Greenhouse and field studies on Cr, Cu, Pb and Zn phytoextraction by Brassica napus from contaminated soils in the Apulia region, Southern Italy

Geoderma 160 (2011) 517–523

Contents lists available at ScienceDirect

Geoderma j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / g e o d e r m a

Greenhouse and field studies on Cr, Cu, Pb and Zn phytoextraction by Brassica napus from contaminated soils in the Apulia region, Southern Italy Gennaro Brunetti a,1, Karam Farrag b,c,d,⁎, Pedro Soler Rovira e,2, Franco Nigro f, Nicola Senesi a,3 a

Dipartimento di Biologia e Chimica Agroforestale e Ambientale, Università di Bari, Via Amendola, 165/A, 70126 Bari, Italy Central Lab for Environmental Quality Monitoring (CLEQM), Egypt National Water Research Center (NWRC), Egypt d Ministry of Water Resources and Irrigation (MWRI), Egypt e Centro de Ciencias Medioambientales, Consejo Superior de Investigaciones Científicas, Serrano 115 dpdo., 28006 Madrid, Spain f Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università di Bari, Via Amendola, 165/A, 70126 Bari, Italy b c

a r t i c l e

i n f o

Article history: Received 23 June 2010 Received in revised form 23 September 2010 Accepted 16 October 2010 Available online 26 November 2010 Keywords: Phytoextraction Heavy metals Brassica napus, compost Bacillus licheniformis

a b s t r a c t In the framework of a project aiming to phytoremediate heavy metal contaminated soils in the Apulia region, Southern Italy, a series of greenhouse experiments followed by field trials were performed in order to optimize heavy metal phytoextraction by Brassica napus. The effects of root colonization by Bacillus licheniformis BLMB1 and of addition of municipal solid waste (MSW) composts on the capacity of B. napus to tolerate and accumulate Cr, Cu, Pb and Zn were evaluated. B. napus was able to accumulate high amount of metals in greenhouse conditions, whereas it grew with difficulty or not at all in the open field, and metal accumulation in plant fractions was relatively low. The accumulation of metals in the plant fractions was in the order: Cr N Zn N Cu N Pb. The presence of either compost or B. licheniformis BLMB1 strain enhanced metal accumulation, Cr in particular, in the experimental conditions used. This effect can be useful in the phytoextraction of Cr from contaminated soils. © 2010 Elsevier B.V. All rights reserved.

1. Introduction The accumulation of heavy metals in agricultural soils is of increasing concern due to food safety issues and potential health risks as well as detrimental effects on soil ecosystems (McLaughlin et al., 1999). Phytoremediation can be defined as the combined use of plants, soil amendments and agronomic practices to remove pollutants from the environment or decrease their toxicity (Salt et al., 1998). This technique may be employed using various approaches, including, phytoextraction, phytovolatilization and phytostabilization (Chaney et al., 1997). In particular, phytoextraction refers to the ability of hyperaccumulator plants to uptake metals from soil and transport them to the above ground parts, which are able to accumulate concentrations up to 100-fold greater than those normally found in non-accumulators species (Baker and Brooks, 1989; Baker et al., 2000; McGrath and Zhao, 2003).

⁎ Corresponding author. Central Lab for Environmental Quality Monitoring (CLEQM), National Water Research Center (NWRC), Egypt. Tel.: +20101022229; fax: +20222035083. E-mail address: [email protected] (K. Farrag). 1 Tel.: + 39 080 5442953; fax: +39 080 5442850. 2 Tel.: + 34 917452500. 3 Tel.: + 39 080 5442853; fax: +39 080 5442850. 0016-7061/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.geoderma.2010.10.023

Finding optimal plant species for remediating a specific soil and selection of appropriate soil amendments able to improve soil conditions allowing plant survival and growth, are the key aspect to achieve this objective (Clemente et al., 2005). Among many fastgrowing and high biomass-accumulating plant species suitable for phytoextraction brassicaceae have received considerable attention (Prasad and Freitas, 2003) based on their capacity to uptake and accumulate Cr and other heavy metals in amounts higher than those of other plant species (Kumar et al., 1995). B. juncea is one of the most promising, non hyperaccumulating plant species for extracting heavy metals from contaminated soils, however other species of the Brassica genus, such as B. campestris, B. carinata, B. napus, B. nigra, B. oleracea and B. rapa, have also been studied (Gisbert et al., 2006; Kumar et al., 1995; Marchiol et al., 2004; Meers et al., 2005). The effect of organic amendment on heavy metal bioavailability depends on its nature, microbial degradability, salt content and influence on soil pH and redox potential, as well as on the particular soil type and metal concerned (Shuman, 1999a,b; Walker et al., 2003, 2004). In particular, the effect of different kinds of municipal solid waste (MSW) composts has been studied for their capacity of increasing metal availability in soil and their uptake by plants (Maftoun et al., 2004; Murphy and Warman, 2001; Pinamonti et al., 1999; Sebastiao et al., 2000; Warman and Rodd, 1998; Zheljazkov and Warman, 2004a,b). Another possibility to enhance metal bioavailability is the use of soil microorganisms and plant root-associated bacteria, which are

