Ecotoxicology and Environmental Safety 168 (2019) 1–8

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Optimization of combined phytoremediation for heavy metal contaminated T

mine tailings by a field-scale orthogonal experiment
⁎
Xinxin Li, Xinlei Wang, Yuedong Chen, Xiaoyong Yang, Zhaojie Cui
School of Environmental Science and Engineering, Shandong University, Qingdao 266000, China

A R T I C LE I N FO A B S T R A C T

Keywords: The combined application of plant, microorganism, and amendment on the phytoremediation of heavy metals
Plant was optimized as a remediation technique for mine tailings by a field-scale orthogonal (L16) experiment, aimed
Microorganism to achieve the maximum of phytoremediation effect. Soybean, M. Circinelloides, and A3 amendment (organic
Amendment fertilizer: rice husk: biochar: ceramsite = 1:1:2:1) were recommended as the best plant, microorganism, and
Phytoremediation
amendment materials, respectively. With the combined plant, microorganism, amendment application, effective
Orthogonal experiment
fractions of Cu, Zn, Pb, Cd, Mn were immobilized for decreased bioavailability, indicating the phytostabilization
served as a major repair pathway. Plant length and biomass in the treatments were significantly higher than that
in the control, indicating their phytoremediation potentials were enhanced. The final contents of heavy metals in
soil were decreased, and the removal rates of soil heavy metals were in the order of Pb＞Cd＞Cu＞Zn＞Mn.
Temporal variations of soil microorganism populations indicated that the abundance of soil microorganism in
the treatments was significantly higher than that in the control, and bacteria became the dominant microbial
species. Results showed that the soil organic matter and catalase, urease, phosphatase activities of the treatments
were all significantly higher than that of the control. This study provided optimized combined plant, micro-
organism, amendment materials in the enhanced phytoremediation field to make up the deficiencies of the long-
term phytoremediation for heavy metals.

1. Introduction Lombardi et al., 2010). Several metals such copper (Cu), zinc (Zn), and
manganese (Mn) are essential for biological systems and provide es-
Mining activities alone in China had generated total mineral re- sential cofactors for metallo-proteins and enzymes in a certain con-
sidues of about 5 billion tons by the end of the year 2010 and the figure centration range. However, metals accumulation can act in a deleter-
was increasing at an alarming rate of 500 million tons per year (Wang ious manner by blocking essential functional groups, displacing other
et al., 2014). The excessive mineral residues can lead to soil erosion, air metal ions, or modifying the active conformation of biological mole-
and water pollution, geo-environmental disasters and diseases, the loss cules (Garba et al., 2016).
of cultivated land, forest and pasture land, and the overall loss of Phytoremediation refers to the use of green plants and their asso-
production, biodiversity, and ultimately loss of economic wealth (Xia ciated microbiota, soil amendments, and agronomic techniques to re-
and Cai, 2002; Wong, 2003). Among these problems, heavy metal (HM) move, contain, or render harmless environmental contaminants
contamination, as the one of the most serious and potential threats in (Cunningham and Ow, 1996). This technique is environmentally
mineral residues, tends to inhibit soil-reforming processes and plant friendly, potentially cheap, visually unobstructive and offers the pos-
growth, and eventually threatened the ecosystem via food chain. sibility of bio-recovery of the HMs. It basically includes phytostabil-
Compared with organic pollutants, HMs cannot be decomposed through ization, phytoextraction, phytodegradation, rhizofiltration, and phyto-
microbial action, instead they will result in contamination even at re- volatilization. Plants, depending on their species and genotype, differ in
latively low-level emissions as their accumulation (Sun et al., 2018). their efficiency of phytoremediation (Baird and Cann, 1997). For soil
HMs, such as lead (Pb) and cadmium (Cd), are known to be toxic with contaminated by HMs, metal toxicity would restrict the growth of all
the potential to cause chronic poisoning, including impaired develop- but the more tolerant plants. These plants can be used to remove,
ment, reduced intelligence, short-term amnesia, cognitive disorder, transfer, stabilize and/or degrade HM soil contaminants. Phenolic
cardiovascular diseases, and so on (Dauvin, 2008; Flora et al., 2008; compounds and 20% of the assimilated carbon are two important class

⁎
Corresponding author.
E-mail address: [email protected] (Z. Cui).

https://doi.org/10.1016/j.ecoenv.2018.10.012
Received 31 May 2018; Received in revised form 25 September 2018; Accepted 4 October 2018
0147-6513/ © 2018 Elsevier Inc. All rights reserved.
X. Li et al. Ecotoxicology and Environmental Safety 168 (2019) 1–8

Fig. 1. Site selection diagram of the demonstration project. The location of the experimental phytoremediation area (a), the boundary of the experimental area (b),
and the distribution of phytoremediation plots (c).

