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Environmental Science and Pollution Research

, Volume 25, Issue 15, pp 14773–14788 | Cite as

Bacterial community structure and diversity responses to the direct revegetation of an artisanal zinc smelting slag after 5 years

  • Youfa Luo
  • Yonggui Wu
  • Hu Wang
  • Rongrong Xing
  • Zhilin Zheng
  • Jing Qiu
  • Lian Yang
Research Article

Abstract

This comparative field study examined the responses of bacterial community structure and diversity to the revegetation of zinc (Zn) smelting waste slag with eight plant species after 5 years. The microbial community structure of waste slag with and without vegetation was evaluated using high-throughput sequencing. The physiochemical properties of Zn smelting slag after revegetation with eight plant rhizospheres for 5 years were improved compared to those of bulk slag. Revegetation significantly increased the microbial community diversity in plant rhizospheres, and at the phylum level, Proteobacteria, Acidobacteria, and Bacteroidetes were notably more abundant in rhizosphere slags than those in bulk waste slag. Additionally, revegetation increased the relative abundance of plant growth-promoting rhizobacteria such as Flavobacterium, Streptomyces, and Arthrobacter as well as symbiotic N2 fixers such as Bradyrhizobium. Three dominant native plant species (Arundo donax, Broussonetia papyrifera, and Robinia pseudoacacia) greatly increased the quality of the rhizosphere slags. Canonical correspondence analysis showed that the differences in bacterial community structure between the bulk and rhizosphere slags were explained by slag properties, i.e., pH, available copper (Cu) and lead (Pb), moisture, available nitrogen (N), phosphorus (P), and potassium (K), and organic matter (OM); however, available Zn and cadmium (Cd) contents were the slag parameters that best explained the differences between the rhizosphere communities of the eight plant species. The results suggested that revegetation plays an important role in enhancing bacterial community abundance and diversity in rhizosphere slags and that revegetation may also regulate microbiological properties and diversity mainly through changes in heavy metal bioavailability and physiochemical slag characteristics.

Keywords

Zinc smelting waste slag Direct revegetation Environmental factors Bacterial community structure Response High-throughput sequencing 

Introduction

In north-western Guizhou, China, artisanal zinc (Zn) smelting has a long history and has been widely applied to extract useful metal elements. Without appropriate treatment, Zn smelting generates a large amount of abandoned waste slag containing high levels of heavy metals. These bare waste slag sites are especially susceptible to eolian dispersion and water erosion and thus pose environmental risks for surrounding ecosystems and populations due to adverse effects on soil properties, water quality, and plant growth (Bradshaw 1997; Feng et al. 2004; Zhang et al. 2012). Therefore, remediation and stabilization actions at these sites must be undertaken to control heavy metal diffusion and to reduce potential toxicity and risks to environmental and human health. In recent years, revegetation has often been considered an ideal co-effective strategy for stabilizing tailings and preventing the diffusion of metal-contaminated small (micrometer-sized) particles by eolian dispersion and water erosion as well as surface runoff and leaching by the plant canopy and roots (Gratão et al. 2005; Mendez and Maier 2008). Thus, revegetation has been widely used for the ecological restoration of mine tailings sites (Mendez and Maier 2008; Parra et al. 2016; Yang et al. 2016).

Many researchers have suggested that the establishment of a self-sustained microbial and plant community is the ultimate goal of the ecological restoration of mine tailings (Mendez and Maier 2008; Rosario et al. 2007). Revegetation success is judged not only based on visually distinguishable aboveground indicators and soil physicochemical properties but also on microbiological properties; however, microbiological properties have had limited consideration (Mummey et al. 2002). Several studies have suggested that the above- and belowground biota are closely ecologically linked (Van der Putten et al. 2001; Wardle et al. 2004), e.g., there are highly positive correlations between vegetation community stability and microbial abundance and composition (Mendez and Maier 2008). Microbes play important roles in facilitating plant growth by affecting heavy metal speciation, reducing phytotoxicity for plants (Mastretta et al. 2006; Salt et al. 1999), and maintaining the stability of soil ecological functions (Loreau 2010). Recovery of the composition and activity of the microbial community may be key to the remediation of mine ecosystems (Kohler et al. 2016). Thus, the critical community composition and activities of a microbial population may be indicators for improving soil quality and ecosystem restoration (Banning et al. 2011; Honeker et al. 2017; Yin et al. 2000).

In the early years, microbial community diversity was investigated using the traditional cultivation techniques. However, these techniques only provided information about cultivable organisms, which account for less than 1% of all organisms and are not representative of the total diversity of microbes (Amann et al. 1995; McCaig et al. 2001). In recent years, certain methods of evaluating microbial community structure have gradually come into use, including phospholipid fatty acid (PLFA) analysis (Frostegård and Bååth 1996), community-level physiological profile (CLPP) analysis (Garland 1997), and nucleic acid-based techniques such as PCR amplification combined with genetic fingerprinting methods (Muyzer and Smalla 1998). PCR-based techniques, including denaturing gradient gel electrophoresis (DGGE) as well as cloning and sequencing of 16S rRNA gene fragments, have commonly been used to investigate the microbial communities in mine tailings (Li et al. 2011; Pepper et al. 2012; Touceda-González et al. 2017; Wakelin et al. 2012; Zhang et al. 2007b). These techniques, however, have failed to provide detailed information regarding the relationships between the vegetation community and microbial diversity in revegetated mine tailings (Roesch et al. 2007). A high-throughput sequencing approach, which is beneficial for the analysis of a large number of detailed taxonomic profiles in samples (Kozich et al. 2013), has been proposed for the in-depth investigation of microbial community composition and diversity in complex environmental samples (Hong et al. 2015; Li et al. 2014, 2016).

