Mixotrophic acidophiles increase cadmium soluble fraction and phytoextraction efficiency from cadmium contaminated soils

Science of the Total Environment 655 (2019) 347–355

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Mixotrophic acidophiles increase cadmium soluble fraction and phytoextraction efficiency from cadmium contaminated soils Xiaodong Hao a,b,c, Ping Zhu b,c, Huaizu Zhang b,c, Yili Liang b,c, Huaqun Yin b,c, Xueduan Liu b,c, Lianyang Bai a, Hongwei Liu b,c,⁎, Huidan Jiang a,⁎⁎ a b c

Biotechnology Research Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China Key Laboratory of Biometallurgy, Ministry of Education, Changsha 410083, China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Cd-contaminated soils were bioremediated using mixotrophic acidophiles. • The mixotrophic acidophiles significantly increased the Cd removal from the soils. • The mixotrophic acidophiles increased Cd uptake into water spinach tissues. • The consortium did not alter soil structure and microbial community composition.

a r t i c l e

i n f o

Article history: Received 22 July 2018 Received in revised form 15 November 2018 Accepted 15 November 2018 Available online 17 November 2018 Editor: Charlotte Poschenrieder Keywords: Bioleaching Cd-contaminated soils Mixotrophic acidophiles Microbially-assisted phytoextraction

a b s t r a c t A profound concern in developing microbially-assisted phytoextraction is that introduced microbes not only remove heavy metals from contaminated soils but also enhance metal uptake into plant tissues from the treated soils. Cadmium (Cd) removal efficiencies were compared after leaching with deionized water (CK), acidified basal salts medium (acid control), cell-free spent medium (spent bioleaching) and mixotrophic acidophiles (two-step bioleaching). Two-step bioleaching using the mixotrophic acidophiles removed 34% of total Cd and 87% of available Cd, significantly more than CK (3% and 4%), acid control (12% and 51%) and spent bioleaching (26% and 75%). Pot experiments of water spinach growing in four treated soils were conducted to evaluate the Cd uptake performance in plants. Notably, the mixotrophic acidophiles increased Cd concentration in plant tissues by 78% compared to the CK group. More interestingly, the mixotrophic acidophiles were not colonized in soils but caused the compositional increase of indigenous microbes such as the genera of Alicyclobacillus, Clostridium sensu strict and Streptacidiphilus. Meanwhile, two-step bioleaching had little effects on soil structure and physicochemical properties determined by the spectroscopy characteristics analysis. These results implied that the mixotrophic acidophiles facilitated the development of microbially-assisted phytoextraction technology. © 2018 Elsevier B.V. All rights reserved.

⁎ Correspondence to: H. Liu, School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (H. Liu), [email protected] (H. Jiang).

https://doi.org/10.1016/j.scitotenv.2018.11.221 0048-9697/© 2018 Elsevier B.V. All rights reserved.

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1. Introduction Cadmium (Cd) is one of the most toxic and carcinogenic heavy metals in agricultural soils (WHO, 1992). Cd is readily accumulated by plant tissues and a threat to human life through the food chain. The immobilization and mobilization techniques can be used for the remediation of Cd-contaminated soils. Immobilization techniques reduce the Cd mobility via adding passivators to contaminated soils, and passivators react with Cd through adsorption, chelation and complexation (Fonti et al., 2016). However, Cd can be released back to the environment when metabolites (e.g., organic acids, enzymes and surfactants) secreted by soil microbes and plant roots dissolve Cd precipitates (Yang et al., 2016; Zhang et al., 2017). Therefore, mobilization techniques that fundamentally decrease the environmental risks and human hazards of Cd are sustainable for remediation of Cd-contaminated soils. Currently, phytoextraction has been applied to the in situ bioremediation of Cd-contaminated agricultural soils due to its low cost and ecofriendliness. Nevertheless, full-scale applications of this technique are very limited owing to poor Cd plant availability in soils and long remediation time (Robinson et al., 2015). In recent years, bioleaching technology has received considerable attention for enhancing the mobility of metals from dredged sediments, sewage sludge, sulphide ores and spent batteries (Bayat and Sari, 2010; Ma et al., 2017; Xin et al., 2012; Zeng et al., 2015). The acidophilic microbes with Fe/S/organic matter oxidizing metabolism not only remove total and available Cd but also mobilize Cd from soils, which facilitate the phytoextraction of Cd. Therefore, microbially-assisted phytoextraction has been considered as a promising technique for the restoration of Cd-contaminated soils. Several microbial species including genera of Aspergillus, Penicillium, Rhodobacter and Acidithiobacillus have been reported to remove heavy metals from contaminated soils. For example, Penicillium chrysogenum was investigated for the bioleaching of soils contaminated with smelting slag (Deng et al., 2013). Rhodobacter sphaeroides was isolated from oil field injection water and used for the bioremediation of lead (Pb) contaminated soils (Li et al., 2016). Aspergillus niger was reported for the bioleaching of red mud from the mining and metallurgy industries (Vakilchap et al., 2016). Acidithiobacillus bacteria were performed for the bioleaching of toxic metals with the co-inoculation of biosurfactant-producing yeast Meyerozyma guilliermondii (Camargo et al., 2018). However, research on bioremediation of Cdcontaminated soils is lacking. The mixotrophic acidophiles including autotrophic and heterotrophic species have better environmental adaptability and are capable of performing more complicated tasks than pure species or their co-cultures in natural soils (Beolchini et al., 2009; Brune and Bayer, 2012; Kang et al., 2016; Latorre et al., 2016). In addition, most previous studies have focused on the effects of microbial inoculation on the fractional distribution and removal efficiency of heavy metals from contaminated soils, rendering the potential effects on indigenous microbial community and soil physicochemical properties largely uninvestigated (Li et al., 2017). Table 1 Physicochemical properties of soil obtained from an abandoned vegetable field in Xiangtan City, Hunan Province of China. Item