of compounds exuded by plant roots into the rhizosphere, which could had developed hypha and could colonize in the rhizosphere to enhance
be utilized as a direct source of energy for soil microorganisms and plant growth and maintain soil beneficial bacteria (Viterbo et al., 2010;
metal ion chelating agents. Soybean (Glycine max (L.) Merr.), Rainbow Tripathi et al., 2013). Some common organic materials with large
pink (Dianthus chinensis L.), and Kochia (Bassia scoparia (L.) AJ Scott) surface areas and excellent optical properties, such as biochar, have
were used as model plants due to their rapid growth, tolerance to been widely used as soil amendments. They can adsorb and fix HMs in
various environments and soil types, and their common use in phy- soil, which acts to reduce metals and their compounds in the environ-
toremediation studies. ment by altering their availability in three ways: by adsorption onto the
Although phytoremediation has been widely accepted as an en- surfaces of mineral particles, by complexation by humic substances in
vironmentally harmonious remediation technique to extract, absorb, or organic particles, and by precipitation reactions (Bradshaw and
detoxify pollutants, it takes longer to implement compared with other Chadwick, 1980). In addition, they were also used for remediating the
treatments. Therefore, one or more of these approaches is often com- physical and chemical properties of the soils, and the provision of plant
bined for more cost-effective treatment. The establishment of a syner- nutrients.
gism between plants and microorganisms has been suggested as a The aim of this study was 1) to optimize the plant, microorganism,
means of accelerating HM phytoremediation process (Ma et al., 2017). and amendment materials in application to the phytoremediation for
Such stimulation of soil microbial communities by root exudates could HM-contaminated soil, 2) to investigate the interactions of plant, mi-
benefit plants through increased availability of soil-bound nutrients and croorganism, and amendment in combination involved in the promo-
degradation of phytotoxic soil contaminants. The mycorrhizal hyphae tion of phytoremediation efficiency. The potential was determined by
network is more extensive than a root system, which can increase up- the growth changes in different plants and their parts, the removal rates
take of pollutants due to the ability to penetrate relatively small soil and speciation distributions of HMs in contaminated soil, and the
pores (Garcíasánchez et al., 2018). Mycorrhizal associations increased changes in soil microorganism populations as well as organic matter
the absorptive surface area of the plant due to extra-matrical fungal and enzyme activities. To find the optimal plant-microorganism-
hyphae exploring rhizospheres beyond the root hair zone, which in turn amendment remediation strategy effectively, this study was designed
enhanced water and mineral uptake. In this research, the selected through an orthogonal experiment (L16) and subsequently carried out in
strains Mortierella sp., Trichoderma asperellum and Mucor circinelloides the field.

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X. Li et al. Ecotoxicology and Environmental Safety 168 (2019) 1–8

2. Material and methods 2.3. Plant length and biomass

2.1. Site description and experimental design Three replicates for each plot were harvested at the 105th day by
cutting the shoots at the soil surface and carefully separating the roots
An experimental plot (approximate trapezoidal with 32-m short from the soil. The roots and shoots were washed thoroughly with dis-
side, 65-m long side, and 35.4-m height) was established on metal- tilled water, and then dried with blotting paper. The length and biomass
contaminated mine tailings from the Anshan Mining Group Corporation of the plant samples in shoot and root were measured.
in Liaoning Province, northeast China (E 122°10′–123°41′, N
40°27′–41°34′). The basic properties of the tailings were: pH 8.49; or-
ganic matter 0.95%; total nitrogen (N) 0.11 g kg−1; effectual phos- 2.4. Soil bacterial, fungal, and actinomycetes populations
phorus (P) 2.19 mg kg−1; effectual potassium (K) 48.60 mg kg−1. The
total contents of target HMs, namely, Cu, Zn, Pb, Cd, and Mn were 51.7, The 5.0 g-fresh soil samples were mixed with 50 mL sterile water
151.5, 43.6, 0.8, and 1960.0 mg kg−1, respectively. and oscillated for 10 min in a Shaker (NHWY-1102C, 70 r min −1,
The design of L16 (4 × 32) matrix was adopted to investigate the 25 °C). The microorganism suspensions were diluted to 10−3 mL by ten
effect of different combinations on phytoremediation. Units such as times method. Each 1 mL suspension of bacterial, fungal, actinomycetes
plant (factor P), microorganism (factor M) and amendment (factor A) was inoculated with beef paste peptone agar medium (containing beef
were optimized through the orthogonal experiment. Levels and factors extract 3.0 g, peptone 10.0 g, NaCl 5.0 g, agar 15.0–25.0 g per liter of
were listed in Supplement 1. The experimental design was comprised of distilled water, pH = 7.4–7.6), potato dextrose agar medium (con-
17 phytoremediation plots corresponding with 16 treatments and the taining potato 200.0 g, glucose 20.0 g, agar 15.0–20.0 g per liter of
control as shown in Supplement 2. Each plot was 5.4 × 16.2 m2 with distilled water), Gao's No.1 agar medium (containing starch 20.0 g,
three replicates and protection line for each plot was 1-cm wide. The KNO3 1.0 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, NaCl 0.5 g, FeSO4·7H2O
site selection and distribution of phytoremediation plots were shown in 0.01 g, agar 20.0 g per liter of distilled water, pH = 7.4–7.6), respec-
Fig. 1. The experiment began in May of 2017 and was run for 105 days. tively. The corresponding suspensions were cultured in an incubator
The experimental temperature was at 18–34 ℃ during the day and (NHWY-1102C; Changzhou Nuoji Instrument Co., Ltd) before colony
9–25 ℃ at night. The sterile distilled water was sprayed in morning counting at 37 °C, pH 7 for 24 h, 48 h, 72 h, respectively. The popula-
through a rotary distributor to keep the soil moisture content level at tions of bacterial, fungal, and actinomycetes were calculated using the
60% of the maximum water holding capacity. following formula (Meyer and Linderman, 1986):
The bacterial/ fungal/ actinomycetes population
(CFU g−1) = average colony number (CFU)·dilution times·fresh weight
2.2. Amendment, plant, and microorganism soil (g)−1

