**2.2 Environment protection - Phytoremediation**

Phytoremediation of soil and waters is a task of numerous research projects and technological undertakings. Such attempts base on an unique feature of *Salix viminalis* i. e. the ability to effective uptake, deactivation and accumulation of relatively high amounts of

Energetic Willow (*Salix viminalis*) – Unconventional Applications 185

Knop's medium (Reski & Abel, 1985) containing copper salt at 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mM stabilized with quartz sand in hydroponic pots. It is also visible that roots and rods (stems) i. e. plant parts responsible for metal ion transportation accumulate copper ions more intensively that new shots and leaves. The latter parts are rather a final location of metal ions and do not participate significantly in the ion transportation. Table 5 informs about biometric changes of the plants exposed to Cu2+ infiltration. The plants were still living but shots, leaves and roots underwent a gradual degradation consisting in reduction of mass and/or dimensions. Table 6 (Mleczek et al., 2009) considers the dependence between the kind of metal ion and its accumulation in different tissues. No strict correlation is visible except general tendency to intensive accumulation of cadmium

Fig. 1. Sampling wood material from *Salix viminalis* species planted in metal ions solutions.

and chromium.

(Łukaszewicz et al., 2009).

heavy metals without loosing its vitality. The efficiency of metal ion accumulation is extraordinarily high if compared to other plants and microorganisms. Therefore *Salix viminalis* is often called "hyper-accumulator". This point let to state that *Salix viminalis* is an unique plant among other energetic plants which mainly offer only a high growth rate and mass production but are poor metal ion accumulators.

Memon et al., 2001 citing other authors stated that retention of heavy metals may be accounted to one the below mentioned technologies (Salt et al., 1995; Pilon-Smits & Pilon, 2000):


In the case of *Salix viminalis* the process of metal ion accumulation proceeds through a root system and ion transport involving vascular tissues in stems and differentiated distribution in the whole plant body. Permeation of ions into roots is a typical way of efficient metal ion collection by *Salix viminalis*. This a basis for practical utilization of *Salix viminalis* for purification of various matrixes (soli, water, etc.) being in contact with roots of the plant. Planting of *Salix viminalis* on metal contaminated soils and/or bringing the plant in contact with contaminated waters lead to slow but constant removal of the metal impurities and finally remediation of soil and waters.

According to Baker & Walker, 1990 plants may follow three pathways when they grow on metal contaminated soils.


Table 5 shows that example heavy metal ions (Cu2+) penetrate all important parts of *Salix viminalis*. The ion penetration and the resulting copper accumulation increase with increasing concentration of Cu2+ in an artificial soil. Plants were incubated in complete

heavy metals without loosing its vitality. The efficiency of metal ion accumulation is extraordinarily high if compared to other plants and microorganisms. Therefore *Salix viminalis* is often called "hyper-accumulator". This point let to state that *Salix viminalis* is an unique plant among other energetic plants which mainly offer only a high growth rate and

Memon et al., 2001 citing other authors stated that retention of heavy metals may be accounted to one the below mentioned technologies (Salt et al., 1995; Pilon-Smits & Pilon,

1. Phytoextraction, in which metal-accumulating plants are used to transport and concentrate metals from soil into the harvestable parts of roots and above-ground

2. Rhizofiltration, in which plant roots absorb, precipitate and concentrate toxic metals

3. Phytostabilization, in which heavy metal tolerant plants are used to reduce the mobility of heavy metals, thereby reducing the risk of further environmental degradation by leaching into the ground water or by airborne spread (Smith & Bradshaw, 1979; Kumar

4. Plant assisted bioremediation, in which plant roots in conjunction with their rhizopheric microorganisms are used to remediate soils contaminated with organics (Walton &

In the case of *Salix viminalis* the process of metal ion accumulation proceeds through a root system and ion transport involving vascular tissues in stems and differentiated distribution in the whole plant body. Permeation of ions into roots is a typical way of efficient metal ion collection by *Salix viminalis*. This a basis for practical utilization of *Salix viminalis* for purification of various matrixes (soli, water, etc.) being in contact with roots of the plant. Planting of *Salix viminalis* on metal contaminated soils and/or bringing the plant in contact with contaminated waters lead to slow but constant removal of the metal impurities and

According to Baker & Walker, 1990 plants may follow three pathways when they grow on