Mean valuea

Cd (mg/kg soil) Pb (mg/kg soil) Cr (mg/kg soil) Mn (mg/kg soil) Cu (mg/kg soil) pH ORP (mV) TOC (%) TON (%) Total P (mg/kg soil) Total K (g/kg soil)

3.1 ± 0.6 30.3 ± 0.8 109.0 ± 1.8 671.0 ± 11.9 35.6 ± 0.5 6.4 ± 0.1 331.4 ± 16.4 1.8 ± 0.04 0.2 ± 0.001 682.0 ± 10.9 11.7 ± 0.1

a

Mean ± standard deviation (n = 3).

Here, bioremediation of Cd-contaminated soils using mixotrophic acidophiles was studied. The effectiveness of the bacterial consortium on enhancing Cd phytoextraction by water spinach was also examined. Water spinach was employed due to its high Cd uptake. The hypothesis was that the mixotrophic acidophiles could remove Cd from contaminated soils and improve phytoextraction efficiencies of bioremediated soils. The objectives of this study were to (i) evaluate the effects of mixotrophic acidophiles on total and available Cd removal; (ii) identify the effects of mixotrophic acidophiles on soil microbial community and physicochemical properties; (iii) investigate the effects of mixotrophic acidophiles on enhancing Cd phytoextraction. 2. Materials and methods 2.1. Soil sampling and soil properties Soil samples were obtained from Xiangtan City, Hunan Province of China (27°42′ N, 112°57′ E). The contaminated site was an abandoned vegetable field around an industrial smelting factory and was contaminated by industry waste water for about 10 years. Surface soils (0–20 cm) were collected via the T-type soil sampler in July 2017. After sampling, soils were separated into two parts. One part was stored in 4 °C freezer for soil physiochemical analyses. The other part was airdried for the subsequent leaching experiments. Soil samples were digested by an acid mixture (HNO3, HF and HClO4) on an electric heating plate (XJS20-42, Laboratory Instrument Equipment Co. Ltd., Tianjin, China) and total element contents were measured by ICP-OES (Optima 5300DV, PerkinElmer, Shelton, USA). Soil pH and oxidation reduction potential (ORP) were determined with a soil:water ratio of 1:2.5 (wt/vol) using a pH meter (BPH-220, Bell Instrument Equipment Co. Ltd., Dalian, China). The total organic carbon (TOC) and organic nitrogen (TON) in soils were investigated by Elemental Analyzer (Elementar Vario EL III, Elementar Analysensysteme, Hanau, Germany). The properties of the soils are listed in Table 1. The heavy metal contents in this soil were below the Environmental Quality Standard for Soils of China (EPM, 1995) except Cd. The total Cd content was 10 times higher than soil standard threshold. Therefore, it is an urgent need for remediation of this soil. 2.2. Preparation of mixotrophic acidophiles Mixotrophic acidophiles were enriched from acid mine drainage (AMD) and contaminated soils used in this study. The AMD samples were collected from Dexing Copper Mine of Jiangxi Province in China. The AMD (100 mL) and fresh soils (10 g) were combined and then inoculated into 900 mL basal salts medium in 3 L glass flask. The basal salts medium contained (NH4)2SO4 (3 g/L), KCl (0.1 g/L), K2HPO4 (0.5 g/L), MgSO4·7H2O (0.5 g/L) and Ca(NO3)2 (0.01 g/L). Elemental sulfur (1 g/L), glucose (0.7 g/L) and yeast extract (0.3 g/L) were also added to provide energy sources. The initial solution pH was adjusted to 3.5 with 9 M sulfuric acid. Cultures were incubated on a rotary shaker (175 rpm) at 32 °C. When solution pH dropped down to 2.5, the next subculturing was prepared via transferring the culture (100 mL) into fresh 900 mL basal salts medium with the same aforementioned culture condition. Subculturing was done as soon as solution pH value was below 2.5. The duration of incubation of each subculture ranged from four to 5 days. The cultivation of mixotrophic acidophiles included 20 times. Finally, the bacteria solution of mixotrophic acidophiles was used in subsequent bioleaching experiments. The properties of the bacteria solution and basal salts medium adjusted to pH 2.5 with 9 M sulfuric acid are listed in Table 2. 2.3. Soil column bioleaching experiments The polyethylene column (30 cm tall and 20 cm diameter) was used for the bioleaching experiments. There were numerous 0.3 cm holes at

