Immobilization of copper and cadmium by hydroxyapatite combined with phytoextraction and changes in microbial community structure in a smelter-impacted soil

Abstract

Applying hydroxyapatite to immobilize heavy metals may cause a decline in soil quality, whereas phytoextraction has been shown to be a slow process under normal agronomic conditions. The benefits of combining hydroxyapatite application and phytoextraction are unclear for improving the soil ecosystem at the same time as remediating heavy metals. A three-year field experiment was conducted to test the combined effects of hydroxyapatite application and phytoextraction (Sedum plumbizincicola, Elsholtzia splendens, and Pennisetum sp.) on remediating cadmium (Cd) and copper (Cu) and improving the soil microbial community structure in a smelter-impacted site. Total Cd and Cu were fractionated using a sequential extraction procedure. Soil microbial biomass and community composition were measured by phospholipid-derived fatty acid (PLFA) analysis. The results showed that hydroxyapatite addition (1%) increased soil pH and decreased the exchangeable fraction of Cu and Cd compared with the control. Combining with hydroxyapatite application, the growth of phytoextractors increased organic matter content and decreased total Cu and Cd concentrations in the soil. Hydroxyapatite application disturbed the soil microbial PLFA composition by increasing fungal PLFA, whereas a combination with plant growth reduced the trend of soil fungal growth. Combining hydroxyapatite with Pennisetum sp. particularly improved Gram-negative and Gram-positive bacterial biomass and reduced the ratio of fungi to bacteria. In conclusion, combining hydroxyapatite with suitable phytoextractors is a promising approach for remediating heavy metals (e.g., Cu and Cd) and simultaneously improving soil microbial community structure and the ecological environment in contaminated areas.

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Introduction

Soil contamination with heavy metals is a growing problem worldwide due to intensive urbanization and industrialization, many manufacturing and agricultural activities, and improper waste disposal.^1–3 Different from organic contaminants, heavy metals cannot be degraded under natural conditions and thus accumulate in soils, posing a great negative impact on the environment.⁴ The pollution state of Cu and Cd in soil is serious around the world. According to statistics, there are about 470 thousand hectares of farmland soil contaminated by heavy metals in Japan, and the heavy metal responsible for the most serious contamination of agricultural land is Cd.^5,6 In Europe, 2.55 million sites have risk of contamination, and heavy metals such as Cu and Cd are the major contaminants.⁷ In China, the pollution situation is even more severe, 16.1 percent soils have been contaminated in all of the points in the surveyed soils, among all of the contamination, Cd and Cu accounted for 7.0% and 2.1%, respectively.⁸ Heavy metal contamination in soils is of concern in agricultural production due to its adverse effects on food quality, crop growth, and environmental health. Therefore, numerous physical, chemical, and biological remediation techniques have been developed to minimize the risk of harm from heavy metal contamination.⁹

In situ immobilization of heavy metals by chemical amendments is a cost-effective and environment-friendly remediation technique that reduces metal bioavailability in soils. Various soil amendments, both inorganic and organic, have been used to immobilize heavy metals in contaminated soils.¹⁰ Hydroxyapatite [Ca₅(PO₄)₃OH], a phosphate-containing amendment, has been shown to effectively immobilize heavy metals such as copper (Cu) and cadmium (Cd) in contaminated sites.^11,12 Phytoextraction of heavy metals by plants, known as hyperaccumulators, has been proved successful in remediating heavy metal,^13,14 with low cost, and without adverse effects on the environment.¹⁵ Hyperaccumulators can grow normally in soils contaminated with heavy metals and accumulate these metals in the harvested parts.¹⁶ A plant species is believed to have the potential to phytoextract Cu when the Cu concentrations in the plant body exceed 1000 mg kg⁻¹ shoot parts dry weight, and the standard for Cd is 100 mg kg⁻¹.¹⁶ In addition to hyperaccumulators, plants characterized by large biomass, fast growth, and strong resistance have the potential to be used as phytoextractors, such as Switchgras, Giantreed and Pennisetum sp.¹⁷ Despite the advantages it offers, phytoextraction has been shown to be a slow-working process due to the low amounts of metals that can be annually removed from the soil under normal agronomic conditions.¹⁸

A holistic approach is demanded to remediate heavy metals in a contaminated site, and reduce or eliminate the environmental risks, as well as restore the ecological functions of soil.¹⁹ In this regard, hydroxyapatite application can immobilize heavy metals, enhance soil enzymatic activities, and improve soil biological properties; growing plants also has multiple beneficial effects such as accumulating heavy metals, reducing soil erosion, and improving soil quality and function.²⁰ A few studies have assessed the probability of combining hydroxyapatite application with phytoextraction for soil remediation, devoting to the fate of heavy metals in soil. There is an evidence that hydroxyapatite to some extent prevents the uptake of heavy metals by plants, leading to a decrease in the recovery of heavy metals in plant tissue.²¹ However, the combined benefits of hydroxyapatite and phytoextraction on soil quality have not been estimated in prior studies solving the interaction between hydroxyapatite and metalliferous plants.