Zn smelting waste slag is characterized by poor moisture retention, nutrient and organic matter (OM) deficiency, and low microbial activity coupled with elevated concentrations of heavy metals. Such waste slags generally do not support plant growth resulting in numerous waste slag sites being devoid of vegetation. We promoted the successful revegetation of a Zn smelting waste slag site by trees, shrubs, and grasses under field conditions in Weining Country of Guizhou Province, China, from 2012 to 2016 through substrate amendment and the selection of suitable plant species (Luo et al. 2017). In previous studies, enzyme activity and microbiological properties (e.g., culturable microbial communities and populations) have been used as indicators of slag/soil quality to evaluate the success of revegetation in Zn smelting regions (Luo et al. 2017; Zhang et al. 2006, 2007a). Thus far, there is limited information available regarding the succession of microbial community structure and diversity based on high-throughput sequencing methods to monitor long-term field conditions in artisanal Zn smelting waste slag after revegetation with trees, shrubs, and grasses. Therefore, the objective of this study was to investigate the effects of a combination of organic amendments and plant establishment on the chemical properties and microbial community structure and diversity of a Zn smelting waste slag site with established vegetation for 5 years. It has been hypothesized that remediation approaches (amendment application and plant cover development) will improve the micro-environment of Zn smelting waste slag, i.e., promote nutrient accumulation, increase moisture, and reduce heavy metals bioavailability. Changes in these key environmental factors will drive shifts in the structure and diversity of rhizosphere microorganisms during the revegetation of Zn smelting waste slag. To evaluate this hypothesis, we examined and compared the differences in microbial community structure and diversity in rhizosphere slags and bulk slag using a high-throughput sequencing approach. Additionally, we investigated the responses of microbial community structure and diversity to waste slag properties influenced by direct revegetation. The findings of this study provide insight into the efficiency of direct revegetation for ameliorating the biological capacity and functions of waste slag. This will be beneficial for the selection of suitable native plant communities to speed up the process of revegetation in artisanal Zn smelting waste slag sites.

Materials and methods

Site description

The Zn smelting slag site is located in north-western Guizhou, China (26° 41′ N, 104° 43′ E) (Fig. 1). This area has a subtropical humid climate with an average temperature of 10 °C and an annual average rainfall of 900–1000 mm. The main soil types in the study area are yellow brown, yellow, and lime soils. The yellow soil is characterized by slightly acidic to acid pH, and the lime soil is characterized by slightly alkaline to alkaline pH. High concentrations of active iron and aluminum in the yellow soil and high levels of calcium and magnesium in the lime soil cause these soils to lack phosphorus (P). The natural vegetation in this region is composed predominantly of herbs and shrubs, such as Cyclobalanopsis glauca, Pinus massoniana, Cryptomeria fortunei, Cinnamomum camphora, Robinia pseudoacacia, Broussonetia papyrifera, and Arundo donax. This area, in Hezhang and Weining counties, is one of the most important artificial Zn mining regions in China. Zn smelting activities began in the last century but were ceased in 2004 due to serious environmental pollution problems. In this district, the artisanal Zn smelting process used indigenous methods: two major Zn ores, sulfide ore in the form of sphalerite (ZnS), and carbonate Zn ore in the form of smithsonite (ZnCO3), were mixed with coal as a reducing agent, placed in ceramic jars, and simply roasted for a few hours using coal as fuel to generate heat in a furnace to a high temperature (~ 800 °C) to produce liquid metallic Zn.
Fig. 1

Location of the Zn smelting slag deposition site and collection area (samples were collected from the rhizospheres of established plant species and from surrounding bare areas); a and b represent bare slag without vegetation; cf show the establishment of various grasses vs. shrubs vs. trees on the slag over periods of 12, 24, 36, and 48 months, respectively

A vast amount of waste slag was generated by previous smelting processes and dumped on the soil surface. The waste slag consisted of a mixture of ore smelting slag and a small amount of unburned cinder. The high level of heavy metal toxicity substantially restricted spontaneous vegetation succession and restoration. No man-driven revegetation work was carried out at this site. In 2012, a remediation attempt was initiated to revegetate the Zn smelting waste slag using a combination of OM amendments and native plant species. OM such as livestock manure compost and plant litter were applied to amend the waste slag at a rate of 22 t ha−1. Then, suitable native plant species such as the annual and perennial herbs perennial ryegrass and Trifolium repens were sown as pioneer plants to improve the slag conditions, and holes were dug for the artificial planting of fast-growing woody plant species (A. donax, B. papyrifera, R. pseudoacacia, and C. fortunei). Plant species such as Chenopodium ambrosioides and Buddleja davidii showed natural colonization after 6 months, when the substrate quality had been improved by planted herbs and woody plant communities. Finally, the planted grasses, shrubs, and tree communities generated a self-sustained ecosystem across 5000 m2 over a 5-year period (from January 2012 to October 2016). The growth characteristics of these plant species are shown in Table 1.
Table 1

Characteristics of plant species in the revegetated area of a Zn smelting slag site

Plant species

Plant height (cm)

Diameter at breast height (cm)

Ground diameter (cm)

Coverage (%)

Plant classification

T. repens

15

10

Annual or perennial herbs

Perennial ryegrass

25

35

Annual or perennial herbs

C. ambrosioides

160

2.2

3.8

10

Annual or perennial herbs

B. davidii

180

7

10

20

Deciduous broad-leaved shrubs

A. donax

200

2

3

80

Evergreen broad-leaved shrub

C. fortunei

350

7.8

9.8

55

Evergreen coniferous trees

R. pseudoacacia

465

14

20

60

Deciduous broad-leaved trees

B. papyrifera

480

18

20

60

Deciduous broad-leaved trees

Slag sample collection and preparation

A field survey was conducted in October 2016 at a Zn smelting waste slag site, and the study area was divided into three quadrats (area = 40 × 40 m for each quadrat) established as triplicate sampling sites according to the distribution of vegetation and bare areas where no vegetation was present; the same eight plant species (perennial ryegrass, T. repens, C. ambrosioides, B. davidii, A. donax, B. papyrifera, R. pseudoacacia, and C. fortunei) were selected for sample collection. In the vegetated area, waste slag samples were collected from the rhizosphere, and the rhizosphere fraction of the waste slag was obtained from the root surface (0–5 mm, strong adherence to the root). Nine bulk slag samples were randomly collected from the bare area with no vegetation present at a fixed depth of 0–20 cm, and these bulk slag samples were thoroughly homogenized to form three composite samples. In each quadrat, three rhizosphere slag samples were randomly collected from each plant species. The three subsamples in each quadrat were thoroughly homogenized to form a composite sample. Finally, three comprehensive rhizosphere samples from each plant species were collected. Plant litter was removed from the slag surface before the slag samples were collected. The fresh samples were sieved through a 2-mm sieve and stored (i) frozen at − 70 °C for microbial community composition analysis or (ii) air-dried at room temperature for physiochemical analysis.