X. Hao et al. / Science of the Total Environment 655 (2019) 347–355 Table 2 Properties of basal salts medium and mixotrophic bacteria solution. Item

pH ORP (mV) SO2− 4 (g/L) Citric acid (mg/L) Oxalic acid (mg/L) Malic acid (mg/L) Gluconic acid (mg/L) Total P (mg/L) Total K (mg/L) a

Mean valuea Basal salts medium

Mixotrophic bacteria solution

2.5 ± 0.04 594.6 ± 1.1 3.8 ± 0.5 Undetected Undetected Undetected Undetected 54.8 ± 0.2 210.8 ± 0.3

2.5 ± 0.002 487.7 ± 0.3 5.0 ± 0.1 17.7 ± 1.0 20.4 ± 1.1 0.3 ± 0.2 2736.1 ± 36.3 63.5 ± 0.8 261.8 ± 0.9

Mean ± standard deviation (n = 3).

the bottom of column. A piece of gauze was placed at the bottom, then air-dried soil samples (4 kg) were cracked and soil blocks sized at 2–4 cm were weighted into the column to a height of 15 cm. Soil leaching treatments included: (i) deionized water (CK); (ii) pH 2.5 basal salts medium (acid control); (iii) mixotrophic acidophiles cultured and sprayed onto soil columns after lowering pH to 2.5 (twostep bioleaching); and (iv) cell-free spent medium after filtering through 0.22 μm filter membrane (spent bioleaching). Before column leaching experiments, soil columns except CK group were saturated using the acid solution of pH 2.5 (2 L) adjusted by 9 M sulfuric acid, and allowed to equilibrate for 2 days. The leaching solution passed through the soil samples by gravity and recirculated through a side loop with a peristaltic pump, and the spray rate of solution was 0.15 L/h. In four leaching treatments, each leaching solution (500 mL) was used for the test and changed every 2 days. All the experiments were carried out in triplicate. The column bioleaching experiments lasted for 14 days at ambient temperature. Solution pH and ORP values were measured daily. At the end of leaching experiment, soil samples were washed by deionized water (1:1, wt/vol) to reduce the soil adsorption amount of dissolved Cd ion, and then collected for the analyses of organic element contents, Cd fraction distribution, total Cd and available Cd contents. 2.4. Pot experiments Pot experiments were conducted to evaluate microbially-assisted phytoextraction. Water spinach seedling growing experiments were carried out in pots (25 cm length, 15 cm width and 7 cm height) containing the washed soils (1.5 kg) after the CK, acid control, two-step bioleaching and spent bioleaching treatments. Water spinach seeds were purchased from Zhejiang Wuwangnong Seeds Co., Ltd. A total of 12 seeds were sowed into each pot and covered with 2 mm thickness soil. During the cultivating period, deionized water was added to the pots by using a sprinkling can to compensate water loss, and the soil moisture content was maintained at approximately 60% (wt/wt). After 15 days, the water spinach seedlings in each pot were harvested, dried at 60 °C for 24 h in oven and then acid digested with HNO3/HClO4 (5:2, vol/vol) at 140 °C. The Cd concentrations were measured by ICPOES. The experiments were conducted in triplicate. 2.5. Analytical methods Spectroscopy characteristics analysis of soils in four treatments was carried out. X-ray diffraction (XRD) profiles were achieved using the XRD diffractometer (DX-2700, Dandong Instrument Equipment Co. Ltd., Dandong, China) with Cu Kα radiation (40 kV, 35 mA). Fourier transform infrared spectroscopy (FTIR) profiles were measured using a Nicolet iS10 FTIR Spectrometer (Thermo Scientific, Rochester, USA). The organic acid contents in bacteria solution were determined by Agilent 1200 HPLC (Agilent Technologies Co. Ltd., Santa Clara, USA). The diethylenetriaminepentaacetic acid (DTPA) method was used to

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evaluate available Cd content in soils. Briefly, 5 g air-dry soils were added into 100 mL shake flasks containing 25 mL extracting agent. The agent consisted of DTPA (0.005 mol/L), triethanolamine (TEA, 0.1 mol/L) and calcium chloride (CaCl2, 0.01 mol/L), and adjusted to pH 7.3 with 6 M HCl. The shake flasks were incubated on a rotary shaker (180 rpm) at 25 °C for 2 h. The supernatant was separated by centrifugation at 3000 rpm for 5 min. The Cd concentration in solution was measured by ICP-OES. Fractional Cd distribution in soils was carried out by sequential extraction as described in Table A1 (Gleyzes et al., 2002). Between successive extractions, the supernatant was collected by centrifugation at 3000 rpm for 20 min. Before carrying out the next extraction, the residue was washed with 20 mL of distilled water and centrifuged for 20 min, and then this supernatant was discarded. Total Cd and available Cd removal efficiency (%) = (W1 − W2) / W2 × 100, where W1 represented the Cd quantity in soils before bioleaching, and W2 represented the Cd quantity in soils after bioleaching.

