**3.4** *P. corethrurus* **earthworm and** *A. mangium* **interaction on phytoremediation processes**

Previous studies have been reported the interactive role of earthworms in improving plant growth in non-contaminated soils [26]. In addition to this, the benefit effect of earthworms in the remediation of metal contaminated soil has been very well demonstrated in numerous research studies [5, 27, 31, 33, 44], but only few studies have been conducted to assess their role in improving plant metal uptake during phytoremediation in contaminated soils [45].

Our findings have shown that the inoculation of acacia with *P. corethrurus* resulted a highly to very highly significant increase (P < 0.01) in plant height, total dry weight biomass, and metal concentration in plant biomass (**Figure 2**), as compared with the uninoculated treatment. Thus, acacia appeared to exhibit rapid growth and high biomass production when earthworms were present. For instance, the phytoextraction efficiency of the plant inoculated with *P. corethrurus* was enhanced by fivefold for Cr, twofold for Ni, and sevenfold for the Pb, as compared with noninoculated plant (**Table 4**). This increase of growth-stimulating and of the amount of Cr, Ni in *A. mangium* biomass could be caused by the earthworms through their burrowing and casting activities, as suggested by [31], because the earthworms can facilitate metal conversion from the stable to the available form by changing physicochemical and biological status of the soil such as soil pH decreases, production of organic acids, and stimulation of microbial activity, contributing to the increase of Ni, Pb, and Cr availability in soil and as a result increased their bioavailability for plants. Furthermore, the increase of metal accumulation in plant biomass could be due to the interactive action between *A. mangium* and *P. corethrurus.* In fact, some species such as *Acacia* secrete different types and quantities of organic acids into the rhizosphere [46], which were the main source of organic matter used by *P. corethrurus* earthworms in the rhizosphere, according to [13]. So, by decomposing different types of root exudates and organic acids secreted by *A. mangium* into the rhizosphere, P*. corethrurus* can probably reduce the stable form of metal while increasing its mobile form in the rhizosphere, enhancing Cr and Ni bioavailability for plant [31]. However, the higher significant (P < 0.05) content of Pb in plant non-inoculated than in plant inoculated with earthworm (**Figure 2**) could be attributed to the metal speciation in rhizosphere or drilosphere [44]. justified the decrease of Pb concentration in plant inoculated with earthworm, as compared with earthworm inoculation, by the fact that earthworm can also reduce the amount of Pb associated with the soluble and exchangeable fraction and subsequently plant uptake.

In addition, the higher content of Cr (29 mg.kg<sup>1</sup> ) and Pb (4.9 mg.kg<sup>1</sup> ) in RS and Ni in DS (5.6 mg.kg<sup>1</sup> ) (**Table 3**) than in plant biomass could be related to the physiological character of *Acacia* species, which here seems to exclude a metal in its shoot tissue as demonstrated in previous studies [3, 4, 39, 46].

These results suggest that, although earthworms have the potential to improve the efficiency of plant phytoremediation in metal-contaminated soils, its effectiveness depends on the nature of the plant, its behavior toward metals, metal speciation in soil, rhizosphere function involved in the phytoremediation process.

### **3.5 Bradyrhizobium and** *A. mangium* **interaction on phytoremediation processes**

Symbiosis between leguminous and rhizobacteria improves plant growth, nutrition and reduces the stress of plants, facilitating their development in

metal-contaminated areas [47, 48]. Previous studies, specially studies from symbiotic microorganism, have demonstrated that rhizobia contribute to plant adaptation to multiple biotic and abiotic stresses, especially under metal-contaminated soils [41, 47, 49]. Among the rhizobacteria obtained from areas contaminated with different metals, there are strains of the genus *Rhizobium* sp., *Sinorhizobium, Mesorhizobium*, *Bradyrhizobium,* and *Azorhizobium* [48]. These strains are recognized as plant growth-promoting rhizobacteria [50].

