five randomly selected points, using a PVC core of 50 mm diameter. The soil cores were taken to the laboratory, where composite samples for each plot were sieved (1 mm).
Soil bulk density was measured in 0-15-cm depth using the core method (Blake & Hartge, 1986). Gravimetric moisture content was determined by oven-drying the soil samples at 105°C and expressed as water-filled pore space using the measured bulk density and particle density of 2.65 g cm− 3. Soil pH (H2O) was analyzed in a 1:2.5 soil-to-water ratio. Since the soils have developed on Triassic limestone and contain carbonates (see soil pH in Table 1), the carbonates were removed by acid fumigation prior to analysis of total soil organic C (Harris et al., 2001). Air-dried, ground and acid-fumigated soil samples were analyzed for total C and N using a CN analyzer (Elementar Vario EL; Elementar Analysis Systems GmbH, Hanau, Germany).
Soil microbial biomass C and N in the top 15-cm depth were determined using the CHCl3 fumigation-extraction method. From a soil sample taken at a sampling point, part was extracted immediately with 0.5 M K2SO4 and part was fumigated with CHCl3 for 5 days and then extracted. Organic C in the extracts was analyzed by UV-enhanced persulfate oxidation using a Total Organic Carbon Analyzer (TOC-Vwp, Shimadzu Europa GmbH, Duisburg, Germany). Organic N in the extracts was determined by UV-enhanced persulfate digestion followed hydrazine sulfate reduction using continuous flow injection colorimetry (Method G-157-96; SEAL Analytical AA3, SEAL Analytical GmbH, Norderstedt, Germany). Microbial biomass C and N were calculated as the difference in extractable organic C and N between the fumigated and unfumigated soils divided by kC = 0.45 and kN = 0.68 for a 5-day fumigation period (Brookes et al. 1985). From the soil extracts of the unfumigated soils, NH4+ and NO3− concentrations were determined by continuous flow injection colorimetry (SEAL Analytical AA3, SEAL Analytical GmbH, Norderstadt, Germany), using a salicylate and dicloroisocyanuric acid reaction for NH4+ determination (Autoanalyzer Method G-102-93) and the cadmium reduction method with NH4Cl buffer for NO3− analysis (Autoanalyzer Method G-254-02).
Soil greenhouse gas fluxes were measured using static chamber method. Four gas samples were removed at 2, 12, 22 and 32 minutes after chamber closure and stored in pre-evacuated glass vials (12-mL Exetainer; Labco Limited, Lampeter, United Kingdom). Gas samples were analyzed using a gas chromatograph (SRI 8610C, SRI Instruments Europe GmbH, Bad Honnef, Germany) equipped with a flame ionization detector (for CO2 and CH4 determinations), an electron capture detector (for N2O determination) and an autosampler. Gas concentrations were determined by comparison of integrated peak areas of samples and three to four standard gases (317, 503, 1000 and 2992 ppb N2O; Deuste Steiniger GmbH, Germany). Gas fluxes were calculated from the linear increase of gas concentration in the chamber versus time, and were adjusted for air temperature and atmospheric pressure measured at the time of sampling:
where Φ = flux (g N m− 2 h-1), V = chamber volume (L), A = chamber area (m²), P = atmospheric pressure (Pa), R = ideal gas constant (8.315 Pa m³ mol− 1 K− 1), T = temperature (K), M = molar mass of N2O-N, CH4-C or CO2-C (g mol− 1), δc/δt = rate of gas concentration change within the chamber (ppm h− 1 = µL L− 1 h− 1) and f = conversion factor (10 − 9 m³ µL− 1). Positive gas fluxes indicate emission from the soil; negative fluxes indicate consumption of the gas by the soil.
The effects of different variables were analyzed by means of five replications for each plot type. At first, data were checked for normal distribution (using Shapiro–Wilk tests), and if necessary, further analyses (Tukey HSD) were done for post hoc analysis. The assessment of significant differences between land uses or sites was carried out using analysis of variance (ANOVA) with Tukey’s HSD test (P ≤ 0.05). In order to find a correlation among variables, cor.test were carried out. All the analyses were done using R 4.2.1 (R Development Core Team, 2022).