2.6. Microbial community analysis 2.6.1. DNA extraction, amplification and sequencing of 16S rRNA gene The genomic DNA of mixotrophic acidophiles used for column bioleaching was extracted using E.Z.N.A. Water DNA kit (Omega BioTek Inc., USA). The genomic DNA of treated soils using four leaching methods was extracted using E.Z.N.A. Soil DNA kit (Omega Bio-Tek Inc., USA). For bacterial and archaeal 16S rRNA genes, the V4 region was amplified with the primer pair 515F (5′-GTGCCAGCMGCCGCG GTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) together with the Illumina adapter sequence. PCR amplification was performed on Applied Biosystems 2720 Thermal Cycler (ABI Inc., California, USA). PCR system (25 μL) consisted of 1 μL (10 nM) of each primer, 1 μL of template DNA, 12.5 μL of 2 × Taq PCR Master Mix (Vazyme, Piscataway, USA) and 9.5 μL of DNase-Free deionized water. Three replicates of amplifications for each sample were carried out and blended after PCR amplification to minimize potential biases of amplification. PCR procedure was as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles of 94 °C for 45 s, 62 °C for 45 s and 72 °C for 30 s, with final extension at 72 °C for 10 min. PCR products were separated by 1.5% (w/v) agarose gel electrophoresis, and recovered using E.Z.N.A. TM Gel Extraction Kit (Omega Bio-Tek Inc., USA). The quality of recovered PCR products was measured using the Nanodrop Spectrophotometer (ND-1000, Thermo Fisher Scientific, Waltham, USA). The sequencing library was constructed via combining approximately equimolar amounts of PCR products (150 ng) in each sample. The MiSeq 500 cycles kit was used for 2 × 250 bp paired-ends sequencing on Illumina MiSeq sequencing platform.

2.6.2. Sequence preprocessing The raw data of 16S rRNA gene from MiSeq sequencing was in FASTQ format. The Illumina adapter and other specific sequences were cut before the process. Sequence processing was performed on the Galaxy pipeline developed by Prof. Zhou's lab (http://zhoulab5.rccc.ou.edu/) at University of Oklahoma. Reads were recognized and assigned into different samples according to barcodes files. Primers were removed from reads with 1.5 allowed mismatches. Btrim program was used with the threshold of QC N 20 over 5 bp window size to filter the unqualified sequences (Kong, 2011). Forward and reverse reads of same sequence with at least 30 bp overlap and b5% mismatches were combined using FLASH program (Mago and Salzberg, 2011). The fastq data after trimming was transformed to fasta format, and then sequences with clustering threshold of 97% were assigned to the same operational taxonomic unit (OTU) using UPARSE (Edgar, 2013). Taxonomic assignment was performed using RDP classifier with minimal 50% confidence score. When the similarity was b50%, the unit was assigned to unclassified.

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2.7. Statistical analysis Alpha-diversity indexes (Shannon index, Simpson index, Pielou evenness and Simpson evenness) were determined by the Institute for Environmental Genomics (IEG), University of Oklahoma website (http://ieg.ou.edu/). Principal correspondence analysis (PCA) for comparing different group sites was conducted on the R statistical platform with the ‘Vegan’ Package. The Venn diagram was drawn using the ‘Venny 2.1’ (http://bioinfogp.cnb.csic.es/tools/venny/index.html). Other statistical analyses were performed using IBM SPSS Statistics 21.0 software. One-way Analyses of Variance (ANOVA) were carried out to identify the difference in Cd removal, soil properties, soil microbial diversity and Cd uptake after four leaching treatments. The least significant difference test (LSD) was used to identify significant (P b 0.05) differences between treatment means. 3. Results and discussion 3.1. Removal efficiencies of total and available Cd in soils The total Cd and available Cd removal efficiencies of the soils using four leaching treatments on day 14 are shown in Fig. 1. The leaching efficiency of total Cd was 34% for two-step bioleaching, which was higher than that of CK, acid control and spent bioleaching (Fig. 1a). No

Fig. 1. Total (a) and available (b) Cd removal efficiencies in soils using different leaching treatments (means ± SD). Figures represent total Cd (a) and available (b) Cd contents in soils; Means with the same letters are not significantly different according to the LSD test (p N 0.05).