Four amendments with different composition ratios labeled A1

(organic fertilizer: rice husk: biochar: ceramsite = 2:1:1:1), A2 (organic 2.5. Soil organic matter and enzyme activities
fertilizer: rice husk: biochar: ceramsite = 1:2:1:1), A3 (organic ferti-
lizer: rice husk: biochar: ceramsite = 1:1:2:1), A4 (organic fertilizer: Soil organic matter (SOM) was determined using the method of
rice husk: biochar: ceramsite = 1:1:1:2) were selected as amendment Peech et al. (1947). The organic carbon in soil was oxidized with a
materials. The organic matter contents of A1, A2, A3, and A4 were mixture of potassium dichromate (K2Cr2O7) and sulphuric acid without
35.39, 42.98, 68.83, and 87.97 g kg−1, respectively, while there were external heating. The excess K2Cr2O7 was titrated with ferrous sulphate.
no heavy metals in the amendments. The organic fertilizer, biochar, and Soil urease (URE) activity was determined using the method of
ceramsite were purchased from Saibo company (Jinan, China). The rice Tabatabai and Bremner (1972) and was expressed as NH4-N mg kg−1
husk was collected from an agricultural field in Anshan of Liaoning h−1. Catalase (CAT) activity was determined using the method of
province, northeast China. Each raw material was air-dried, passed Johnson and Temple (1964). The residual of the hydrogen peroxide
through 1-mm sieve, and then mixed by certain ratios. They were added (H2O2) in the filtrate was titrated with 0.2 mol L−1 potassium per-
to 10% soil weight (soil thickness was 20 cm). manganate (KMnO4) solutions and was expressed as 0.2 mol L−1
Three typical plants labeled as P1(Soybean), P2(Rainbow pink), KMnO4 mL g−1 h−1. Phosphatase (PHO) activity was determined using
P3(Kochia) were selected as plant materials. All sample seeds were the disodium phenyl phosphate colorimetric method (Kramer and
purchased from the Fruitful Seed Company (Shouguang, China). The Yerdei, 1959). It was assayed by treating 10 g soil with 2.5 mL toluene
seeds were pre-germinated by soaking in sterile water at 37 °C for 24 h and 10 mL p-nitrophenol phosphatase solution (0.1 mol L−1, pH 11).
and then placed in an incubator (PGX-35048, temperature of 28 °C, After 1 h of incubation at 37 °C, 0.5 mol L−1 calcium chloride (CaCl2)
relative humidity of 80%) until the seeds turned white. After germi- and 0.5 mol L−1 sodium hydroxide (NaOH) solution were added. The
nation, similar-looking seedlings were selected and transferred to each suspension was filtered, and the color intensities of filtrates were
phytoremediation plot. The planting density was 30 plants m−2. measured at 420 nm.
Three typical microorganisms labeled M1(M. Circinelloides), M2(T.
asperellum), M3(Mortierella sp.) were selected as microorganism mate-
rials. All strains were isolated from mine tailings in Anshan of Liaoning 2.6. Heavy metal contents and their speciation distributions in soil
Province, northeast China (E 122°10′-123°41′, N 40°27′-41°34′). The
isolation and identification of the three strains were introduced in our By the end of the experiment, soil samples were collected from the
previous study (Sun et al., 2017; Zhang et al., 2018). The strains were surface (0–10 cm) of the experimental plot in the rhizosphere. After air-
activated in an incubator (NHWY-1102C; Changzhou Nuoji Instrument dried, soil samples were polished and selected by 0.1-mm sieve. The
Co., Ltd) at 30 °C, pH 7 for 2 days before being inoculated into a Martin total contents of HMs in soil were digested with HClO4/ HNO3 (1:4,
culture medium in sterile Erlenmeyer flasks and oscillated for 24 h in a vol/vol). The speciation distributions of HMs in soil were determined
Shaker (NHWY-1102C, 220 r min −1, 25 °C). The fungal spore suspen- by Tessier five-step sequential extraction method (Tessier et al., 1979).
sion (1 × 107 CFU mL−1) was added into each phytoremediation plot The contents of Cu, Mn, and Zn from these samples were analyzed by a
with 80 mL m−2 applied amount from T1 to T16. The equal amount flame atomic absorption spectrophotometer (GB17138-1997,
sterile distilled water was added into the control plot. WA2081), and the contents of Pb and Cd were analyzed by a graphite
furnace atomic spectrophotometer (GB17141-1997, TAS-990).