Our findings have shown that the inoculation of acacia with *Bradyrhizobium* (T2) resulted a highly to very highly significant increase (P < 0.01) in plant height, total dry weight biomass, and metal concentration in plant biomass (**Figure 2**), as compared with the non-inoculated plant. Thus, acacia appeared to exhibit rapid growth performance (seven-fold) when *Bradyrhizobium* was present. This growth stimulation could be attributed to the interactive action between *A. mangium* and with symbiotic rhizobia such as *Bradyrhizobium,* which have the capacity to form symbiotic association with *A. mangium.* and consequently influence positively plant P nutrition and growth and then soil microbial activities [51, 52].

Furthermore, positive effects from inoculation with *Bradyrhizobium* on metal uptake by *A. mangium* in metal-contaminated soil have been observed. The inoculation of *A. mangium* with *Bradyrhizobium* (T2) increased very highly significant (P < 0.001) Cr, Ni, and Pb amounts by four-, three-, and twofold, respectively, as compared with the control treatment (T0). In addition, the phytoextraction efficiency of the plant inoculated with *Bradyrhizobium* was enhanced by 32-fold for Cr, 4-fold for Ni, and 8-fold for the Pb, as compared with non-inoculated plant (**Table 4**). This positive effect may attribute to *Bradyrhizobium* Sp., which can increase the availability of soil metal through the production of metal chelating, agents siderophores, and organic acid [47, 53], and can also modify heavy metals speciation and metal/organic matter interaction by transformation of organic compounds [42], consequently increasing their bioavailability for plants. Ours findings showed that *Bradyrhizobium* effectively enhances *A.mangium* growth, its metal uptake, and also their accumulation in root than shoot tissues. Ours results indicated also that *Bradyrhizobium* improves metal bioavailability in soil and subsequently for plant. So, according to [47], this is possible because *Bradyrhizobium* can decrease the toxicity of metal contamination in plant by transforming pollutants into nontoxic or less toxic form and also by enhancing antioxidant defense in plants exposed to metal-contaminated soils. Similar reports also demonstrated that the inoculation with *Bradyrhizobium* higher increases Cu concentrations in *soybean* and especially in white *lupin* in inoculated plants [53, 54], showed that *Methylobacterium* sp. notably enhances the bioaccumulation of As in *Acacia farnesiana* biomass mainly in shoots. In contrast [55], showed that *Bradyrhizobium* Sp. reduced Ni and Zn uptake by Greengrass plants, which was probably due to the ability of *Bradyrhizobium* to protect plants against the inhibitory toxic effects of Ni and Zn.

Ours results demonstrated that under inoculated with *Bradyrhizobium* treatment (T2), Cr, Ni, and Pb amounts were very highly significant (P < 0.001), 8, 10, to 15-fold greater in roots tissue than in shoot tissue (**Figure 3**). Likewise, the bioaccumulation factors (BCFs) and the translocation factors (TFs) of Cr, Ni, and Pb, which were < 1 respectively, revealed that the presence of *Bradyrhizobium* improved better the uptake of Cr and Ni mainly in roots. While for Pb, the presence of *Bradyrhizobium* improved Pb accumulating in roots. The presence of *Bradyrhizobium* modified the phytoextractor potential of non-inoculated plant to act as Pb phytoexcluders. Thus, in the presence of *Bradyrhizobium*, *A. mangium* is considered to *Combined Effects of Earthworms and Plant Growth-Promoting Rhizobacteria (PGPR)… DOI: http://dx.doi.org/10.5772/intechopen.108825*

have great potential for the phytoimmobilization of Cr, Ni, and Pb. A similar effect has been observed by [56], which after inoculation with *Cupriavidus taiwanensis*, *Mimosa pudica* showed higher capacity of metal uptake and improved Pb, Cu, and Cd accumulating mainly in roots.

These findings supported the ability of *Bradyrhizobium* to protect *A. mangium* plants against the inhibitory toxic effects of Ni, Cr, and Pb, as demonstrated by [57].

