Concentrations of metals in the soil samples
As expected, the Pb, Zn, and Cd concentrations in the soil samples increased with increasing distances from the abandoned Pb smelter plant (Table 2). The contamination levels (L1, L2, and L3, respectively) for the metals were 195.43, 1000.58, and 5529.08 mg kg− 1 (Pb); 1.45, 8.00, and 89.30 mg kg− 1 (Cd); and 218.53; 490.68 and 1043.63 mg kg− 1 (Zn). The high metal concentrations found seriously threaten soil quality in the region.
Table 2
Prevention (PV) and investigation values (IV) for metals in two soil use scenarios (R = residential; I = industrial), and Cd, Pb and Zn concentrations in soil samples collected in different sites around the Pb smelter plant in Santo Amaro, Ba state, Brazil
Metal | PV | IV (R; I) | L1 | L2 | L3 |
Cd (mg kg− 1) | 1.3 | 8; 20 | 1.45 | 8.00 | 89.30 |
Pb (mg kg− 1) | 72 | 300; 900 | 195.43 | 1004.58 | 5529.08 |
Zn (mg kg− 1) | 300 | 1000; 2000 | 218.53 | 490.68 | 1043.63 |
The Cd, Pb and Zn contents in the L3 were 446, 140 and 83 times higher than the background concentration of these metals for soils in the region (Dos Santos et al. 2017). These figures are much higher than the prevention and/or investigation values established by the Brazilian resolution for permissible levels of metals in agricultural soils (Conama, 2009). The concentrations of these metals in the soil collected at the shortest distance from the smelter plant had values 70-, 76- and 3.5-fold, respectively, above the prevention value (PV). Even the L1 soil farthest from the waste disposal focus and with the lowest contamination level had a PV for Pb above the allowable concentration. The concentrations for Pb in L2 and L3, and for Cd in L3, were also above the investigation value (IV), which indicates the maximum tolerable concentrations for soils under residential or industrial use (Conama, 2009).
Plant biomass and nodulation
The increasing contamination levels did not affect the jack bean shoots biomass (Table 3). Inoculation of plants with the BR 2811 strain promoted an over 40% increase in shoots dry matter of the plants growing in L1 compared to non-inoculated plants; such an increase was significantly higher than plants inoculated with the BR 7606 strain. On the other hand, the mucuna mean biomass was diminished in L3 and showed no significant inoculation effect with the different strains or contamination levels.
Table 3
Shoots biomass of jack bean and mucuna inoculated with diazotrophic bacteria or non-inoculated cultivated in soil samples with different contamination levels collected in Santo Amaro, Bahia, Brazil.
Shoots biomass | Jack beans |
L1 | L2 | L3 | Mean |
Non-inoculated | 3.69 ab | 5.50 | 3.48 | 4.22 |
BR 2811 | 5.27 a | 4.33 | 3.24 | 4.28 |
BR 3251 | 2.81 ab | 3.10 | 4.46 | 3.46 |
BR 7606 | 1.15 b | 2.87 | 1.99 | 2.01 |
BR 10026 | 3.58 ab | 4.85 | 3.76 | 4.01 |
BR 10247 | 3.16 ab | 2.75 | 2.89 | 2.93 |
Mean | 3.28 | 3.89 | 3.30 | |
Shoots biomass | Mucuna |
L1 | L2 | L3 | Mean |
Non- inoculated | 5.05 | 5.05 | 3.55 | 4.53 |
BR 2811 | 3.38 | 3.89 | 3.92 | 3.73 |
BR 3251 | 3.93 | 3.80 | 3.73 | 3.82 |
BR 7606 | 4.25 | 4.64 | 2.01 | 3.63 |
BR 10026 | 4.78 | 6.05 | 2.93 | 4.59 |
BR 10247 | 4.30 | 4.41 | 4.36 | 4.36 |
Mean | 4.28 A | 4.64 A | 3.42 B | |
L1- lowest contamination level; L2 = intermediate contamination level; L3 = highest contamination level. Means followed by the same capital letters in lines and lowercase letters in columns are not significantly different at 5% probability by Tukey’s test. |
The inoculations did not influence the N concentration in the shoot tissues. All plants of both species produced nodules in all treatments. However, Jack beans had the highest number of nodules in the L2 (intermediate contamination level). In contrast, mucuna nodulation was reduced in the soil with the highest contamination level (Fig. 1).