Physiochemical analysis

pH and electrical conductivity (EC) were measured in slag deionized water 1:2.5 (w/v). Slag moisture was measured by a mass loss of approximately 10 g from fresh slag samples after drying at 105 °C for at least 8 h. Nutrient (N, P, and K) availability and OM content were determined according to Bao (2000). Available N was measured by the alkali solution diffusion method. Available P was extracted by using 0.5 mol l−1 NaHCO3. OM content was measured by dichromate oxidation and titration with ferrous ammonium sulfate. Available K was extracted with 1 mol l−1 NH4OAc (pH 7). The available metals were determined as previously reported (Lindsay and Norvell 1978). The following procedure was used for total heavy metal measurements in the slag: 0.1000 g samples of granite were added to Teflon vessels, and a 3-ml mixture of HNO3 and HCl (1:3 v/v) was added to each sample. The sealed vessels were then placed in an electric oven and heated to 180 °C for 12 h (overnight). The vessels were placed on a heating plate at approximately 105 °C to evaporate the acid and keep the samples from cooling. The solution samples retained a volume of approximately 1 ml after they evaporated to dryness. The final digestion solution samples were filtered and reconstituted up to a 50-ml volume with deionized water. Quality control of waste slag material was performed by using a certified appropriate material (GBW07404) to verify the effectiveness and accuracy of the extraction analysis. The abovementioned tests were performed in duplicate. The available K and heavy metals were measured using atomic absorption spectrophotometry (AAS, ICE3500, Thermo Fisher Scientific, USA).

DNA extraction and sequencing

Considering heterogeneity of waste slag, three composite samples of the bulk slag and each rhizosphere were thoroughly homogenized to a composite sample for determining the microbial community structure and diversity. Microbial DNA was extracted from 0.5-g slag samples according to the manufacturer’s instructions using a E.Z.N.A® Soil DNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA). DNA density and purity were determined by microspectrophotometry (NanoDrop 2000, Thermo Scientific, Wilmington, USA). Extracted DNA quality was verified by agarose gel electrophoresis (1%). During DNA extraction, blanks that did not contain slag samples were extracted along with each batch of samples for quality control and subsequently sequenced.

The V3–V4 hypervariable regions of the bacterial 16S rRNA genes were amplified and sequenced using the primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) (Xu et al. 2016). PCR was performed in triplicate with a 20-μl mixture, which included 4 μl of 5× Fast Pfu buffer, 2 μl of 2.5 mM dNTPs, 0.8 μl of each primer (5 μM), 0.4 μl of Fast Pfu DNA polymerase, and 0.4 μl of diluted DNA template (10 ng) using a PCR Gene Amp 9700 thermocycler (Applied Biosystems, Foster City, CA, USA). The following amplification protocol was used: 95 °C for 3 min, followed by 27 cycles at 95 °C for 30 s, 55 °C for 30 s, and at 72 °C for 30 s, and a final extension at 72 °C for 5 min. All the samples were amplified and purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer’s instructions. Quantification of PCR products was performed using a QuantiFluor™-ST (Promega, Madison, WI, USA). Purified amplified fragments were used to construct a PE 2 × 300 library according to the standard operating procedures of the Illumina MiSeq platform (Illumina, San Diego, CA, USA). PCR products for sequencing were carried out using an Illumina’s MiSeq PE300 platform at Majorbio Bio-pharm Technology Co., Ltd., Shanghai, China. The raw high-throughput sequencing data were deposited in the NCBI Sequence Read Archive database (accession number: SRP115062).

Sequencing data analysis

Raw FASTQ files were de-multiplexed and quality-filtered using Trimmomatic (version 0.30) with the following criteria: (i) The reads were truncated at any site that received an average quality score of < 20 over a 50-bp sliding window, and truncated reads shorter than 50 bp were discarded. (ii) Primers were exactly matched, with two nucleotide mismatches allowed, and reads containing ambiguous characters were removed. (iii) Only overlapping sequences longer than 10 bp were merged according to their overlapping sequence. Operational taxonomic units (OTUs) were clustered with a 97% similarity cutoff using UPARSE (version 7.1, http://drive5.com/uparse/), and chimeric sequences were identified and removed using UCHIME.

Microbial community diversity and richness indices, such as the Chao, ACE, Shannon-Wiener, and Simpson indices, were calculated based on the OTUs using the MOTHUR program. Microbial community structure and composition at the phylum and genus levels were compiled from the SILVA database and published studies and compared with those of the present study. Pearson’s correlations were calculated between waste slag physiochemical properties and microbial community diversity. Principal component analysis (PCA) for microbial community composition at the phylum level in the slag with and without plant cover was carried out using CANOCO 4.5. Canonical correspondence analysis (CCA), carried out using CANOCO 4.5, was used to evaluate the effects of environmental factors (pH, EC, moisture, available nutrient, OM, and available heavy metals) that changed during the revegetation process on the microbial community structure. Graphics were drawn using Sigmaplot 12.5.

Results

Physiochemical properties

The moisture of the bulk waste slag was the lowest among all the samples. The presence of plants significantly (p < 0.05) increased the moisture of the waste slag, and the moisture in the rhizosphere of B. papyrifera was the highest, as shown in Table 2. Compared to the bulk slag, revegetation slightly decreased the pH values but significantly (p < 0.05) increased the EC except for the grasses perennial ryegrass and T. repens; the EC of B. papyrifera and A. donax were the highest, followed by C. ambrosioides and C. fortunei. Additionally, Table 3 shows that, compared to the bulk slag, the presence of eight plant species significantly decreased Cu availability and increased Pb availability; however, the effects of the eight plant species on Zn and Cd availability can be divided into two groups. First, the presence of T. repens, B. davidii, R. pseudoacacia, and C. fortunei decreased the availability of Cd, and the presence of B. davidii, R. pseudoacacia, and C. fortunei decreased the availability of Zn; second, the presence of perennial ryegrass, C. ambrosioides, A. donax, and B. papyrifera increased the availability of Zn and Cd. The total contents of heavy metals such as Cu, Pb, and Cd, in the rhizospheres of the eight plant species were lower than those in the bulk slag, with the exception of the Cd content in the perennial ryegrass rhizosphere. In contrast, the total Zn content in the rhizospheres of the eight plant species was higher than that in the bulk slag, which indicated enhanced Zn accumulation in the rhizospheres of the eight plant species. The available nutrients (N, P, and K) and OM of bulk waste slag were significantly (p < 0.05) lower than those in the rhizosphere waste slags (Table 2). The strength of the effects of revegetation on the available nutrients and OM depended on the plant species: A. donax was associated with the largest increase in available P, the presence of T. repens or B. papyrifera was associated with the largest increase in available N, the presence of T. repens was associated with the largest increase in OM, and the presence of A. donax or B. papyrifera was associated with the largest increase in available K.
Table 2