### **3.6** *P. corethrurus* **earthworms and Bradyrhizobium interactions on Pb, Ni, and Cr uptake by** *A. mangium*

Earthworms and rhizobacteria are essential for nutrient cycling and organic matter dynamics in terrestrial ecosystems. In soils, they tightly interact especially in the rhizosphere. In our experiment, we tested the effects of earthworm *P. corethrurus* and *Bradyrhizobium* on the growth performance of *A. mangium* and also its metal uptake in metal-contaminated soil. We observed a significant (P < 0.05) positive effect of both inoculants (earthworm *P. corethrurus* and *Bradyrhizobium*) on *A. mangium* growth and total biomass, as compared with plant non-inoculated and also with plant inoculated with earthworm *P. corethrurus* or *Bradyrhizobium* only (**Figure 2**), as demonstrated in numerous reports [26, 51, 52]. This growth stimulation in the presence of both inoculants could be related to [1] earthworm *P. corethrurus* activity, which by increasing the mineralization of soil organic matter enhances nutrient availability, stimulates microbial activities [2]; the production of plant growth regulator substances through the stimulation of microbial activity [3, 58]; the stimulation of plant symbionts in the soil rhizosphere [4]; the plant genotype *A.mangium,* a leguminous, which used as symbiont *Bradyrhizobium* recognized as a plant growth-promoting bacteria (PGPR) [50], and [4] to the bio-control of metal stress by earthworm [27, 31, 33, 44] and by *Bradyrhizobium* [41, 47, 48, 59] only and also by the combined action with both inoculants [45]. This synergistic interaction is probably due to the stimulation of plant growth-promoting rhizobacteria, such as *Bradyrhizobium*, population in the presence of earthworms [60].

Furthermore, a significant (P < 0.05) greater amounts of Cr, Ni, and Pb in total biomass of plant have been observed in the presence of both inoculants, which was increased by 2–10-fold for Cr, 2–3-fold for Ni, and 2–4-fold for the Pb, as compared with non-inoculated and individual inoculated plants (**Figure 2**). Likewise, the phytoextraction efficiency of the plant inoculated with both inoculants was enhanced by 2–9-fold for Cr, 2–3-fold for Ni, and 2-fold for the Pb, as compared with individual inoculation (**Table 3**). This increase could be attributed to the combined activities of the two inoculants that have the ability to enhance metal uptake in plant tissues and to protect plants against toxic effects have been demonstrated in previous studies [31, 42]. The improvement of metal uptake by *A. mangium* inoculated with both inoculants, as compared with its inoculation with *Bradyrhizobium* or earthworm individually, could be linked to the relationship between earthworm and rhizobacteria (*Bradyrhizobium*). This finding suggests that combined inoculants consisting of *Bradyrhizobium* and earthworms have potential for enhancing metal uptake by *A. mangium,* confirming our hypothesis. The results indicate that metal uptake by this tolerant plant species was greatly facilitated by the interactions among these organisms, most likely due to the concomitant stimulation of metal immobilization and biomass production, as demonstrated by [13, 42].

In the presence of both inoculants (*P. corethrurus* earthworms and *Bradyrhizobium*), *A. mangium* preferentially accumulated Pb, Cr, and Ni in the roots than in shoots tissue (**Figure 3**) with TF < 1 (**Table 3**) and BCF < 1, indicating that for Pb, Ni, and Cr, *A. mangium* promotes the phytoimmobilization process.

