Phytoremediation abilities of maize (Zea mays L.) inoculated with plant growth promoting rhizobacteria in Zinc and Cadmium contaminated soils Helena Moreira , Ana P. G. C. Marques , António O. S. S. Rangel , Paula M. L. Castro CBQF – Centro de Biotecnologia e Química Fina, Escola Superior de Biotecnologia, Centro Regional do Porto da Universidade Católica Portuguesa, Rua D. António Bernardino de Almeida, 4200-072 Porto, Portugal hgmoreira@porto.ucp.pt O etalsbacteria Introduction Methodology Results and Conclusions Acknowledgements Heavy metal contaminated soils are a worldwide problem. Efforts to reduce their high impact by using sustainable and low-cost strategies such as phytoremediation may be a promising path in remediation techniques. Maize (Zea mays L.) is a crop that grows widely throughout the world with important attributes to be considered a plant suitable for this purpose such as:  high biomass yield per hectare;  quick, vigorous and tall (2-3 m) growing cereal capable of continuous phytoextraction of metals from contaminated soils;  accumulator and tolerant for Cd and Zn.. Plant stress associated with phytoremediation strategies can be reduced by plant growth promoting rhizobacteria (PGPR). The use of these bacteria may increase the maize performance concerning its overall economical aspects, such as an increase of biomass production, that can be used for the generation of energy. Ralstonia eutropha (B1) and Cryseobacterium humi (B2) are PGPR and metal resistant rhizobacteria isolated from a metal contaminated site and showed to be able to enhance biomass and growth production in maize by up 360 and 47 % respectively, in previous experiments. The aim of the present work was to assess the influence of the inoculation with selected PGPR on the biomass production and metal accumulation by Zea mays in Zn and Cd contaminated soils. 0 1 2 3 4 5 6 7 8 Cd 0 Cd 10 Cd 20 Cd 30 (g) [Cd] soil (mg.Kg -1 ) Root Biomass B0 B1 B2 a a a,b b a b a a a b a b Cd Zn Substrate Zea mays seeds Inocula This work was supported by Fundação para a Ciência e a Tecnologia and Fundo Social Europeu (III Quadro Comunitário de apoio), research grants of Helena Moreira (SFRH/BD/64584/2009) and Ana Marques (SFRH/BPD/34585/2007) and by National Funds through FCT – Fundação para a Ciência e Tecnologia under the project PEst-OE/EQB/LA0016/2011. Table 1: Cd accumulation in roots and shoots of Zea mays Zn (mg kg -1 ) Treatment Roots Shoots No bacteria B1 B2 No bacteria B1 B2 0 87 ± 11 a 35 ± 4 b 75 ± 5 b 18 ± 1b 24 ± 2 a 18 ± 1 a 100 260 ± 17 b 336 ± 45 a 338 ± 41 b 695 ± 82 b 836 ± 59 b 968 ± 102 a 500 809 ± 55 a 805 ± 92 a 820 ± 113 a 430 ± 47 a 415 ± 43 b 422 ± 52 b 1000 2225 ± 304 a 1653 ± 127 b 2143 ± 159 a 640 ± 61 a,b 565 ± 61 b 681 ± 39 a Zn and Cd accumulation in plant tissues – shown in tables 1 and 2 - and dry biomass - shown in figures 2 to 5 - were determined in order to infere on the influence of the bacterial inoculation and degree of metal contamination on plant development and remediation capacities. Figure 5: Shoot biomass in plants exposed to Zn; results are shown as means ±S.D (n=4). Means with different letters in concentration are significantly different from each other (P < 0.05) according to the Duncan test. Figure 3: Shoot biomass in plants exposed to Cd; results are shown as means ±S.D (n=4). Means with different letters in concentration are significantly different from each other (P < 0.05) according to the Duncan test. Figure 2: Root biomass in plants exposed to Cd; results are shown as means ±S.D (n=4). Means with different letters in concentration are significantly different from each other (P < 0.05) according to the Duncan test. Cd was not detected in plants grown in control soil, therefore treatment was omited from the table. Results are shown as means ±S.D (n=4). Means with different letters in each plant line are significantly different from each other (P < 0.05) according to the Duncan test. - Roots: in general bacteria increased root biomass, however this was not always significant; - Shoots: Bacteria generally had do influence on shoot biomass production; - There was no trend for bacterial influence on Zn accumulation at the roots and shoots. Table 2: Zn accumulation in roots and shoots of Zea mays Results are shown as