significant (P N 0.05) differences were observed between the plots for spent bioleaching and acid control, for spent bioleaching and two-step bioleaching, but significant (P b 0.05) difference was found among CK, acid control and two-step bioleaching methods. The removal efficiency of available Cd in two-step bioleaching was more than CK, acid control and spent bioleaching and (Fig. 1b). The removal efficiency in twostep bioleaching was significantly (P b 0.05) higher than that of the other three leaching treatments. Compared with acid control, cell-free spent medium (spent bioleaching) had a great effect on total Cd and available Cd removal, which indicated the metabolites produced by mixotrophic acidophiles in microbial growth process played an important role in Cd removal from the soils (Camargo et al., 2018; Tang et al., 2017). Previous studies showed that organic acid molecules containing multiple ligands had the influence on the Cd removal through the chelation (Wuana et al., 2010). As shown in Table 2, the concentrations of citric acid, oxalic acid, malic acid and gluconic acid in bacteria solution were 17.7 mg/L, 20.4 mg/L, 0.3 mg/L and 2736.1 mg/L, respectively. Ren et al. (2009) used gluconic acid, citric acid, oxalic acid, malic acid and a mixture of the four acids to determine the chemical leaching of soils contaminated by heavy metals. Gluconic acid and citric acid leached 100% Cd from metalscontaminated soils. The Cd removals using oxalic acid and malic acid were below 20%. It was interesting to find that low Cd removal was obtained using the mixture of acids. These results suggested that lower Cd removal (62%) by the mixture of four acids compared to gluconic and citric acids might be due to the precipitation of Cd as their oxalates

Fig. 2. Variations of pH (a) and ORP (b) values in solution using different leaching treatments. Each leaching solution changed every 2 days.

X. Hao et al. / Science of the Total Environment 655 (2019) 347–355

Fig. 3. Relative Cd fractions in soils using different leaching treatments. Figures represent the contents of each Cd fraction in soils.

and metal complexes. Although the basal salts medium (Acid control) and bacteria solution had similar pH values, the organic acids showed a good acid soluble effect and could enhance the Cd extraction in soils. The removal percentages of total and available Cd using two-step bioleaching was higher than that of spent bioleaching, which suggested that mixotrophic acidophiles played other important roles in mobilizing or removing Cd from the soils. In addition, the results also showed the bacteria solution could be directly applied in the soil remediation without the procedure of separating the microbes from bacteria solution.

3.2. Solution pH and ORP Four different solutions were used to leach contaminated soils and were changed every 2 days. A similar pH trend was observed, except for the CK treatment, during the leaching process (Fig. 2a). In the CK group, the solution pH maintained from 6.6 to 6.8 over the course of the experiment. However, a sudden pH increase occurred after fresh leaching solutions were added into each leaching system, which resulted from the neutralization of hydronium ions by hydroxide and carbonate. The solution pH values were up to 4.3, 4.1 and 4.0 on day 4 in acid control, spent bioleaching and two-step bioleaching, respectively. Thereafter, highest pH values decreased with time and declined to 3.8, 3.7 and 3.6 on day 14, which were due to the alkalinity of the soils consumed by protons. pH was an important factor affecting the environmental behaviors of heavy metals in soils and the extent of metal solubilisation (Sauvé et al., 2000; Kumar and Nagendran, 2007). The low pH of leaching solution could not only increase the mobility of heavy metals in soils, but was needed for metals released from the soils to be stable in solution phase (Chen and Lin, 2001). Lower pH values were observed in two-step bioleaching compared with acid control and spent bioleaching. This is attributed to mixotrophic acidophiles added into soils producing acids through metabolism (e.g., the oxidation of glucose and S0). This may explain the high removal efficiencies of total and available Cd were obtained in two-step bioleaching.

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The variations of solution ORP during leaching process are shown in Fig. 2b. The initial ORP value of leaching solution in acid control (594.6 mV) was higher than spent bioleaching (499.9 mV) and twostep bioleaching (487.7 mV). The redox potentials decreased rapidly in 2 days and final ORP values were 442.3 mV, 439.2 mV and 455.5 mV on day 14, respectively. In CK group, solution ORP maintained from 280 mV to 320 mV over the course of experiment. ORP was another important factor controlling heavy metals mobility in soils. It had been published that high ORP favored the solubilisation of metals associated with oxidizable fraction (Fonti et al., 2013). When ORP in soils increased, the dissolution efficiencies of metal sulfides and degradation rates of organic compounds correspondingly improved, further accelerated the release of adsorbed/complexing heavy metals (Peng et al., 2009). The reaction was expressed as: MS2 + 3.5O2 + 3H2O → M(OH)2 + 2SO42− + 4H+ (M represented the heavy metals). The release of H+ into porewater would decrease the soil pH and then cause a secondary release of heavy metals. Kelderman and Osman (2007) showed that, with ORP increased, the stable metal fraction of Cd bound to organic sulfide would decrease from 65% to 30% and form a more mobile fraction. 3.3. Cd fraction distribution analysis in soils Fraction distribution (acid-soluble fraction, reducible fraction, oxidizable fraction and residual fraction) of Cd in soils using four leaching treatments is shown in Fig. 3. In CK group, residual fraction (79%) was the dominant phase and the following phases were reducible fraction (10%) and acid-soluble fraction (9%), while oxidizable fraction (2%) only accounted for a small proportion. For these four forms of Cd in soils, oxidizable fraction and residual fraction were relatively stable and non-leachable. Conversely, acid-soluble fraction and reducible fraction with high activity and bioavailability could lead to large environmental risks. Compared with CK group, the differences on the Cd partitioning were observed in the other three leaching treatments. The acid-soluble and reducible fraction percentages of Cd declined. Meanwhile, residual and oxidizable fraction percentages of Cd increased. Biological toxicity of Cd was not only determined by the total count, but also more dependent on the fraction distribution presented in soils. In two-step bioleaching, more residual fraction and oxidizable fraction percentages and less acid-soluble fraction and reducible fraction percentages were obtained compared to the other three leaching methods. 3.4. Soil microbial diversity and community composition Microbial community composition and dynamics were examined by 16S rRNA amplification. The microbial diversity of soils in four different treatments on day 14 is shown in Table 3. Alpha-diversity indexes in treated soils of CK group, including Shannon index, Simpson index, Pielou evenness, Simpson evenness and observed OTU number, were significantly (P b 0.05) higher than that of treated soils in acid control, spent bioleaching and two-step bioleaching. However, the diversity did not show significant difference in these three treatments. This result might suggest the low pH of three leaching solution inhibited the growth of some microbes in soils, and thereby decreased microbial diversity. Yet even so, highest Cd removal efficiency was still obtained in two-step bioleaching (Fig. 1), which indicated the decrease of microbial

