CSAWAC 46 (2) (2018) · Vol. 46 · No. 2 · February 2018
CLEAN Soil Air Water Renewables Sustainability Environmental Monitoring
2 | 2018
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RESEARCH PAPER Soil
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Microbial-Enhanced Phytoremediation of Lead Contaminated Calcareous Soil by Centaurea cyanus L. Akbar Karimi, Habib Khodaverdiloo,* and Mir Hassan Rasouli-Sadaghiani Potentially toxic elements are non-biodegradable; therefore, remediation of PTEs-contaminated environments is particularly challenging.[6] Phytoremediation, the use of plants along with their microbial association for remediation of contaminated soils, is a promising solar-driven, cost-effective and environmentally suitable alternative for restoration of PTEscontaminated soils.[7,8] However, most hyperaccumulator plants usually have low biomass and slow growth, which limits their application in large-scale operations.[9,10] Furthermore, while the total Pb concentrations are high in many of the contaminated sites, the bioavailable fraction of Pb is often very low in the soil environment, especially in calcareous soils due to complexation with various organic and inorganic soil colloids, sorption on oxides and clays, and precipitation as carbonate minerals.[5] Since plants have the ability to extract soluble or free forms of Pb rather than the bound ones, mobilized Pb fraction usually is a major limiting factor for phytoremediation of Pb-contaminated soils. Among alternatives to overcome these limitations is the use of specific microorganisms, such as arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR) that form symbiotic associations with most land plants.[11] AMF can improve growth and tolerance to stress conditions of host plants[12] and increase the bioavailability of PTEs in soil, metal uptake by plants from soil and root-to-shoot translocation of absorbed metals.[12] Therefore, AMF has been emerged as the most prominent symbiotic fungi for contribution to phytoremediation.[11,12] PGPR include a diverse group of rhizospheric bacteria that can enhance plant growth and development in PTEs-contaminated soils by mitigating toxic effects of PTEs on the plants.[9] Furthermore, PGPR can improve the phytoremediation of PTEs through mobilizing nutrients in soils, producing numerous plant growth regulators, enhancing the metal bioavailability by release of metabolites (e.g., organic acids, siderophores) and oxidation/ reduction reactions.[9] Kamran et al.[13] reported that inoculation of Eruca sativa with Pseudomonas putida enhanced the shoot and root biomass and Cd uptake by the plant. Although inoculation of autochthonous AMF and PGPR have shown to be an effective strategy in bioremediation of sterilized
Microbe-assisted phytoremediation is a promising technology for remediation of potentially toxic element contaminated soils. A greenhouse study was conducted to assess the effect of Pseudomonas spp. and Glomus spp. on phytoremediation of a Pb-contaminated calcareous soil by Centaurea cyanus L. The study was carried out as a factorial experiment arranged in a randomized complete block design with three replications. Factors are microbial inoculation in three levels (mix inoculation with Glomus spp. or Pseudomonas spp. and non-inoculated) and four Pb concentrations (0, 250, 500, and 1000 mg kg1) in soil. The results revealed that microbial inoculation significantly increased the shoot dry weight and Pb accumulation in C. cyanus compared to non-inoculated plants. Comparison of microbial treatments indicated that higher shoot Pb concentration, shoot modified bioconcentration factor (shoot mBCF, 5.33–5.63) and translocation factor (TF, 1.09–1.2) are obtained for Pseudomonas spp. inoculation, while higher plant biomass, plant Pb accumulation, root Pb concentration, root mBCF (5.91– 6.46) and lower TF (0.77–0.99) are recorded for Glomus spp. inoculation. These results show that Pseudomonas spp. and Glomus spp. are more effective in phyotoextraction and phytostabilization of Pb by C. cyanus, respectively. It could be concluded that Pseudomonas spp. and Glomus spp. would be a promising strategy in bioremediation of Pb-contaminated soils, especially at medium levels of soil Pb contamination (250 and 500 mg kg1).
