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Phytoextraction of polluted soils

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Phytoextraction of polluted soils Powered By Docstoc
					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
Introduction
                                        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)
                                       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
                                        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
                                     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
                                                    Introduction

                         Objective
Hirschfeldia incana            Pseudomonas maltophilia
  (hyperaccumulating plant)     (heavy metal resistant bacterium)




Association Pseudomonas maltophilia in the plant
                  rizosphere




      Phytoextraction when they grow in Zn
               contaminated soils
Materials and methods
                               Materials and methods

  Isolation of Zinc resistant microorganisms
and 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)
                                                     Materials and methods


   MICs (Minimal inhibitory concentrations)

                                       1 ml

            Each erlenmeyer flasks
            was inoculated with 1 ml




Culture grown 37 ºC               Erlenmeyer flasks with 50 ml de TSA
24 h (0.5 mM Zn2+)                plus different concentrations of Zn2+
                                  were incubated with shaking at 37ºC for
                                  24 h

   -Bacterial growth was monitored by optical
   density measuring (OD660 nm)
                                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.
                               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.
                             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.
                               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.
                              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.
Results and discussion
                                                 Results and discussion

 Isolation of Zn resistant microorganisms
Several bacterial strains were isolated from sludge
samples.
Pseudomonas maltophilia was the most resistant Zn2+
 Heavy metal     Maximum
               resistant level
                    (mM)         The resistance level of heavy
     Zn2+           9.9          metals on solid culture medium
     Ni2+           2,8          are represented in Table 1.
     Co2+           0,4
     Cu2+           1,1
                                 The results indicate that this
    VO2+            3,1
                                 strain is strongly multi-
    TeO32-          23,5
     Pb2+           4,4
                                 resistant.
     Hg2+           0,04
    CrO42-         240,9

 Table 1. Resistence of Pseudomonas maltophilia to several heavy metals
                                                  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).

        Concentration of Zn2+    DO660 nm      Percentage of growth
                                                    diminution
         Control (sin metal)      1.402                100
              0,4 mM              0.881                63
              1,1 mM              0.809                58
              1,8 mM              0.616                44
              2,2 mM              0.104                 7
              3,3 mM              0.132                 9
              4,4 mM              0.138                10



   Table 2. Minimal inhibitory concentration to Zn2+ by Pseudomonas putida
                                                 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)


       Concentration of Cr2+    DO660 nm    Percentage of growth
                                                 diminution
        Control (sin metal)      1.55               100
             1,4 mM              1.227               79
             2,7 mM              1.018               66
              4 mM               0.799               51
             5,4 mM              0.727               47
             6,7 mM              0.712               46



 Table 3. Minimal inhibitory concentration to Cr2+ by Pseudomonas putida
                                                  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.

  Time               UFC/g soil

  1 day after         6 x 108     The number of released bacterium
  watering with Zn
                                  in the leached was not significant
  8 day after         4 x 109     (3x103 UFC/ml)
  watering with Zn
  14 day after        8 x 109
  watering with Zn
                                  Table 4. UFC/g of soil in       the
  21 day after        8 x 109     associations plant-bacterium.
  watering with Zn
                                                  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).


            Total fresh     Total dry      Root dry      Shoot dry
            biomass (g)    biomass (g)    biomass (g)    biomass (g)
   Plant    11.31 ± 1.40   1.05 ± 0.29   0.05 ± 0.015    0.98 ± 0.21
  Plant +  11.09 ± 2.88    1.15 ± 0.33   0.045 ± 0.025   1.08 ± 0.32
 bacterium


               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)
                                        Results and discussion

Phytoextraction capacity by plant and plant-
          bacterium associations

          Plant                Plant + bacterium
  g Zn2+/g dry weight        g Zn2+/g dry weight
   Soil     Root    Shoot      Soil     Root      Shoot

 445,32 ±   40,29 ± 230,34 ± 389,34 ±   48,72 ±   172,28 ±
  86,03      4,44    46,38    42,56      35,79     61,67


 Table 6. Zn2+ concentracion in soil and plant and
 plant-bacterium associations
                                   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).
                             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.
                                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.
                              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.

				
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