Environ Sci Pollut Res DOI 10.1007/s11356-014-2848-1
RESEARCH ARTICLE
Phytomanagement of Cd-contaminated soils using maize (Zea mays L.) assisted by plant growth-promoting rhizobacteria Helena Moreira & Ana P. G. C. Marques & Albina R. Franco & António O. S. S. Rangel & Paula M. L. Castro
Received: 22 September 2013 / Accepted: 28 March 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Zea mays (L.) is a crop widely cultivated throughout the world and can be considered suitable for phytomanagement due to its metal resistance and energetic value. In this study, the effect of two plant growth-promoting rhizobacteria, Ralstonia eutropha and Chryseobacterium humi, on growth and metal uptake of Z. mays plants in soils contaminated with up to 30 mg Cd kg−1 was evaluated. Bacterial inoculation increased plant biomass up to 63 % and led to a decrease of up to 81 % in Cd shoot levels (4– 88 mg Cd kg−1) and to an increase of up to 186 % in accumulation in the roots (52–134 mg Cd kg−1). The rhizosphere community structure changed throughout the experiment and varied with different levels of Cd soil contamination, as revealed by molecular biology techniques. Z. mays plants inoculated with either of the tested strains may have potential application in a strategy of soil remediation, in particular short-term phytostabilization, coupled with biomass production for energy purposes.
Responsible editor: Elena Maestri H. Moreira : A. P. G. C. Marques : A. R. Franco : A. O. S. S. Rangel : P. M. L. Castro (*) CBQF—Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa/Porto, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal e-mail: [email protected] H. Moreira e-mail: [email protected] A. P. G. C. Marques e-mail: [email protected] A. R. Franco e-mail: [email protected] A. O. S. S. Rangel e-mail: [email protected]
Keywords Zea mays . Soil . PGPR . Phytomanagement . Cadmium . Biomass production . Remediation
Introduction Soil pollution by heavy metals is one of the main ecological problems worldwide. Cadmium is among the main metal contaminants and is one of the most toxic substances to living organisms, affecting plants, animals, and humans (Glick 2010; Kirkham 2006). This toxic metal has been released into the soil due to several anthropogenic processes, namely mining activity, production of batteries and fertilizers, and disposal of contaminated waste (Cui and Wang 2006; Lagriffoul et al. 1998). In plants particularly, high levels of this nonessential element can induce several negative effects, diminishing their growth and nutrient uptake and inhibiting photosynthesis (Prasad 1995; Toppi and Gabbrielli 1999). Depending on total concentration levels of Cd in soil, polluted areas are impaired for several uses, namely for residential, commercial, and, particularly, agricultural purposes, which have the lowest acceptable threshold level. Remediation techniques are therefore required to reduce or attenuate environmental risks. Phytoremediation, the use of plants to restore heavy metal-contaminated land, has arisen as a promising alternative technology to the use of classical chemical or physical cleaning methods (Juwarkar et al. 2010; Marques et al. 2009) in not severely polluted areas (Kavamura and Esposito 2010; Khan 2005). Although this technique is less expensive and less harmful to soil microbial diversity (Glick 2010), a long time is required to achieve acceptable metal levels. A soil remediation strategy known as phytomanagement can compensate such a time by adding value to the plants used for remediation and preventing movement of contaminants through the environment while reducing contamination (Fässler et al. 2010). This kind of
Environ Sci Pollut Res
management of contaminated soil also contributes to enhance soil quality and productivity. Fast-growing plants with high yield, biomass, and metal tolerance such as maize (Zea mays L.), a cadmium-tolerant plant, have been explored as alternatives in soil remediation applications (Meers et al. 2005, 2010; Thewys et al. 2010). While mitigating contamination, these plants can be used for biomass production, which is considered one of the most promising renewable energy options (Meers et al. 2010; Mleczek et al. 2010; Ruiz et al. 2009). However, there are some restrictive issues concerning the remediation process of metal-contaminated soils using plants, such as the bioavailability of the metals in soil, which is often a limiting factor for their extraction by plants (Li and Ramakrishna 2011). An alternative to chemical amendments, namely the use of chelating agents (Gunawardana et al. 2010), is the use of metal-resistant rhizosphere bacteria which may also enhance metal bioavailability (Rajkumar et al. 2010). The limited growth of plants exposed to high Cd levels, which can induce several toxic symptoms (Prasad 1995), and the Cd content on the plant harvestable parts (McKendry 2002) are also problems in plant-based remediation strategies. Plant growth-promoting rhizobacteria (PGPR) have been reported to reduce heavy metal stress and to promote phytoremediation and biomass production in contaminated soils (Ma et al. 2011; Marques et al. 2013; Miransari 2011; Saharan and Nehra 2011). Growth-promoting mechanisms include the production of phytohormones (auxins, cytokinins, and gibberellins), the