Effects of revegetation on the physiochemical parameters of waste slag

Samples

Moisture (%)

pH

EC (μS cm−1)

Available P (mg kg−1)

Available N (mg kg−1)

Available K (mg kg−1)

Organic matter (g kg-1)

Bulk slag

13.2 ± 0.4a

7.96 ± 0.21c

234.33 ± 17.90a

0.81 ± 0.06a

27.65 ± 2.97a

52.71 ± 0.04a

80.30 ± 8.52a

T. repens

25.1 ± 1.6cd

7.34 ± 0.10ab

292.67 ± 35.73ab

13.11 ± 1.52e

151.55 ± 21.83f

217.84 ± 27.46bc

241.36 ± 31.56e

Perennial ryegrass

21.7 ± 3.4b‡

7.72 ± 0.06bc

284.67 ± 35.23ab

7.24 ± 1.03c

94.85 ± 9.81cd

249.98 ± 26.65cd

135.97 ± 17.61b

C. ambrosioides

21.5 ± 1.4b

7.43 ± 0.30ab

624.67 ± 74.81c

11.60 ± 1.43de

126.82 ± 10.68e

245.90 ± 37.79cd

191.48 ± 16.00cd

B. davidii

21.2 ± 0.7b

7.48 ± 0.20ab

364.00 ± 58.04b

6.94 ± 1.04c

79.57 ± 12.18bc

212.89 ± 31.70bc

204.28 ± 19.13cd

A. donax

22.4 ± 1.5bc

7.32 ± 0.37a

825.33 ± 60.45d

15.67 ± 2.07f

79.57 ± 9.85bc

292.27 ± 46.33de

179.87 ± 21.45cd

C. fortunei

22.7 ± 1.0bc

7.54 ± 0.02ab

549.00 ± 72.06c

7.09 ± 0.95c

101.15 ± 13.27d

188.55 ± 23.82b

171.38 ± 25.89c

R. pseudoacacia

21.4 ± 0.8b

7.70 ± 0.11abc

338.33 ± 24.03b

5.40 ± 0.66b

66.85 ± 8.89b

220.71 ± 34.76bc

210.14 ± 31.23d

B. papyrifera

26.4 ± 1.9d

7.61 ± 0.12abc

922.67 ± 98.51d

11.24 ± 1.06d

140.23 ± 15.71ef

306.72 ± 46.22e

199.97 ± 27.89cd

Treatment means in a column followed by the same letter(s) are not significantly different at p < 0.05

EC electrical conductivity

Values are the mean ± SE

Table 3

Effects of revegetation on the heavy metal bioavailability of waste slag

Samples

Available heavy metal contents

Total heavy metal contents

Available Cu (mg kg−1)

Available Pb (mg kg−1)

Available Zn (mg kg−1)

Available Cd (mg kg−1)

Total Cu (mg kg−1)

Total Pb (mg kg−1)

Total Zn (mg kg−1)

Total Cd (mg kg−1)

Bulk slag

77.96 ± 1.24g

117.56 ± 4.11a

282.57 ± 29.27d

8.61 ± 0.66c

1808.30 ± 132.91f

16,882.51 ± 1332.30d

10,327.61 ± 174.14a

98.61 ± 0.78d

T repens

14.94 ± 2.27a

211.94 ± 12.21cd

310.51 ± 23.91de

6.54 ± 0.75b

649.46 ± 36.94a

7344.44 ± 574.37a

13,913.54 ± 1990.50b

70.78 ± 6.78ab

Perennial ryegrass

66.27 ± 9.11f

195.79 ± 25.80bc

376.40 ± 25.35fg

11.74 ± 0.97e

1049.80 ± 25.06e

9057.78 ± 556.38bc

11,354.76 ± 1175.64a

110.54 ± 8.29e

C. ambrosioides

53.57 ± 7.99e

171.92 ± 24.88b

384.24 ± 16.87g

10.94 ± 1.40de

1107.12 ± 15.09e

9592.27 ± 115.17c

11,756.74 ± 104.30ab

89.58 ± 3.59cd

B. davidii

39.61 ± 5.86d

136.61 ± 19.06a

190.63 ± 28.12b

5.90 ± 0.81ab

1151.49 ± 35.37e

7956.54 ± 929.93ab

11,004.68 ± 909.05a

64.61 ± 3.57a

A. donax

47.91 + 7.13e

141.58 ± 21.25a

334.69 ± 49.06ef

9.89 ± 1.38cd

908.56 ± 48.62d

8254.21 ± 666.48ab

11,655.22 ± 2051.37ab

87.58 ± 6.84c

C. fortunei

16.79 ± 2.45ab

127.31 ± 8.52a

132.73 ± 20.35a

4.57 ± 0.55a

876.76 ± 66.59cd

8530.45 ± 267.80abc

10,901.80 ± 779.00a

75.80 ± 6.70b

R. pseudoacacia

23.06 ± 3.68bc

234.39 ± 34.31de

234.73 ± 23.21c

5.85 ± 0.78ab

790.60 ± 37.58bc

7923.33 ± 715.49ab

11,059.27 ± 980.09a

86.39 ± 6.80c

B. papyrifera

26.15 ± 3.40c

257.32 ± 27.64e

334.50 ± 53.38ef

9.09 ± 1.40c

735.36 ± 117.12ab

9029.24 ± 525.51bc

11,319.35 ± 1375.65a

68.24 ± 2.78ab

Treatment means in a column followed by the same letter(s) are not significantly different at p < 0.05