518

G. Brunetti et al. / Geoderma 160 (2011) 517–523

stimulated by root exudates including a wide range of organic molecules (Kamnev and van der Leile, 2000; van der Lelie, 1998). The use of rhizobacteria in combination with plants is expected to provide high efficiency in phytoremediation (Abou-Shanab et al., 2003; Whiting et al., 2001). In particular, plant growth promoting rhizobacteria (PGPR) are now being considered to play an important role in phytoremediation technologies (Mayak et al., 2004). The PGPR can promote plant growth in contaminated sites and enhance arid soil detoxification by conferring resistance to water stress (Burd et al., 2000; Mayak et al., 2004). According to Khan (2005), PGPR can improve the growth of plants used for phytoremediation, e.g., by increasing plant nutrition and health and biomass production, and reducing the level of contaminant uptake. Bacillus licheniformis, a Gram-positive, spore-forming soil bacterium, is regarded as a plant growth promoting rhizobacterium and has been classified as GRAS (generally recognized as safe). Previous researches demonstrated that different microbial strains of B. licheniformis can improve the growth and development of the host plant in heavy metal contaminated soils by mitigating toxic effects of heavy metals on the plants (McLean et al., 1990; 1992; Ramos et al., 2003; Yakimov et al., 1995). However, research in this area is very limited and requires field studies to support greenhouse or growth chamber results (Lucy et al., 2004). Thus, combining the use of PGPR with MSW composts may be a good means for increasing the efficiency of phytoremediation. This work is part of a series of studies initiated in 2007 to evaluate the feasibility of the phytoremediation technique to remediate soils contaminated by heavy metals (Cr in particular) in the Park of Alta Murgia, in Apulia region, Southern Italy (Brunetti et al., 2009a,b; Farrag et al., 2008, 2009). The main objective of this study was to evaluate the potential role of a MSW compost and B. licheniformis BLMB1 on the metal phytoextraction capacity of Brassica napus. In particular, the availability, accumulation, uptake and removal of Cr, Cu, Pb and Zn from polluted soils will be evaluated. 2. Materials and methods 2.1. Soil and pot experiment The contaminated soil (A) used in this experiment was a Typic Haploxeralfs, fine-loamy, mixed, thermic (Soil Taxonomy, 2003) collected in the Park of Alta Murgia, which features total concentrations of Cr, Cu, Pb and Zn largely exceeding the Italian maximum levels permitted for agricultural soils (Italy, 2006). An uncontaminated agricultural soil (B) collected from the same area was used as a reference. Soils were collected after grass cover removal from the top 20 cm, air-dried, gently ground to pass through a 2-mm sieve, homogenised and used to fill the pots (3 kg soil per pot). A MSW compost (10% w/w) and an aqueous cell suspension (108 cells ml−1) of B. licheniformis BLMB1 isolated from a semicommercial formulate (10% v/w) were used as amendments by thorough mixing with the soil. The heavy metal content of the compost used was within the Italian limits (Iwegbue et al., 2007). The pot experiment was carried out from July to October 2007 on B. napus plants grown in a greenhouse covered with a screen without supplementary light or heat. The average temperature of the greenhouse ranged from 29.6 ± 5.60 °C (day) to 14.5 ± 4.32 °C (night), and the relative humidity was 65.5 ± 10.9%, with an average of 12 h photoperiod per day. To prevent emergence failures, twenty seeds were sown in each pot initially. Then, when the first pair of true leaves appeared only 10 uniform seedlings per pot were allowed to grow for 90 days after sowing, and were then harvested. The set-up consisted of 15 pots made of polyvinyl chloride (PVC) with a diameter of 20 cm and a depth of 20 cm. The design included T1 (uncontaminated soil B), T2 (contaminated soil A), T3 (T2 + compost at 10%), T4 (T2 + B. licheniformis BLMB1 10%), and T5 (T2 + compost at 10% + B. licheniformis BLMB1 10%). All treatments were carried out

in triplicate. All pots were watered and kept at the field capacity moisture throughout the growing season. 2.2. Field experiment The field experiment was carried out in the Altamura site of the Park of Alta Murgia from February to August 2008 in plots of 35 m × 35 m which were established in two contaminated (A1, A2) and one non-contaminated (B1) locations. Each plot was divided into two portions, an uphill one (35 m × 60 m) with no amendment, and a downhill one (35 m × 20 m) treated with B. licheniformis BLMB1 (1 L diluted in 10 L of water then sprayed on the soil). A distance of 5 m separates the two portions. The experiments were performed in triplicate. The B. napus plants grown on these plots were harvested after 150 days from sowing. For each selected plot n. 7 soil subsamples were collected and composite. 2.3. Analytical procedures 2.3.1. Soil Soil analyses were carried out following internationally recommended procedures and the Italian official methods (Italy, 1999). Soil pH was determined by a glass electrode in distilled water (pH H2O) suspensions at 1:2.5 soil to liquid ratio. Electrical conductivity (EC) was measured using a conductimeter in filtrates from suspensions of 1:2 soil to water ratio. Texture and particle size distribution were determined by the pipette method after dispersing the soil sample in a sodium hexametaphosphate and sodium carbonate solution. Total organic carbon (TOC) was measured by the Walkley–Black method (Walkley and Black, 1934). Available phosphorus (Pava) was determined using a spectrophotometer UV/VIS on sodium bicarbonate and sodium hydroxide soil extracts according to the Olsen method (Olsen et al., 1954). The total content of heavy metals were determined in microwave assisted digests (Multiwave Perkin Elmer 3000) of soil samples added with a suprapure HNO3:H2O2:HCl mixture (5:1:1 v/v). The metal extractable fraction in soil was estimated on soil extracts by diethylenetriamine pentaacetic acid (DTPA)–CaCl2–triethanolamine (TEA) buffered at pH 7.3 (Lindsay and Norvell, 1978). This protocol is generally recommended for alkaline calcareous soils and excludes the effects of carbonate dissolution. The contents of heavy metals (Cd, Cr, Cu, Ni, Pb and Zn) in both acid-digested and DTPA extracts were determined using an inductively coupled plasma optical emission spectrometer (ICP-OES ICAP 6300 Thermo Electron). All chemicals were of analytical reagent grade and distilled water Milli-Q was used for solution preparation and dilution. Reagent blanks and laboratory NIST certified standards were used and routinely checked during ICP-OES determinations. 2.3.2. Plant To evaluate the heavy metal concentrations, B. napus plants were harvested and separated into roots and shoots (all the above ground parts). Roots collected extended down to 0–20 cm soil depth. Successively, the separated fractions were thoroughly washed with tap water to remove all visible fine soil particles, rinsed with deionised water, oven dried at 60 °C for 3 days, and finally ground to a powder using a Retsch MM200 mixer mill and kitchen miller. To determine the total heavy metal contents the plant fractions were subjected to microwave assisted digestion with a suprapure HNO3: H2O2:HCl mixture (5:1:1 v/v) and spectroscopic measurement as described for soil samples. The Bioconcentration Factor (BF) of each metal in plants was calculated by dividing the total content in shoots by the total content in soil (Brooks, 1998). Further, the Translocation Factor (TF) was calculated by dividing the total metal content in shoots by the total metal content in roots (Brooks, 1998). Both factors were calculated on a dry mass basis.

G. Brunetti et al. / Geoderma 160 (2011) 517–523

2.3.3. Compost The compost has been analyzed using standard methods (Collana Ambiente, 1998; Italy, 1999) after air-drying, grinding with a mixer mill, and passing through a 1 mm sieve. The pH was measured in distilled water at a 1:10 sample to water ratio using a pH meter. Electrical conductivity (EC) was determined on the filtrate at 1:10 sample to water ratio by a conductimeter. The total organic carbon was determined using the Walkley–Black method. Total nitrogen (Ntot) was determined by the Kjeldahl method. Heavy metal concentrations were determined as described for soil samples.