Table 3 Alpha-diversity indexes of soils using different treatments on day 14. The indexes were calculated from OTU relative abundance of each replicate. Item

CK

Acid control

Spent bioleaching

Two-step bioleachinga

Shannon index Simpson index Pielou evenness Simpson evenness Observed OTU number

6.5 ± 0.1 a 234.2 ± 79.2 a 0.9 ± 0.02 a 0.1 ± 0.042 a 1777 ± 58 a

4.1 ± 0.2 b 17.6 ± 2.8 b 0.60 ± 0.02 b 0.022 ± 0.002 b 812 ± 53 b

3.9 ± 0.5 b 15.0 ± 4.4 b 0.59 ± 0.06 b 0.018 ± 0.003 b 817 ± 127 b

3.9 ± 0.1 b 14.4 ± 0.8 b 0.59 ± 0.01 b 0.019 ± 0.001 b 769 ± 12 b

a

Mean ± standard deviation (n = 3). Means within a row followed by the same letter are not significantly different according to the LSD test (p N 0.05).

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diversity had little effect on Cd removal from soils. The shift in ratios of survived functional species adapted to low pH would play the important role in the Cd bioleaching performance. The venn diagram showed 139 core genera (38%) among the four types of samples. The soils in CK, acid control, spent bioleaching and two-step bioleaching had 56, 11, 12 and 19 unique genera, respectively

Fig. 4. Venn diagram (a), principal correspondence analysis (PCA) (b) and relative abundance of microbial community structure of inoculum (mixotrophic acidophiles) and dynamics of soils using different treatments (c).

(Fig. 4a). The numbers of shared OTU with CK group in acid control, spent bioleaching and two-step bioleaching groups were only 9, 16 and 8, respectively. These results also indicated the decrease of alphadiversity in three treated soils. PCA analysis showed all replicates in treated soils on day 14 were clustered together and separated from soil samples of CK group, indicating that the microbial composition changed in three leaching process (Fig. 4b). Microbial community structure of inoculum (mixotrophic acidophiles), soil sample in CK group on the 14th day and dynamics at the genus level in the other three treated soils are illustrated in Fig. 4c. The most abundant nine genera (N 2%) of initial inoculum including autotrophic and heterotrophic microbes are shown in the pie chart. Mycobacterium (18%) and Sulfobacillus (17%) were dominant species, and the other seven genera had relatively lower abundance ranging from 2% to 5%. In CK group, N50% of 16s rRNA sequences could not be classified into genera. The percentages of low relative abundance genera (b2%) were 32%. Moreover, the genera Gp6, Nitrososphaera and Gp16 were only accounted for 8%, 6% and 3%, respectively. However, most of the genera in three treated soils showed different abundance compared with mixotrophic acidophiles and CK soils. Major genera were Alicyclobacillus, Clostridium sensu strict and Streptacidiphilus in whole processes of three leaching treatments. The relative abundance of Acidiphilium and Metallibacterium increased gradually with time in acid control, spent bioleaching and two-step bioleaching. As shown in Fig. 4c and Fig. A1, the dominant microbial genera in initial inoculum all had low percentages (b2%) in soils of two-step bioleaching, which suggests mixotrophic acidophiles added in soils were not colonized in treated soils. These results indicate introduced bacteria only had a weak effect on indigenous microbial community composition, and potential environmental risks such as continuous soil acidification could be avoided. Instead, compared with CK, three leaching treatments affected proportional change of indigenous microbes (Fig. 4c and Fig. A2). The relative abundances of genus Alicyclobacillus (0.01%), Clostridium sensu strict (0.11%) and Streptacidiphilus (0.03%) existed in CK soils increased to approximately 40%, 9% and 7% in the whole process of three treated soils. Previous studies indicated the roles of these three genera were as follows: Alicyclobacillus were acidophilic and Gram-positive spore formers, and several Alicyclobacillus isolates are capable of mixotrophic growth (Kaksonen et al., 2016). For example, Al. tolerans could use organic compounds, Fe2+ and sulfide minerals to support their growth (Karavaiko et al., 2005; Yahya et al., 2008). Wang et al. (2016) found that genus Alicyclobacillus had a similar habitat with Sulfobacillus. They proposed a synergy that Sulfobacillus strains oxidized Fe2+ into Fe3+ and produced metabolites that could be consumed by the Alicyclobacillus strains. Both of the two strains could benefit from this synergy, eventually enhancing the heavy metals removal rates. Alexandrino et al. (2014) indicated Clostridium spp. presented significant metabolic activity and metal-resistance in mixed sulfidogenic cultures. Clostridium spp. could initiate heavy metals bioremoval and reduce the toxicity of heavy metals to heavy metals-sensitive microbes. The genus Streptacidiphilus belonging to acidophilic actinobacteria could utilize glycerol and sucrose as energy sources in a broad pH range between 3.5 and 6.5 (Cho et al., 2008). Williams and Robinson (1981) reported that the important role of Streptacidiphilus was to decompose fungal biomass in acid litters and soils. Unlike the three genera above, the Acidiphilium and Metallibacterium ratios gradually increased in three treated soils. Most of the species belonging to the genus Metallibacterium were isolated from acidic biofilm and water of metal mine (Ziegler et al., 2013). The Metallibacterium was acid-tolerant and had the heterotrophic iron oxidation and reduction abilities (Kay et al., 2014). Acidiphilium also had a wide range of pH tolerance. Previous studies indicated that Acidiphilium not only served as a consumer of organic matters produced by autotrophic Acidithiobacillus sp. and Sulfobacillus sp. (Hao et al., 2017; Johnson and Hallberg, 2003), but also used S0 and reducing inorganic sulfur compounds (RISCs) as energy. Acidiphilium sp. in pure or