1. Introduction In recent years, human activities such as mining, wastewater irrigation, application of agrochemicals, emissions from waste incinerators and cars and discharge of large amounts of metalenriched solid and effluents accelerated the soil contamination.[1] Potentially toxic elements (PTEs) contamination of soils is of particular attention due to food security issues and several reported health risks to both human and living organisms.[2,3] Lead (Pb) is among highly toxic and most widely PTEs at contaminated sites. It originates from various anthropogenic sources and causes a variety of health, environmental, and ecological problems.[4,5] A. Karimi, Dr. H. Khodaverdiloo, Prof. M. H. Rasouli-Sadaghiani Faculty of Agriculture Department of Soil Science Urmia University Urmia 57135-165, Iran E-mail:
[email protected]
DOI: 10.1002/clen.201700665
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soils artificially spiked by PTEs, the effect of microbial inoculation on PTEs phytoremediation depends upon various factors such as plant genotype, microbial species, types and concentrations of PTEs, and physicochemical properties of soils.[14] While researches have been conducted to study these effects, investigations on various AMF/PGPR species are rare for calcareous soils and wild plants, suggesting a promising research field that needs to be further studied. Centaurea cyanus L. (corn flower) is a vigorous (and not palatable for animals) plant with relatively fast growth rate from the Asteraceae family that has the potential for PTEs phytoremediation.[15] As a droughtand stress-tolerant plant, it is widely distributed in arid lands, disturbed sites and roadsides. The plants not palatable for animals, such as C. cyanus L., are promising for their potential application for phytoremediation of contaminated soils, since they possess lower risk of food chain contamination. In the case of phytoremediation of calcareous soils in arid and semiarid zones, the plants that are tolerant to both drought and PTEs are preferred. Therefore, the objective of this study was to evaluate the effect of inoculation with selected AMF (a mixture of Glomus sp. including G. intraradices, G. mosseae, and G. fasciculatum) and PGPR (a mixture of Pseudomonas sp. including P. putida, P. fluorescens, and P. aeruginosa) which can produce siderophores and organic acids, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, on phytoremediation of a Pb-contaminated calcareous soil by C. cyanus.
2. Experimental Section 2.1. Soil Physicochemical Analyses The experimental soil was collected from the Western Azerbaijan province, NW Iran. The selected soil was classified as typic Endoaquepts according to USDA Soil Taxonomy.[16] Airdried subsamples (2 mm) of the soil were used for physicochemical analysis. Particle size was determined by the hydrometer method.[17] The pH of the soil was determined using soil to 0.01 M CaCl2 suspension, 1:5, with a glass electrode. The total carbonates expressed as calcium carbonate equivalent (CCE) was determined by a rapid titration method[18] which is based on the reaction of HCl with carbonates. Particularly, 1 g of dry soil sample was added to 10 mL of 1 M HCl, shaken and placed in a water bath for 30 min at 70 C and CCE was determined by back titration of remaining HCl with 1 M NaOH.[18] Organic matter (OM) was determined by dichromate oxidation,[19] in which 0.5 g of 0.5 mm soil sample was reacted with 10 mL of potassium dichromate (K2Cr2O7) and 20 mL of concentrated sulfuric acid (H2SO4) by shaking for 30 min and then adding 100 mL of distilled water and 1 mL of diphenylamine. Then, the organic matter content was determined by volumetric titration of the soil suspension with ammonium ferrous sulfate (Fe (NH4)2 (SO4)2 6 H2O).[19] Soil electrical conductivity (EC) and soluble ions were determined in a saturated paste extract.[20] The cation exchange capacity (CEC) was measured using sodium acetate (1 M NaOAc) at pH 8.2,[21] so that 5 g of soil sample was placed in a 100-mL centrifuge tube and 33 mL of NaOAc (at pH 8.2) was added to the soil. After shaking the suspension for 5 min, it was centrifuged and the
Clean – Soil, Air, Water 2018, 46, 1700665
