Uv vis 1601 pc

To acquire the rhizosphere, the dug roots were progressively stirred to separate them from the bulk soils. Measurements were made of the concentrations of phenolic, oxalic and citric acids [38 (link),39 (link)]. Organic acids (citric and oxalic acids) were gathered in 0.1% phosphoric acid that also contained butylated hydroxyanisole, using ribitol as an internal reference. Filtrates were used for HPLC quantification using a LaChrom L-7455 diode array (Merck-Hitachi, Barcelona, Spain), as described by de Sousa et al. [40 (link)], following centrifugation. According to Zhang et al. [41 (link)], the phenolic concentration was calculated using spectrophotometry (Shimadzu UV-Vis 1601 PC, Kyoto, Japan).

After gently shaking the roots, 10 g of rhizosphere soil was swirled in double distilled water. The total phenolic content of the soil extract was determined spectrophotometrically using Zhang et al. (2006) (link) technique. Citric acid was determined by HPLC, as described by De Sousa et al. (2016) (link). According to Zhang et al. (2006) (link), ten grams of rhizosphere were forcefully mixed in distilled water, and the filtrates were used to spectrophotometrically assess the phenolic content (Shimadzu UV-Vis 1601 PC, Japan). To measure the negative effect of heavy metals on microbial population in the soil, ten grams of each soil sample was added to 90 mL of 0.1% (w/v) solution of sodium pyrophosphate. After homogenization for 30 min, this solution was decimally diluted (10⁻¹ to 10⁻⁷) and aliquots of the resulting solutions were plated on appropriate culture media. After incubation at 25 or 30°C, for up to 10 days, the colony-forming units (CFUs) were counted.

Chemical properties of the soil (pH and electrical conductivity) were measured in diluted aqueous soil extract (1:5 w/v) using a pH meter (AD 3000) and a conductivity meter (Jenway 3305), respectively. Following Zhang et al. [31 (link)]), the phenolic content was estimated spectrophotometrically (Shimadzu UV-Vis 1601 PC, Kyoto, Japan). Twenty grams of soil were stirred vigorously with bi-distilled water followed by filtration and the filtrate was used for estimation of phenolic content. The content of mineral nutrients in soil rhizosphere were determined by shaking the excavated root very gently to separate from the bulk soil; then, the soil attached to the fine roots (2 mm thick layer) was obtained by brushing. About 20 g of soil rhizosphere were extracted in aqueous HNO₃ solution (80%) and were detected by using ICP-MS (Finnigan Element XR, Scientific, Beremen, Germany) [32 ]. A total of 20 g of soil was mixed in a flask with 100 mL of distilled water and shaken for 12 h. Then, the soil extract was filtered and used to determine organic acid and phenolic compounds [33 (link)]. The percentage of CaCO₃ was estimated according to Brown et al. [34 (link)]. The contents of carbon (C) and nitrogen (N) were measured by CN element analyzer (NC-2100, Carlo Erba Instruments, Milan, Italy).