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Table 4 Physicochemical properties of soils receiving different leaching treatments. Item

CK

Acid control

Spent bioleaching

Two-step bioleachinga

pH ORP (mV) TOC (%) TON (%) Total P (mg/kg soil) Total K (g/kg soil)

6.5 ± 0.06 a 329.1 ± 12.7 c 1.7 ± 0.03 b 0.20 ± 0.001 c 678.1 ± 9.4 c 11.6 ± 0.04 ab

5.7 ± 0.03 b 352.7 ± 11.1 bc 1.9 ± 0.04 b 0.25 ± 0.004 b 801.3 ± 23.2 b 11.9 ± 0.12 a

5.6 ± 0.09 b 364.9 ± 11.9 ab 2.0 ± 0.02 a 0.27 ± 0.004 a 856.9 ± 25.2 a 11.8 ± 0.23 ab

5.7 ± 0.13 b 383.8 ± 1.3 a 1.9 ± 0.01 b 0.28 ± 0.007 a 831.5 ± 32.2 ab 11.5 ± 0.32 b

a

Mean ± standard deviation (n = 3). Means within a row followed by the same letter are not significantly different according to the LSD test (p N 0.05).

mixed-species culture with Acidithiobacillus sp. had significant heavy metals leaching capacity in solids such as sulphide ores, sludges and electronic wastes. From the above, most of increased genera in treated soils were mixotrophic or facultatively heterotrophic acidophilus or acid-resistant microbes. 3.5. Physicochemical and spectral characteristics of soils

that the addition of this consortium increased total (34%) and available Cd (87%) removal efficiencies (Fig. 1). In agreement with our hypothesis, mixotrophic acidophiles enhanced Cd contents in plant tissues of water spinach by approximately 78% compared to CK group. The increased Cd uptake could be attributed to a beneficial effect of the consortium on Cd mobilization in contaminated soils. From a practical perspective, hyperaccumulators such as Cardamine hupingshanesis and