clear supernatant was discarded. This process was done three times. Excess salt of NaOAc was washed out by adding a mixture of water and ethanol until the EC of the supernatant was 0.05) in comparison with Pb0 (Table 3). The highest and lowest relative shoot and root dry matter yields were observed in AMF and control plants, respectively. Although, there were no significant (p 0.05) differences in relative root yield among all treatments, the relative shoot dry matter yield of AMF and PGPR plants was significantly (p 0.05) higher than that of the non-inoculated (control) plants. In general, Pb tolerance (relative dry matter yield) of plants in all treatments was in the order: AMF > PGPR > control. High concentration of Pb causes imbalance of mineral nutrients in growing plants. Pb toxicity in plant leads to damage on the photosynthesis apparatus and inhibition of photosynthesis and cellular respiration, and interferes with a wide range of physiological and biochemical processes in cells.[30] Pb may replace some essential elements in pigments and enzymes, thus disturbing their activity. Similarly, Punamiya et al.[4] reported shoot dry matter of vetiver decreased significantly as the concentration of Pb in soil increased. In this research, the highest shoot dry matter was observed in AMF plants (Table 3). It has been well demonstrated that AMF may improve nutritional status, uptake of water and growth of host plants through the large surface area of their hyphae and the increase of root length density and the enhancement of soil enzymes, especially acid phosphatase activity.[11] In addition, AMF may favor absorption of phosphorus, which could explain the higher tolerance of plants grown in contaminated environments.[31] The
Clean – Soil, Air, Water 2018, 46, 1700665
0
3.66 0.22a,c
4.51 0.21a,b
6.67 0.30a,a
250
3.32 0.19
b,b
3.95 0.14
6.41 0.28a,a
500
2.39 0.25b,c
3.19 0.06c,b
6.07 0.09b,a
1000
1.41 0.06
1.65 0.06
2.73 0.29c,a
Average
a,c
b,b
2.70 C
d,b
3.33 B
5.47 A
Relative shoot dry matter yield 0
1.00 0.06a,c
1.23 0.07a,b
1.82 0.04a,a
250
0.91 0.05
a,c
1.08 0.04
1.75 0.04a,a
500
b,c
0.65 0.06
0.87 0.01
1000
0.38 0.04
0.45 0.07
Average
c,b
0.73 C
ab,b b,b c,b
0.91 B
1.38 0.05b,a 0.75 0.05b,a 1.43 A
1
Root dry matter yield (g pot ) 0
1.06 0.11a,a
1.22 0.18a,a
1.38 0.23a,a
250
0.95 0.14
ab,a
1.06 0.08
1.26 0.15a,a
500
0.77 0.09b,a
0.93 0.12ab,a
1.08 0.06b,a
1000
0.68 0.12
0.76 0.09
0.94 0.09b,a
Average
ab,a
c,a
0.87 A
a,a
0.99 A
1.17 A
Relative root dry matter yield 0
1.00 0.11a,a
1.15 0.18a,a
1.30 0.23a,a
250
0.90 0.14
1.00 0.08
1.18 0.15ab,a
500
0.73 0.09
0.88 0.12
1.02 0.06bc,a
1000
0.64 0.12
0.72 0.09
0.89 0.09c,a
Average
ab,a b,a b,a
0.82 A
ab,a b,a b,a
0.94 A
1.10 A
a) Native Pb was 21.42 mg kg1. For each data, mean SE followed by the first and second same letters is not significantly different among soil Pb levels and among control, AMF and PGPR treatments, respectively, according to the Duncan’s test at 5% probability level (n ¼ 3).
competition or functional complementarity among AMF species might have impacts on growth and Pb accumulation in host plants. Jansa et al.[32] reported that in Medicago truncatula and Allium porrum, multispecies mixtures of AMF provided more P and supported greater plant growth than single AMF. This supports results of the predominant AMF in C. cyanus under Pb contamination. Inoculation of C. cyanus with PGPR increased plant biomass under Pb toxicity condition compared to the corresponding controls. PGPR enhanced the biomass and increased the dry matter yield of plants by reducing ethylene levels and production of different phytohormones, that is, gibberellins and auxin under PTEs contamination.[3] It is also well documented that PGPR containing ACC-deaminase is helpful in decreasing the ethylene levels in plants by converting ACC (ethylene precursor) to ammonia and α-ketobutyrate.[9,14] Thus selected PGPR which possess ACC-deaminase facilitates C. cyanus growth by reducing ethylene stress, and decreasing Pb
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stress in plants. PGPR directly facilitates the growth of their host plants under PTEs stress. The mechanisms include solubilization of nutrients, and production of siderophores and phytohormones. PGPR may alleviate PTEs toxicity and enhance plant development through one or more of these mechanisms.[9] The results of this study are in agreement with the previous study conducted by Kamran et al.,[3] who reported that the PGPR strengthen the plant growth by increasing shoot yield in E. sativa under PTEs stress.