In order to accurately assess the efficiency and effectiveness of remediation, soil quality indicators must be properly selected. Soil microbial community structure has been shown to be a sensitive indicator of soil quality, which rapidly responses to the environmental changes after land use and management practices.^22–24 The use of soil microbial community structure as a soil quality indicator has been proposed, either by itself or combined with other physical, chemical and biological properties of the soil.²⁵ To our knowledge, only a few studies have assessed the individual effects of hydroxyapatite application and phytoextraction on soil microbial community structure.^20,26,27

In this study, we hypothesized that soil biochemical quality benefits from the combination of hydroxyapatite application and phytoextraction, despite the efficiency of phytoextraction being reduced by hydroxyapatite use. We also hypothesized that different phytoextractors (Sedum plumbizincicola, Elsholtzia splendens, and Pennisetum sp.) had different effects on the soil microbial community structure. To test these hypotheses, a three-year field experiment was conducted in a smelter-impacted soil with combined hydroxyapatite application and phytoextraction for remediation purposes. The objectives were to: (1) determine the immobilization and phytoextraction of heavy metals by analyzing soil Cu and Cd fractions, as well as plant biomass and metal accumulation and (2) assess the soil quality by monitoring the changes in soil microbial community structure after treatment.

Materials and methods

Study site

The study was conducted in a smelter-impacted area in Guixi City, Jiangxi Province, China. The area has a subtropical monsoon climate, with an average annual precipitation of 1808 mm. In this area, farmers had used wastewater discharged from a copper smelter for irrigation. Consequently, more than 130 ha of farmland area has suffered heavy metal contamination (mainly Cu and Cd), resulting in high Cd concentrations in rice exceeding the acceptable level.^28,29 Presently, most of the surrounding farmland is abandoned, with serious pollution, barren land, and desertification in local areas. The soil texture is sandy loam; the basic physicochemical properties of the soil are listed in Table 1.

Table 1 Physicochemical properties of the tested soil

pH	Organic C g kg^−1	Available N mg kg^−1	Available P mg kg^−1	Available K mg kg^−1	Total Cu mg kg^−1	Total Cd mg kg^−1
4.35	28.5	162	83.8	143	632	0.412

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Reagent and plants

Hydroxyapatite (purity > 96.0%, pH 7.71) was purchased from Emperor Nano Material Co. Ltd (Nanjing, China). The major properties of the hydroxyapatite were: particle size, 3 μm; specific surface area, 45.7 m² g⁻¹; calcium: phosphorous molar ratio, 1.72; Cd and Cu concentration were 3.83 × 10⁻² and 5.85 mg kg⁻¹ respectively.

Three phytoextractors were selected, including a Cu-tolerant plant (Elsholtzia splendens), a Cd hyperaccumulator (Sedum plumbizincicola), and an energy plant (Pennisetum sp.). All of the plants were derived from indoor-grown seedlings.

Experimental design

Five treatments were included in the study: untreated soil (CK), hydroxyapatite and native Setaria lutescens (MW), hydroxyapatite and Elsholtzia splendens (ME), hydroxyapatite and Sedum plumbizincicola (MS), and hydroxyapatite and Pennisetum sp. (MP). The plot area was 5 m (length) × 4 m (width) each, and plots were separated by plastic plates.

Each plot received one application of 1% hydroxyapatite (based on the 0–17 cm soil weight) on 23 December 2012. The hydroxyapatite was fully mixed into the soil by plowing. S. lutescens, an indigenous plant, was growing after hydroxyapatite application. Therefore, the MW treatment was considered as a hydroxyapatite-only treatment. For the other three treatments (ME, MS, and MP), E. splendens, S. plumbizincicola, and Pennisetum sp. were planted on 26 April each year (2013, 2014, and 2015). Weeds (mainly S. lutescens) were cleared from all plots before planting every year, and no weeding was carried out thereafter. The planting density was 20 cm × 20 cm for E. splendens and S. plumbizincicola plants, and 50 cm × 50 cm for Pennisetum sp. plants. All plots were managed using the same field management.

Sample collection

S. plumbizincicola was harvested in mid-July every year, while the other plants were harvested in mid-December every year. Only the above-ground parts (shoots) of the plants were collected. The plant samples were washed with tap water and then rinsed with ultrapure water. Thereafter, the samples were dried to constant weight in an oven at 80 °C and stored at room temperature before metal determination.

Soil samples (about 1 kilogram) were collected from the 0–17 cm depth at five representative locations per plot after harvest and then mixed together to form a composite sample. The soil samples were divided into two portions: one portion was aired and ground for physicochemical analysis, and the other portion kept at −80 °C for microbial analysis.

Soil physicochemical analysis

Soil pH was measured by mixing 2 g of soil sample with 5 mL of deionized water. The suspension mixture was stirred at room temperature (25 °C) for 0.5 h before the pH was measured using a pH meter (PHS-3CW-CN, Bante instrument, Shanghai, China). Soil organic carbon (SOC) was measured using the Walkley–Black procedure.³⁰ Soil available phosphate (P) and nitrogen (N) were measured in accordance with Bingham³¹ and soil available potassium (K) was measured according to Olsen.³²

Heavy metal analysis

Total soil Cu and Cd were measured by atomic absorption spectrophotometry (SpectrAA-220) after sample (0.4–0.5 ± 0.0001 g, 0.149 mm) digestion with hydrofluoric acid (HF), nitric acid (HNO₃), and perchloric acid (HClO₄) (10 : 5 : 5 mL) on an electric heating plate. A standard soil sample (GBW07405, National Research Center for Certified Reference Materials, China) was used to ensure the reliability of the experimental data. A sequential extraction procedure was used to fractionate Cu and Cd according to previous studies^29,33 with slight modifications. The metals were divided into five operationally defined fractions through the following steps:

(1) Exchangeable (EXC) fraction: 16 mL of 1 mol L⁻¹ MgCl₂ (pH 7.0) was added to a 50 mL centrifuge tube containing 2 ± 0.0001g of soil. The extraction was carried out under stirring at 120 rpm for 2 h, at the temperature of 25 ± 1 °C. After centrifugation (10 min, 4000 rpm), the supernatant was filtered with a 0.45 μm membrane. The filtrate was transferred into a 15 mL centrifuge tube and stored in refrigerator at 4 °C.