siderophores, the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, and the fixation of atmospheric nitrogen (Glick 2010; Ma et al. 2011; Saharan and Nehra 2011). There are some studies involving Z. mays in phytoremediation (Li et al. 2009; Meers et al. 2005; Thewys et al. 2010; Wang et al. 2007; Wójcik and Tukiendorf 2005) and biomass production strategies (Dhugga 2007). Studies to elucidate the benefits of PGPR for growth of maize in Cd-contaminated soil towards achieving a phytomanagement/remediation strategy are valuable. In the present study, two bacterial strains, Ralstonia eutropha and Chryseobacterium humi (a novel species) (Pires 2010), isolated from a heavy metal-contaminated site, which had shown to promote the growth of Z. mays plants in agricultural soil (Marques et al. 2010), were tested. The effects of these bacterial strains on the growth and metal accumulation of Z. mays plants grown in Cd-contaminated soils, as well as changes in the rhizosphere community, were investigated.
Materials and methods Soil preparation Agricultural sandy loam soil with no detectable Cd and average pH of 6.71, 3.1 % organic content, 4.8 % water content,
1,736 mg kg−1 of N, 2,600 mg kg−1 of P, and 10,600 mg kg−1 of K (soil dry weight (DW)) was sampled randomly from a depth of 0–20 cm (other soil properties have been previously described in Marques et al. 2013). Root and litter materials were removed, and samples were air dried, passed through a 2-mm sieve, and mixed uniformly. Cadmium-treated soil was prepared by autoclaving soil (120 °C for 70 min in two consecutive days), drying in an oven at 40 °C for 4 days, and mixing with CdCl2 to achieve concentrations of 0, 10, 20, and 30 mg Cd kg−1 to mimic different contamination levels. Soil moisture content was maintained at 60 % of the water holding capacity by adding deionized sterile water. Spiked soil was subjected to three cycles of wet and dry processes for approximately 4 weeks in the greenhouse and was mixed once a week so that Cd was evenly dispersed in the soil (Blaylock et al. 1997). Experimental plan Two PGPR strains, R. eutropha (B1) and C. humi (B2), have been isolated from sediment samples collected from an industrially contaminated site in northern Portugal and shown to be able to tolerate Cd concentrations up to 500 mg L−1 in liquid cultures (Pires 2010). A pot experiment was conducted in a controlled growth room (12-h photoperiod, 450 μmol m−2 s−1 photosynthetically active radiation, 18–21 °C temperature range, 50–60 % relative humidity range) in order to test the effect of the selected PGPR on Z. mays plants grown in Cd-treated soil. Treatments in the greenhouse included assays with non-spiked soil and soil spiked with Cd at three concentrations, 10, 20, and 30 mg kg−1. Each treatment was subjected to a different type of inoculation: control (no bacteria), B1 (R. eutropha), and B2 (C. humi) strains. Four replicates were used for each Cd level/ inoculation type treatment. Z. mays seeds (variety Aveline, purchased from Lusosem, Portugal) were surface sterilized with 0.5 % (v/v) NaOCl for 10 min and were rinsed several times with deionized sterile water. Seeds were germinated in plastic pots (8-cm diameter and 10-cm height) with 400 g of the testing soils. Each pot received six seeds which were placed at 2-cm depth. Pots were randomly rearranged in the greenhouse every 2 weeks during the experiment. After germination, seedlings were reduced to four per pot. Ten milliliters of each bacterial strain suspension (108 CFU mL−1) in nutrient broth was used for the inoculation, by spraying soil surfaces (Marques et al. 2010), 2 weeks after germination. To the control pots, 10 mL of sterile nutrient broth was added. Plants were harvested after 12 weeks, separated in roots and shoot and washed with tap water, followed by washes with a 0.1 M HCl solution, and deionized sterile water. Shoot and root biomass was determined after oven drying at 70 °C for 48 h, grinding, and sieving to
RESEARCH ARTICLE
Phytomanagement of Cd-contaminated soils using maize (Zea mays L.) assisted by plant growth-promoting rhizobacteria Helena Moreira & Ana P. G. C. Marques & Albina R. Franco & António O. S. S. Rangel & Paula M. L. Castro
Received: 22 September 2013 / Accepted: 28 March 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Zea mays (L.) is a crop widely cultivated throughout the world and can be considered suitable for phytomanagement due to its metal resistance and energetic value. In this study, the effect of two plant growth-promoting rhizobacteria, Ralstonia eutropha and Chryseobacterium humi, on growth and metal uptake of Z. mays plants in soils contaminated with up to 30 mg Cd kg−1 was evaluated. Bacterial inoculation increased plant biomass up to 63 % and led to a decrease of up to 81 % in Cd shoot levels (4– 88 mg Cd kg−1) and to an increase of up to 186 % in accumulation in the roots (52–134 mg Cd kg−1). The rhizosphere community structure changed throughout the experiment and varied with different levels of Cd soil contamination, as revealed by molecular biology techniques. Z. mays plants inoculated with either of the tested strains may have potential application in a strategy of soil remediation, in particular short-term phytostabilization, coupled with biomass production for energy purposes.