In addition, the content of Cr was higher in the DS compartment (23 mg.kg<sup>1</sup> dry soil) than in the RS (12 mg.kg<sup>1</sup> dry soil) in the presence of both inoculants (earthworms and *Bradyrhizobium*) (**Table 4**). Our results also showed that the concentration of Cr was higher in the root (12 mg.kg<sup>1</sup> ) than in the shoot tissue (0.7 mg.kg<sup>1</sup> ). This finding suggested that Cr mobilized in the DS compartment (23.2 mg.kg<sup>1</sup> dry soil), was preferentially transferred to RS compartment and then to the plant root tissue. While, the content of Pb and Ni was significantly higher in the RS compartment, ranging from 16.4 and 12.7 mg.kg<sup>1</sup> dry soil, respectively (**Table 4**), than in the DS compartment (range 3–4 mg.kg<sup>1</sup> dry soil). Despite the highest content of Pb and Ni in the RS compartment, the concentrations of Pb and Ni were lower in the plant shoot biomass. The translocation of Pb and Ni from the root to the shoot tissue was weak. This phenomenon could be linked to the behavior of *A. mangium,* which, in the presence of both bioinoculants, behaved as Pb, Cr, and Ni-excluder plant and promoted the phytoimmobilzation process for Cr, Pb, and Ni.

Efficiency of the different phytoremediation treatments applied.

Our results showed that inoculation of *A. mangium* with *Bradyrhizobium* or earthworms only and with both inoculants significantly increased (P < 0.05) in the height (twofold), total dry biomass weight (7–15-fold), and metal uptake of the plant (2–10 fold), as compared with the non-inoculated plant. However, the presence of *Bradyrhizobium* and earthworms increases twofold the total plant biomass and two- to fivefold metal accumulation in plant biomass, as compared with inoculated with earthworms or *Bradyrhizobium*.

Furthermore, irrespective of the heavy doses, the phytoextraction efficiency (PEE) percentage rank was in the order T3 > T2 > T1 > T0 (**Table 3**). The PEE percentage of *A. mangium* increased significantly in the presence of earthworms and *Bradyrhizobium*, demonstrating values of 18% for Pb, 9% for Cr, and 12.6% for Ni, followed by T2 (when *A. mangium* was inoculated with *Bradyrhizobium* only) with 8% for Pb, 6.4% for Cr, and 6% for Ni and by T1 with 7% for Pb, 1% for Cr, and 5% for Ni (**Table 3**). We found strong evidence that the inoculation of plant with PGPR and earthworm enhanced soil Pb/Ni/Cr mobility and bioavailability in metalcontaminated soil, facilitating their transfer and absorption by plant.

This result indicated that the phytoremediation capacity of *A. mangium* was improved in response to the inoculation and optimally improved in the presence of both inoculants. So, our finding reveled that it is possible to use the combination of metal-tolerant plant and soil organisms (*Bradyrhizobium* and earthworms) as a potential bioaugmentation tool to accelerate metal phytoremediation efficiency in metalcontaminated soils.

### **4. Conclusion and recommendation**

Beneficial effects of combined inoculation with *P. corethrurus* earthworms and *Bradyrhizobium* and of individual inoculation with *P. corethrurus* earthworms or *Bradyrhizobium* on A. mangium growth and its Pb, Ni, and Cr uptake in metalcontaminated soil have been observed in this study. Ours results revealed that the concomitant stimulation of metal immobilization and biomass production in the presence of these organisms and also that the inoculation of plant with PGPR (*Bradyrhizobium*) and earthworm enhanced soil Pb/Ni/Cr mobility and bioavailability *Combined Effects of Earthworms and Plant Growth-Promoting Rhizobacteria (PGPR)… DOI: http://dx.doi.org/10.5772/intechopen.108825*

in metal-contaminated soil, facilitating their transfer and absorption by plant. However, the growth stimulation and the metal accumulation in plant were increase twofold for the total plant biomass and two- to fivefold for metal amount in plant biomass, as compared with inoculated with earthworms or *Bradyrhizobium*. In addition, the presence of these organisms promoted the phytoimmobilization process of Ni, Cr, and Pb preferentially in *A. mangium* roots than in shoot tissue. Our experiments highlight the importance of soil organisms on the phytoremediation efficiency. It appears that earthworms and/or PGPR (*Bradyrhizobium*) have the potential to enhance the phytoextraction efficiency of plants in metal-contaminated soil.