The ideal plant for phytoextraction use must have the ability to hyperaccumulate metals, preferably in the shoot, tolerance to high concentrations of metals in the soil, rapid growth and high biomass and easy harvesting (Marchiol et al., 2004; Nascimento & Xing, 2006). Jack beans and mucuna are nodulating legumes capable of producing large amounts of aboveground biomass and providing symbiotically fixed nitrogen to systems in short growing cycles (Hauser & Nolte 2002; Dantas et al., 2019), but are still poorly studied in relation to tolerance to heavy metal contamination in Brazilian soils (Melo et al. 2006; Da Silva et al., 2018). Despite the symptoms presented, the two species survived and managed to produce aboveground biomass at the three soil contamination levels (Table 3), demonstrating that they are relatively tolerant to high concentrations of heavy metals in the soils, which qualifies them for phytoremediation programs in the studied soils (Nascimento et al. 2021).
Both jack beans and mucuna nodulated in all treatments (Fig. 2), even at the highest soil contamination level, demonstrating the presence of naturally established rhizobia populations in the contaminated soils. All collected nodules were apparently active (evaluation by reddish color indicative of the presence of leg-hemoglobin), indicating the occurrence of BNF (Uheda & Syono, 1982). As they are tolerant to excess Cd, Pb and Zn, these native bacteria demonstrated competitiveness and efficiency in forming symbiosis with the two legume species, indicating a potential for future isolation and selection of specific strains to recommend inoculation in contaminated areas.
Concentration of metals in plants
The Cd, Pb, and Zn concentrations in the shoots and roots of both plant species increased significantly with the soil contamination levels (Table 3). Lead was more concentrated in roots than in shoots. Inoculation had significant effects on further increasing the concentration of metals in the plants, especially in the highest level of contamination. The BR 7606 strain had the most consistent effect for the two legumes at L3, which had higher Cd, Pb and Zn concentrations in shoots when compared to non-inoculated plants and/or those inoculated with the other strains.
Inoculation at L1 and L2 only influenced the Zn concentration in the jack bean roots, while plants inoculated with the BR 10247 strain had lower concentrations than non-inoculated plants. For mucuna at L1 and L2, the inoculation effect on Zn concentrations was only observed in the shoots, with plants inoculated with BR 3051 and BR 7606 strains having lower Zn concentrations (Table 3).
Our results showed for the first time that although the BR3051 strain does not increase the production of jack bean biomass (Table 3), it promotes an increase in the Cd concentration (Table 4), which may occur due to increased solubility. The jack bean generally concentrated more Cd in the shoots than in the roots, while the mucuna preferentially concentrated Cd in the roots. On the other hand, the immobilization of Cd in plant roots can also be associated with protection mechanisms activated by plants, including protection of the photosynthetic apparatus; in legumes, this mechanism can be used to protect the nitrogenase enzyme (Gómez-Sagasti & Marino 2015; Barba-Brioso et al., 2023). Cadmium normally shows high mobility in soil and plants (López-Millán et al. 2009); in the soils from Santo Amaro, Cd was mainly found associated with organic matter (labile fraction), providing greater potential for Cd availability (Da Silva et al. 2017), which may also have influenced the bioavailability of Cd in the soil. The highest Cd concentrations were observed in the roots of jack beans which received inoculation with the BR 7606 strain in soil samples in L3 (Table 4).
Table 4
Total concentration of Cd, Pb and Zn in shoots and roots of jack bean and mucuna, inoculated with diazotrophic bacteria or non-inoculated, cultivated in soil samples with different contamination levels collected in Santo Amaro (Bahia, Brazil).