Responsible editor: Elena Maestri H. Moreira : A. P. G. C. Marques : A. R. Franco : A. O. S. S. Rangel : P. M. L. Castro (*) CBQF—Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa/Porto, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal e-mail: [email protected] H. Moreira e-mail: [email protected] A. P. G. C. Marques e-mail: [email protected] A. R. Franco e-mail: [email protected] A. O. S. S. Rangel e-mail: [email protected]
Keywords Zea mays . Soil . PGPR . Phytomanagement . Cadmium . Biomass production . Remediation
Introduction Soil pollution by heavy metals is one of the main ecological problems worldwide. Cadmium is among the main metal contaminants and is one of the most toxic substances to living organisms, affecting plants, animals, and humans (Glick 2010; Kirkham 2006). This toxic metal has been released into the soil due to several anthropogenic processes, namely mining activity, production of batteries and fertilizers, and disposal of contaminated waste (Cui and Wang 2006; Lagriffoul et al. 1998). In plants particularly, high levels of this nonessential element can induce several negative effects, diminishing their growth and nutrient uptake and inhibiting photosynthesis (Prasad 1995; Toppi and Gabbrielli 1999). Depending on total concentration levels of Cd in soil, polluted areas are impaired for several uses, namely for residential, commercial, and, particularly, agricultural purposes, which have the lowest acceptable threshold level. Remediation techniques are therefore required to reduce or attenuate environmental risks. Phytoremediation, the use of plants to restore heavy metal-contaminated land, has arisen as a promising alternative technology to the use of classical chemical or physical cleaning methods (Juwarkar et al. 2010; Marques et al. 2009) in not severely polluted areas (Kavamura and Esposito 2010; Khan 2005). Although this technique is less expensive and less harmful to soil microbial diversity (Glick 2010), a long time is required to achieve acceptable metal levels. A soil remediation strategy known as phytomanagement can compensate such a time by adding value to the plants used for remediation and preventing movement of contaminants through the environment while reducing contamination (Fässler et al. 2010). This kind of
Environ Sci Pollut Res
management of contaminated soil also contributes to enhance soil quality and productivity. Fast-growing plants with high yield, biomass, and metal tolerance such as maize (Zea mays L.), a cadmium-tolerant plant, have been explored as alternatives in soil remediation applications (Meers et al. 2005, 2010; Thewys et al. 2010). While mitigating contamination, these plants can be used for biomass production, which is considered one of the most promising renewable energy options (Meers et al. 2010; Mleczek et al. 2010; Ruiz et al. 2009). However, there are some restrictive issues concerning the remediation process of metal-contaminated soils using plants, such as the bioavailability of the metals in soil, which is often a limiting factor for their extraction by plants (Li and Ramakrishna 2011). An alternative to chemical amendments, namely the use of chelating agents (Gunawardana et al. 2010), is the use of metal-resistant rhizosphere bacteria which may also enhance metal bioavailability (Rajkumar et al. 2010). The limited growth of plants exposed to high Cd levels, which can induce several toxic symptoms (Prasad 1995), and the Cd content on the plant harvestable parts (McKendry 2002) are also problems in plant-based remediation strategies. Plant growth-promoting rhizobacteria (PGPR) have been reported to reduce heavy metal stress and to promote phytoremediation and biomass production in contaminated soils (Ma et al. 2011; Marques et al. 2013; Miransari 2011; Saharan and Nehra 2011). Growth-promoting mechanisms include the production of phytohormones (auxins, cytokinins, and gibberellins), the