519

the colonies developed, those belonging to the Bacillus genus were identified based on morphological characters and biological procedures. 2.4. Statistical analyses Statistical analysis of data was made by using the Statgraphics Plus 5.1. software. One-way analysis of variance (ANOVA) and Tukey's test were applied. Means and standard deviations were calculated using Microsoft Excel.

2.3.4. Bacterium B. licheniformis MBBL1 was grown in nutrient agar plate for 48 h at 30 °C. The agar medium consisted of beef extract (5 g), peptone (10 g) and agar (20 g) in 1 L distilled water. The pH value of the medium was adjusted to 7.2 with 10% (w/v) NaOH and 10% (w/v) HCl. A loop of the bacterial culture was then inoculated in 300 ml of the nutrient medium and the flasks were incubated at 30 °C for 48 h. The cells grown were centrifuged at 6000 rpm for 10 min, washed three times with phosphate buffer, and suspended in 300 ml of the same buffer. To assess the population of sporogenous bacillaceae (expressed as log CFU g−1, CFU = Colony Forming Unit), soils samples were collected from the treated pots both before B. napus seeding and at the end of its vegetative cycle. According to Chilcott and Wigley (1993), soil samples were taken from the sub-surface (2–15 cm — plant's root zone) using a sterile spatula. All samples (250 g each) were transported in sterile plastic bags to the laboratory, allowed to dry for 3 days in the air, and then 2 mm — sieved to remove all large particles and plant debris. Successively, 1 g of each soil sample was suspended in 10 ml of sterile distilled water and shaken vigorously for 2 min. Then the samples were treated at 70 °C for 60 min in a water bath. Aliquots (100 μl) from several 10-fold serial dilutions of the heat-treated sample were spread onto nutrient agar plates and incubated in the dark at 37 °C for 24 h. Pure colonies were obtained by repetitive dilution and triple streaking on nutrient agar. Among

3. Results and discussion 3.1. Total and available metal contents in soils The main properties, total and DTPA-extractable heavy metal levels of the soils used, A, B, A1, A2 and B1 and compost, are shown in Table 1. Data show that the soil texture was silty loam (SL), except the silty clay loam (SCL) from the B2 site. The slightly alkaline pH values, the high percentages of OM and the presence of carbonates suggest that an important retention of heavy metals is to be expected in these soils (Greger, 2003; Kabata-Pendias and Pendias, 1992; Larlson et al., 2000; Shuman, 1999a,b). More details on these soils can be found in Brunetti et al., 2009a. The total contents of Cr, Cu, Pb and Zn in the polluted soils were higher and that of Ni and Cd lower than the maximum admissible levels for agricultural soils (Italy, 2006). In particular, the value recorded for Cr (1977.8 mg kg−1) was 13 times higher than the maximum admissible limits in Italy. Based on their content in soils, the metals studied can be classified, from the higher to the lower pollution level, as follows: Cr N Zn N Pb N Cu. However, metal availability in these soils appeared to be low (Brunetti et al., 2009a), as shown by the DTPA-extractable contents (Table 1), and in the order: Zn N Cu N Pb N Cr.

Table 1 General properties and contents of total and DTPA-extractable heavy metals in soils and compost studied. Parameters

Units

Soilsa

Compost

A

B

A1

A2

B1

Texture pH H2O EC CO3tot CO3act TOC Ntot Pava

– – ds m− 1 g kg− 1 g kg− 1 g kg− 1 mg kg− 1 mg kg− 1

SL 8.5 0.32 89 83 65.9 8.4 83

SL 8.7 0.22 85 30 41.7 6.1 71

SL 8.2 ± 0.2 0.41 ± 0.04 135 ± 22 108 ± 38 89.9 ± 8.3 11.3 ± 2.8 150 ± 24

SL 8.0 ± 0.1 0.42 ± 0.04 76 ± 38 69 ± 29 71.2 ± 17 12.1 ± 3.1 147 ± 18

SCL 8.0 ± 0.3 0.22 ± 0.02 36 ± 13 31 ± 8 21.5 ± 2 2.2 ± 0.2 23 ± 5

– 8.7 1.3 * * 232 14.8 83

Total concentrations Cd Cr Cu Ni Pb Zn

mg kg− 1 mg kg− 1 mg kg− 1 mg kg− 1 mg kg− 1 mg kg− 1

1.6 (2)b 1977.8 (150) 188.8 (120) 74.8 (120) 202.3 (100) 679.5 (150)

0.9 66.3 28.7 39.2 58.5 114.7

1.2 ± 0.1 1812.4 ± 183 351.4 ± 20.8 59.0 ± 4.7 257.4 ± 42 851.4 ± 64.3

0.9 ± 0.2 1391.1 ± 38.2 162.5 ± 27.8 52.4 ± 6.3 810.9 ± 29.6 514.9 ± 167.5

0.7 ± 0.1 54.5 ± 4.4 32.2 ± 2.4 37.8 ± 2.3 46.4 ± 2.7 119.1 ± 7.4

b 0.001(105)c 48(100) 105(300) 39(50) 67(140) 230(500)

0.18 0.06 6.93 1.37 4.57 60.45

0.09 0.002 0.67 0.18 0.60 0.44

0.14 ± 0.03 0.09 ± 0.01 8.56 ± 1.80 1.25 ± 0.36 4.92 ± 0.71 82.5 ± 18.2

0.12 ± 0.02 0.11 ± 0.02 5.22 ± 1.20 1.12 ± 0.28 6.68 ± 1.20 76.64 ± 14.20

0.08 ± 0.01 0.004 ± 0.001 0.48 ± 0.05 0.22 ± 0.03 0.52 ± 0.06 0.64 ± 0.09

DTPA-extractable contents Cd mg kg− 1 Cr mg kg− 1 Cu mg kg− 1 Ni mg kg− 1 Pb mg kg− 1 Zn mg kg− 1

EC, electrical conductivity; CO3, carbonates; TOC, total organic carbon; Ntot, total nitrogen; Pava, available phosphorus; SL, silty loam; SCL, silty clay loam; *, not determined. a Soil values of A1, A2 and B1 are mean ± STD (n = 7), a distance of 5 km separates the field experiment locations (A1, A2, B1) and the locations of soils collected for greenhouse experiment (A, B). b Values in parentheses are the maximum total contents in soils for public and private green areas and residential sites (Italy, 2006). c Values in parentheses are the maximum metal total contents in compost according to the Italian limits (Vander derf et al., 2002).