means ±S.D (n=4). Means with different letters in each plant line are significantly different from each other (P < 0.05) according to the Duncan test. Figure 4: Root biomass in plants exposed to Zn; results are shown as means ±S.D (n=4). Means with different letters in concentration are significantly different from each other (P < 0.05) according to the Duncan test.. -Roots: bacteria generally increased roots biomass, but had no influence on Cd accumulation ; - Shoots: in general bacteria decreased Cd accumulation in shoots and had no influence on its biomass. Cd (mg kg -1 ) Treatment Roots Shoots No bacteria B1 B2 No bacteria B1 B2 10 52 ± 2 a 25 ± 6 b 71 ± 23 a 21 ± 4 a 6 ± 2 b 4 ± 1 b 20 80 ± 6 a 82 ± 6 a 82 ± 7 a 32 ± 3 a 30 ± 3 a 21 ± 2 b 30 122 ± 11 a 131 ± 13 a 134 ± 11 a 88 ± 5 a 81 ± 5 b 82 ± 1 a,b 0 1 2 3 4 5 6 7 8 9 10 Cd 10 Cd 20 Cd 30 Bfroots [Cd] soil (mg.Kg-1) Bioconcentration Factor - Roots B0 B1 B2 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Cd 10 Cd 20 Cd 30 Tf [Cd] soil (mg.Kg -1 ) Translocation factor B0 B1 B2 0 5 10 15 20 25 Cd 0 Cd 10 Cd 20 Cd 30 (g) [Cd] soil (mg.Kg -1 ) Shoot Biomass B0 B1 B2 a a a a a a a a a a a b 0 1 2 3 4 5 6 7 8 Zn/Cd0 Zn 100 Zn 500 Zn 1000 (g) [Zn] soil (mg.Kg -1 ) Root Biomass B0 B1 B2 a a a a b b a,b a,b b a b b 0 5 10 15 20 25 Zn/Cd0 Zn 100 Zn 500 Zn 1000 (g) [Zn] soil (mg.Kg -1 ) Shoot Biomass B0 B1 B2 b a a a b a a a a a a a Bioconcentration and Translocation Factors (BCF/TF) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Zn 0 Zn 100 Zn 500 Zn 1000 BF roots [Zn] soil (mg.Kg-1) Bioconcentration Factor - Roots B0 B1 B2 0 1 2 3 4 5 6 7 8 9 Zn 0 Zn 100 Zn 500 Zn 1000 BF Shoots [Zn] soil (mg.Kg -1 ) Bioconcentration Factor - Shoots B0 B1 B2 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Zn 0 Zn 100 Zn 500 Zn 1000 TF [Zn] Soil (mg.Kg -1 ) Translocation Factor B0 B1 B2 No bacteria (B0),B1, B2 0, 100, 500 and 1000 mg Zn kg -1 soil 0, 10, 20, 30 mg Cd kg -1 soil 12 weeks after seeding Biomass (roots and shoots) Zn/Cd accumulation (roots,shoots) Figure 1: Scheme of the experimental work - At low concentration (10 mg.kg -1 ) BF on roots was decreased by strain B1 and was increased significantly by B2. However these bacteria had no influence on it at higher concentrations; - Bacterial inocula decreased significantly BCF on shoots to values below 1 but had no significant influence at higher concentrations; - TF<1 at all concentrations, but at 10 mg.kg -1 , B2 decreased it significantly. - Bacterial inocula increased significantly BCF on roots and shoots at a soil concentration of 100 mg Zn Kg -1 but generally had no influence at higher concentrations; - B1 had influence in TF only in plants grown in non spiked soil. Cd Zn a a a a a a a a a a a a a,b b a a a b a b a a a a b c a a a a a a a a a a b b b a a a a a a a 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Cd 10 Cd 20 Cd 30 BF shoots [Cd] soil (mg.Kg -1 ) Bionconcentration Factor - Shoots B0 B1 B2 b b b c Figure 6: Bionconcentration factors (BF) of roots; results are shown as means ±S.D (n=4). Means with different letters in concentration are significantly different from each other (P < 0.05) according to the Duncan test. Figure 7: Bionconcentration factors (BF) of roots; results are shown as means ±S.D (n=4). Means with different letters in concentration are significantly different from each other (P < 0.05) according to the Duncan test.. Figure 8: Translocation factors (TF); results are shown as means ±S.D (n=4). Means with different letters in concentration are significantly different from each other (P < 0.05) according to the Duncan test.. Figure 9: Bionconcentration factors (BF) of shoots; results are shown as means ±S.D (n=4). Means with different letters in concentration are significantly different from each other (P < 0.05) according to the Duncan test.. Figure 10: Bionconcentration factors (BF) of roots; results are shown as means ±S.D (n=4). Means with different letters in concentration are significantly different from each other (P < 0.05) according to the Duncan test.. Figure 11: Translocation Factor (TF) results are shown as means ±S.D (n=4). Means with different letters in concentration are significantly different from each other (P < 0.05) according to the Duncan test.. a a a a b a a a a a a b b