(2) Carbonate-bound (CA) fraction: 16 mL of 1 mol L⁻¹ CH₃COONa (adjusted to pH 5.0 with CH₃COOH) was added to the residue soil from step (1) and shaken in a reciprocating oscillator for 3 h at 25 ± 1 °C.

(3) Fe–Mn oxides-bound (Fe–Mn) fraction. The residue from step (2) was shaken with 40 mL of 0.04 mol L⁻¹ NH₂OH·HCl in 25% (v/v) CH₃COOH followed by occasional agitation for 6 h at 96 ± 3 °C.

(4) Organic matter-bound (OM) fraction: 6 mL of 0.02 mol L⁻¹ HNO₃ and 10 mL of 30% H₂O₂ (adjusted to pH 2.0 with HNO₃) were added to the residue from step (3), and the mixture was heated at 85 ± 3 °C for 2 h in a water bath. Then, 6 mL of 30% H₂O₂ (adjusted to pH 2.0 with HNO₃) was added, and the mixture was heated at 85 ± 3 °C for 3 h. After cooling, 10 mL of 3.2 mol L⁻¹ CH₃COONH₄ in 20% (v/v) HNO₃ was added and the mixture was continuously agitated for 30 min at 25 ± 1 °C.

(5) Residual (RES) fraction: The residue from step (4) was digested as performed for total Cu and Cd analysis.

Plant samples (0.25 g) were digested by using a mixture of 4 mL HClO₄ and 6 mL HNO₃ on an electric heating plate, and the metal contents were measured by atomic absorption spectroscopy. Blanks, replicate samples, and a certified reference material (GBW07401, provided by the Institute of Geophysical and Geochemical Exploration, Langfang, Hebei Province, China) were used to ensure the reliability of the experimental data.

Soil microbial analysis

Soil microbial phospholipid-derived fatty acids (PLFAs) were extracted as previously described.^34,35 Microbial species were identified by the type of fatty acid using the Sherlock Microbial Identification System (MIDI Inc, Newark, DE, USA). The total content of PLFAs and monomer fatty acids was determined using a 19:0 internal standard. Only fatty acids with carbon (C) chains less than 20, and mol% and content of more than 1% were analyzed.

Bacteria were shown by i13:0, i14:0, i15:0, a15:0, i16:0, i17:0, i18:0, i19:0, a16:0, a17:0, a19:0, 13:1ω3c, 16:1ω6c, 16:1ω7c, 17:1ω8c, 18:1ω3c, 18:1ω5c, 18:1ω6c, 18:1ω7c, cy17:0, cy19:0, 19:1ω6c, 20:1ω9c, 21:1ω3c, 21:1ω8c, 22:1ω3c, 22:1ω9c, and 24:1ω7c. Fungi were shown by 18:2ω6c and 16:1ω5c. The ratio of fungi to bacteria was shown by F/B. Actinomycetes were shown by 10Me16:0, 10Me17:0, 10Me18:0, and 10Me18:1ω7c. Gram-positive bacteria (G⁺) were shown by saturated fatty acids i13:0, i14:0, i15:0, a15:0, i16:0, i17:0, i18:0, i19:0, a16:0, a17:0, a19:0. Gram-negative bacteria (G⁻) were shown by 13:1ω3c, 16:1ω6c, 16:1ω7c, 17:1ω8c, 18:1ω3c, 18:1ω5c, 18:1ω6c, 18:1ω7c, cy17:0, cy19:0, 19:1ω6c, 20:1ω9c, 21:1ω3c, 21:1ω8c, 22:1ω3c, 22:1ω9c, and 24:1ω7c. The ratio of Gram-positive to Gram-negative bacteria was shown by G⁺/G⁻.^36,37

Statistical analysis

All the treatments were carried out in triplicate. The means and standard deviations of each treatment were calculated and presented. Differences between the means of treatments was estimated using one-way ANOVA at a significance level of 0.05 using SPSS 20.0 (IBM SPSS, Somers, NY, USA) when necessary. PLFA data were analyzed by canonical correspondence analysis (CCA) using ArcGIS 10 (ERIS, Redlands, CA, USA) and Visual Basic.