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2.7. Calculation of removal rates of heavy metals 3.2. Analysis of soil microorganism populations

To evaluate the capability of the plants to absorb the metal ions The significant changes of the microorganism populations in dif-
from soil, the removal rate (%) of heavy metals was calculated by the ferent treatments were determined by the bacterial, fungal, and acti-
formula (Orhan and Buyukgungor, 1993): nomycetes populations (Fig. 3). As the experiment progressed, the
bacteria became the dominant one of the three microorganisms with
Removal rate(%) = (C0 − C)/C0 × 100% the highest magnitude (103 CFU·g−1), then followed by fungi (102
CFU g−1) and actinomycetes (10 CFU g−1). The three microorganism
where C0 and C represented the heavy metal concentrations (mg kg−1)
populations (MPs) were stimulated overall throughout the whole ex-
of before and after the experiment, respectively.
periment, except the fungal populations of the control and T6 treatment
were decreased from the 45th day to the 60th day. This inhibition may
2.8. Statistical analysis be associated with the soil nutrition consumption. For example, Torsvik
and Ovreas (2002) reported that soil with low nutrients showed posi-
Multi-factor analysis of variance was used to study the effect of tive selection for k-proteobacteria, which is selection for bacteria with
plant, microorganism, and amendment on the phytoremediation. One- lower growth potential. According to Fig. 3, populations in T7 and T13
way analysis of variance (ANOVA) were performed for all quantitative treatments were notably stimulated, while there were no significant
data using SPSS (ver. 20.0) and least significant difference (LSD) test differences among other treatments. On the 60th day, the actinomy-
was used to analyze the significant differences between the treatments cetes populations in T3 and T11 treatments were increased notably
and the control. Graphs and tables were produced using Origin 9.0 and compared with others.
Microsoft Excel 2013. All of the results in the present study were per-
formed in triplicate. 3.3. Analysis of soil organic matter and enzyme activities