520

G. Brunetti et al. / Geoderma 160 (2011) 517–523

3.2. Metal concentrations in plants grown in greenhouse pots In general, the total concentration of heavy metals in plants grown on polluted soil substrate in greenhouse pots (T2) was relatively high (Table 2), thus showing accumulation and high tolerance of B. napus as it was expected. The highest values measured (mg kg−1) in shoots and roots for Cr were 254.8 and 51.3 followed by Zn with values of 156.1 and 39.1, respectively. Further, data in Table 2 indicated that the type of soil amendment affected significantly and differently the concentration of heavy metals in B. napus. Although treatment T5 showed the highest concentrations of metals in shoots, no statistically significant effect was found among T3, T4 and T5, except for Ni. In agreement with Baker (1981), the accumulation of metals was generally higher in shoots than in roots, which is a typical behavior of accumulator species. The content of metals in the roots of B. napus, for treatments T3, T4 and T5 were significantly higher than in T2. The highest metal contents and the highest differences were found for treatment T4 and T5 where bacillus was added. Further, at the end of .B. napus vegetative cycle, a significantly higher population of sporogenous bacillaceae was found in the soil amended with bacillus (T4), as compared to the untreated polluted soil (T2) (Fig. 1). The highest population was found in the soil amended with both the bacillus and compost (T5), as compared to the other treatments (Fig. 1), whereas a similar population of sporogenous bacillaceae was found for treatments T3 and T4. These results suggested that B. licheniformis BLMB1 is a promising candidate strain for enhancing plant metal accumulation in multi metal contaminated soils. This effect may be achieved by various mechanisms including: increased mobilization of metals (Chen et al., 2005; Gadd, 1990; Idris et al., 2004), reduced toxicity of metals transformed into less toxic forms, immobilization of metals on the cell surface or in intracellular polymers, and metal precipitation or biomethylation (Silver, 1996). Similar to bacillus, compost addition caused significant metal accumulation in shoots and roots of B. napus (Table 2), as previously reported by various authors (Maftoun et al., 2004; Murphy and Warman, 2001; Pinamonti et al., 1999; Warman and Rodd, 1998; Zheljazkov and Warman, 2004a,b). For all treatments the BF values of B. napus were lower than 0.2 for Cr, Cu, Ni and Pb, between 0.35 and 0.43 for Cd, and between 0.23 and 0.29 for Zn (Table 3). In general, BF was reported to decrease with increasing soil metal concentration (Zhao et al., 2003), and values lower than 0.2 are considered normal when plants are grown on polluted soils (McGrath and Zhao, 2003). In contrast, the TF values were higher than 1 for treatments T2, T3, T4 and T5 (Table 3), with a maximum value of 5.04 for Cr and Pb. As tolerant plants have TF values less than 1 and hyperaccumulators higher than 1 (Baker, 1981), the TF values measured here showed that B. napus behaved as a highly metal accumulator plant in greenhouse conditions.

Fig. 1. Population of sporogenous bacillaceae (expressed as log CFU g−1) in control soil (B) and polluted soil (A) before seeding B. napus, and control soil (T1), polluted soil (T2), polluted soil + compost (T3), polluted soil + B. licheniformis MBBL1 (T4), and soil + compost 10% + B. licheniformis MBBL1 (T5) at the end of the vegetative cycle of the plants. One-way variance analysis (ANOVA), Tukey test, 95% confidence level (p ≤ 0.05).

3.3. Metal concentrations in plants grown in the field In the field experiment an acceptable growth of B. napus was observed in experiment B1, whereas it failed to grow properly in experiment A2 and no growth was observed in experiment A1. One possible explanation for these results is that the contamination levels of soil A1 (e.g., Cr mean concentration was 1812.4 mg kg−1) was higher than that of soil A2. Although B. napus successfully accumulated relatively high amounts of metals in greenhouse conditions, it failed to uptake the same amount of metals in field conditions, possibly because plants explore potted soil more intensely (Delorme et al., 2000). According to Conesa et al. (2007), the differences in metal uptake between field and controlled pot conditions may be attributed to the different physiological state of the plant and/or to some modification of soil properties and climate parameters in pot conditions. B. napus appeared to accumulate most metals in the root system, whereas the shoots content was relatively low. Thus B. napus cannot be considered an accumulator plant in field conditions but a metal tolerant plant that possesses mechanisms allowing to cope with high metal concentration in soil. The order of accumulation of the six metals analyzed by B. napus was: Zn N Cr N Cu N Pb N Ni N Cd. Generally, the metal contents in shoots and roots of B. napus collected from soil A2 were higher than those collected from soil B1(Table 4). Cr uptake was relatively low in the field experiment where B. napus, like many other plants, may express an exclusion strategy (Khan, 2001) accumulating Cr in roots more

Table 2 Concentration of heavy metals (mg kg−1 d.w.) in shoots and roots of Brassica napus grown in greenhouse pots. Treat.

Cd

Shoots T1 T2 T3 T4 T5

0.39 ± 0.02 0.56 ± 0.05 0.67 ± 0.01 0.68 ± 0.02 0.67 ± 0.02

Roots T1 T2 T3 T4 T5

0.30 ± 0.03 0.32 ± 0.03 0.43 ± 0.03 0.57 ± 0.03 0.61 ± 0.06

Cr

Cu

Ni

Pb

Zn

a b c c c

1.9 ± 0.3 a 254.8 ± 24.5 b 349.3 ± 3.5 c 366.7 ± 13.4 c 372.2 ± 7.7 c

5.6 ± 0.9 a 23.2 ± 1.7 b 27.2 ± 0.4 c 27.4 ± 0.8 c 28.4 ± 0.6 c

0.7 ± 0.1 a 10.7 ± 0.6 b 13.2 ± 0.4 bc 14.0 ± 0.7 c 15.0 ± 2.1 c

0.6 ± 0.1 19.6 ± 1.9 23.4 ± 0.4 23.6 ± 0.8 24.6 ± 0.7

a b c c c

30.7 ± 2.1 a 156.1 ± 12.3 b 186.9 ± 2.0 c 190.4 ± 5.5 c 195.6 ± 2.1 c

a a b c c

4.9 ± 1.2 a 51.3 ± 6.5 a 129.5 ± 15.2 b 229.8 ± 23.2 c 254.2 ± 31.2 c

9.1 ± 1.4 a 10.9 ± 0.7 a 16.3 ± 1.2 b 20.7 ± 1.5 c 22.0 ± 2.4 c

1.9 ± 0.4 a 2.9 ± 0.2 a 5.2 ± 0.5 b 7.9 ± 0.5 c 8.2 ± 0.8 c

1.2 ± 0.2 3.9 ± 0.4 9.1 ± 1.1 15.2 ± 1.3 16.6 ± 2.2

a a b c c

19.7 ± 1.8 a 39.1 ± 3.1 a 78.9 ± 11.7 b 118.8 ± 11.4 c 127.5 ± 14.0 c

Analysis of variance (ANOVA): Tukey's test with 95% of significance (values in the same vertical column and same plant tissues followed by the same letter are not significantly different at p ≤ 0.05).