Values are the mean ± SE

Changes in bacterial community diversity

The bacterial community diversity in Zn smelting slag and in the rhizospheres of plant species in revegetated areas is shown in Fig. 2 and Table 4. A total of 209,416 reads and 13,389 observed OTUs were obtained from the bulk slag and rhizospheres of plant species in revegetated areas. The observed OTUs in the rhizospheres of plant species were higher than those in the bulk slag. Among all the rhizosphere slag samples, the observable number of bacterial OTUs was highest for A. donax, whereas that of T. repens was the lowest. The Abundance-based Coverage Estimator (ACE) and Chao indices are two critical indices used to measure microbial community abundance; the Shannon-Wiener and Simpson indices are the two critical indices used to measure microbial community diversity. The bacterial community abundance and diversity indices (ACE, Chao, and Shannon-Wiener) were higher in the rhizosphere slags than those in the bulk slag, whereas the Simpson indices were higher in the bulk slag than those in the rhizosphere slags. Among the slag samples from the rhizospheres of the eight plant species, bacterial community abundance (ACE and Chao indices) in the rhizosphere of A. donax and the diversity (Shannon-Wiener index) in the rhizospheres of R. pseudoacacia were highest, whereas those in the rhizospheres of T. repens and perennial ryegrass, respectively, were the lowest. The Simpson index and Shannon-Wiener index values contrasted in the rhizospheres of the plant species.
Fig. 2

Rarefaction curves of OTUs recovered from the Zn smelting waste slag and from the rhizospheres of plant species in revegetated areas

Table 4

Sequencing information and bacterial diversity indices based on 97% identity of 16S rRNA gene sequences

Samples

Sequencing results

Diversity estimates

Total sequences

Observed OTU

ACE

Chao

Shannon

Simpson

Bulk slag

29,631

574

710.53

751.40

4.78

0.021

T. repens

20,800

1450

1638.81

1659.26

6.23

0.005

Perennial ryegrass

18,000

1494

1784.89

1787.60

6.09

0.009

C. ambrosioides

20,200

1507

1813.28

1838.38

6.18

0.005

B. davidii

24,200

1564

1817.63

1809.49

6.13

0.005

A. donax

27,400

1801

2019.52

2073.50

6.47

0.003

C. fortunei

23,200

1665

1919.10

1935.95

6.32

0.004

R. pseudoacacia

23,185

1748

1980.20

1983.65

6.48

0.003

B. papyrifera

22,800

1586

1818.55

1816.95

6.42

0.003

Abundance-based coverage estimator (ACE); Chao Chao’s species richness; Shannon Shannon-Weiner Index; Simpson Simpson Index

Species level, similarity of greater than 97% level used to define operational taxonomic units (OTUs)

Changes in bacterial community composition

Bacterial community composition in Zn smelting slag and in the rhizospheres of the plant species in the revegetated areas is shown in Fig. 3. The seven most abundant phyla in the bulk slag and rhizosphere slags were Proteobacteria (27.4–49.0%), Actinobacteria (16.7–21.5%), Acidobacteria (4.3–24.1%), Chloroflexi (8.0–21.9%), Bacteroidetes (2.4–7.7%), Gemmatimonadetes (3.5–8.2%), and Saccharibacteria (1.1–5.9%). The relative abundances of Proteobacteria, Bacteroidetes, Acidobacteria, and Saccharibacteria were higher in the rhizospheres of plant species compared to those in the bulk slag (Fig. 3). At the class level, within the proteobacteria, the presence of vegetation resulted in the highest relative abundance values of Alphaproteobacteria followed by Betaproteobacteria and Gammaproteobacteria, and the relative abundance of Deltaproteobacteria was lowest. The relative abundances of Alphaproteobacteria and Deltaproteobacteria in the bulk slag were lower than those in the rhizospheres of plant species. The relative abundances of Alphaproteobacteria and Deltaproteobacteria in the rhizosphere of B. davidii were the highest, followed by those in the rhizospheres of T. repens and B. papyrifera, with no differences among the other plant species. The relative abundances of Betaproteobacteria and Gammaproteobacteria in the bulk slag were higher than those in the rhizosphere slags, and the relative abundances of Betaproteobacteria and Gammaproteobacteria were higher in the rhizospheres of B. davidii and B. papyrifera, respectively, than those in the rhizospheres of the other plant species. The relative abundance of Bacteroidetes in the rhizosphere of T. repens was the highest, followed by those in the rhizospheres of R. pseudoacacia, A. donax, and B. papyrifera. The relative abundance of Saccharibacteria in the rhizosphere of A. donax was highest, while their abundance in the rhizosphere of B. papyrifera was the lowest. The relative abundance of Saccharibacteria in the rhizospheres of the other six plants showed no obvious difference. Planctomycetes was primarily found in the rhizosphere of R. pseudoacacia. The presence of vegetation was associated with a decrease in the relative abundances of Chloroflexi, Gemmatimonadetes, Nitrospirae, Unclassified bacteria, and WCHB1-60 in the rhizosphere slags compared to the bulk slag.
Fig. 3

Relative abundances of the major bacterial phyla in Zn smelting slag and in the rhizospheres of plant species in revegetated areas. The bacterial phyla with low frequencies (< 1%) in all the samples were pooled and denoted “others”

The abundances of the 50 major genera (relative abundance > 1% in at least one sample) are summarized in a heat map (Fig. 4). The heat map shows that the distribution patterns of microbial genera in the different plant rhizospheres were similar but strongly differed from those of the bulk slag, which suggests that at the genus level, the plant species, in general, strongly affected the bacterial community in waste slag, but that there were only minor differences in the different rhizospheres. Genera such as Pedomicrobium, Bradyrhizobium, Arthrobacter, Flavobacterium, Acidibacter, Nocardioides, Rhodoplanes, Terrimonas, Streptomyces, Hyphomicrobium, Mycobacterium, and Devosia were dominant in most rhizosphere samples. Among these genera, some bacterial taxa can be related to specific functions, e.g., plant growth-promoting rhizobacteria (PGPR) such as Flavobacterium, Streptomyces, and Arthrobacter as well as symbiotic N2 fixers such as Bradyrhizobium. In contrast, Gemmatimonas, Gaiella, Nitrospira, and Acidiferrobacter were observed primarily in the bulk slag.
Fig. 4

Heat map showing the bacterial composition of the 50 most abundant genera among the slags samples in this study. The abundance of genera is indicated, and a cluster analysis of the community composition between the samples is shown. The color change from − 0.5 to 3.0 is based on logarithmic values (lg)

