Phytoextraction of polluted soils with heavy metals using plant-bacterium associations

1. Phytoextraction of polluted soils with heavy metals using plant-bacterium associations Fernández-Santander A, Casillas JL, Sotelo C and Romero C. Departamento de Química y Medio Ambiente Universidad Europea de Madrid Villaviciosa de Odón, 28670 Madrid

2. Introduction

3. Introduction Heavy metal pollution - Problem of the industrial societies - Long-term persistance in the environment - Highly toxic, for instance, Cr (VI) is mutagenic and carcinogenic (Losi et al., 1994)

4. Introduction How do toxic metals species remove from contaminated soils? - Physical elimination and carriage to rubbish dump - Metal immobilization to soil - Washing soil All of them are expensive and pollutant

5. Introduction Some organisms have achieved to survive in heavy metals polluted environments - Some bacterium: isolated from contaminated soils, waters and sediments (Valls and Lorenzo, 2002) - Some plants: characterized by highly efficient uptake and high tolerance (Cunningham and Ow, 1996) Very useful for phytoremediation

6. Introduction What is phytoremediation? - A natural way to decontaminate polluted soils and waters - It uses living plants to extract heavy metals from contaminated soils and waters. - It is cheaper and less pollutant

7. Introduction Objective Hirschfeldia incana (hyperaccumulating plant) Pseudomonas maltophilia (heavy metal resistant bacterium) AssociationPseudomonas maltophilia in the plant rizosphere Phytoextraction when they grow in Zn contaminated soils

8. Materials and methods

9. Materials and methods Isolation of Zinc resistant microorganismsand determination of maximum resistant level - From sludges samples - Samples were grown in TSA plus increasing levels of Zn2+ - Incubation: 37ºC 48 h -Identification: Gram staining and biochemical analysis (API micromethod)

10. Materials and methods MICs (Minimal inhibitory concentrations) 1 ml Each erlenmeyer flasks was inoculated with 1 ml Culture grown 37 ºC 24 h (0.5 mM Zn2+) Erlenmeyer flaskswith 50 ml de TSA plus different concentrations of Zn2+ were incubated with shaking at 37ºC for 24 h -Bacterial growth was monitored by optical density measuring (OD660 nm)

11. Materials and methods Establishment of plant-bacterium associations Selection of hyperaccumulating plant Hirschfeldia incana They were seeded individually in flowerpot with 150 g of soil.They grew until 30 cm height. It grows in a great variety of climatic conditions of the Iberian Peninsula.

12. Materials and methods Establishment of plant-bacterium associations Selection of heavy metal resistant bacterium - Bacterium cultures were grown 24 h in TSA medium plus 0.5 mM Zn2+. - Cultures were centrifuged and bacterium were suspended in hidroponic solution. - Each plant was watered with this solution: 108 UFC/g soil.

13. Materials and methods Capacity of Zn acumulation from plant-bacterium associations - Each plant was watered 7 days with a non-metal water at room temperature. - Later, they were watered with an increasing Zn2+ solution two times per week for eight weeks - 5 repetitions for each one of them were made.

14. Materials and methods Capacity of Zn acumulation from plant-bacterium associations - A count of viable from soil sample (3 cm of depth) was made in order to determine the number of UFC/g soil. - It was determinated UFC/ml of leached during first two weeks in which plants were watered with Zn2+ solution.

15. Materials and methods Chemical analysis Root and shoot were dried and weighted. Zn2+ contents of each samples (soils and plants) were extracted using modified nitric acid method (EPA 3500). Zn2+ concentrations were quantified using Atomic Absorption Spectrophotometry and expressed as µg Zn/g dry weight of plant tissue or soil.