G. Brunetti et al. / Geoderma 160 (2011) 517–523

521

Table 3 BF and TF of studied metals in Brassica napus for different treatments in greenhouse pots. Metal

Factors

T1

T2

T3

T4

T5

Cd

BF TF BF TF BF TF BF TF BF TF BF TF

0.43 ± 0.02 b 1.32 ± 0.19 a 0.03 ± 0.00 a 0.41 ± 0.10 a 0.19 ± 0.03 b 0.62 ± 0.15 a 0.02 ± 0.00 a 0.41 ± 0.08 a 0.01 ± 0.00 a 0.51 ± 0.11 a 0.27 ± 0.02 b 1.57 ± 0.23 a

0.35 ± 0.03 a 1.78 ± 0.28 b 0.13 ± 0.01 b 5.04 ± 0.94 c 0.12 ± 0.01 a 2.13 ± 0.24 c 0.14 ± 0.01 ab 3.73 ± 0.53 c 0.10 ± 0.01 ab 5.04 ± 0.95 c 0.23 ± 0.02 a 4.02 ± 0.57 b

0.41 ± 0.01 b 1.57 ± 0.10 ab 0.18 ± 0.00 c 2.73 ± 0.35 b 0.14 ± 0.00 a 1.67 ± 0.10 b 0.18 ± 0.01 b 2.55 ± 0.26 b 0.12 ± 0.00 b 2.60 ± 0.38 b 0.28 ± 0.00 b 2.41 ± 0.37 a

0.42 ± 0.01 b 1.21 ± 0.04 a 0.19 ± 0.01 c 1.60 ± 0.11 ab 0.15 ± 0.00 a 1.33 ± 0.06 b 0.19 ± 0.01 c 1.77 ± 0.07 b 0.12 ± 0.00 c 1.55 ± 0.08 ab 0.28 ± 0.01 b 1.61 ± 0.11 a

0.43 ± 0.01 b 1.16 ± 0.12 a 0.19 ± 0.00 c 1.48 ± 0.18 a 0.15 ± 0.00 a 1.30 ± 0.12 b 0.20 ± 0.03 c 1.84 ± 0.35 b 0.12 ± 0.00 c 1.50 ± 0.20 ab 0.29 ± 0.00 b 1.55 ± 0.16 a

Cr Cu Ni Pb Zn

Analysis of variance (ANOVA): Tukey's test with 95% of significance (values in the same horizontal row and same factor followed by the same letter are not significantly different at p b 0.05).

tion and metal BF. These authors concluded that phytoextraction is not feasible using plants having a low BF for metals, regardless of how large is the achievable biomass. In particular, the BF should be 20 or greater to achieve a halving of soil metal contents in less than 10 crop harvests, assuming that the metal is taken up by plants from the top 20-cm soil. Further, with a biomass of 20 t ha−1 per crop, a BF greater than 10 would be required (McGrath and Zhao, 2003). Data in Table 6 show that hundreds of years would be needed to respect Italian regulation limits. Due to this, phytoremediation by B. napus cannot be considered the appropriate choice for metal polluted soils in the studied area.

than shoots, thus behaving as a typical tolerant plant. The Cu level in both roots and shoots of B. napus grown on soil A2 was lower than that on soil B1 showing no effect due to bacillus amendment, whereas Pb concentration was slightly greater in A2. Zn concentration in shoots and roots of B. napus grown on B1 was much lower than that of A2 and 2–3 times greater than that in B. Statically, different effects of the bacillus were observed on the accumulation efficiency of metals in B. napus grown on soils A2 and B1 (Table 4). In soil B1, the bacillus enhanced shoots uptake of Cu, whereas in soil A2 the bacillus enhanced the shoots accumulation of Cr and Pb. No significant effect was observed in metal uptake by roots in B1 and A2 soils after the addition of bacillus. Thus, more specific studies are needed to distinguish the role of this strain on metal mobility in field conditions. The BF values of B. napus were lower than 1 for all tested metals (Table 5), whereas with few exceptions, the TF values were generally lower than 1, except for Cd. This result explains the high concentration of metals found in roots. In general, B. napus in field appeared to behave as a tolerant plant with relatively high efficiency in accumulating and translocating different metals.

4. Conclusions B. napus showed a different metal tolerance and accumulation in field conditions compared to the pot experiment. In the pot experiment, a significantly high accumulation of Cr, Cu, Pb and Zn was found in shoots and roots of B. napus. The accumulation of metals was found to be higher in shoots than in roots, which is the typical behavior of accumulator species. In the field experiment, B. napus failed to achieve the same amount of accumulated metals achieved under the greenhouse conditions. The accumulation of studied metals in plant parts was relatively low, roots accumulated more than shoots showing tolerance mechanisms allowing B. napus to cope with high metal concentration in soil. Data obtained also indicated that the type of soil amendment affected significantly the concentrations of metals in B. napus. Both compost and B. licheniformis BLMB1 strain enhanced B. napus accumulation of metals, especially Cr, in pot experiment. Generally, B. licheniformis BLMB1 showed high resistance to high concentrations of Cr. Further researches are needed to elucidate the mechanisms of this strain against toxic elements, Cr in particular, in soil and plant, to improve the knowledge about the potential for field application, and to reduce time needed for soil recovery.

3.4. Suitability of B. napus for metals phytoextraction Based on the results of greenhouse experiments, the metal phytoextraction by B. napus for one hectare of contaminated soil has been estimated to need several centuries (Table 6). The amount of metals that B. napus is able to extract from soil was calculated from the metal concentration in the plant and the total plant biomass, considering 5 t ha−1 per year as the average harvest recorded for B. napus in the studied area, and one crop per year. The amount of metals to be extracted from soil was calculated as the difference between the metal concentration (Cr, Cu, Pb and Zn) in soil, considering 20 cm top soil (bulk density 1.3 g cm−1), and the limits established by the Italian law. According to McGrath and Zhao (2003), the efficiency of phytoextraction is determined by two key factors: biomass produc-

Table 4 Concentration of heavy metals (mg kg−1) in shoots and roots of Brassica napus grown in field conditions.