Results and discussion

Effects of hydroxyapatite application and plant growth on soil pH and SOC

In the untreated soil (CK), the pH slightly decreased from 4.24 in the first year to 4.20 in the third year (Table 2). With hydroxyapatite application only (MW), the soil pH was greatly increased to 5.17 in the first year than the control soil. This may be due to the high pH (7.71) of HAP, and it is consistent with Cui et al. who found soil pH was increased with the additions of micro-HAP and nano-HAP.³⁸ However, the soil pH remained almost constant in the second year (5.16) and then decreased in the third year (5.03). This may be attributable to the location of the area in an acid rain settlement region, where the H⁺ ions from acid rain resulted in the reduction of soil pH over time.³⁹ Moreover, the growth of phytoextractors in the presence of hydroxyapatite did not significantly change the soil pH (Table 2), although the plant might secreted some weak organic acid ions, sugars, amino acids, vitamins, and inorganic ions (HCO₃⁻, OH⁻, and H⁺) by roots could change the soil pH.⁴⁰

Table 2 Effects of hydroxyapatite application and phytoextraction on soil pH. CK = untreated soil, MW = hydroxyapatite + Setaria lutescens, ME = hydroxyapatite + Elsholtzia splendens, MS = hydroxyapatite + Sedum plumbizincicola, MP = hydroxyapatite + Pennisetum sp. Different lowercase letters indicate significant differences between treatments at the same time (n = 3, P < 0.05)

pH
Treatment	2013	2014	2015
CK	4.24 ± 0.213b	4.23 ± 0.113b	4.20 ± 0.276b
MW	5.17 ± 0.118a	5.16 ± 0.134a	5.03 ± 0.140a
ME	5.33 ± 0.054a	5.24 ± 0.258a	5.26 ± 0.168a
MS	5.19 ± 0.107a	5.13 ± 0.204a	5.13 ± 0.164a
MP	5.15 ± 0.143a	5.14 ± 0.252a	5.07 ± 0.104a

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After three years of remediation, the SOC content barely changed with hydroxyapatite application (MW) relative to the control (CK); however, significant increases occurred in the three combination treatments (ME, MS, MP Fig. 1). The highest SOC content was obtained with the MP treatment, which did not significantly differ from the results of ME and MS. The growing of plants could increase the amount of litter and fine roots, and change the structure of soil aggregates, leading to an increase in SOC content.^41–43 This means that the plants growth is effective to increase the nutrient supply for microbial activities.

Fig. 1 Soil organic carbon content in 2015. CK = untreated soil, MW = hydroxyapatite + Setaria lutescens, ME = hydroxyapatite + Elsholtzia splendens, MS = hydroxyapatite + Sedum plumbizincicola, MP = hydroxyapatite + Pennisetum sp. Different lowercase letters indicate significant differences between treatments after three years (n = 3, P < 0.05).

Changes in total concentrations and different fractions of Cd and Cu in soil

Heavy metals in soils can be classified into solid and solution phases; however, the metals are more likely to exist in a solid phase. Solid-phase heavy metals can be further divided into different fractions, whose solubility, mobility, bioavailability, and potential environmental toxicity cannot be fully reflected by any single-step extraction method. In the present study, a sequential extraction procedure was used to solve the problem. The total concentrations and different fractions of Cu and Cd in the soil are listed in Tables 3 and 4, respectively. The percentage distributions of the Cu and Cd fractions in the soil are shown in Fig. 2a and b, respectively.

Table 3 Effects of hydroxyapatite application and phytoextraction on Cu fractions. CK = untreated soil, MW = hydroxyapatite + Setaria lutescens, ME = hydroxyapatite + Elsholtzia splendens, MS = hydroxyapatite + Sedum plumbizincicola, MP = hydroxyapatite + Pennisetum sp., EXC = exchangeable fraction, CA = carbonate-bound fraction, Fe–Mn = Fe–Mn oxides-bound fraction, OM = organic matter-bound fraction, RES = residual fraction. Different lowercase letters indicate significant differences between treatments after three years (n = 3, P < 0.05)

Treatment	EXC mg kg^−1	CA mg kg^−1	Fe–Mn mg kg^−1	OM mg kg^−1	RES mg kg^−1	Total mg kg^−1
CK	211 ± 11.1a	87.2 ± 1.73b	143 ± 5.03b	118 ± 4.42a	98.2 ± 11.5b	668 ± 11.7a
MW	75.2 ± 14.8b	123 ± 7.22a	171 ± 8.48ab	126 ± 4.22a	135 ± 9.91ab	658 ± 24.5a
ME	65.5 ± 19.4b	117 ± 16.7ab	173 ± 9.17ab	128 ± 10.9a	121 ± 16.4ab	616 ± 8.56b
MS	68.4 ± 12.6b	118 ± 17.5ab	196 ± 20.6a	125 ± 4.73a	113 ± 17.3ab	631 ± 8.54ab
MP	69.1 ± 8.53b	117 ± 9.83ab	173 ± 23.0ab	131 ± 8.16a	149 ± 12.7a	649 ± 13.8ab

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Table 4 Effects of hydroxyapatite application and phytoextraction on Cd fractions. CK = untreated soil, MW = hydroxyapatite + Setaria lutescens, ME = hydroxyapatite + Elsholtzia splendens, MS = hydroxyapatite + Sedum plumbizincicola, MP = hydroxyapatite + Pennisetum sp., EXC = exchangeable fraction, CA = carbonate-bound fraction, Fe–Mn = Fe–Mn oxides-bound fraction, OM = organic matter-bound fraction, RES = residual fraction. Different lowercase letters indicate significant differences between treatments after three years (n = 3, P < 0.05)

