Bacterial culture collection
The pure culture of bacterial strain Pseudomonas aeruginosa (ABPL-251), obtained from the Agricultural Biotechnology and Phytopathology Laboratory, Department of Botany, University of Karachi, was used later in the experiment. The bacteria were grown using King's B broth media. This broth was incubated for ten days and had attained a cell density of approx. 8x108cells/mL.
A screen house, made at the Department of Botany, the University of Karachi, experimented in the clay pots of 10 cm diameter having a capacity of 1 kg soil with pH = 7.2 and EC= 4mS/m in September-November. Before sowing, the pH of amended soil was 7.6, and electrical conductivity was 5.2mS/m. Neemex powder (@1%) was mixed in the soil seven to ten days before sowing and was kept moist. A local market provided the sunflower seeds (Helianthus annuus L.) that were held on for 2-3 minutes in 1% sodium hypochlorite solution for surface sterilization and afterward washed thrice with distilled water and then air-dried. Ten seeds in each pot were sown, drenched with 25 mL broth of P. aeruginosa to respective treatment banks, and 25 ml of distilled water for control. After a week of germination, four seedlings per pot were treated with different concentrations (0.2mM and 0.5mM) of lead by using lead nitrate salt [Pb(NO3)2]. When the seedlings attained four leaves stage (two weeks of sowing), lead treatment started using lead nitrate solutions via irrigation in the morning. They were treated on alternate days during the first week but later treated daily till the 45th day. Each treatment was replicated four times for the study, and the treatments included were:
Ps= P. aeruginosa inoculation in soil
N= Neem cake amendment in soil
Ps+N= Neem cake amendment + P. aeruginosa inoculation
0.2 mM= 0.2 mM Pb stress
0.5 mM= 0.5 mM Pb stress
Ps+0.2 mM= P. aeruginosa inoculation + 0.2 mM Pb stress
Ps+0.5 mM= P. aeruginosa inoculation + 0.5 mM Pb stress
N+0.2 mM= Neem cake amendment + 0.2 mM Pb stress
N+0.5 mM= Neem cake amendment + 0.5 mM Pb stress
Ps+N +0.2 mM= Neem cake amendment + P. aeruginosa inoculation + 0.2 mM Pb stress
Ps+N +0.5 mM= Neem cake amendment + P. aeruginosa inoculation + 0.5 mM Pb stress
The replicated data of the fresh weight and root and shoot lengths of all treatments were noted to demonstrate their growth, then oven-dried at 80°C for 48 hours to obtain dry weights of plants.
Determination of Phytoremediation of Lead
The capacity of Pseudomonas aeruginosa to immobilize lead and reduce its bioavailability in a liquid medium was determined by culturing LB broth medium (50 mL), incubated for 60 minutes in a rotary shaker at 150 rpm (7.0 pH and 37°C) (Marzan et al. 2017). After achieving the optical density of 0.6 at 600 nm, 200 ppm Pb solution was added using [Pb (NO3)2] salt in a flask and then incubated for 24 hours. The culture was then centrifuged at 2000 g for 15 minutes to separate bacterial colonies and media. The pellet discarded and supernatants collected were mixed with double volume of concentrated HNO3 to carry out acid digestion of the lead present in the supernatant. The mixtures were heated on a hot plate to reduce them to the initial volume. The lead concentration was analyzed using atomic absorption spectrophotometer. Phytoremediation of lead by P. aeruginosa, was quantified and compared with control by the formula
Phytoremediation (%) = (Lead in broth - Lead in culture) / (Lead added to broth) × 100
Estimation of carbohydrates
Anthrone reagent determined carbohydrates content (Hamid et al. 2011). Dried leaf samples were homogenized in ethanol ratio (1:10) and centrifuged at 1300 g for 10 minutes. The 1 mL supernatant with 3 mL of Anthrone reagent (prepared by mixing 0.2 g in 100 mL conc. H2SO4) was kept in a water bath for 30 minutes. The cooled contents were then read at 680 nm using a spectrophotometer. The distilled water and anthrone reagent made the control. A calibration curve of carbohydrate content (µg/mL) was prepared by glucose as a standard.
Determination of protein
The Bradford method (1976) determined the protein content. The leaf material was extracted using phosphate buffer (0.1 M with pH 7) and centrifuged at 1300 g for 10 minutes. For estimation, 1 mL chilled extract and 5 mL Bradford assay reagent were mixed, and the optical density was read at 595 nm, taking Bradford and phosphate buffer as blank. BSA (Bovine Serum Albumin) was used as a standard.
Total phenolic content
Gallic acid was used as a standard to estimate total phenolic content (Rahman et al. 2017). Leaf aliquots (0.1 mL) mixed with 2 mL 2% Na2CO3 were left at room temperature. After 2 minutes, 50% Folin-Ciocalteau Phenol reagent (0.1 mL) was added and then kept the samples in darkness for 30 minutes at 720 nm optical density.
The ascorbic acid was extracted and analyzed as described protocol (Sofy et al. 2020). To estimate ascorbic acid, 1 ml leaf extract and 2 mL 2, 4 dinitrophenyl hydrazine (2% in concentrated H2SO4) were mixed. One drop of 10% thiourea (1 g in 10 mL distilled water) was prepared and kept in a water bath for 15 minutes. After chilling, 5 mL 80% H2SO4 was added and read at 530 nm by a spectrophotometer.
Peroxidase activity was estimated as described by Reddy et al. (1995). 20% homogenate was prepared in 0.1 M phosphate buffer with 6.5 pH to extract the enzyme. 0.1 mL enzyme extract was made with 3 mL of 0.05 M pyrogallol solution to determine peroxidase activity. 0.5 mL of 1% H2O2 solution (made in 0.1 M potassium phosphate buffer) was added to the test solution, and the absorbance was read every 30 seconds up to 3 minutes at 430 nm.
Percentage of H2O2 scavenging activity
Leaf aliquot (10 mg/mL) and 0.6 mL of 40 mM H2O2 solution (prepared in 0.1 M phosphate buffer of 7.4 pH) were mixed, and phosphate buffer was applied to obtain the final volume of 3 mL to determine H2O2 scavenging activity (Ruch et al. 1989). A blank test tube was prepared without H2O2, and O.D was measured at 230 nm. The following formula calculated the percent scavenging activity of H2O2.
H2O2 scavenging activity (%) = (A0 – A1) / A0 × 100
A0 = Absorbance of control
A1 = Absorbance of plant extract
For statistical analysis, one-way ANOVA means triplicated data, and standard errors were performed using the program COSTAT. The mean values are of significant difference by LSD at p<0.05.