Vanadate molybdate reagent

Microbial biomass was determined by vacuum filtration of the samples collected at the end of each repeated batch cycle, washed with distilled H₂O, and then dried at 70 °C to a constant weight. The immobilized biomass in the polyurethane foam cubes was estimated from the difference between the total carrier cell concentration and the initial weight of the carrier. Glucose concentration was measured by enzymatic test combination (R-Biopharm, Darmstadt, Germany). The concentration of soluble phosphate was determined by the molybdo-vanado method using vanadate molybdate reagent (Sigma-Aldrich Cat. No 94686). pH was measured by pH-meter Mettler Toledo FiveEasy F20. All fermentation experiments were performed in triplicate (3 flasks per treatment) and values were analysed and presented as mean ± standard deviation.
Shoot and root dry weights were recorded after drying at 70 °C. Shoot P content was determined by the molybdo-vanado method described by Lachica et al. (1973 ). Soil–plant experiments were arranged in completely randomized designs with four repetitions. Data were submitted to analysis of variance and treatment means were compared using the Tukey test (P < 0.05).

Phosphorus (P) uptake was analyzed according to a modified method after Goehler and Drews (1978) , Ehrenberger (1991) (link), and Ghanem et al. (2014) (link). Dried and pulverized randomly selected samples of shoot tissues (250 mg per sample) were subjected to an acid digestion (adding 2 mL of 65% HNO₃ directly to the sample with 2 mL of 30% hydrogen peroxide and 5 mL H₂O placed outside the sample vessel) at 200°C for 15 min in a microwave (μPREP-A microwave system, MLS GmbH, Leutkirch, Germany). The digested solution was diluted to 50 mL with deionized water. To an aliquot of 5 mL of the sample solution, 10 mL of vanadate–molybdate reagent (Sigma-Aldrich, Steinheim, Germany) was added. After 5 min incubation, P concentration was determined at 440 nm using a V-550 spectrophotometer (Jasco GmbH, Grossumstadt, Germany). Results were calculated in mg P × g^-1 dry weight.