Treatment	EXC mg kg^−1	CA mg kg^−1	Fe–Mn mg kg^−1	OM mg kg^−1	RES mg kg^−1	Total mg kg^−1
CK	0.174 ± 9.91 × 10^−3a	3.12 × 10^−2 ± 3.52 × 10^−3b	4.46 × 10^−2 ± 4.64 × 10^−3b	7.64 × 10^−2 ± 5.43 × 10^−4b	0.174 ± 5.24 × 10^−3ab	0.406 ± 154 × 10^−2a
MW	0.0876 ± 1.23 × 10^−2b	5.14 × 10^−2 ± 3.78 × 10^−3a	6.92 × 10^−2 ± 4.23 × 10^−3a	1.26 × 10^−2 ± 2.11 × 10^−3a	0.185 ± 1.47 × 10^−2a	0.389 ± 1.24 × 10^−2ab
ME	0.0816 ± 9.23 × 10^−3b	5.30 × 10^−2 ± 1.01 × 10^−2a	6.63 × 10^−2 ± 5.87 × 10^−3a	1.05 × 10^−2 ± 1.66 × 10^−3ab	0.188 ± 2.04 × 10^−2a	0.364 ± 5.34 × 10^−2b
MS	0.0674 ± 1.20 × 10^−2b	5.24 × 10^−2 ± 8.28 × 10^−3a	6.48 × 10^−2 ± 5.92 × 10^−3a	1.19 × 10^−2 ± 1.43 × 10^−3a	0.158 ± 1.13 × 10^−2ab	0.350 ± 1.20 × 10^−2b
MP	0.0788 ± 1.20 × 10^−2b	4.85 × 10^−2 ± 6.18 × 10^−3ab	6.03 × 10^−2 ± 6.23 × 10^−3a	9.53 × 10^−3 ± 7.46 × 10^−4ab	0.149 ± 6.01 × 10^−3b	0.350 ± 2.27 × 10^−2b

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Fig. 2 Effects of hydroxyapatite application and phytoextraction on percentage of (a) Cu and (b) Cd fractions after three years. CK = untreated soil, MW = hydroxyapatite + Setaria lutescens, ME = hydroxyapatit e + Elsholtzia splendens, MS = hydroxyapatite + Sedum plumbizincicola, MP = hydroxyapatite + Pennisetum sp., EXC = exchangeable fraction, CA = carbonate-bound fraction, Fe–Mn = Fe–Mn oxides-bound fraction, OM = organic matter-bound fraction, RES = residual fraction.

Without any treatment (CK), the total Cu concentration was 668 mg kg⁻¹ in the soil. The EXC fraction of Cu (211 mg kg⁻¹, 31.6%) was most abundant, followed by the Fe–Mn fraction (143 mg kg⁻¹, 21.4%) and OM fraction (118 mg kg⁻¹, 17.7%); the CA fraction (87.2 mg kg⁻¹, 13.0%) and RES fraction (98.2 mg kg⁻¹, 14.7%) were the lowest. With hydroxyapatite application (MW), the total Cu concentration was slightly but not significantly decreased to 658 mg kg⁻¹. The EXC fraction was reduced to 75.2 mg kg⁻¹ (11.4%), while the CA fraction was increased to 123 mg kg⁻¹ (18.7%); no significant differences were found in the other factions compared with the data of CK. On the basis of hydroxyapatite application, the growth of E. splendens (ME), S. plumbizincicola (MS), and Pennisetum sp. (MP) further reduced the total Cu concentration to varying degrees, without significantly affecting the distribution of Cu in different fractions. After three years of combined remediation, the total Cu concentrations were decreased by 52.8 mg kg⁻¹ (P < 0.05), 37.9 mg kg⁻¹ (P > 0.05), and 19.0 mg kg⁻¹ (P > 0.05), respectively, for ME, MS, and MP compared with CK.

The total Cd concentration was 0.406 mg kg⁻¹ in the soil without treatment (CK). The Cd in the untreated soil mainly occurred in the EXC fraction (0.174 mg kg⁻¹, 42.9%), similar to the distribution of Cu. Additionally, the RES fraction (0.174 mg kg⁻¹, 43.0%) was abundant and markedly greater than the OM fraction (7.64 × 10⁻² mg kg⁻¹, 1.9%). With hydroxyapatite application (MW), the total Cd concentration was slightly, but not significantly decreased to 0.389 mg kg⁻¹ in the soil. The EXC fraction of Cd was significantly reduced to 0.0876 mg kg⁻¹ (22.5%), while the CA, Fe–Mn, and OM fractions were markedly increased to 5.14 × 10⁻² mg kg⁻¹ (13.2%), 6.92 × 10⁻² mg kg⁻¹ (17.8%), and 1.26 × 10⁻² mg kg⁻¹ (3.2%), respectively. In the presence of hydroxyapatite, the growth of phytoextractors slightly reduced the total Cu concentration, without changing the distribution of Cu in various fractions; an exception was that the RES fraction was significantly reduced for MP compared with MW. After 3 years, the total concentration of Cd was significantly reduced for the three combination treatments (ME, MS, and MP) compared with the control (CK).

The exchangeable fraction of heavy metals is considered easily mobile and available.³³ Additionally, the carbonate-, Fe–Mn oxides-, and organic matter-bound fractions of heavy metals are potentially available to plants and microorganisms, while the residual fraction is dominant among the five fractions. The exchangeable and carbonate-bound fractions of Cu are often present in uncontaminated soil at low concentrations (<10% of total Cu).⁴⁴ In the present study, the exchangeable and carbonate-bound fractions of Cu constituted 44.7% of the total Cu in the untreated soil, which was inappropriate for agricultural use. We conjectured that the source of Cu was mainly artificial and introduced from the exhaust gas, dust and wastewater from the nearby copper smelter.