3. Results By the end of the experiment, responses of SOM, CAT, PHO, and
URE to different plant, microorganism, and amendment species in soil
3.1. Analysis of plant growth changes were shown in Fig. 4. Soil had higher levels of SOM, CAT, PHO, URE no
matter which plant, microorganism and amendment were combined
The plant length and biomass differed considerably in different and added into soil as compared with the control. Maximum values of
plants (Soybean, Rainbow pink, and Kochia) and as a result, the three SOM, CAT, PHO, and URE after 105 days in different treatments ranged
plants responded widely in terms of their growth changes to different from 102.6% (p < 0.001), 43.5% (p < 0.001), 42.2% (p < 0.01),
microorganism and amendment application (Fig. 2). To find out the and 59.7% (p < 0.001), respectively. The increases in SOM and three
best plant in phytoremediation efficiency, we made comparisons among enzyme activities also varied with the types of plant, microorganism
Soybean, Rainbow pink and Kochia which were enhanced by the same and amendment. The highest values were all observed in the Soybean-
combination of microorganism and amendment (M×A). Compared planted soils. The maximum increases of SOM, CAT, PHO, and URE
with T16, T11, T9, T13 treatments, higher lengths were found by 60.0 were found with addition of M1 × A4, M1 × A3, M1 × A3, and
(9.7), 17.2 (31.4), 20.5 (51.8), 81.0 (20.5) % in shoot (root) in T1, T3, M1 × A2 combinations, respectively.
T4, T7 treatments, respectively. The corresponding values for biomass
were 29.2 (36.2), 13.1 (21.7), 130.9 (56.8), 125.2 (151.9) %, respec- 3.4. Analysis of HM bioavailability
tively. Obviously, plant growth was better in Soybean than Rainbow
pink and Kochia. Furthermore, M×A interaction for plant length and The effect of plant, microorganism, and amendment on the bioa-
biomass confirmed the type differences of microorganism and amend- vailability of HMs in soil after the harvest was shown in Fig. 5. Bioa-
ment in response to plant growth promotion. Significantly, the length vailability is the plant-effective fraction of an ingested dose that be-
and biomass in shoot (root) were 9.1 (10.3) % and 10.8 (10.2) % (all comes available for distribution to different organs (Epa, 2007). The
p < 0.05) higher in the plant inoculated with M. Circinelloides relative Tessier sequential extractions were conducted for five fractions: ex-
to T. asperellum (T4-T6), as well as 37.9 (20.7) % and 15.9 (26.0) % (all changeable, carbonate bound, Fe/Mn oxide, organic bound, and re-
p < 0.05) higher relative to Mortierella sp. (T1-T2, T3-T8), respec- sidual, and the first four fractions can be used as indictors for soil HM
tively. In addition, A3 application increased the higher length and bioavailability (HMB). The final contents of HMs extracted from the
biomass in shoot (root) by 11.8 (15.2) % and 32.1(13.7) %, 26.6 (24.4) control varied in the following order: Mn (1952.2 mg kg−1) > Zn
% and 30.5 (17.5) %, 13.5 (16.8) % and 29.5 (48.7) % compared to A1 (149.5 mg kg−1) > Cu (50.1 mg kg−1) > Pb(42.1 mg kg−1) > Cd
−1
(T3-T7), A2 (T4-T7, T5-T6), A4 (T13-T16) amendments, respectively. (0.8 mg kg ). Zn was the most bioavailable metal that the bioavailable

Fig. 2. Comparison of the length (a) and bio-

mass (b) in shoot and root in different treat-
ments. T1 to T8 represented Soybean, T9 to
T12 represented Rainbow pink, and T13 to T16
represented Kochia. Error bars represented the
standard deviation of three replicates. The *,
**, *** above the columns represented that the
values are significantly different at p < 0.05,
p < 0.01, and p < 0.001, respectively.

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Fig. 3. Temporal variation of soil bacterial (a), fungal (b), and actinomycetes (c) populations in different treatments.

phase was accounting for 66.6–74.5% by the end of the experiment 21.9–183.9% (p < 0.01), 13.6–27.3% (p < 0.05), and 0.1–16.4%
even with application of plant, microorganism, and amendment. By (p > 0.05) in Cu, Zn, Pb, Cd, and Mn, respectively, after the addition of
contrast, the least bioavailable metal was Pb with only 20.1–57.2% plant, microorganism, and amendment to soil. The lowest bioavail-
bioavailable fractions, and the bioavailability of Mn was also very low. ability in Cu, Zn, Pb, Cd, and Mn was found in T7 treatment with ad-
Compared with the control, the percentage of bioavailable HMs de- dition of P1, M1, and A3 in combination, while the highest ones was
creased by 4.7–20.5% (p < 0.05), 4.3–11.9%(p > 0.05), found in T14 treatment with addition of P3, M3, and A2 in

Fig. 4. Soil organic matter (a) and catalase(b), phosphatase (c), and urease (d) activity. Error bars represented the standard deviation of three replicates. The *, **,
*** above the columns represented that the values are significantly different at p < 0.05, p < 0.01, and p < 0.001, respectively.

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Fig. 5. Percentage of each fraction in soil Cu (a), Zn(b), Pb(c), Cd(d), Mn(e).

combination. contamination of agriculture land of China (Z et al., 2014), while no

significant differences were observed in the control (p > 0.05). It was
partly due to dilution of amendment. The remedial effect of plant,
3.5. Analysis of total contents and removal rates of HMs in soil microorganism, and amendment on the HMR varied, depending on the
additive properties and HM types. The removal rate of Mn in soil ranged
By the end of the experiment, the total contents and removal rates of from 11.2% to 28.9%. This was the minimum range among these five
HMs (HMR) in soil under different treatments were listed in Table 1. HMs. The highest removal rates of Cu, Zn, Pb, Cd, Mn were 41.9%,
The total contents of Cu, Zn, Pb, Cd, and Mn were significantly de- 37.9%, 65.1%, 56.6%, and 35.1% by adding M1 to the soil; therefore,
creased (all p < 0.05) and met the risk control standard for soil

Table 1
Final total contents and Removal rates of soil heavy metals in different treatments.
Total content (mg kg-1) Removal rate (%)