Shoots

Roots

Plots

Cd

Cr

Cu

Ni

Pb

Zn

B1-uphill B1-dowhill A2-uphill A2-dowhill B1-uphill B1-dowhill A2-uphill A2-dowhill

0.52 ± 0.08 b 0.65 ± 0.10 b 0.18 ± 0.05 a 0.21 ± 0.0 a 0.28 ± 0.02 b 0.36 ± 0.08 b 0.19 ± 0.01 a 0.19 ± 0.02 a

0.50 ± 0.13 a 0.58 ± 0.09 a 22.59 ± 10.71 b 32.76 ± 1.6 c 0.84 ± 0.13 a 0.41 ± 0.04 a 57.04 ± 18.43 c 39.02 ± 2.71 b

5.48 ± 0.89 a 9.82 ± 0.52 c 6.89 ± 1.09 ab 7.20 ± 0.9 b 15.38 ± 1.48 b 14.49 ± 1.01 b 7.66 ± 0.99 a 7.42 ± 0.50 a

0.58 ± 0.05 a 0.64 ± 0.05 ab 0.77 ± 0.15 b 0.80 ± 0.1 b 1.25 ± 0.16 b 0.65 ± 0.05 a 1.45 ± 0.28 b 1.42 ± 0.07 b

0.96 ± 0.10 a 0.93 ± 0.10 a 1.73 ± 0.37 b 2.25 ± 0.4 c 1.49 ± 0.26 a 1.42 ± 0.08 a 2.57 ± 0.61 b 2.35 ± 0.25 b

25.96 ± 6.48 a 31.92 ± 1.46 a 73.79 ± 7.05 b 69.60 ± 1.7 b 23.44 ± 0.95 a 26.44 ± 2.31 a 100.72 ± 15.29 c 71.09 ± 2.08 b

The values in the table are mean ± STD. B1-dowhill: control soil + bacillus; A2-dowhill: contaminated soil + bacillus. Analysis of variance (ANOVA): Tukey's test with 95% of significance (values in the same column followed by the same letter are not significantly different at p b 0.05).

522

G. Brunetti et al. / Geoderma 160 (2011) 517–523

Table 5 BF and TF of studied metals in Brassica napus in field condition.

BF

TF

Plots

Cd

Cr

Cu

Ni

Pb

Zn

B1-uphill B1-dowhill A2-uphill A2-dowhill B1-uphill B1-dowhill A2-uphill A2-dowhill

0.72 ± 0.16 b 1.01 ± 0.16 c 0.19 ± 0.05 a 0.23 ± 0.02 a 1.75 ± 0.30 b 1.80 ± 0.09 b 0.93 ± 0.23 a 1.17 ± 0.18 a

0.01 ± 0.00 a 0.01 ± 0.00 a 0.02 ± 0.01 b 0.03 ± 0.00 c 0.64 ± 0.10 ab 1.35 ± 0.08 c 0.40 ± 0.02 a 0.84 ± 0.06 b

0.17 ± 0.04 b 0.32 ± 0.02 c 0.04 ± 0.01 a 0.05 ± 0.01 a 0.42 ± 0.05 a 2.16 ± 0.26 c 0.90 ± 0.11 b 0.97 ± 0.20 b

0.02 ± 0.00 a 0.02 ± 0.00 a 0.02 ± 0.00 a 0.01 ± 0.00 a 0.49 ± 0.08a 0.96 ± 0.20 b 0.57 ± 0.15 a 0.56 ± 0.05 a

0.02 ± 0.00 b 0.02 ± 0.00 b 0.002 ± 0.004 a 0.003 ± 0.004 a 0.66 ± 0.04 a 0.68 ± 0.03 a 0.65 ± 0.04 a 0.96 ± 0.15 b

0.22 ± 0.05 b 0.28 ± 0.01 c 0.14 ± 0.03 a 0.14 ± 0.03 a 1.13 ± 0.23 a 1.14 ± 0.27 a 0.74 ± 0.07 a 0.98 ± 0.12 a

The values in the table are mean ± STD. B1-dowhill: control soil + bacillus; A2-dowhill: contaminated soil + bacillus. Analysis of variance (ANOVA): Tukey's test with 95% of significance (values in the same column followed by the same letter are not significantly different at p b 0.05).

Table 6 Time remediation (years) needed by Brassica napus for different treatments in greenhouse pots. Treatment T2 (polluted T3 (polluted T4 (polluted T5 (polluted

soil) soil + compost) soil + bacillus) soil + compost + bacillus)