Additionally, the exchangeable fraction can be used to evaluate the bioavailability and environmental toxicity of heavy metals.⁴⁵ In the current study, the concentrations of Cu and Cd were decreased in the exchangeable fraction but increased in the Fe–Mn oxides-bound and residual fractions after hydroxyapatite application compared with the control. The results illustrate that hydroxyapatite can significantly reduce the bioavailability of Cu and Cd in the contaminated soil. The exchangeable fraction of Cd (42.9%) was greater than Cu (31.6%) in the untreated soil, indicating a higher mobility of Cd than that of Cu in this contaminated site. The exchangeable fraction (concentrations and percentages) of both Cd and Cu was significantly decreased after hydroxyapatite addition compared with the untreated soil. This demonstrates that hydroxyapatite can decrease the bioavailability of Cd and Cu, and thus is effective in remediating soils contaminated with the two metals.

The possible mechanism of the immobilization of heavy metals by hydroxyapatite can be attributed to the following points: (1) ion exchange reaction on the surface of hydroxyapatite (eqn (1));⁴⁶ (2) surface complexation reaction of hydroxyapatite (eqn (2));⁴⁷ (3) co-precipitation (eqn (3) and (4));⁴⁸ and (4) precipitation of amorphous to poorly crystalline, mixed metal phosphates.⁴⁹

Ca₁₀(PO₄)₆(OH)₂ + xCd²⁺ = Ca_10−x(PO₄)₆ + xCa²⁺ (1)

≡POH + Cd²⁺ = ≡POCd⁺ + H⁺ (2)

xCd²⁺ + (10 − x)Ca²⁺ + 6H₂PO₄⁻ + 2H₂O = Ca_10−xCd_x(PO₄)₆(OH)₂ + 14H⁺ (3)

xCd²⁺ + (10 − x)Ca²⁺ + 6HPO₄²⁻ + 2H₂O = Ca_10−xCd_x(PO₄)₆(OH)₂ + 8H⁺ (4)

During plant growth, the root system provides an interface between the plant and soil. The roots are not only an absorption and metabolism organ, but also a powerful secretor.⁵⁰ Respiration, absorption, and secretion of organic compounds by the roots greatly impact the physicochemical properties of the soil, as well as the migration and conversion of heavy metals in the contaminated soil. In the present study, the growth of phytoextractors such as E. splendens, S. plumbizincicola, and Pennisetum sp. reduced the total Cd and Cu concentrations in the soil with hydroxyapatite application (Tables 3 and 4). This effect could be attributable to the strong absorption and transport capacity of these plants for heavy metals (Table 5). Furthermore, root exudates and litter could increase the soil organic carbon (SOC) content, enhance the chelation of organic matter and heavy metals, and decrease the exchangeable fraction of heavy metals.⁵¹ Owing to the differences in plant types, the secretion of organic compounds could vary considerably, affect the soil pH, redox potential, and, eventually, the fractions of Cu and Cd.⁵²

Table 5 Shoot biomass and Cu and Cd accumulation in each plant during phytoextraction. CK = untreated soil, MW = hydroxyapatite + Setaria lutescens, ME = hydroxyapatite + Elsholtzia splendens, MS = hydroxyapatite + Sedum plumbizincicola, MP = hydroxyapatite + Pennisetum sp. Different lowercase letters indicate significant differences between treatments in the same year (n = 3, P < 0.05). — indicates no plant growth

Treatment	Shoot biomass (kg dry weight per plot per year)			Metal accumulation (mg per plot per year)
				Cu			Cd
	2013	2014	2015	2013	2014	2015	2013	2014	2015
CK	—	—	—	—	—	—	—	—	—
MW	20.2 ± 9.81bc	17.1 ± 3.04bc	10.4 ± 1.12c	4.72 × 10^2c	5.69 × 10^2b	4.47 × 10^2b	20.7b	20.5b	13.0c
ME	30.1 ± 8.33ab	25.1 ± 2.69b	28.7 ± 8.44b	5.47 × 10^3a	5.08 × 10^3a	5.85 × 10^3a	78.3a	64.1a	75.1b
MS	4.50 ± 0.725c	4.20 ± 0.422c	5.43 ± 0.936c	2.05 × 10^3c	1.82 × 10^3b	2.56 × 10^3ab	59.9ab	58.9a	76.1ab
MP	44.6 ± 6.67a	58.4 ± 12.2a	75.3 ± 8.18a	3.75 × 10^3b	5.96 × 10^3a	7.62 × 10^3a	58.2ab	79.5a	104a

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Changes in shoot biomass and metal accumulation in different plant species

In the present study, the native S. lutescens and the three phytoextractors were able to grow normally after hydroxyapatite application. Although S. lutescens also appeared in the untreated soil, the plants grew slowly and gradually withered—a typical symptom of plants suffering from heavy-metal poisoning.⁵³ Among the three phytoextractors, Pennisetum sp. had the largest biomass every year and reached 75.3 kg per plot in 2015, which was 15 times that of S. plumbizincicola. E. splendens also had a larger biomass than S. lutescens and remained about 28.0 kg per plot in the three years. S. plumbizincicola had the lowest biomass, with a mean value of 4.71 kg per plot over the three years. The biomass data ranked MP > ME > MW > MS (Table 5).