Inoculation treatmen | Jack bean | Mucuna | |
L1 | L2 | L3 | L1 | L2 | L3 | |
Cd in Shoots (mg.kg − 1) | |
Non-inoculated | 5.43 aB | 40.92 aB | 244.36 abA | 1.63 aB | 6.09 aB | 90.16 bcA | |
BR 2811 | 8.83 aB | 21.68 aB | 186.86 bA | 2.10 aB | 9.86 aB | 102.59 bcA | |
BR 3051 | 3.25 aB | 13.71 aB | 285.52 aA | 1.24 aB | 5.49 aB | 98.44 bcA | |
BR 7606 | 3.96 aB | 20.51 a B | 189.23 bA | 1.49 aB | 8.34 aB | 183.58 aA | |
BR 10026 | 3.38 aA | 27.75 aA | 2.14 cA | 1.67 aB | 10.82 aB | 84.37 cA | |
BR 10247 | 3.39 aB | 23.45 aB | 223.98 abA | 1.34 aB | 4.33 aB | 124.97 bA | |
| Cd in Roots (mg.kg − 1) | |
Non-inoculated | 1.33 aB | 11.36 aAB | 37.28 bA | 2.36 aB | 10.42 aB | 76.84 bcA | |
BR 2811 | 1.26 aB | 3.85 aB | 65.43 abA | 0.95 aB | 4.18 aB | 121.17 abA | |
BR 3051 | 0.8 aB | 4.45 aB | 67.13 abA | 1.05 aB | 8.41 aB | 92.28 abcA | |
BR 7606 | 1.21 aB | 7.22 aB | 100.11 aA | 1.33 aB | 13.53 aB | 140.85 aA | |
BR 10026 | 0.55 aB | 14.80 aAB | 38.89 bA | 1.30 aB | 13.77 aB | 60.00 cA | |
BR 10247 | 1.34 aB | 4.33 aB | 34. 93 bA | 0.50 aB | 6.13 aB | 77.84 bcA | |
| Pb in Shoots (mg.kg − 1) | |
Non-inoculated | 2.89 aB | 23.72 aB | 127.99 bcA | 0.33 aB | 1.35 aB | 37.30 bA | |
BR 2811 | 13.85 aB | 11.48 aB | 165.10 bcA | 0.63 aB | 2.15 aB | 26.02 dA | |
BR 3051 | 2.29 aB | 6.74 aB | 202.30 bA | 0.25 aB | 1.12 aB | 26.42 dA | |
BR 7606 | 2.58 aB | 11.15 aB | 404.57 aA | 0.37 aB | 1.58 aB | 62.80 aA | |
BR 10026 | 11.35 aB | 16.01 aB | 107.86 cA | 0.35 aB | 1.45 aB | 28.72 bcA | |
BR 10247 | 20.27 aB | 7.91 aB | 161.91 bcA | 0.32 aB | 0.82 aB | 36.37 cdA | |
| Pb in roots (mg.kg − 1) | |
Non-inoculated | 24.21 aB | 319.69 aAB | 695.14 aA | 2.72 aB | 17.68 aB | 292.45 aA | |
BR 2811 | 101.51 aB | 78.9B | 1085.98 abA | 5.11 aB | 9.93 aB | 226.40 abA | |
BR 3051 | 27.70 aB | 47.27 aB | 1062. 41 abA | 2.73 aB | 3.60 aB | 201.32 abA | |
BR 7606 | 14.20 aB | 78.61 aB | 1430.96 aA | 3.68 aB | 8.48 aB | 139.68 bA | |
BR 10026 | 9.60 aB | 254.24 aAB | 448.65 bA | 2.90 aB | 10.77 aB | 202.90 abA | |
BR 10247 | 17.36 aB | 141.3 aB | 714.10 bA | 0.30 aA | 17. 68 aA | 4.5 cA | |
| Zn in shoots (mg.kg − 1) |
Non-inoculated | 148.91 aB | 138.59 aB | 490.11 aA | 27.39 bB | 99.69 aA | 94.18 bA |
BR 2811 | 143.27 aB | 119.96 aB | 460.74 aA | 142.01 aA | 29.21 bB | 131.13 abA |
BR 3051 | 85.83 a B | 67.59 aB | 584.41 aA | 43.53 bB | 16.47 bB | 131.31 abA |
BR 7606 | 111.46 aB | 105.96 aB | 503.99 aA | 41.25 bB | 31.31 bB | 149.74 aA |
BR 10026 | 56.46 aB | 149.36 aAB | 241.63 bA | 19.09 bB | 99.81 aA | 154.94 aA |
BR 10247 | 79.30 aB | 83.48 aB | 550.41 aA | 20.55 bB | 38.89 bB | 179.80 aA |
| Zn in roots (mg.kg − 1) |
Non-inoculated | 27. 39 bB | 99.70 aA | 92.18 bA | 71.21 aB | 160.99 aB | 440.97 abA |
BR 2811 | 142.01 aA | 29.21bB | 131.13 abA | 152.92 aB | 130.91 aB | 507.28 abA |
BR 3051 | 43.53 bB | 16.47 bB | 131.31 abA | 48.07 aB | 35.54 aB | 532.30 abA |
BR 7606 | 41.25 aB | 31.31 bB | 149.74 aA | 82.02 aB | 161.39 aB | 374.01 bA |
BR 10026 | 19.09 bC | 99.69 aB | 154.94 aA | 133.11 aB | 138.16 aB | 571.40 aA |
BR 10247 | 20.55 bB | 38.89 bB | 179.80 aA | 20.78 aB | 201.41 aA | 76.95 cAB |
Means followed by the same lowercase letter in columns and uppercase letter in lines, for each legume species, are not significantly different at 5% probability by Tukey’s test. |