siderophores, the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, and the fixation of atmospheric nitrogen (Glick 2010; Ma et al. 2011; Saharan and Nehra 2011). There are some studies involving Z. mays in phytoremediation (Li et al. 2009; Meers et al. 2005; Thewys et al. 2010; Wang et al. 2007; Wójcik and Tukiendorf 2005) and biomass production strategies (Dhugga 2007). Studies to elucidate the benefits of PGPR for growth of maize in Cd-contaminated soil towards achieving a phytomanagement/remediation strategy are valuable. In the present study, two bacterial strains, Ralstonia eutropha and Chryseobacterium humi (a novel species) (Pires 2010), isolated from a heavy metal-contaminated site, which had shown to promote the growth of Z. mays plants in agricultural soil (Marques et al. 2010), were tested. The effects of these bacterial strains on the growth and metal accumulation of Z. mays plants grown in Cd-contaminated soils, as well as changes in the rhizosphere community, were investigated.
Materials and methods Soil preparation Agricultural sandy loam soil with no detectable Cd and average pH of 6.71, 3.1 % organic content, 4.8 % water content,
1,736 mg kg−1 of N, 2,600 mg kg−1 of P, and 10,600 mg kg−1 of K (soil dry weight (DW)) was sampled randomly from a depth of 0–20 cm (other soil properties have been previously described in Marques et al. 2013). Root and litter materials were removed, and samples were air dried, passed through a 2-mm sieve, and mixed uniformly. Cadmium-treated soil was prepared by autoclaving soil (120 °C for 70 min in two consecutive days), drying in an oven at 40 °C for 4 days, and mixing with CdCl2 to achieve concentrations of 0, 10, 20, and 30 mg Cd kg−1 to mimic different contamination levels. Soil moisture content was maintained at 60 % of the water holding capacity by adding deionized sterile water. Spiked soil was subjected to three cycles of wet and dry processes for approximately 4 weeks in the greenhouse and was mixed once a week so that Cd was evenly dispersed in the soil (Blaylock et al. 1997). Experimental plan Two PGPR strains, R. eutropha (B1) and C. humi (B2), have been isolated from sediment samples collected from an industrially contaminated site in northern Portugal and shown to be able to tolerate Cd concentrations up to 500 mg L−1 in liquid cultures (Pires 2010). A pot experiment was conducted in a controlled growth room (12-h photoperiod, 450 μmol m−2 s−1 photosynthetically active radiation, 18–21 °C temperature range, 50–60 % relative humidity range) in order to test the effect of the selected PGPR on Z. mays plants grown in Cd-treated soil. Treatments in the greenhouse included assays with non-spiked soil and soil spiked with Cd at three concentrations, 10, 20, and 30 mg kg−1. Each treatment was subjected to a different type of inoculation: control (no bacteria), B1 (R. eutropha), and B2 (C. humi) strains. Four replicates were used for each Cd level/ inoculation type treatment. Z. mays seeds (variety Aveline, purchased from Lusosem, Portugal) were surface sterilized with 0.5 % (v/v) NaOCl for 10 min and were rinsed several times with deionized sterile water. Seeds were germinated in plastic pots (8-cm diameter and 10-cm height) with 400 g of the testing soils. Each pot received six seeds which were placed at 2-cm depth. Pots were randomly rearranged in the greenhouse every 2 weeks during the experiment. After germination, seedlings were reduced to four per pot. Ten milliliters of each bacterial strain suspension (108 CFU mL−1) in nutrient broth was used for the inoculation, by spraying soil surfaces (Marques et al. 2010), 2 weeks after germination. To the control pots, 10 mL of sterile nutrient broth was added. Plants were harvested after 12 weeks, separated in roots and shoot and washed with tap water, followed by washes with a 0.1 M HCl solution, and deionized sterile water. Shoot and root biomass was determined after oven drying at 70 °C for 48 h, grinding, and sieving to
