Description of the Experimental Site
This potted experiment was conducted at the Teaching and Research Farm, Landmark University, Omu-Aran Kwara State, Nigeria, located at Latitude 8° 9′N and Longitude 5° 61′E.
Soil Collection
The soil was obtained from the screen house, Teaching and Research Farm, Landmark University from the top 0 – 15 cm depth. The soil samples were air-dried for 5 days and sieved using 2 mm mesh gauze to remove debris and stones. The sieved soil was sterilized by potting soil mixed with a bit of water into the electrical soil sterilizer and then sterilized for one hour. The soil is then poured out and bagged to weigh 10 kg per pot.
Sources of Materials
Siam seeds were gotten from around the Teaching and Research farm. The Bacteria isolates (Pseudomonas and Bacillus spp.) were obtained from the Entomology lab, Federal Institute of Industrial Research Oshodi (FIIRO), Oshodi, Lagos State, Nigeria. Lead nitrate was obtained from the Chemistry laboratory, Landmark University Omu-Aran, Kwara State Nigeria.
Experimental design and soil treatment application
The experiment was a 3×3 factorial laid out in a Completely Randomized Design (CRD), and each treatment was replicated three times. This consist of soil was polluted at three different levels (10ppm, 20ppm, and 30ppm) and three (3) microbial inoculant (Pseudomonas and Bacillus spp and Non-inoculant). Pollutant was obtained by preparing a stock solution of 1M lead nitrate, and the 10ppm, 20ppm, and 30ppm concentrations were obtained from this stock solution. Each bag of soil was thoroughly mixed in the lead nitrate as expected
Microbial Inoculant Preparation
The bacterial isolates (Pseudomonas and Bacillus subtilis) were cultured for 48 hrs in Luria-Bertani Broth (LB) medium, and then cells were harvested by centrifugation at 12000g for 10 minutes. Each of the Pseudomona aeruginosa and Bacillus subtilis was suspended in sterile distilled water, and the concentration was adjusted to give 108cells/ml
Seed sowing and plant inoculation
All methods were performed in accordance with the relevant guidelines and regulations. Siam seeds were sown one week after the pollution of the soil. Planting was done at 3 seeds per bag. Twenty mls (20 mls) of the prepared microbial inoculants (Pseudomonas and Bacillus spp.) were then introduced into the soil at the plant root two weeks after planting.
Preparation of plant samples for analysis
Methods were performed in accordance with the relevant guidelines and regulations. Twelve (12) weeks after planting, the plant were harvested and carefully rinsed under running tap water to remove clogged soil particles. Water droplets were removed from the plant roots using blotting papers. Then, the plant samples were separated into roots and shoots.
Determination of heavy metal (Pb) in Soil
The soil samples were collected twelve (12) weeks after planting. Five gram (5 g) of soil was weighed into 100 ml plastic bottle. 50 ml of 0.1 m HCl was added and shaken for 30 min. Soil suspension was filtered. Pb content of the soil was determined using Atomic Absorption Spectrometer (AAS) (Model number: AA320N).
Determination of Pb in plant material
All methods followed the relevant guidelines and regulations. [28] acid digestion method was employed to digest grounded plant samples (shoot and root). One g of the plant materia was weighed into a beaker of 50 ml capacity. Then 10 ml mixture of analytical grade acids: NO3;H2SO4; HClO4 in the ratio 1:1:1. Was added. The beakers containing the samples were covered with watch glasses and left overnight. The digestion was carried out at temperature of 70°C until about 4 ml was left in the beaker. Then, another 10 ml of the acid mixture was added. This mixture was allowed to evaporate to a volume of about 4 ml. After cooling, the solution was filtered to remove small quantities of waxy solids and distilled water was added to make up to a final volume of 50 ml. Lead concentrations in the samples were analyzed with AAS (Model number: AA320N)
Determination of Physical and Chemical Properties of Soil of the Study Site.
As shown in table1, the physical and chemical properties measured were; Soil pH using Kent pH meter model 7020, organic matter content using the wet oxidation method as described by [29]. The hydrometer method of [30] was employed to determine particle size. Total nitrogen was estimated by the macro Kjel-dahl method [31], available phosphorus (P) was determined by Bray-1 extraction method [32], and to determine ECEC, the method of [33] was employed.
Determination of bio concentration and translocation factor
Bio-concentration factor (BCF) and translocation factor (TF) were calculated using the formula of [34] and [35] as
Statistical analysis
Data collected were subjected to analysis of variance using SPSS (version 21). Means were separated using Duncan Multiple Range Tests at a significant level of P<0.05