Despite its low biomass, S. plumbizincicola (a Cd hyperaccumulator) showed the highest absorption capacity for Cu (451.5 mg kg⁻¹) and Cd (13.7 mg kg⁻¹), which was 13.8 and 11.8 times that of S. lutescens, respectively. E. splendens (a Cu-tolerant plant) also showed high absorption capacity for Cu and Cd, which reached 202 mg kg⁻¹ and 2.59 mg kg⁻¹, respectively(Table 5, Fig. 3). The Cd adsorption capacity of S. plumbizincicola was only 14.1 mg kg⁻¹ (Fig. 3), much lower than the value reported in a previous study, i.e., up to 320 mg kg⁻¹.⁵⁴ This discrepancy might be due to a higher Cd concentration in the soil used in the previous study, which reached 14.7 mg kg⁻¹. Moreover, 1% hydroxyapatite was added to the soil to promote the growth of plants in the present study. Therefore, the bioavailability of Cu and Cd was decreased in the soil, which, together with the plant physiological properties, might have influenced metal uptake by S. plumbizincicola.⁵⁵

Fig. 3 Concentrations of (a) Cu and (b) Cd in the shoots of each plant. MW = Setaria lutescens, ME = Elsholtzia splendens, MS = Sedum plumbizincicola, MP = Pennisetum sp. Different lowercase letters indicate significant differences in the same treatment among the three years (n = 3, P < 0.05).

The three-year data of Cu and Cd in plants showed a significant increase in the concentrations of Cd and Cu in S. lutescens (a native plant) and Pennisetum sp. (an energy plant) over time, while this phenomenon was absent in E. splendens and S. plumbizincicola. The absorption of heavy metals by plants may have been increased due to the elevation of Cu and Cd concentrations in the soil with the decrease in soil pH over time.⁵⁶ On the other hand, E. splendens and S. plumbizincicola have a better buffering capacity for the environmental changes than S. lutescens and Pennisetum sp.^57,58

There was a significant decrease in the biomass of MW treatment in the three years (Table 5), from 20.2 to 10.4 kg dry weight per plot per year. This also might be related to the increase of the concentration of heavy metals in soil. A study conducted in this area by Cui⁵⁹ showed that, after the application of 5 kinds of amendments, the ryegrass could grow in the first year, but the biomass of ryegrass decreased year by year during the three years. There were only two kinds of amendments (lime, apatite) could make the ryegrass grow after three years. These results proved that, plants could grow normally only in the treatments of application the amendments, and the effect of the amendments would decrease year by year, which lead to the increase of heavy metal activity and the reduction of plant biomass. The present study supports these findings, there was no plant in the treatment of no hydroxyapatite application and as time going by, the biomass of S. lutescens was gradually reduced in the MW treatment.

With regard to the absolute accumulation of Cu and Cd, Pennisetum sp. showed the greatest advantage, with the three-year cumulative amounts of 1.73 × 10⁴ and 242 mg, respectively (Table 5). The high accumulation of heavy metals by Pennisetum sp. plants is mainly ascribed to their large biomass.⁶⁰ The integrated results of plant biomass and accumulation capability showed that E. splendens and S. plumbizincicola had similar absolute accumulation of Cu and Cd. The poor biomass and accumulation ability of S. lutescens showed that this native plant species had the lowest absolute accumulation. In terms of absolute accumulation concentration, the remediation efficiency of different plants ranked MP > ME > MS > MW.

Changes in soil microbial community structure

The PLFA composition of soil microbial communities can be used as a fingerprint to characterize the patterns of soil microbial community structure.⁶¹ After three years of remediation, substantial improvements were observed in the total PLFA (PLFA_total) of the soil (>40.0 nmol g⁻¹) compared with the control (36.3 nmol g⁻¹), except for the ME treatment (38.1 nmol g⁻¹) (Table 6). The addition of hydroxyapatite probably depressed the bioavailability of heavy metals in the soil and thus attenuated their toxicity to soil microorganisms to some extent. Meanwhile, the increase in soil pH (due to hydroxyapatite application) and SOC (due to plant growth) could reduce the soil exchangeable acid and exchangeable Al, which are beneficial to restore the soil microbial community structure.^62,63

Table 6 Effects of hydroxyapatite application and phytoextraction on soil microbial PLFA composition. CK = untreated soil, MW = hydroxyapatite + Setaria lutescens, ME = hydroxyapatite + Elsholtzia splendens, MS = hydroxyapatite + Sedum plumbizincicola, MP = hydroxyapatite + Pennisetum sp. Different lowercase letters indicate significant differences between treatments after three years (n = 3, P < 0.05)