Cr

Cu

Pb

Zn

3730 2721 2592 2554

1540 1317 1305 1260

2715 2277 2256 2161

1764 1473 1446 1408

Acknowledgements This work has been funded by the Regione Puglia (Italy) through the research project POR Puglia 2000–2006, Misura 1.8-Azione 4: “Monitoraggio siti inquinati.” Supporto scientifico alle attività di recupero funzionale ed il ripristino ambientale del sito inquinato dell'Alta Murgia. P. Soler-Rovira is a recipient of a contract from JAEDoc program of CSIC. References Abou-Shanab, R.A., Angle, J.S., Delorme, T.A., Chaney, R.L., van Berkum, P., Moawad, H., Ghanem, K., Ghozlan, H.A., 2003. Rhizobacterial effects on nickel extraction from soil and uptake by Alyssum murale. New Phytol. 158 (1), 219–224. Baker, A.J.M., 1981. Accumulators and excluders-strategies in the response of plants to heavy metals. J. Plant Nutr. 3, 643–654. Baker, A.J.M., Brooks, R.R., 1989. Terrestrial higher plants which hyperaccumulate metallic elements—a review of their distribution, ecology and phytochemistry. Biorecovery 1, 81–126. Baker, A.J.M., McGrath, S.P., Reeves, R.D., Smith, J.A.C., 2000. Metal hyperaccumulator plants: a review of ecology and physiology of a biological resource for phytoremediation of metal-polluted soils. In: Terry, N., Banuelos, G. (Eds.), Phytoremediation of Contaminated Soil and Water. Lewis Publishers, Boca Raton, FL, pp. 85–107. Brooks, R.R., 1998. Plants that Hyperaccumulate Heavy Metals. CAB International, Wallingford, UK. Brunetti, G., Farrag, K., Senesi, N., 2009a. Time frame and effectiveness of phytoremediation for heavy metal decontamination of soils in the Apulia region, southern Italy. In: Senesi, N., Bergheim, W. (Eds.), Book of Abstract of the 15th International Symposium MESAEP 2009 “Environmental Pollution and its Impact on Life in the Mediterranean Region”, Bari-Italy. ISBN: 978-3-936175-12-7, p. 55. Brunetti, G., Soler-Rovira, P., Farrag, K., Senesi, N., 2009b. Tolerance and accumulation of heavy metals by wild plant species grown in contaminated soils in Apulia region, Southern Italy. Plant Soil 318, 285–298. doi:10.1007/s11104-008-9838-3. Burd, G.I., Dixon, D.G., Glick, R.R., 2000. Plant growth promoting bacteria that decrease heavy metal toxicity in plants. Can. J. Microbiol. 46, 237–245. Chaney, R.L., Malik, M., Li, Y.M., Brown, S.L., Angle, J.S., Baker, A.J.M., 1997. Phytoremediation of soil metals. Curr. Opin. Biotechnol. 8, 279–284. Chen, Y.X., Wang, Y.P., Lin, Q., Luo, Y.M., 2005. Effect of copper-tolerant rhizosphere bacteria on mobility of copper in soil and copper accumulation by Elsholtzia splendens. Environ. Int. 31, 861–866. Chilcott, C.N., Wigley, P.J., 1993. Isolation and toxicity of Bacillus thuringensis from soil and insect habitats in New Zealand. J. Invertebr. Pathol. 61, 244–247. Clemente, R., Walker, D.J., Bernal, M.P., 2005. Environ. Pollut. 138, 46–58. Collana Ambiente, 1998. Metodi di analisi dei Compost- determinazioni chimiche fisiche, biologiche e microbiologiche, Analisi Merceologica dei rifiuti. Collana Ambiente, vol. 6/1998 -Regione Piemonte, Assessorato all'ambiente. Conesa, H.M., Robinson, B.H., Schulin, R., Nowack, B., 2007. Growth of Lygeum spartum in acid mine tailings: response of plants developed from seedlings, rhizomes and at field conditions. Environ. Pollut. 145, 700–707. Delorme, T.A., Angle, J.S., Coale, F.J., Chaney, R.L., 2000. Phytoremediation of phosphorous-enriched soils. Int. J. Phytoremediation 2, 173–181.

Farrag, K., Brunetti, G., Petrozza, A., Palma, A., 2008. Tolerance and accumulation of heavy metals by durum wheat and barley grown in contaminated soils in Apulia region, south Italy. Riassunti del II Congresso Nazionale, Qualità del Suolo, Alimenti e Salute. Bari-Italy, p. 26. Farrag, K., Brunetti, G., Senesi, N., 2009. Potential of Brassica napus for the phytoremediation of heavy metals contaminated soils in Apulia region, southern Italy. In: Senesi, N., Bergheim, W. (Eds.), Book of Abstract of the 15th International Symposium MESAEP 2009 “Environmental Pollution and Its Impact on Life in the Mediterranean Region”, Bari-Italy. ISBN: 978-3-936175-12-7, p. 56. Gadd, G.M., 1990. Heavy metal accumulation by bacteria and other microorganisms. Experientia 46, 834–840. Gisbert, C., Clemente, R., Navarro-Aviñó, J., Baixauli, C., Giner, A., Serrano, R., Walker, D.J., Bernal, M.P., 2006. Tolerance and accumulation of heavy metals by Brassicaceae species grown in contaminated soils from Mediterranean regions of Spain. Environ. Exp. Bot. 56, 19–27. Greger, M., 2003. Metal availability, uptake, transport and accumulation in plants. In: Prasad, M.N.V. (Ed.), Heavy Metal Stress in Plants: From Biomolecules to Ecosystems. Springer, pp. 1–28. Idris, R., Trifonova, R., Puschenreiter, M., Wenzel, W.W., Sessitsch, A., 2004. Bacterial communities associated with flowering plants of the Ni hyperaccumulator Thaspi goesingense. Appl. Environ. Microbiol. 70, 2667–2677. Italy, 1999. Ministero per le Politiche Agricole, Metodi ufficiali di analisi chimica del suolo. Decreto Ministeriale del 13 Settembre1999. Gazzetta Ufficciale n 248 del 21.10.1999. Italy, 2006. Decreto Legislativo 3 aprile 2006, n. 152. Norme in materia ambientale Parte IV –Titolo V- Bonifica dei siti contaminati. Supplemento Ordinario alla Gazzetta Ufficiale n. 88 del 14.04.2006. Iwegbue, C.M.A., EMuh, F.N., Isirimah, N.O., Egun, A.C., 2007. Fractionation characterization and speciation of heavy metals in composts and compost-amended soils. Afr. J. Biotecnol. 6 (2), 067–078. Kabata-Pendias, A., Pendias, H., 1992. Trace Elements in Soils and Plants. CRC Press, Boca Raton, FL. Kamnev, A.A., van der Leile, D., 2000. Chemical and biological parameters as tools to evaluate and improve heavy metal phytoremediation. Biosci. Rep. 20 (4), 239–258. Khan, A.G., 2001. Relationships between chromium biomagnifications ratio, accumulation factor, and mycorrhizae in plants growing on tannery effluent polluted soil. Environ. Int. 26, 417–423. Khan, A.G., 2005. Role of soil microbes in the rhizospheres of plants growing on trace metal contaminated soils in phytoremediation. J. Trace Elem. Med. Biol. 18, 355–364. Kumar, N.P.B.A., Dushenkov, V., Motto, H., Raskin, I., 1995. Phytoextraction: the use of plants to remove heavy metals from soils. Environ. Sci. Technol. 29, 1232–1238. Larlson, J., Likens, G., Fitzpatrick, J., Crock, J., 2000. Cadmium toxicity among wildlife in the Colorado Rocky Mountains. Nature 406, 181–183. Lindsay, W.L., Norvell, W.A., 1978. Development of a DPTA soil test for zinc, iron, manganese and copper. Soil Sci. Soc. Am. J. 42, 421–428. Lucy, M., Reed, E., Glick, B.R., 2004. Application of free living plant growth promoting rhizobacteria. Antonie van Leeuwenhoek. Int. J. Gen. Mol. Microbiol. 86, 1–25. Maftoun, M., Moshiri, F., Karimian, N., Ronaghi, A., 2004. Effects of two organic wastes in combination with phosphorus on growth and chemical composition of spinach and soil properties. J. Plant Nutr. 27 (9), 1635–1651. Marchiol, L., Sacco, P., Assolari, S., Zerbi, G., 2004. Reclamation of polluted soil: phytoremediation potential of crop-related Brassica species. Water Air Soil Pollut. 158, 345–356. Mayak, S., Tirosh, S., Glick, B.R., 2004. Plant growth promoting bacteria that confer resistance to water stress in tomatoes and peppers. Plant Physiol. 166 (2), 525–530. McGrath, S.P., Zhao, F.J., 2003. Phytoextraction of metals and metalloids from contaminated soils. Curr. Opin. Biotechnol. 14, 277–282. McLaughlin, M.J., Parker, D.R., Clarke, J.M., 1999. Metals and micronutrients—food safety issues. Field Crop Res. 60, 143–163. McLean, R.J.C., Beauchemin, D., Beveridge, T.J., 1992. Influence of oxidation state on iron binding by Bacillus licheniformis capsule. Appl. Environ. Microbiol. 58 (1), 405–408. McLean, R.J.C., Beauchemin, D., Claphan, L., Beveridge, T.J., 1990. Metal-binding characteristics of the Q-glutamyl capsular polymer of Bacillus licheniformis ATCC 9945. Appl. Environ. Microbiol. 56, 3671–3677. Meers, E., Ruttens, A., Hopgood, M., Lesage, E., Tack, F.M.G., 2005. Potential of Brassica rapa, Cannabis sativa, Helianthus annuus and Zea mays for phytoextraction of heavy metals from calcareous dredged sediment derived soils. Chemosphere 61, 561–572.

