**2.4 Plant harvest and analysis**

At the end of the experiment (90 days), three plants for each treatment were harvested. The rhizosphere soil (RS) and the drilosphere soil (DS) were collected. The *Combined Effects of Earthworms and Plant Growth-Promoting Rhizobacteria (PGPR)… DOI: http://dx.doi.org/10.5772/intechopen.108825*

soil that remained attached to the roots after gentle shaking was collected as rhizospheric soil (RS). Drilospheric soil is earthworm's structure (casts). The remaining bulk soil was the rest after collecting rhizospheric soils and drilospheric soils [23].

Growth parameters such as shoot length, fresh weight, and dry weight of the plants were measured. The height of acacia was measured for each treatment and each replicate. Shoots (leaves and stems) were harvested, and roots were carefully removed from the soil, rinsed with tap water, and washed three times with deionized water; nodules were detached and counted. The fresh weight of plant was determined for each plant part (shoots and roots) and then the plant part was dried at 60°C for 72 h, weighed, and stored for analysis. The total dry weight of biomass (shoots + roots) of each plant per pot was determined. Rhizobial infection was evaluated by counting the number of nodules per plant. All the different soil compartments were air-dried and stored prior to the analyses. The earthworms were hand-collected, counted, and weighed. Ni, Cr, and Pb concentrations in plant shoots (leaves and stems), roots, and the different soil compartments (RS and DS) were dosed by inductively coupled plasma atomic emission spectrometry (ICP-AES, Spectroblue) after total digestion of plant or soil samples.

The ability of the plant to accumulate metals from the soil and transfer metals from the roots to the shoots was estimated by the bioconcentration factor (BCF) and translocation factor (TF), respectively, as described by [3]. BCF is the ratio of the metal concentration in the total plants biomass to that in the soil used to fill into pot experiment. TF is the ratio of the metal concentration in the shoots to that in the roots of plants.

$$\text{Bioconcentration Factor (BCF)}:\text{BCF}\_{\text{ETM}} = \frac{[\text{ETM}] \text{total} \text{Plant biomass } \left(\text{mg/kg dry material}\right)}{[\text{ETM}] \text{soil} \text{std of filled} \text{input} \ (\text{mg/kg dry soil})} \tag{1}$$

$$\text{Translation Factor (TF)}:\text{FT}\_{\text{ETM}} = \frac{[\text{ETM}] \dot{m} \text{Shoots } \left(\text{mg/kg dry material}\right)}{[\text{ETM}] \dot{m} \text{Rotos } \left(\text{mg/kg dry material}\right)}\tag{2}$$

According to [24], plants with both factors (TF and BCF) > 1 are suitable for phytoextraction while, plants with both factors <1 are suitable for phytoimmobilization. Plants with TF > 1 promote the phytoextraction process, while plants with TF < 1 are suitable for phytoimmobilization process [3]. Moreover, plants with BCF > 1 are qualified as a hyperaccumulator [3].

The phytoextraction efficiency (PEE) by *acacia* under different treatments was calculated as suggested in studies [25]:

$$\text{(PEE\%)} = \frac{[\text{ETM}] \text{in plant tissue } (\text{mgkg} - 1) \ge \text{Wplant dry weight } (\text{g})}{[\text{ETM}] \text{in soil } (\text{mgkg} - 1) \ge \text{Wsoil used to fill into } \text{pot}(\text{g})} \text{ x100.} \tag{3}$$

where:

½ � ETM in plant tissue= metal (Pb, Ni or Cr) concentration in plant tissue (mg kg�<sup>1</sup> ).

Wplant dry weight= total plant dry biomass (g).

½ � ETM insoil= metal (Pb, Ni or Cr) concentration in polluted soil for pot experiment (mg.kg�<sup>1</sup> ).

W soil used to fill into pot gð Þ = Weight of soil used to fill the pot (g).