3.3. Pb Accumulation in Root and Shoot
Table 4. Shoot and root Pb concentration, shoot Pb extraction and root Pb stabilization in control, AMF or PGPR treatments at different levels of soil Pb contamination. Shoot Pb concentration (mg kg1) Control
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AMF
Total Pb added to soil (mg kg ) 0
2.04 0.18d,b
2.81 0.16d,a
2.32 0.08a,ab
250
17.12 1.23
c,a
27.62 1.58
23.27 1.17b,b
500
25.38 1.98b,c
47.19 2.15b,a
31.68 0.67c,b
1000
35.74 0.04
59.01 6.95
41.89 0.77d,b
Average
Pb concentration in roots and shoots increased significantly (p 0.05) in all treatments with increasing Pb concentration in soil (Table 4). Inoculation with AMF and PGPR increased significantly (p 0.05) the Pb concentration in roots and shoots compared to the control treatment. There were significant (p 0.05) differences in shoots and roots Pb concentration among all treatments. In general, the shoots Pb concentration was in the order: PGPR > AMF > control whereas the roots Pb concentration was in the order: AMF > PGPR > control (Table 4). In all treatments, shoots Pb extraction increased significantly (p 0.05) with increasing Pb concentration in the soil up to 500 mg kg1 but decreased at 1000 mg Pb kg1 (Table 4). Shoots Pb extraction in AMF and PGPR plants was up to 2.67 and 2.11 times higher than in control plants (mean of all Pb concentrations in soil). In all treatments, Pb stabilized in roots significantly (p 0.05) increased as soil Pb raised (Table 4). The highest Pb stabilized in roots (0.067 mg pot1) was recorded in AMF plants grown in Pb1000. The Pb stabilized in roots was significantly (p 0.05) higher in plants inoculated with AMF compared to either control plants or those inoculated with PGPR (Table 4). Mean roots Pb stabilization in plants inoculated with AMF (0.042 mg pot1) and PGPR (0.027 mg pot1) was, respectively, up to 2.1 and 1.35 times higher than in the control plants (0.020 mg pot1). The average concentration of Pb was higher in shoots (34.16 mg kg1) of PGPR inoculated plants than in roots (30.69 mg kg1), whereas in AMF and control treatments the average of Pb concentration in roots was higher than in shoots. While the shoots Pb concentration in PGPR plants was significantly (p 0.05) higher than in AMF plants, Pb extracted in AMF plants was higher than in PGPR plants (Table 4 and Figure 5), because shoots dry matter in AMF plants was higher than in PGPR plants (Table 3). In addition, in all treatments, Pb accumulated in plants increased with increasing Pb concentration in the soil up to 500 mg kg1 but decreased at 1000 mg Pb kg1 (Figure 5). In general, the Pb accumulated in plants was in the order: AMF > PGPR > control (Figure 5). The highest (0.246 mg pot1) and lowest (0.012 mg pot1) Pb accumulated in plants was observed in AMF plants at Pb500 mg kg1 and non-inoculated plants at 0 Pb mg kg1, respectively (Figure 5). The results of the present study also showed that AMF and PGPR inoculation alleviated Pb toxicity in C. cyanus L. (Table 4). Various detoxification mechanisms of PTEs in plants symbioses with AMFare reported such as the plasma membrane functions as a selective barrier in plants, activation of specific or non-specific transporters and pores in the plasma membrane in plants,
PGPR 1 a)
c,c
a,c
2.70 C
a,a
34.16 A
24.79 B
Root Pb concentration (mg kg1) 0
4.16 0.07d,b
4.52 0.12d,a
4.64 0.02d,a
250
c,c
14.00 2.38
c,b
24.22 3.37
32.77 2.49c,a
500
b,c
31.98 0.88
b,b
39.64 2.89
50.18 4.04b,a
1000
46.57 1.83
Average
a,c
24.18 C
54.38 1.48
a,b
30.69 B
71.14 0.96a,a 39.68 A
1
Shoot Pb extraction (mg pot ) 0
0.008 0.001b,b
0.013 0.001c,a
0.016 0.001d,a
250
0.057 0.005
0.109 0.010
b,b
0.149 0.008b,a
500
0.061 0.008a,c
0.151 0.009a,b
0.192 n0.004a,a
1000
0.050 0.012
0.099 0.013
0.114 0.010c,a
Average
a,c
a,b
0.044 C
b,a
0.093 B
0.118 A
Root Pb stabilization (mg pot1) 0
0.004 0.000c,b
0.006 0.001c,a
0.006 0.001d,a
250
0.017 0.006
b,b
0.026 0.005
0.041 0.003c,a
500
0.025 0.003
ab,b
1000
0.032 0.007
Average
0.020 B
a,b
b,b
0.037 0.006
a,b
0.054 0.007b,a
0.041 0.005
a,b
0.067 0.008a,a
0.027 B
0.042 A
a) Native Pb was 21.42 mg kg1. For each data, mean SE followed by the first and second same letters is not significantly different among soil Pb levels and among control, AMF and PGPR treatments, respectively, according to the Duncan’stest at 5% probability level (n ¼ 3).