16. Results and discussion

17. Results and discussion Isolation of Zn resistant microorganisms Several bacterial strains were isolated from sludge samples. Pseudomonas maltophiliawas the most resistant Zn2+ The resistance level of heavy metals on solid culture medium are represented in Table 1. The results indicate that this strain is strongly multi-resistant. Table 1. Resistence of Pseudomonas maltophilia to several heavy metals

18. Results and discussion Zn Minimal inhibitory concentration (MICs) MIC values were 2 mM to Zn2+ (Table 2). These values are similar when compared with results reported by several authors (Filali et al., 2000). Table 2. Minimal inhibitory concentration to Zn2+ by Pseudomonas putida

19. Results and discussion Cr Minimal inhibitory concentration (MICs) MIC values were 4 mM to Cr2+ (Table 2). These values are higher when compared with results reported by several authors (Viti et al., 2003) Table 3. Minimal inhibitory concentration to Cr2+ by Pseudomonas putida

20. Results and discussion Phytoextraction capacity by associations Hirschfeldia incana and Pseudomonas maltophilia The value of UFC/g of soil was 106 before establishing the associations plant-bacterium The values of UFC/g of soil in the associations plant-bacterium (table 4) indicate that the number of bacteria stays throughout the time during in which the plants are watered with the dissolution of Zn. The number of released bacterium in the leached was not significant (3x103UFC/ml) Table 4. UFC/g of soil in the associations plant-bacterium.

21. Results and discussion Phytoextraction capacity by associations Hirschfeldia incana and Pseudomonas maltophilia Plant growth parameters indicated that there were no differences when soil was inoculated with heavy metal resistant bacterium (table 5). Table 5. Plant growth parameters Other authors have demostrated that when sunflowers were grown in Zn contaminated soils and inoculated with heavy metal resistant bacterium the growth was higher (Benlloch et al., 2002)

22. Results and discussion Phytoextraction capacity by plant and plant-bacterium associations Table 6. Zn2+ concentracion in soil and plant and plant-bacterium associations

23. Results and discussion Phytoextraction capacity by plant The analytical system had a 85% recovery efficiency and detection limit of 1 ppm. Plant acumulated 0.02% Zn2+ of their dry weight: 99% heavy metal was acumulated in the shoot. Similar results are found by other authors in other Zn hyperacumulating plant as Thlaspi caerulenscens (Cunningham and Ow, 1996). Studies with other heavy metals indicated similar acumulate rates, for instance, Ipoemoea alpina with Cu (Cunningham and Ow, 1996) and Silene with Pb (Verklei et al., 1991).

24. Results and discussion Phytoextraction rate by plant-bacterium associations Plant-bacterium associations acumulated 0.016% Zn2+ of their dry weighth: 99% heavy metal was acumulated in the shoot. There were not significant differences in the acumulating heavy metal rate between plant and plant-bacterium associations. The introduction of bacterium in the soil did not modify phytoextraction capacity plant.

25. Results and discussion Phytoextraction rate by plant and plant-bacterium associations Other authors have described 21% increase phytoextraction capacity in sunflower-bacterium associations (Benlloch et al., 2002). In this case the origin of bacterium was plant rizosphere. Although Pseudomonas maltophilia is a very resistant Zn2+ bacterium, it was isolated from sludge. Perhaps that is the reason would explain the not correct association with Hirschfeldia incana.

26. References -Benlloch M., Sancho E., Tena M. 2002. Fitorremediación de suelos contaminados del área de Aznalcollar. Servicio Publicaciones Universidad de Córdoba. -Cunningham S.D., Ow. 1996. Promises of phytoremediation. Plant Physiol 110:715-719 -Filali B.K., Taoufik J., Zeroual Y., Dzairi, F.Z., Talbi, M., Blaghen, M. Waste water bacterial isolated resistant to heavy metals and antibiotics. 2000. Current Microbiology 41:151-156. -Losi ME, Amrhein C., Frankenberger WTJ. 1994. Environmental biochemistry of chromium. Rev Environ Contam Toxicol 136:91-131. -Valls M., Lorenzo de V. 2002. Exploiting the genetic and biochemical capacities of bacteria for the remediation of heavy metal pollution. FEMS Microbiology Reviews 26: 327-338. -Viti C., Pace A., Giovannetti L. 2003. Characterization of Cr(VI)-Resistant Bacteria Isolated from Chromium-Contaminated Soil by Tannery Activity. Current Microbiology 46:1-5. -Verklei, J.A.C., Lolkema, P.C., de Neeling, A.L. y Harmens, H. (1991)Heavy-metal resistance in higher plants: biochemical and genetics aspects. In Ecological Responses to Environmental Stresses, ed. J. Rozema and J.A.C. Verkleij, 8-19. London: Kluwer Academic.