1. Metal excluders: aerial parts of these plants are free from metal contamination despite

2. Metal indicators: such plants accumulate metals in their aerial parts and the

3. Accumulators and hyperaccumulators: These plants concentrate metals in their aerial part but the metal content in the tissues exceeds metal content in the soil. A plant capable to accumulate more than 0.1% of Ni, Co, Cu, Cr or Pb or 1% of Zn (despite of differences in metal content in the soil) in its leaves (dry mass) is called a hyperaccumulator. *Salix viminalis*, according to our earlier studies, may be accounted to the accumulators / hyperaccumulators category. Figs 1 and 2 present (Łukaszewicz et al., 2009) some of our results on the concentration of selected metal ions (Zn2+, Cu2+, Cr3+) in different parts of *Salix viminalis* rods after a certain time of contact with water

Table 5 shows that example heavy metal ions (Cu2+) penetrate all important parts of *Salix viminalis*. The ion penetration and the resulting copper accumulation increase with increasing concentration of Cu2+ in an artificial soil. Plants were incubated in complete

of high concentration of them in the soil and in the roots.

concentration of metals depends on the metal content in the soil.

from polluted effluents (Smith & Bradshaw, 1979); Dushenkov et al., 1995).

mass production but are poor metal ion accumulators.

shoots (Brown et al., 1994; Kumar et al., 1995).

Anderson, 1992; Anderson et al., 1993).

finally remediation of soil and waters.

metal contaminated soils.

solutions of the ions.

2000):

et al., 1995).

Knop's medium (Reski & Abel, 1985) containing copper salt at 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mM stabilized with quartz sand in hydroponic pots. It is also visible that roots and rods (stems) i. e. plant parts responsible for metal ion transportation accumulate copper ions more intensively that new shots and leaves. The latter parts are rather a final location of metal ions and do not participate significantly in the ion transportation. Table 5 informs about biometric changes of the plants exposed to Cu2+ infiltration. The plants were still living but shots, leaves and roots underwent a gradual degradation consisting in reduction of mass and/or dimensions. Table 6 (Mleczek et al., 2009) considers the dependence between the kind of metal ion and its accumulation in different tissues. No strict correlation is visible except general tendency to intensive accumulation of cadmium and chromium.

Fig. 1. Sampling wood material from *Salix viminalis* species planted in metal ions solutions. (Łukaszewicz et al., 2009).

Energetic Willow (*Salix viminalis*) – Unconventional Applications 187

length [cm]

[g] Leaves Shoots Roots Rods

0 0.22 0.28 0.48 1.47 6.58 194.9 9.69 9.76 11.57

0.5 0.84 1.69 3.27 3.89 4.24 182.0 8.91 6.18 3.39

1.0 1.65 2.46 5.38 5.25 3.97 181.3 8.77 5.69 2.66

1.5 2.79 2.93 8.84 6.37 3.71 179.0 8.13 4.36 2.37

2.0 3.46 3.56 10.61 7.44 3.65 174.7 7.47 4.34 1.84

2.5 4.22 3.90 13.51 8.17 3.42 174.6 7.55 3.99 1.82

3.0 4.30 4.69 15.38 9.46 3.41 171.8 5.93 3.82 0.82

Tissue Decreasing metal accumulation abilities of tissues (from left to Wright)

Root Zn Cd Pb Cu Co Cr Ni

Bark Cu Cd Zn Co Pb Cr Ni

Leaf Ni Cr Co Zn Pb Cu Cd

Petioles Cd Cr Zn Cu Pb Ni Co

Shoot (0.1 m) Cu Cr Zn Cd Pb Ni Co

Shoot (1 m) Cd Cr Cu Pb Ni Zn Co

Table 6. Heavy metal accumulation in different tissues of *Salix* materials. The table content

Table 5. Copper accumulation in *Salix viminalis* L. organs and biomass parameters for subsequent levels of copper addition to the growing medium – mean values (n=3). Selected

Total leaves surface [cm2]

Shoots length [cm]

Roots length [cm]

Roots biomass

[mg/ kg] Leaves

Cu accumulation (dry weight)

data cited after (Gąsecka et al., 2010).

cited after (Mleczek et al. 2009) with no changes.

Cu addition [mM/ dm3]

Fig. 2. Metal content in following segments of shoots (distance from rhizosphere: 1.1-5 cm; 2.55-60 cm; 3.110-115 cm; 4.165-170 cm; 5.220-225 cm). (Łukaszewicz et al., 2009).