chelation in the cytosol including the use of metallothioneins (plants and fungi), organic acids, amino acids, and compoundspecific chaperones, active exportation by specific and non-specific pathways in plants and fungal cells.[31] In addition, symbioses with AMF provided the host plant with nutrients such as phosphorus which may be involved in plant Pb detoxification by means of molecules of phytates that can neutralize excess of metals.[11] Alleviation of Pb toxicity in PGPR plants may be due to enhancement of antioxidant enzymes activity in plants which is thought to be a detoxification mechanism. The data reported in the present work indicated that inoculation of AMF increased Pb accumulation in shoots and roots of Centaurea plant (Table 4 and Figure 5). AMF can affect the phyto-availability and mobility of metals by producing chelating factors. The ability of AMF in producing PTEs chelating factors, such as metallothioneins and glomalin, increases the availability of PTEs like Pb for plants, thus
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Moreover, the results indicated that Pb accumulation in Centaurea increased through PGPR inoculation (Table 4 and Figure 5). The increase in Pb uptake and concentration of PGPR plants might be due to the increase in Pb bioavailability. The metabolites released by inoculated PGPR (e.g. siderophores, organic acids, plant growth regulators, ACC deaminase, etc.) can alter the uptake of Pb indirectly through their effects on plant growth dynamics, and directly, through chelation, acidification and oxidation-reduction reactions in the rhizosphere. Therefore, PGPR may alleviate Pb toxicity and enhance plant growth through one or more of these mechanisms. Similar results have been also reported by Aksorn and Chitsomboonal,[34] indicating that PGPR enhanced the Pb accumulation in Vetiveria zizanioides.
Table 5. Shoot and root Pb mBCF and TF, in control, AMF or PGPR treatments at different levels of soil Pb contamination. Shoot mBCF Control
PGPR
AMF
1 a)
Total Pb added to soil (mg kg ) 0
1.30 0.18b,a
1.46 0.20b,a
1.26 0.30c,a
250
4.30 0.45
a,b
5.44 0.26
4.59 0.34a,b
500
4.32 0.31
a,b
5.63 0.38
3.72 0.31b,b
1000
4.00 0.17
a,b
5.33 0.59
3.73 0.11b,b
Average
a,a a,a a,a
3.48 B
4.47 A
3.33 B
Root mBCF 0
2.64 0.28c,a
2.35 0.20b,a
2.53 0.61b,a
250
4.45 0.09
4.77 0.16
a,b
6.46 0.45a,a
500
5.44 0.12a,b
4.73 0.26a,c
5.91 0.66a,a
1000
5.22 0.30
4.89 0.41
6.33 0.20
Average
b,b
a,b
4.44 B
a,b
3.4. Modified Bioconcentration Factor (mBCF)
a,a
4.19 B
In all treatments, with increasing soil Pb from Pb0 to Pb250, shoots mBCF significantly (p 0.05) increased, however, at higher concentrations (Pb500, Pb1000) no significant changes were observed in PGPR and control treatment, whereas, in AMF treatments shoots mBCF significantly (p 0.05) decreased (Table 5). The values of shoots mBCF in AMF and control treatments were not significantly (p > 0.05) different at all levels of soil Pb. The values of roots mBCF increased as the levels of Pb increased (Table 5). Inoculation of plants with PGPR caused a significant (p 0.05) increase (26.5–33.2%) in roots mBCF at Pb levels of 250, 500, and 1000 mg kg1. No matter the soil Pb level, the roots mBCF were significantly (p 0.05) higher in AMF plants compared to PGPR and control plants (Table 5). The highest root smBCF (6.46) was recorded in AMF plants at Pb250. In this study, roots and shoots mBCF obtained for all treatments and different levels of Pb in soil were above unity, indicating that the plant is able to take up and accumulate Pb (Table 5). At all concentrations of soil Pb, the maximum roots and shoots mBCF was observed in AMF and PGPR plants, respectively. According to mBCF, C. cyanus L. has the potential to