Lead was more concentrated in roots when compared to metal extraction in shoot biomass (Table 4). It was previously verified that Cd and Pb accumulation preferentially occurs in the roots (Yang et al., 2014; Guarino & Sciarrillo, 2017;). The relatively low Pb concentrations in the shoots are due to the low Pb mobility and also its high toxicity to plants, which can reduce the transport of Pb to shoots as a tolerance strategy (Aslam et al., 2021; Steliga & Kluk, 2020). Lead compounds also have low solubility in the soil of Santo Amaro, which is retained in poorly available soil fractions, mainly associated with iron oxides and residual fractions (Da Silva et al. 2017). Mucuna plants which received inoculation with the BR 3051 strain and jack bean plants that received the BR 7606 strain immobilized greatest amounts of Pb, suggesting these strains are able to solubilize Pb from the soil.
The average Zn levels were higher in the shoots than roots (Table 4). As Zn is an essential element, its translocation to shoots is facilitated compared to Pb and Cd. We observed that inoculated jack bean plants were more efficient in accumulating Zn in the root system than plants which did not receive inoculation. It is likely that the presence of bacteria in the soil has altered the rhizosphere of plants, modifying the solubility of heavy metals (Boechat et al., 2017; Zheng et al., 2023); however, more targeted studies of the rhizosphere of plants are needed to elucidate the immobilization of metals in roots. Plant-associated bacteria may also have influenced the Zn translocation in the plant, as they can aid phytoextraction by increasing the transport of heavy metals in plants (Boechat et al., 2017).
Phytoextraction efficiency
The phytoextraction efficiency was estimated based on the net removal of metals from the soil (Table 4). The phytoextraction efficiency followed the metal concentration order in shoot biomass (Pb > Zn > Cd). Cadmium removal by jack beans and mucuna was not influenced by inoculation when cultivated in the soil with lower contamination levels. However, inoculation influenced metal phytoextraction in L3, which increased or decreased efficiency according to the inoculated strain. Inoculation in jack beans with BR 3051 and BR 7606 strains had no effect on Cd removal; interestingly, the other strains caused a lower Cd accumulation in the jack beans than the non-inoculated plants. All inoculants in the mucuna promoted greater net removal of Cd than control, and plants inoculated with the BR 3051 and BR 2811 strains showed significantly greater removals.
The treatment without inoculation showed greater net removal of Pb by the jack bean plants in L2 (Table 4). Yet, the net removal of Pb in L3 was greater in plants with the BR 3051 inoculant. It was also observed that the BR 10026 strain inoculation in the mucuna promoted the greatest net removal of Pb in L2, while inoculated plants differed significantly from non-inoculated plants in the L3 with higher net metal removals. Mucuna inoculations with the tested strains generally caused a Pb removal of up to 46% compared to non-inoculated plants.