During the experiment young shoots of *Salix viminalis* after defoliation were put into vessels with water and left until fresh leaves and root sprouted. Selected plants were moved to glass vessels filled with Cu, Cr and Zn salts solutions (0.01 M each). Additionally, chelation agent i. e. EDTA in water solution was added in the amount calculated basing on the assumption that EDTA was capable to form bichelates. According to some authors (Blaylock et al., 1997) plants should be more tolerant to chelated metal ions since after complexation their toxicity is lower. However, there is no common agreement about the positive influence of chelation on the metal ion uptake by *Salix viminalis.* After 7 days the plants were taken form the vessels and appropriate parts of stems (wood samples) were cut and subjected to elemental analysis (fig. 2). It is visible that metal ions enter the aerial part of *Salix viminalis* but the concentration of metals depends on the height above the ground level. Some studies point out differentiated distribution of metal ions in roots, stem, leaves etc. Tables 5 and 6 present such a kind of data (Gąsecka et al., 2010).

Fig. 2. Metal content in following segments of shoots (distance from rhizosphere: 1.1-5 cm;

During the experiment young shoots of *Salix viminalis* after defoliation were put into vessels with water and left until fresh leaves and root sprouted. Selected plants were moved to glass vessels filled with Cu, Cr and Zn salts solutions (0.01 M each). Additionally, chelation agent i. e. EDTA in water solution was added in the amount calculated basing on the assumption that EDTA was capable to form bichelates. According to some authors (Blaylock et al., 1997) plants should be more tolerant to chelated metal ions since after complexation their toxicity is lower. However, there is no common agreement about the positive influence of chelation on the metal ion uptake by *Salix viminalis.* After 7 days the plants were taken form the vessels and appropriate parts of stems (wood samples) were cut and subjected to elemental analysis (fig. 2). It is visible that metal ions enter the aerial part of *Salix viminalis* but the concentration of metals depends on the height above the ground level. Some studies point out differentiated distribution of metal ions in roots, stem, leaves etc. Tables 5 and 6 present

2.55-60 cm; 3.110-115 cm; 4.165-170 cm; 5.220-225 cm). (Łukaszewicz et al., 2009).

such a kind of data (Gąsecka et al., 2010).


Table 5. Copper accumulation in *Salix viminalis* L. organs and biomass parameters for subsequent levels of copper addition to the growing medium – mean values (n=3). Selected data cited after (Gąsecka et al., 2010).


Table 6. Heavy metal accumulation in different tissues of *Salix* materials. The table content cited after (Mleczek et al. 2009) with no changes.

Energetic Willow (*Salix viminalis*) – Unconventional Applications 189

bind metal ions in metal-thiolate clusters. Over 50 metallothioneins has been identified so far in plants. Organic acids and amino acids because of N and O content may chelate intensively various metal ions. Shah & Nongkynrih, 2007 claim that "citrate, malate, and oxalate have been implicated in a range of processes, including differential metal tolerance, metal transport through xylem and vacuolar metal sequestration". Salicylic acid and its derivatives which are definitely present in *Salix viminalis* tissues, has been also identified as chelating agent in some plants. For *Salix viminalis* naturally high concentration of the latter species is probably the key

Fig. 3. A model of the mechanisms that occur in plant cell upon exposure to metals: metal ion uptake, chelation, transport, sequestration, signalling and signal transduction. The diagram shows the uptake of metal ions by K+ efflux and transporter proteins, their sequestration by formation of PCs by enzyme PC synthase and GSH in vacuoles, the subsequent degradation of PC-peptides by peptidases to release GSH, the generation of ROI species, the contribution of Ca2+ towards activation of Ca2+/calmodulin kinase(s) and MAP kinase(s) cascade leading to defense gene activation in nucleus, the effect of ROI on natural plant defense pathways like octadecanoid pathway (JA) and phenyl propanoid pathway (SA) biosynthesis that lead to defense and cell protectant gene activation is also included to correlate the induced metal stress defense with natural plant defense mechanism. AOS - allene oxide synthase; APX ascorbate peroxidase; BA - benzoic acid; BA-2H - benzoic acid 2-hydroxylase; CAT - catalase; GSH - glutathione; JA - jasmonic acid; M2+ - metal ions; MAPK - mitogen activated protein

kinase; 12-oxo PDA reductase - 12-oxo-cis-10,15-phytodienoic acid reductase; PC phyochelatin; PL - phospholipase; POX - peroxidase; SA - salicylic acid; SOD - superoxide dismutase. The figure and the caption cited with no changes after Shah & Nongkynrih, 2007.

factor providing hyperaccumulating properties of the plant.