Cu Zn (×10) Pb Cd(×10-2) Mn (×102) Cu Zn Pb Cd Mn

C 50.1 ± 4.5 15.0 ± 3.9 42.1 ± 2.7 79.0 ± 5.9 19.5 ± 1.8 3.1 ± 0.7 1.3 ± 0.5 3.4 ± 0.8 1.3 ± 0.5 0.4 ± 0.1
T1 34.5 ± 3.2* 10.6 ± 2.7* 28.3 ± 1.3* 49.4 ± 4.2* 16.0 ± 2.1* 33.3 ± 1.1* 30.0 ± 1.0** 35.0 ± 1.1* 38.3 ± 1.2* 18.4 ± 0.7*
T2 38.7 ± 1.6* 11.7 ± 3.1* 32.2 ± 1.1* 55.0 ± 2.9* 17.4 ± 1.8* 25.2 ± 1.1 22.7 ± 0.9 26.2 ± 0.9 31.3 ± 1.0 11.2 ± 0.6
T3 32.1 ± 2.6* 10.1 ± 3.7* 22.1 ± 2.1* 39.7 ± 4.3* 13.8 ± 2.4* 38.0 ± 1.9** 33.2 ± 1.0** 49.4 ± 2.0** 50.4 ± 1.3** 29.5 ± 1.0**
T4 33.2 ± 3.7* 10.4 ± 2.6* 25.2 ± 1.1* 44.6 ± 3.4* 14.9 ± 2.3* 35.7 ± 0.9** 31.7 ± 0.9** 42.1 ± 1.5* 44.3 ± 1.2* 23.9 ± 1.1**
T5 33.7 ± 2.1* 10.7 ± 3.2** 22.1 ± 1.1** 39.0 ± 2.7* 13.9 ± 1.5* 34.8 ± 1.9** 29.5 ± 0.8** 49.4 ± 2.0** 51.2 ± 1.5** 28.9 ± 1.3**
T6 36.2 ± 2.4 11.2 ± 3.1* 28.3 ± 2.4* 48.7 ± 4.6* 16.1 ± 2.3* 30.0 ± 1.4* 26.3 ± 0.9* 35.0 ± 1.6* 39.1 ± 1.2* 17.8 ± 1.4*
T7 30.8 ± 3.2* 9.9 ± 5.2* 18.9 ± 1.3* 34.7 ± 2.1* 12.7 ± 1.4* 40.4 ± 2.0*** 34.9 ± 1.4*** 56.7 ± 2.7*** 56.6 ± 2.0*** 35.1 ± 2.0***
T8 36.3 ± 3.4* 11.2 ± 4.2* 25.9 ± 2.1* 45.2 ± 3.5* 15.2 ± 1.6* 29.8 ± 1.5* 25.9 ± 1.0* 40.6 ± 1.7* 43.5 ± 1.0* 22.3 ± 1.6*
T9 31.2 ± 3.1* 10.8 ± 4.5* 25.0 ± 1.2* 42.9 ± 2.7* 14.8 ± 1.6* 39.6 ± 1.6*** 28.5 ± 1.1* 42.6 ± 1.3* 46.4 ± 1.6** 24.4 ± 1.4**
T10 35.3 ± 2.3* 11.9 ± 2.9* 31.3 ± 1.4* 52.0 ± 2.5* 17.1 ± 1.1* 31.7 ± 0.7* 21.6 ± 0.7 28.2 ± 1.2 35.0 ± 1.3* 12.7 ± 0.7*
T11 30.0 ± 2.5* 10.6 ± 4.2* 21.9 ± 1.5* 38.0 ± 3.7* 13.7 ± 1.9* 41.9 ± 1.1*** 30.0 ± 1.4** 49.8 ± 1.7** 52.5 ± 1.6** 30.0 ± 1.3**
T12 33.0 ± 2.4* 11.5 ± 3.7* 22.5 ± 1.3* 38.7 ± 2.1* 14.0 ± 1.7* 36.1 ± 1.0** 24.3 ± 0.9* 48.3 ± 1.9** 51.6 ± 1.3** 28.5 ± 1.8**
T13 30.3 ± 2.3** 9.4 ± 3.1* 15.2 ± 1.4* 35.6 ± 2.7* 14.2 ± 1.7* 41.4 ± 1.5*** 37.9 ± 1.7*** 65.1 ± 2.3*** 55.5 ± 1.0*** 27.3 ± 1.0***
T14 37.0 ± 3.4* 11.0 ± 4.2* 25.3 ± 1.5* 51.0 ± 3.8* 15.9 ± 1.6* 28.5 ± 1.0* 27.3 ± 1.2* 41.9 ± 1.6* 36.2 ± 1.2* 19.0 ± 0.9*
T15 34.4 ± 2.1* 10.5 ± 4.3* 21.5 ± 1.7* 44.8 ± 2.6* 14.6 ± 1.9* 33.5 ± 1.0* 30.9 ± 1.0** 50.7 ± 1.4** 44.0 ± 1.3* 25.5 ± 1.2**
T16 34.0 ± 2.4* 10.1 ± 3.5* 24.6 ± 1.3* 50.2 ± 3.2* 15.6 ± 1.7* 34.3 ± 1.4** 33.1 ± 1.3** 43.5 ± 1.4* 37.2 ± 1.7* 20.6 ± 1.4*

Each value represented a mean ± SD of three independent experiments of each treatment in triplicate. The *, **, *** within the columns represented that the values
are significantly different at p < 0.05, p < 0.01, and p < 0.001, respectively.