G. Brunetti et al. / Geoderma 160 (2011) 517–523 Murphy, C., Warman, P.R., 2001. Effect of MSW Compost Applications on Low-bush Blueberry Soil and Leaf Tissue Trace Elements. Proceedings of the 6th International Conference on the Biogeochemistry of Trace Elements, Guelph, ON, p. 166. Olsen, S.R., Cole, C.V., Watanabe, F.S., Dean, L.A., 1954. Estimation of Available Phosphorus in Soils by Extraction with Sodium Bicarbonate. United States Department of Agriculture Circular No. 939. 19 pp. Pinamonti, F., Nicolini, G., Dalpiaz, A., Stringari, G., Zorzi, G., 1999. Compost use in viticulture: effects on heavy metal levels in soil and plants. Commun. Soil Sci. Plan. 30 (9–10), 1531–1549. Prasad, M.N.V., Freitas, H., 2003. Metal hyperaccumulation in plants — biodiversity prospecting for phytoremediation technology. Electron. J. Biotechnol. 6, 285–321. Ramos, B., García, J.A.L., Probanza, A., Barrientos, M.L., Gutièrrez Mañero, F.J., 2003. Alterations in the rhizobacterial community associated with European alder growth when inoculated with PGPR strain Bacillus licheniformis. Environ. Exp. Bot. 49, 61–68. Salt, D.E., Smith, R.D., Raskin, I., 1998. Phytoremediation. Ann. Rev. Plant Physiol. Plant Mol. Biol. 49, 643–668. Sebastiao, M., Queda, A., Campos, L., 2000. Effect of municipal solid waste compost on potato production and heavy metal contamination indifferent types of soil. In: Warman, P.R., Taylor, B. (Eds.), Proceedings of the International Composting Symposium. CBA Press Inc. (Pubs), Halifax/Dartmouth, Nova Scotia, Canada, pp. 760–772. Shuman, L.M., 1999a. Effect of organic waste amendments on Zn adsorption by two soils. Soil Sci. 164, 97–205. Shuman, L.M., 1999b. Organic waste amendments effect on zinc fractions of two soils. J. Environ. Qual. 28, 1442–1447. Silver, S., 1996. Bacterial resistance to toxic metal ions — a review. Gene 179, 9–19. Taxonomy, Soil, 2003. Keys to Soil Taxonomy, Ninth Edition. Soil Survey Division — Natural Resources Conservation Service — U.S. Department of Agriculture, Washington, US (DC). Van der Lelie, D., 1998. Biological interactions: the role of soil bacteria in the bioremediation of heavy metal-polluted soils. In: Vangronsveld, J., Cunningham, S.D. (Eds.), Metal-contaminated Soils. In situ Inactivation and Phytorestoration. Springer-Verlag Berlin Heidelberg, New York, pp. 31–50.

523

Vander derf, P., Carter, C., Browne, G., Hosty, M.D., 2002. Assessment of Evaluation of Outlets of Compost Produced from Municipal Waste. 2000 MS-6-MI Final report. Environ. Protection, Wexford, Ireland. Walker, D.J., Clemente, R., Roig, A., Bernal, M.P., 2003. The effects of soil amendments on heavy metal bioavailability in two contaminated Mediterranean soils. Environ. Pollut. 122, 303–312. Walker, D.J., Clemente, R., Bernal, M.P., 2004. Contrasting effects of manure and compost on soil pH, heavy metal availability and growth of Chenopodium album L. in a soil contaminated by pyritic mine waste. Chemosphere 57, 215–224. Walkley, A., Black, I.A., 1934. An examination of the Degtjareff method for determining organic carbon in soils: effect of variations in digestion conditions and of inorganic soil constituents. Soil Sci. 63, 251–263. Warman, P.R., Rodd, V., 1998. Influence of Source-separated MSW Compost on Vegetable Crop Growth and Soil Properties: Year 3. Proceedings of the 8th Annual Meeting of the Composting Council of Canada, Ottawa, Ontario, November 3 – 5, pp. 263–273. Whiting, S.N., de Souza, M.P., Terry, N., 2001. Rhizosphere bacteria mobilize Zn for hyperaccumulation by Thlaspi caerulescens. Environ. Sci. Technol. 35 (15), 3144–3150. Yakimov, M.M., Timmis, K.N., Wray, V., Fredrickson, H.L., 1995. Characterization of a new lipopeptide surfactant produced by thermotolerant and halotolerant subsurface Bacillus licheniformis BAS50. Appl. Environ. Microbiol. 61, 1706–1713. Zhao, F.J., Lombi, E., McGrath, S.P., 2003. Assessing the potential for zinc and cadmium phytoremediation with the hyperaccumulator Thlaspi caerulescens. Plant Soil 249, 37–43. Zheljazkov, V., Warman, P.R., 2004a. Source-separated municipal soil waste compost application to Swiss chard and basil. J. Environ. Qual. 33, 542–552. Zheljazkov, V., Warman, P.R., 2004b. Phytoavailability and fractionation of copper, manganese, and zinc in soil following application of two composts to four crops. Environ. Pollut. 131, 187–195.