High metal ion accumulation is not an exclusive feature of *Salix viminalis*. Other *Salix* genotypes may exhibit high accumulation capacities towards different heavy metal ions (table 7) with particular emphasis on Zn2+ accumulation (Mleczek et al., 2010).

Table 7. Concentration of heavy metals in young shoots of 12 *Salix* genotypes before and after experiment (hydroponic estimation of heavy metal accumulation). Table cited after (Mleczek et al., 2010) with no changes.

Mechanism of heavy metal intrusion, transportation, deactivation and accumulation has been investigated intensively over many years (Shah & Nongkynrih, 2007; Memon et al., 2001; Lasat, 2001, Clemens, 2006). Fig. 3 illustrates the complex nature of the processes. Shah & Nongkynrih, 2007 recall several basic mechanism of metal ion assimilation among which chelating plays a crucial role. Many substances (chelators) occurring in plant cells contain typical chelating (ligand) atoms like oxygen, nitrogen and sulfur ones. Chelators contribute to metal ion detoxification. Other functional compounds called chaperones specifically deliver metal ions to organelles and metalrequiring. The principal metal chelators in plants are phytochelatins, metallothioneins, organic acids and amino acids. Shah & Nongkynrih, 2007 after some other authors state that phytochelatins are small metal-binding peptides which formation involves glutathione, homoglutathione, hydroxymethyl-glutathione or gammaglutamylcysteine. Metallothioneins are low molecular mass cysteine (cys)-rich proteins, that

High metal ion accumulation is not an exclusive feature of *Salix viminalis*. Other *Salix* genotypes may exhibit high accumulation capacities towards different heavy metal ions

Cd Co Cr Cu Ni Pb Zn

before 0.86 0.134 0.67 6.54 3.05 1.93 65.72 after 1.48 0.218 0.94 7.88 8.45 2.15 70.02

before 1.64 0.112 0.99 5.72 3.08 5.62 91.46 after 1.75 0.240 1.27 6.73 9.28 6.07 96.34

before 2.19 0.050 2.29 7.94 4.66 2.16 57.19 after 2.47 0.098 2.51 7.98 7.49 2.51 60.86

before 1.77 0.058 1.91 7.01 4.18 1.11 58.33 after 2.42 0.122 2.32 7.49 4.86 1.39 60.65

before 1.58 0.050 2.21 4.22 4.25 1.47 97.48 after 1.87 0.095 3.04 6.47 8.44 1.53 103.21

before 1.87 0.054 0.55 8.12 3.74 1.93 94.28 after 2.03 0.124 1.02 11.51 5.46 2.29 98.33

before 0.89 0.057 2.02 9.35 3.51 0.97 81.48 after 1.06 0.129 2.47 10.87 7.75 1.27 84.79

before 1.21 0.093 3.16 5.24 2.28 2.05 62.49 after 1.58 0.174 3.78 8.89 5.46 2.40 68.92

before 1.57 0.026 1.61 7.13 1.97 2.88 91.37 after 1.82 0.049 2.32 10.37 3.24 3.11 98.45

after 0.78 0.048 1.33 10.19 8.45 1.76 112.27

after 1.97 0.151 3.54 8.31 7.58 2.30 89.55

before 0.47 0.037 2.07 4.71 1.19 1.41 37.91 after 0.92 0.059 3.18 6.95 2.73 1.76 44.51

*Salix nigra* Marsch before 0.61 0.036 0.59 8.26 6.42 1.42 102.47

*Salix japonica* before 1.51 0.069 2.74 6.79 3.68 2.07 84.25

Table 7. Concentration of heavy metals in young shoots of 12 *Salix* genotypes before and after experiment (hydroponic estimation of heavy metal accumulation). Table cited after

Mechanism of heavy metal intrusion, transportation, deactivation and accumulation has been investigated intensively over many years (Shah & Nongkynrih, 2007; Memon et al., 2001; Lasat, 2001, Clemens, 2006). Fig. 3 illustrates the complex nature of the processes. Shah & Nongkynrih, 2007 recall several basic mechanism of metal ion assimilation among which chelating plays a crucial role. Many substances (chelators) occurring in plant cells contain typical chelating (ligand) atoms like oxygen, nitrogen and sulfur ones. Chelators contribute to metal ion detoxification. Other functional compounds called chaperones specifically deliver metal ions to organelles and metalrequiring. The principal metal chelators in plants are phytochelatins, metallothioneins, organic acids and amino acids. Shah & Nongkynrih, 2007 after some other authors state that phytochelatins are small metal-binding peptides which formation involves glutathione, homoglutathione, hydroxymethyl-glutathione or gammaglutamylcysteine. Metallothioneins are low molecular mass cysteine (cys)-rich proteins, that

(table 7) with particular emphasis on Zn2+ accumulation (Mleczek et al., 2010).