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M1 was considered to be a good microorganism for phytoremediation increase of concentrations of HMs or the distance away from mining
in a short time frame. The P3 × A3 was the most effective combination tailing center. The results suggested it was feasible to use soil enzymatic
for Zn, Pb, and Mn based on their 51.5%, 108.1%, and 123.3% higher activities to indicate the pollution situation by combined HMs in the
removal rates compared to other combinations, respectively. The soil of mine area. Plenty of researches had reported that organic
highest removal rate of Cu was recorded in P2 × A1 combination as amendments had the ability to enhance the soil enzyme activity
increased by 47.9% and Cd was recorded in P1 × A3 combination as through the following ways: 1) the massive microorganisms in the
increased by 61.3%, respectively. added materials that had the capacity to stimulate the activity of soil
enzymes; 2) the functional groups on the humic substrates, such as
4. Discussion carboxyl, phenolic, alcohol and carbonyl could react with heavy metal
ions in soil by forming metal-humate complexes to reduce the toxicity
Heavy metal pollution is a serious environmental concern, claiming of heavy metal and improve the microbial activity (Nannipieri et al.,
plant productivity in contaminated areas. Although certain plants are 1994; Dick et al., 1997).
able to adapt to some levels of these stresses, their growth rates and According to L16 (4 × 32) matrix, the corresponding HMB and HMR
ultimately survivals are also affected (Srivastava et al., 2008; Ismaiel of each treatment were listed in Fig. 5 and Table 1. The phytor-
et al., 2014). Plant growth, calculated on the basis of plant length and emediation effects of plant, microorganism, and amendment on HM
biomass, has long been used as an important parameter for assessing contaminated soil were determined not only by the fertility improve-
species variation in the efficiency of phytoremediation (Anjum et al., ment of soil, but also the immobilization effects on HMs. The bioa-
2008). The selected plant species were ubiquitous, cosmopolitan and vailability of HMs to plants and soil biota including fauna and micro-
have relatively high biomass. They were optimized for plant growth organisms was controlled by their total contents in soil and their
and phytoremediation potential in this study. The result of the ortho- chemical forms. In this study, with the plant, microorganism, and
gonal experiment (L16) has indicated that plant growth in Soybean was amendment application, the bioavailable fractions of HMs in tailings
relatively better than Rainbow pink and Kochia with combined appli- appeared to have been transformed to more non-available forms and
cation of the same microorganism and amendment (such as the com- difficultly taken up by plants. This variation was influenced by the
parisons of T1-T16, T3-T11, T9-T4, T13-T7); thus, Soybean was iden- contents of these metals in soil and their chemical aggressive reactions
tified as the best plant material for phytoremediation potential, then to the amendments. Amendment of contaminated soils with lime,
followed by Kochia and Rainbow pink. The cause of this overwhelming phosphate and organic acids generally improved the soil pH (8.74) and
growth was likely due to higher energy supply to maintain plant normal reduced the bioavailability of HMs. Compared the contribution values
turgor pressure (Kirst, 2003); good function of the electron transport of different raw materials in the amendment, the order of significant
chain and photosystems (Mehta et al., 2010); or from direct effects on materials was biochar > organic fertilizer > rice husk > ceramsite.
cellular components (Gu et al., 2012). Moreover, the presence of other chemicals which may aggravate or
The orthogonal experiment (L16) was conducted and 16 micro- ameliorate metal toxicity and affect the way plants responding to the
organism-inoculated treatments were carried out. The corresponding toxic metals. High surface areas and plentiful sites of biochar in
MPs, SOM, CAT, PHO, URE of each factor were shown in Fig. 3 and amendment presented a strong adsorption affinity for HMs. The organic
Fig. 4. As mentioned, the M. circinelloides has the most dominant mi- functional groups on the surface of biochar, such as carboxylic and
crobial effect on the phytoremediation in this study, then followed by T. phenolic functional groups that could interact with HMs in tailings and
asperellum and Mortierella sp. The MPs in the plant rhizosphere were produce some complexes (Cheng et al., 2008; Huang et al., 2011). It
usually composed of diverse and synergistic communities rather than could reduce HMs and their compounds in the environment by nega-
single strain. Such abundance required several organisms with dis- tively correlating with HM bioavailability. Hua et al. (2009) used bio-
tinctive enzyme systems, and further promoted remediation of HM- char for composting, showing that incorporation of biochar into sludge
contaminated soil. Jindo et al. (2012) found that biochar addition composting materials significantly reduced the mobility of HMs in
significantly enhanced the diversity of fungi in biochar-amended com- sludge composting material. Otherwise, extracellular polymeric sub-
post, suggesting a change in microbial composition compared to the stances (EPSs) may be produced by microorganism and remained in
non-inoculated compost. Chen et al. (2017) reported that biochar ad- different substrata or cell-to-cell aggregation to bond HMs with func-
dition dramatically influenced the bacterial community structure tional groups such as –NH, –OH, and C–O. Differences in the effect of
during the composting process which significantly correlated with C/N fungal strains on HMB can be explained that each fungal strain had its
ratio, water soluble carbon, and organic matter (P < 0.05) in pile. particular inactivation capacity and tolerance to different HMs. M.
Toxic metals can also adversely affect the number, abundance and ac- Circinelloides was the most effective in immobilizing HMs in soils
tivity of soil organisms, inhibiting SOM decomposition and N miner- meanwhile Mortierella sp. was relatively poor, which was consistent
alization processes. There is a body of evidence to suggest that the with the previous findings of Li et al. (2017). Similarly, Morillo et al.
exogenous metabolic activities of rhizosphere microorganisms may be (2006) reported that Paenibacillus jamilae CECT 5266 grown in aqu-
controlled by plants in return for the provision of root exudates. Soil eous extracts of two-phase olive mill waste produced EPS capable of
organic matter and enzymes, such as CAT, PHO and URE are specific absorbing HMs in the following order from a multi metal sorption
biochemical indicators of soil fertility and played an important role in system: Pb > Cd > Cu > Zn > Ni > Co. Liu et al. (2017) con-
controlling soil N and P mineralization and cycling (Huang et al., 2012; firmed the importance of biochar and microorganism amendment in
Lebrun et al., 2012). To some degree, their activities reflected the ca- reducing not only N losses during composting but also the availability
pacity of organic nutrient mineralization potentials in soil (Schneider of heavy metals in the compost pile, particularly Pb and As, thereby
et al., 2000; Vanlauwe and Giller, 2006; Huang et al., 2011). Thus, improving the composting effectiveness.
taking the organic matters, humic acid, microbiota content in amend-
ments into consideration, compared to the control, the activities of 5. Conclusion
CAT, PHO and URE were promoted with the addition of amendments,
and the corresponding highest activities were all presented in the T7 In the present study, three plant species, Soybean, Rainbow pink,
treatment (M1 × A3). These activities were strongly correlated with Kochia, were selected with the aim to evaluate their potentials for a
SOM and MPs. In this study, with the increased SOM, the bioavailable field-scale experimental phytoremediation. The results of plant growth
content of HMs in soil decreased, resulting in an increase in CAT, PHO, parameters clearly demonstrated a higher efficiency of Soybean to
and URE activities. Liao, Huang (2005) reported that soil dehy- phytoremediation for heavy metals in terms of plant length and biomass
drogenase and urease activities decreased significantly with the production. Soil organic matter, enzyme activities, and microorganism