Zinc removal increased as the contamination level increased in the soil (L3 > L2 > L1) (Table 4). Mucuna bean inoculation with the BR 10026 strain significantly increased the potential of the legume-rhizobia system in removing Zn from L2, while the BR 2811 and BR 3051 strains were more efficient in L3.
The Cd, Pb and Zn removal was proportional to the increase in the soil contamination levels in both the jack and mucuna (Table 5). However, it is important to note that even with the greatest net removal, soils with very high metal contents can make phytoextraction unfeasible due to the time required to bring the metals to regulatory concentrations (Da Silva et al., 2017). Therefore, the best relationship between net removal, metal concentration in the soil, the most suitable rhizobia strain and plant species must be considered to optimize the process. There were no significant differences in L1 soils in relation to inoculations, which indicates that the bacteria studied can help phytoextraction in soils with higher levels of heavy metals.
Table 5
Net removal of of Cd, Pb and Zn on shoots and roots of jack bean and mucuna plants, inoculated with selected rhizobia strains or non-inoculated, cultivated in soil samples with different levels of contamination collected in Santo Amaro, Bahia, Brazil.
Inoculant | Jack bean | | Mucuna |
L1 | L2 | L3 | | L1 | L2 | L3 |
Cd |
Non-inoculated | 0.01 aB | 0.04 aB | 0.25 abA | | 0.01 aB | 0.01 aB | 0.09 abA |
BR 2811 | 0.01 aB | 0.03 aB | 0.18 bA | | 0.01 aB | 0.01 aB | 0.10 abA |
BR 3051 | 0.01 aB | 0.01 aB | 0.28 aA | | 0.01 aB | 0.01 aB | 0.10 abA |
BR 7606 | 0.01 aB | 0.02 aB | 0.19 bA | | 0.01 aB | 0.01 aB | 0.18 aA |
BR 10026 | 0.01 aA | 0.02 aA | 0.01 cA | | 0.01 aB | 0.01 aB | 0.08 bA |
BR 10247 | 0.01 aB | 0.02 aB | 0.22 abA | | 0.01 aB | 0.01 aB | 0.12 bA |
Pb |
Non-inoculated | 0.01 aB | 0.02 aB | 0.13 bcA | | 0.01 aB | 0.02 bB | 0.43 aA |
BR 2811 | 0.01 aB | 0.01 aB | 0.17 bcA | | 0.01 aB | 0.01 bB | 0.35 aA |
BR 3051 | 0.01 aB | 0.01 aB | 0.20 bA | | 0.01 aB | 0.03 abB | 0.41 aA |
BR 7606 | 0.01 aB | 0.01 aB | 0.40 aA | | 0.01 aB | 0.01 abB | 0.39 aA |
BR 10026 | 0.01 aB | 0.02 aB | 0.10 cA | | 0.01 aB | 0.03 aB | 0.44 aA |
BR 10247 | 0.02 aB | 0.01 aB | 0.16 bcA | | 0.01 aB | 0.01 abB | 0.32 aA |
Zn |
Non-inoculated | 0.15 aB | 0.14 aB | 0.49 Aa | | 0.29 aB | 0.30 bB | 0.48 bcA |
BR 2811 | 0.14 aB | 0.12 aB | 0.46 aA | | 0.25 aB | 0.28 bB | 0.72 aA |
BR 3051 | 00.8 aB | 0.08 aB | 0.58 aA | | 0.24 aB | 0.29 bB | 0.64 abA |
BR 7606 | 0.14 aB | 0.11 aB | 0.51 aA | | 0.26 aA | 0.34 bA | 0.37 cA |
BR 10026 | 0.06 aB | 0.15 aAB | 0.24 bA | | 0.26 aB | 0.46 aA | 0.51 bcA |
BR 10247 | 0.07 aB | 0.08 aB | 0.55 aA | | 0.24 aB | 0.30 bB | 0.75 aA |
Means followed by the same lower case letter, in the columns, and uppercase letters, in the lines, are not significantly different at 5% probability by Tukey’s test. L1- lowest contamination level; L2 = intermediate contamination level; L3 = highest contamination level. |