*Salix purpurea var.*  Angustifilia Kerner

*Salix purpurea L*. Nigra longifolia pendula

*Salix purpurea L.*  Green Dicks

*Salix purpurea L.*  Uralensis

*Salix purpurea var*. Schultze Schultze

Salix fragilis L*.* 

*Salix petiolaria*  Rigida

*Salix purpurea* 

*Salix purpurea*  Utilissima

(Mleczek et al., 2010) with no changes.

Kanon

233

*Salix alba L.*  Kanon

*Salix* genotype Metal kontent in *Salix* genotypes [mg / kg] (dry mass)

bind metal ions in metal-thiolate clusters. Over 50 metallothioneins has been identified so far in plants. Organic acids and amino acids because of N and O content may chelate intensively various metal ions. Shah & Nongkynrih, 2007 claim that "citrate, malate, and oxalate have been implicated in a range of processes, including differential metal tolerance, metal transport through xylem and vacuolar metal sequestration". Salicylic acid and its derivatives which are definitely present in *Salix viminalis* tissues, has been also identified as chelating agent in some plants. For *Salix viminalis* naturally high concentration of the latter species is probably the key factor providing hyperaccumulating properties of the plant.

Fig. 3. A model of the mechanisms that occur in plant cell upon exposure to metals: metal ion uptake, chelation, transport, sequestration, signalling and signal transduction. The diagram shows the uptake of metal ions by K+ efflux and transporter proteins, their sequestration by formation of PCs by enzyme PC synthase and GSH in vacuoles, the subsequent degradation of PC-peptides by peptidases to release GSH, the generation of ROI species, the contribution of Ca2+ towards activation of Ca2+/calmodulin kinase(s) and MAP kinase(s) cascade leading to defense gene activation in nucleus, the effect of ROI on natural plant defense pathways like octadecanoid pathway (JA) and phenyl propanoid pathway (SA) biosynthesis that lead to defense and cell protectant gene activation is also included to correlate the induced metal stress defense with natural plant defense mechanism. AOS - allene oxide synthase; APX ascorbate peroxidase; BA - benzoic acid; BA-2H - benzoic acid 2-hydroxylase; CAT - catalase; GSH - glutathione; JA - jasmonic acid; M2+ - metal ions; MAPK - mitogen activated protein kinase; 12-oxo PDA reductase - 12-oxo-cis-10,15-phytodienoic acid reductase; PC phyochelatin; PL - phospholipase; POX - peroxidase; SA - salicylic acid; SOD - superoxide dismutase. The figure and the caption cited with no changes after Shah & Nongkynrih, 2007.

Energetic Willow (*Salix viminalis*) – Unconventional Applications 191

Fig. 4. Nitrogen adsorption isotherm at -196 0C for bare *Salix viminalis* wood finally carbonized at 700 0C. I type isotherm characteristic for the presence of nanopores.

Fig. 5. Nitrogen adsorption isotherm at -196 0C for activated *Salix viminalis* wood

the presence of nanopores.

(phosphoric acid treatment) finally carbonized at 700 0C. I type isotherm characteristic for

Memon et al., claim that the application of biological metal-accumulators and metalhyperaccumulators for purification of soils and waters has several positive features like "low cost, generation of a recyclable metal-rich plant residue, applicability to a range of toxic metals and radionuclides, minimal environmental disturbance, elimination of secondary air or water-borne wastes, and public acceptance". The latter statement applies in full to *Salix viminalis*, too. Table 8 proves that Cd removal from soil is extraordinarily high (217 g/ha) if compared to other phytoaccumulators tested in the study (Porębska & Ostrowska, 1999). Also the concentration of the metal in dry *Salix viminalis* wood was very high (22.1 mg/kg) exceeding values found in our study Łukaszewicz et al., 2009.


Table 8. Estimated removal of Cd with the biomass. Selected data cited and translated after Porębska & Ostrowska, 2009.