7
X. Li et al. Ecotoxicology and Environmental Safety 168 (2019) 1–8

populations were improved significantly with addition of micro- Jindo, K., Suto, K., Matsumoto, K., García, C., Sonoki, T., Sanchezmonedero, M.A., 2012.
organism and amendment in combination. Results of heavy metal Chemical and biochemical characterisation of biochar-blended composts prepared
from poultry manure. Bioresour. Technol. 110, 396–404.
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Li, X., Zhang, X., Cui, Z., 2017. Combined bioremediation for lead in mine tailings by
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Acknowledgements
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zymatic activities in areas polluted by tailings from Pb-Zn-Ag mine. J. Environ. Sci.
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Anshan Iron and Steel Technology Project (11161467), the National Liu, W., Huo, R., Xu, J., Liang, S., Li, J., Zhao, T., Wang, S., 2017. Effects of biochar on
nitrogen transformation and heavy metals in sludge composting. Bioresour. Technol.
Natural Science Foundation of China (20977094), and the Science and 235, 43.
Technology Development Plan Projects of Weifang (2014ZJ1055). The Lombardi, P.E., Peri, S.I., Guerrero, N.R.V., 2010. ALA-D and ALA-D reactivated as bio-
authors would like to thank all the anonymous referees for their con- markers of lead contamination in the fish Prochilodus lineatus. Ecotoxicol. Environ.
Saf. 73, 1704–1711.
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