Correlations between physiochemical properties and bacterial community diversity

Correlations between physiochemical properties and bacterial community diversity are shown in Table 5. Physiochemical properties such as pH, moisture, available K, and OM strongly influenced the abundance (ACE and Chao) and diversity (Shannon-Wiener and Simpson) of the bacterial community. OM, available K, and moisture were significantly positively correlated with the Shannon-Wiener index, ACE, and Chao values. Available N, available P, and EC were not significantly correlated with the Shannon-Wiener index, ACE, and Chao values. pH values were significantly negatively correlated with the Shannon-Wiener index, ACE, and Chao values. Available Cu, Zn, and Cd were observed to negatively correlate with the Shannon-Wiener index, ACE, and Chao values, but these correlations were not significant, with the exception of the Shannon-Wiener index and available Cu. In contrast, correlations with available Pb were the opposite of those with Zn and Cd. There was no significant correlation between available Cd and Shannon-Wiener index values. The relationship between the abovementioned physiochemical properties and the Simpson index values was the opposite of that between the same properties and the Shannon-Wiener index value.
Table 5

Correlations between physiochemical properties and bacterial structural diversity

 

pH

EC

Moisture

Available K

Available N

Available P

Organic matter

Available Cu

Available Pb

Available Zn

Available Cd

Shannon-Wiener

−0.740*

0.589

0.878**

0.875**

0.599

0.572

0.799**

−0.707*

0.561

−0.185

−0.111

Simpson

0.783*

−0.621

−0.872**

−0.840**

−0.617

−0.581

−0.854**

0.753*

−0.510

0.250

0.192

ACE

−0.702**

0.572

0.777**

0.845**

0.476

0.493

0.699*

−0.590

0.423

−0.226

−0.072

Chao

−0.716*

0.591

0.769**

0.845*

0.468

0.519

0.691*

−0.581

0.403

−0.212

−0.060

EC electrical conductivity

Significant at the p < 0.05 probability level

**Significant at the p < 0.01 probability level

Moisture and nutrient content increased in the rhizosphere in waste slag and were positively correlated with bacterial diversity. Moisture, available K, and OM significantly influenced bacterial diversity. However, while the bioavailability of heavy metals (Cu, Zn, and Cd) were lower in the rhizospheres of the plant species than those in the bulk slag, these elevated toxic metals still exerted adverse effects on bacterial diversity.

Principal components analysis of bacterial community structure

PCA of the bacterial community measured in slag with and without vegetation showed that the first principal component (PC1) explained approximately 57.8% of the variance, while the second component (PC2) explained 35.2% (Fig. 5). Rhizosphere slags and bulk slag were dispersed across PC1 and PC2, which indicated that bacterial community composition in the rhizospheres of plant species differed from that in the bulk slag, and the bacterial community structures in the rhizospheres of the plant species was distinguished from that of the bulk slag based on PC2. Community structures in the rhizosphere slags and bulk slag were divided into three groups. Group 1 is represented by the community structure in the bulk slag. The bacterial community included taxa such as Nitrospirae and unclassified bacteria, which were primarily found in the bulk slag but with low relative abundance. Moreover, the bacterial communities in the rhizospheres of the eight plant species clustered into two final subgroups: those in the rhizospheres of T. repens and B. davidii comprised the second group, and those in the rhizospheres of the other plant species comprised the third group.
Fig. 5

PCA of the bacterial communities in Zn smelting waste slag and from the rhizospheres of plant species in revegetated areas. The bacterial populations indicated major microbial community abundance at the phylum level

Relationship between waste slag properties and bacterial community structure

Figure 6 shows that the majority of the selected waste slag properties were closely related to the first axis. The responses of bacterial community structure and diversity in the rhizospheres and bulk waste slag to environmental factors differed. Bacterial community structure in the bulk slag was independently located in the fourth quadrant of the sorting graph. The microbial communities in the plant rhizospheres strongly differed from that of the bulk slag and this difference can be related to slag properties. The first two axes represented 83.5% of the cumulative percentage of variance of the relation between bacterial community composition and slag parameters. Separation between the bacterial community of the bulk slag sample from those of the rhizosphere samples occurred along the first axis. The slag properties along the first axis, including pH; available heavy metals (Cu and Pb); moisture; available nutrients (N, P, and K); and OM; mainly explained the differences between the bacterial communities of these samples. The eight community structures were divided into three clusters, whereby the communities in the rhizospheres of T. repens and B. davidii represented the first cluster. The community structural differences of this cluster from the bulk slag were mainly related to differences in the contents of OM and available Cu. The bacterial community in the rhizosphere of C. ambrosioides was considered a separate community cluster and the remaining five communities represented the third cluster. The differences between bulk slag and the third cluster were mainly explained by moisture; available nutrients (N, P, and K); and available Pb. However, the separation between the bacterial communities in the individual rhizospheres mainly occurred along the second axis, meaning that available Zn and Cd were the slag parameters, which mainly explained the differences between the rhizosphere communities of the eight plant species. The results showed that differences in bacterial community structure between the rhizosphere slags were affected by slag parameters in the eight plant communities (Fig. 6).
Fig. 6

CCA ordination graph showing the bacterial communities in Zn smelting waste slag and the rhizospheres of plant species in revegetated areas as well as the environmental factors in Zn smelting waste slag. The environmental factors primarily include moisture, pH, EC and the bioavailability of nutrients and heavy metals. OM = organic matter; AN = available N; AP = available P; AK = available K; ACu = available Cu; APb = available Pb; AZn = available Zn; ACd = available Cd; EC = electrical conductivity

Discussion

Soil microbes are crucial for the regulation of biogeochemical processes and the maintenance of ecosystem functions. Investigating the microbial diversity in waste slag and the diversity of responses to direct revegetation measures represents a first step towards the purposeful manipulation of microbial communities for waste slag revegetation. This study showed that the bacterial diversity of bulk slag was extremely low due to adverse factors that created a hostile environment for microflora, and many studies have shown that heavy metals are negatively correlated with the composition and diversity of the soil microbial community and have toxic effects on microbial communities (Wang et al. 2007, 2008). Bacterial community structure and diversity changed with direct revegetation measures. The observable bacterial OTU numbers in the rhizosphere of A. donax were the highest among all the plant species (Fig. 2; Table 4), which indicated that the bacterial diversity indices in the rhizosphere of A. donax were the highest. This is likely due to the high biomass of A. donax, which secretes large amounts of root exudates, e.g., organic acid anions, into the rhizosphere and thus exerts a positive effect on the bacterial community by providing carbon sources (Shi et al. 2011; Wu et al. 2017). The assumption of high rhizodeposition by A. donax is supported by our observation of the highest available P content in the rhizosphere of A. donax among all plant species (Table 2); organic acid anions released though ion exchange or complexation are known to increase the available P for plant growth in waste slag with low P levels. Our observation may also be explained by the fact that the high biomass of A. donax secretes more organic acid anions (Hinsinger 2001; Johnson et al. 1994).