5.30 A
TF 0
0.49 0.03b,b
0.62 0.02b,a
0.50 0.01b,b
250
0.99 0.14
a,b
1.16 0.08
0.71 0.09a,b
500
0.79 0.02
a,b
1.20 0.12
0.63 0.08a,b
1000
0.77 0.04
a,b
1.09 0.13
0.59 0.09a,b
Average
a,a a,a a,a
0.76 B
1.02 A
0.61 B
a) Native Pb was 21.42 mg kg1. For each data, mean SE followed by the first and second same letters is not significantly different among soil Pb levels and among control, AMF and PGPR treatments, respectively, according to the Duncan’stest at 5% probability level (n ¼ 3).
increasing accumulation of Pb in plants. Gu et al.[33] has reported similar results reporting that mycorrhizal fungi inoculation significantly increased plant biomass and Pb accumulation in roots and shoots of Hylotelephium spectabile and Tradescantia pallida.
0.27
Root
a
Shoot
Pb uptake in plant (mg pot-1)
0.24 0.21
b
b
b
0.18
c c
0.15 0.12
d
d
0.09
d
0.06 0.03
f
e
e
0 Control PGPR AMF Control PGPR AMF Control PGPR AMF Control PGPR AMF Pb0
Pb250
Pb500
Pb1000
Figure 5. Pb uptake of plant in control, AMF or PGPR treatments at different levels of soil Pb contamination. Different letters above columns indicate significant differences according to the Duncan’s test at 5% probability level (n ¼ 3).
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be used in phytoextraction and phytostabilization of Pb. The low Pb concentration in shoots and roots of C. cyanus L. in this study was attributed to the low bioavailability of Pb in the calcareous soil with high pH (8.1) (Table 1) which had very low bioavailable fraction (1 M NH4NO3 extractable Pb < 12.33 mg kg1). In calcareous soils with high pH, most of the metals in soil are not available to plants, including Pb, which is inherently immobile in these soils.[35] Calcareous soil has high affinity to irreversibly sorb PTEs, through metal-carbonate precipitation.[35] In addition, at all concentrations of Pb in soil, inoculation of AMF and PGPR significantly increased the amount of roots mBCF and shoots mBCF, respectively (Table 5), because of higher shoots Pb concentration in PGPR treatment and higher roots Pb concentration in AMF treatment.
Abbreviations
3.5. Translocation Factor (TF) In all treatments, with increasing soil Pb from Pb0 to Pb250, rootto-shoot TF significantly (p 0.05) increased, while at higher concentrations (Pb500, Pb1000) no significant differences were recorded. Plants inoculated with PGPR had higher TF than AMF and control plants at every concentration of soil Pb. Translocation factor of Pb in plants at all treatments was in the order: PGPR > control > AMF (Table 5). Lower TF in AMF treatment showed that AMF-plant association immobilizes Pb in roots and reduces its translocation toward shoots. This result indicated that symbiosis of Glomus spp. with plant may restrict the transport of Pb to shoots and thus serve as a filtration barrier against transfer of Pb to the shoots of C. cyanus L. Some previous studies suggested that Pb was strongly retained within roots of mycorrhizal plants.[11] AMF has also been found to sequester PTEs in the roots of plants and restrict their translocation to the shoots.[11] It has been reported by Yang et al.[11] that AMF inoculation enhances Pb uptake and accumulation in the root system compared to non-mycorrhizal plants in Pb-contaminated soil, and AMF plays a filtering/sequestering role in Pbdetoxification. The TF for AMF and control treatments was 1) and low TF (