Treatment	PLFAtotal nmol g^−1	PLFAbact nmol g^−1	PLFAfungi nmol g^−1	PLFAact nmol g^−1	G^+ nmol g^−1	G^− nmol g^−1	G^+/G^− ratio	F/B ratio
CK	36.3 ± 1.82b	26.9 ± 1.73c	5.03 ± 0.433b	4.30 ± 0.445c	20.0 ± 1.23c	6.60 ± 0.544b	3.10 ± 0.333a	0.181 ± 2.34 × 10^−2b
MW	45.8 ± 2.74a	32.2 ± 2.04ab	7.70 ± 0.849a	6.02 ± 0.192ab	23.6 ± 0.925ab	8.63 ± 1.26ab	2.82 ± 0.428a	0.240 ± 2.42 × 10^−2a
ME	38.1 ± 2.15b	29.0 ± 1.15bc	4.41 ± 0.443b	4.73 ± 0.743bc	21.5 ± 0.493bc	7.51 ± 0.823b	2.93 ± 0.235a	0.153 ± 1.25 × 10^−2bc
MS	41.8 ± 0.833ab	32.1 ± 0.336b	4.78 ± 0.526b	5.04 ± 0.545bc	23.8 ± 0.396b	8.19 ± 0.110ab	2.87 ± 0.146a	0.151 ± 2.38 × 10^−2bc
MP	47.1 ± 3.08a	36.5 ± 2.23a	4.11 ± 0.507b	6.18 ± 0.537a	27.4 ± 1.82a	9.12 ± 1.01a	3.03 ± 0.433a	0.120 ± 1.41 × 10^−2c

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In the hydroxyapatite only treatment (MW), the PLFA_total was significantly improved in the soil compared with the control (CK). MW particularly improved the fungal PLFA (PLFA_fungi); despite an improvement in the bacterial PLFA (PLFA_bact, mainly G⁺) and actinomycete PLFA (PLFA_act), the F/B ratio was the markedly increased for this treatment (0.240). In the presence of hydroxyapatite, the E. splendens (ME) and S. plumbizincicola (MS) treatments reduced the PLFA_total in the soil compared with MW. This change mainly attributed to a reduction in the PLFA_fungi, which resulted in a lower F/B ratio (0.153 and 0.151). The Pennisetum sp. treatment (MP) did not further change the PLFA_total of the soil compared with MW, however, MP improved the PLFA_bact (G⁺ and G⁻) and decreased the PLFA_fungi, resulting in the lowest F/B ratio (0.120) (Table 6).

The PLFA_bact/PLFA_fungi ratio and the F/B ratio are indices for measuring soil microbial function.⁶⁴ In the present study, hydroxyapatite application alone changed the soil pH significantly and decreased the bioavailable Cu and Cd contents in the contaminated soil; however, the non-biological system has a buffering effect on the soil habitat, leading to vigorous fungal growth, unfavorable for the formation of microbial community structure.⁶⁴ The combined hydroxyapatite application and phytoextraction effectively increased biomass of soil bacterial communities, while the trend of soil fungal growth was alleviated (Table 6). Compared with E. splendens and S. plumbizincicola, Pennisetum sp. had a larger biomass and its root system was widely distributed in the soil; thus, the soil rhizosphere effect was more obvious for the MP treatment, which led to a more significant improvement in the microbial community and biomass.⁶⁵ Pearson's correlation analysis revealed that the SOC content was significantly correlated with the PLFA_bact in the contaminated soil (R² = 0.714, P = 0.05). This indicates that the root metabolism and secretion of plants have the potential to enrich the soil microbial community.

Two-dimensional CCA (Fig. 4) showed the relationship between different treatments and PLFA factors more directly. The corresponding coordinate axes explained 89.3% of the variation of the soil microbial community structure. The microbial communities of three combination treatments (ME, MS, and MP) were clear distinct from those of the hydroxyapatite-only treatment (MW) and the untreated soil (CK). Soil bacterial and fungal communities were sensitive to Cu and Cd contamination, and negatively correlated with the control. The application of hydroxyapatite only was significantly positively correlated with the soil fungal community. Combination of hydroxyapatite application and phytoextraction, particular with Pennisetum sp. (MP), changed the soil microbial community and was significantly positively correlated with soil Gram-positive and Gram-negative bacteria. This indicates that the MP treatment can effectively alleviate the trend of soil fungi growth and improve the bacterial communities and biomass, recovering the soil ecological function.

Fig. 4 Two-dimensional canonical correspondence analysis of different treatment groups and soil microbial communities. MW = Setaria lutescens, ME = Elsholtzia splendens, MS = Sedum plumbizincicola, MP = Pennisetum sp.

Conclusions

This study demonstrates the benefits of combing hydroxyapatite application and phytoextraction for improving soil quality at the time of remediating heavy metals. The application of hydroxyapatite alone increased the soil pH and decreased the bioavailable Cd and Cu concentrations in a smelter-impacted soil. However, the disturbance of soil chemical properties by only hydroxyapatite application caused an imbalance in the soil microbial community structure, which might stimulate fungal growth. With hydroxyapatite application, growth of phytoextractors such as Pennisetum sp, S. plumbizincicola, and E. splendens increased the SOC content, while it reduced the active Cd and Cu fractions, as well as some of the total Cu and Cd contents, leading to a decreased metal availability in the soil. In the presence of hydroxyapatite, growth of S. plumbizincicola and Pennisetum sp. increased the total soil microbial PLFA. Hydroxyapatite addition combined with Pennisetum sp. growth was more conducive to restore the soil micro-ecological system by reducing the ratio of fungi to bacteria. Therefore, hydroxyapatite amendment combined with suitable phytoextractors is an effective way for remediation of heavy metals contaminated soil and simultaneous improvement of soil micro-ecological environment in contaminated areas.

Acknowledgements

The authors acknowledge the National Basic Research Program of China (2013CB934302), the National Science and Technology Support Plan (2015BAD05B01), the Jiangxi Province Talent Project 555, and the National Natural Science Foundation of China (41571461).

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