Previous studies have reported that the establishment of plant communities enhances the development of diverse microorganisms (Li et al. 2014; Pepper et al. 2012; Rosario et al. 2007). In the present study, revegetation was shown to either directly or indirectly drive changes in the bacterial communities in Zn smelting slag, resulting in increases in the relative abundances of Alphaproteobacteria, Bacteroidetes, Acidobacteria, and Deltaproteobacteria and decreases in the relative abundances of Gemmatimonadetes, Chloroflexi, and Nitrospirae (Fig. 3), which is consistent with the observations of Li et al. (2016). The higher relative abundance of Nitrospirae in the bulk slag may be attributable to the complex interactions among root exudates, nutrient elements, and microorganisms (Jason et al. 2013). Root exudates are produced from plants and stimulate heterotrophic growth, which leads to local competition between roots and microorganisms for inorganic nutrients (Klemedtsson et al. 1988; Mukerji et al. 2006). At the phylum level, the most persistent dominant bacterial populations included Proteobacteria, Actinobacteria, Bacteroidetes, and Acidobacteria (Fig. 3). The relative abundances of Proteobacteria and Bacteroidetes were notably higher in the rhizospheres of plant species in revegetated slag than those in the bulk slag, which is consistent with previous studies (Lauber et al. 2009; Rastogi et al. 2010). Proteobacteria and Bacteroidetes are among the most abundant bacterial phyla across terrestrial habitats and are positively correlated with C mineralization (Fierer et al. 2007). Therefore, the remarkably higher relative abundances of Proteobacteria and Bacteroidetes in the present study may be consistent with increased OM content in the rhizospheres of plant species. Additionally, an increase in Nocardioides was observed in the rhizosphere slags, and this genus is closely related to heterotrophic genera that degrade OM, e.g., alkanes and polycyclic aromatic hydrocarbons (Afzal et al. 2013; Cébron et al. 2009). Alphaproteobacteria was the most prevalent class among the four subgroups of Proteobacteria, which is also consistent with other studies (Li et al. 2014; Pepper et al. 2012). At the genus level, revegetation increased PGPR such as Flavobacterium, Streptomyces (Kuffner et al. 2008), and Arthrobacter (Saravanan et al. 2008) as well as certain symbiotic N2 fixers such as Bradyrhizobium (Duhan et al. 1998). These rhizosphere bacteria affect plant growth by enhancing the uptake of nutrients from the surrounding environment (Çakmakçi et al. 2006) and by improving plant stress tolerance to heavy metal toxicity. Previous studies indicated that Flavobacterium species promote biological nitrogen fixation (Gholami et al. 2009; Akhtar and Siddiqui 2009), and Arthrobacter species enhance phosphate solubilization and make it available to plants (Chen et al. 2006; Igual et al. 2001; Zhu et al. 2011). Zhang et al. (2004) suggested that Arthrobacter species are able to adapt to high metal concentrations of tailings by physiological and genetic modifications. In the present study, Fig. 4 shows that the relative abundances of Flavobacterium and Bradyrhizobium were higher in the rhizospheres of A. donax and B. davidii, respectively, than those in the rhizospheres of the other plants. The relative abundance of Arthrobacter in the rhizosphere of T. repens was lower than in that of the other plants. These specific community differences in the rhizospheres of A. donax, B. davidii, and T. repens are related to available nutrients contents. Available N in the rhizospheres of A. donax and B. davidii was lower than that in the rhizospheres of other plants, with the exception of R. pseudoacacia; the content of available P in the rhizosphere of T. repens was lower than that in the rhizosphere of A. donax but higher than that in the rhizospheres of the other plants (Table 2). Some studies have shown that the available nutrients in the rhizospheres of plants also depend on the quantities and qualities of root exudates and plant litter from different plant species (Xuluc-Tolosa et al. 2003; Pfeiffer et al. 2013; Esperschütz et al. 2009). Therefore, the establishment of healthy vegetation and a microbial community within the plant-slag-microbial system enhanced mutual development during the process of revegetation of Zn smelting slag.

Many studies have shown that the soil microbial community structure is affected by multiple environmental factors, including pH (Griffiths et al. 2011), soil texture (Chodak and Niklinska 2010), plant community diversity (Stefanowicz et al. 2012), water content (Moche et al. 2015; Praeg et al. 2014), temperature (Wang et al. 2014), and clay mineral content (Balogh et al. 2011). These factors directly or indirectly affect microbial abundance and activity by regulating the bioavailability of nutrients, pollutants and root exudates (Garbeva et al. 2004; Giller et al. 2009). The bacterial diversity was low in bulk slag, and CCA (Fig. 6) showed that the differences in bacterial community structure between bulk slag and rhizosphere slags were explained by pH; available heavy metals (Cu and Pb); moisture; OM; and available nutrients (N, P, and K). OM content and nutrients (N, P, and K) availability increased by approximately two- to fivefold, whereas the content of available Cu was reduced by one- to fivefold (Tables 2 and 3). This indicated that revegetation caused the positive response to bacterial diversity, which was likely attributable to the coupled effects that reduced the bioavailability of Cu, the neutralization of pH values, and the increased carbon and energy sources provided by the plant-released root exudates through rhizosphere deposition (Renella et al. 2008). Previous studies showed that plants tend to decrease soil pH values due to the secretion of organic acids from plant roots and the release of litter decomposition products (Chodak et al. 2009; Noll and Wellinger 2008; Tscherko et al. 2004). The bioavailability of Cu in the rhizospheres of plant species was reduced compared to that in the bulk slag. This may be attributable to the root exudates and the lower pH in the plant rhizospheres, resulting in the release of micronutrient elements from relatively insoluble components (Chaignon et al. 2009; Kabata-Pendias 1993). The released Cu can easily be leached out or absorbed by plants as a micronutrient element (Asensio et al. 2013). The differences in rhizodeposition and Cu uptake by the different plant species result in different amounts of available Cu distributed in the rhizosphere. Additionally, root exudates and litter degradation products immobilize heavy metals (Bouwman and Vangronsveld 2004). This study showed that moisture, pH, Cu, Pb, OM, and nutrient content (N, P, and K) availability were the main factors causing the changes in the bacterial community structure between the bulk and rhizosphere slags, which is consistent with previous studies (Li et al. 2017; Zhang et al. 2013; Zornoza et al. 2015). The present study showed that revegetation increased the content of available Pb in the rhizosphere slags compared to the bulk slag (Table 3), which is also consistent with previous studies (Yang et al. 2010; Zhang et al. 2006). This increase was likely caused by slag disturbance during the remedial work and subsequent plant rhizosphere processes by which root exudates and microbial activity increase the solubility of metal ions in the rhizosphere (Ruttens et al. 2006; Zhang et al. 2006). Therefore, the significantly positive correlation between available Pb and microbial indices such as Shannon-Wiener, ACE, and Chao values (Table 5) may be explained by the positive effects of root exudates on microbiological properties (Esperschütz et al. 2009; Shi et al. 2011; Wu et al. 2017). There were significant differences among the bacterial communities in the plant rhizospheres, likely attributable to differences in litter quality, root exudates (Li et al. 2011), and the utilization efficiency of water and nutrients in the rhizosphere. Plant species affect microbial activity, particularly root exudates and litter carbon utilization, and different microbial species demonstrate significant differences in the competition for carbon sources (Berg and Smalla 2009). The relative abundance of Acidobacteria in the rhizosphere slags was significantly higher than that in the bulk slag, which was likely caused by the decrease in pH because lower pH environments are beneficial for the growth of Acidobacteria (Jones et al. 2009; Lauber et al. 2009).

