Element analyzer

The physical–chemical characteristics of soil samples were assessed in accordance with the study by Khan et al. (2022a (link)). The total nitrogen (TN) of the soil was determined using an element analyzer (Thermo Scientific TM, Waltham, MA, USA), and the soil pH was measured using a glass electrode with a soil-to-water ratio of 1:2.5 (Lu, 2000 ). Using sodium bicarbonate, soil-available phosphorus (AP) was collected and quantified using the molybdenum blue method. An Extech Portable Sucrose Brix Refractometer was used to determine the% Brix (Mid-State Instruments, San Luis Obispo, CA, USA). The theoretical yield of sugarcane was calculated using the formula (Pang et al., 2021 (link)):

NH₄⁺ and NO₃⁻ were extracted with 2 M KCl at a soil/extractant ratio of 1:5 after shaking for 60 min at 250 rpm and 25°C (26 (link)). Then the extract was filtered through double loop quantitative filter paper (Whatman, China) and was analyzed on a CleverChem ONE spectrophotometer (Alliance company, France) by extraction with KCl solution—automated method with segmented flow analysis (26 (link), 27 (link)). Available phosphorus was analyzed by spectrophotometric determination after alkali fusion (28 (link)). Total nitrogen, total carbon, total phosphorus, and organic carbon were determined using an element analyzer (ThermoFisher, Germany) (29 (link)). Total phosphorus was determined by spectrophotometric determination after alkali fusion (30 (link)). The activities of leucine aminopeptidase (LAP) and urease were measured by a soil enzyme activity assay kit (Jianglai Company, China). Leucine aminopeptidase breaks down L-leucine p-nitroaniline to p-nitroaniline. The maximum absorption peak of the product was shown at 450 nm (31 (link)). Urea hydrolysis by urease can produce NH₃-N. It can react with sodium hypochlorite and phenol to produce water-soluble blue dye indophenol blue in a strong alkaline medium. The product has a characteristic absorption peak at 630 nm (32 (link)).