The relative abundance of Nitrospirae, which is composed of nitrifiers that can oxidize nitrites to nitrates, was primarily found in the bulk slag compared with that in the rhizosphere slags. Mertens et al. (2006) showed that nitrifying populations are well adapted to metal-contaminated sites. Niemeyer et al. (2012) also reported that nitrification is positively correlated with metal stress and is the only microbial process that is significantly increased in the most contaminated soils in the smelting areas. High rates of nitrification may indicate that the balance of nutrient cycling is disturbed, resulting in nutrient loss through the enhanced leaching and runoff of nitrates. However, nitrification rates declined with forest development in consecutive succession stages (Singh et al. 2001). This is consistent with the results obtained in the present study showing that the relative abundance of nitrifying populations in the slag decreased with the development of vegetation. Mummey et al. (2002) investigated soil microbiological properties 20 years after surface mine reclamation and concluded that the spatial characteristics of total biomass, bacterial and fungal biomarkers, microbial biomass C, and soil OM averaged only 20, 16, 28, 44, and 36% of the values found in undisturbed soils. We therefore speculate that the bacterial community diversity in the rhizospheres of plant species significantly increased after 5 years of revegetation but may still be lower than that in uncontaminated soil under the same climatic conditions. This is because the time for revegetation of the Zn smelting slag was short, and the bioavailability of heavy metals in the rhizosphere of plant species was still higher and the OM content was still lower than those in uncontaminated soil. These adverse factors had negative effects on microbial activity and biomass (Gil-Sotres et al. 2005; Hinojosa et al. 2004; Renella et al. 2003). Despite this, the plant-slag-microbial system may still enhance mutual development during the revegetation of Zn smelting slag in the long term. However, the revegetation success may depend on the combination of organic amendment and plant cover to improve the micro-environment and quality of Zn smelting waste, resulting in increased microbial community diversity. Moreover, microbial regulation, which has the capacity to detoxify metals efficiently by transforming them into insoluble salts or relatively nontoxic states, may also promote the development of the micro-environment and promote the establishment of stable vegetation and a microbial community with increased revegetation time (Kuiper et al. 2004; Belimov et al. 2005). This study assessed only the changes in bacterial community structure in the rhizospheres of plant species after 5 years of revegetation of Zn smelting waste slag. Long-term monitoring of the microbial community structure and associated biogeochemical functions should be performed to ensure the absence of adverse environmental impacts. Moreover, the collected rhizosphere samples reveal only the impact of revegetation on the chemical and biological properties at the slag plant interface, which represent the starting point for phytoremediation. It is necessary to collect further core samples between the plants to understand the positive effects of revegetation on total revegetated slag. Information on the spatial heterogeneity of slag microbiological properties is needed to understand the action range and extent of plant species.

Conclusion

After 5 years of revegetation, the physiochemical properties and microbial community diversity of the rhizosphere slags significantly increased. At the genus level, PGPR such as Flavobacterium, Streptomyces, and Arthrobacter and certain symbiotic N2 fixers such as Bradyrhizobium were higher in the rhizosphere slags than those in the bulk slag. The woody plants B. papyrifera, A. donax, and R. pseudoacacia serve as suitable plant species for the revegetation of Zn smelting slag due to their rapid plant growth with high biomass production, their higher efficiency in reducing metal toxicity and their capacity to improve slag fertility and enhance microbial community diversity. Canonical correspondence analysis showed that bacterial community structure and diversity in the rhizosphere slag were significantly different from those of the bulk slag and that the bacterial community structures in the rhizospheres of different plant species used for revegetation of the waste slag were influenced by different waste slag properties. This study suggests that revegetation plays an important role in improving bacterial community structure and diversity in Zn smelting slag. The results indicate that revegetation potentially regulates microbiological properties through shifts in heavy metal bioavailability and physiochemical slag properties and that the plant-slag-microbe system enhances mutual development during the revegetation of Zn smelting slag in the long term.

Notes

Funding

The study was funded by a grant from the National Natural Science Foundation of China (no. 41663009), the United Found of the Guizhou Province Government and the Natural Science Foundation of China (no. U1612442), and the Natural and Science Project of the Education Department of Guizhou Province (no. KY[2016]011).

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Youfa Luo
    • 1
  • Yonggui Wu
    • 1
    • 2
  • Hu Wang
    • 1
  • Rongrong Xing
    • 1
  • Zhilin Zheng
    • 1
  • Jing Qiu
    • 1
  • Lian Yang
    • 1
  1. 1.College of Resource and Environmental EngineeringGuizhou UniversityGuiyangChina
  2. 2.Institute of Applied EcologyGuizhou UniversityGuiyangChina

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