The potential effects of four different treatments on soil physicochemical properties on day 14 are shown in Table 4. Soil pH values in three treatments were significantly (P b 0.05) lower than the CK group. The significantly (P b 0.05) higher ORP values were observed in three treated soils compared to CK. The results of pH and ORP variations in treated soils were in accordance with the variations in leaching solution (Fig. 2), i.e., three leaching solution decreased soil pH and increased soil ORP, and facilitated the Cd solubility. The plots for the three treatments also show significant (P b 0.05) differences in total P and TON compared to CK. This might be due to the leaching solution added into soils increased total P and TON contents (Table 2). The other selected physiochemical properties (TOC and total K) of the soils showed little difference between the treatments. The spectroscopy characteristics analysis of soils was carried out to determine the effects of different treatments on soil composition and structure. As shown in Fig. 5a, a XRD characteristic peak at 26.78° with line spacing of 3.2187 Å showed quartz in CK soils as the main composition. The peak position did not appear offset and main peaks for quartz (3.2180 Å, 3.2179 Å and 3.2183 Å) maintained stable indicating the bioleaching process using mixotrophic acidophiles did not alter soil composition and structure. For the FTIR curves of CK soils (Fig. 5b), the infrared absorption peak around 500 cm−1 corresponded to the M-O (M refers to Fe, Ni and Mn). The spectral curves of the soils after three leaching treatments did not change from that of CK soils. The leaching process could not affect the chemical bonds in soils. 3.6. Plant growth and Cd uptake To evaluate the effects of different leaching treatments on plants growth and Cd uptake, pot experiments were conducted with water spinach sowed into the treated soils (Fig. 6). As shown in Fig. 6a, the height of water spinach tissues on day 15 was around 12 cm in acid control, spent bioleaching and two-step bioleaching, and was shorter than that of growing in CK soils (12.5–13 cm). After treatments, the soils had minor effect on the growth of water spinach. However, there were significant influences on Cd uptake in water spinach tissues (Fig. 6b). Cd content in plant tissues in two-step bioleaching was significantly (P b 0.05) higher than that of CK, acid control and spent bioleaching. The introduced bacteria with two-step bioleaching increased the Cd uptake of plants, and cumulative Cd content was 4.4fold higher than CK group. Unlike other soil genera tested by the previous studies (Lebeau et al., 2008; Sessitsch et al., 2013), the mixotrophic acidophiles were enriched from AMD and Cd-contaminated soils. The microbes in this consortium and generated metabolites were thought to be directly relevant to the Cd-containing compounds oxidation and might thereby mobilize Cd. Indeed, the results based on the two-step bioleaching experiment showed

Fig. 5. XRD (a) and FTIR curves (b) in soils using different leaching treatments.

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Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2018.11.221. References

Fig. 6. The growth of water spinach (a) and Cd uptake by water spinach tissues (b) on day 15 harvested from soils using different leaching treatments. Means with the same letters are not significantly different according to the LSD test (p N 0.05).

Rorippa globosa (Turcz. ex Fisch. & C.A. Mey.) Hayek replacing water spinach could be utilized to bioextract the remaining Cd. It is therefore believed to expect the two-step bioleaching to be effective for Cd removal and a promising technology towards a microbially-assisted phytoremediation of Cd-contaminated soils. 4. Conclusions This study showed that the two-step bioleaching using mixotrophic acidophiles removed an estimated 34% of total Cd and 87% of available Cd from Cd-contaminated soils. Meanwhile, the two-step bioleaching decreased Cd acid-soluble fraction and reducible fraction percentages, and increased oxidizable fraction and residual fraction percentages in soils. More significantly, two-step bioleaching increased Cd uptake in plant tissues of water spinach by 78% compared to CK group. The mixotrophic acidophiles had minor effects on soil structure and physicochemical properties, and only affected the proportional change of indigenous soil microbial population. Acknowledgments This work was funded by the National Natural Science Foundation of China (31570113 and 51504298), the Postdoctoral Research Funding Plan in Central South University for HW Liu (10300502042003), the Hunan Provincial Natural Science Foundation of China (2017JJ3160) and the Hunan Provincial Key Research and Development Plan (2018NK2112).

Alexandrino, M., Costa, R., Canário, A.V.M., Costa, M.C., 2014. Clostridia initiate heavy metal bioremoval in mixed sulfidogenic cultures. Environ. Sci. Technol. 48, 3378–3385. Bayat, B., Sari, B., 2010. Comparative evaluation of microbial and chemical leaching processes for heavy metal removal from dewatered metal plating sludge. J. Hazard. Mater. 174, 763–769. Beolchini, F., Dell Anno, A., De Propris, L., Ubaldini, S., Cerrone, F., Danovaro, R., 2009. Auto- and heterotrophic acidophilic bacteria enhance the bioremediation efficiency of sediments contaminated by heavy metals. Chemosphere 74, 1321–1326. Brune, K.D., Bayer, T.S., 2012. Engineering microbial consortia to enhance biomining and bioremediation. Front. Microbiol. 3, 203. Camargo, F.P., Prado, P., Tonello, P.S., Dos, A.S., Duarte, I., 2018. Bioleaching of toxic metals from sewage sludge by co-inoculation of Acidithiobacillus and the biosurfactantproducing yeast Meyerozyma guilliermondii. J. Environ. Manag. 211, 28–35. Chen, S.Y., Lin, J.G., 2001. Bioleaching of heavy metals from sediment: significance of pH. Chemosphere 44, 1093–1102. Cho, S.H., Han, J.H., Ko, H.Y., Kim, S.B., 2008. Streptacidiphilus anmyonensis sp. nov., Streptacidiphilus rugosus sp. nov. and Streptacidiphilus melanogenes sp. nov., acidophilic actinobacteria isolated from Pinus soils. Int. J. Syst. Evol. Microbiol. 58, 1566–1570. Deng, X.H., Chai, L.Y., Yang, Z.H., Tang, C.J., Wang, Y.Y., Shi, Y., 2013. Bioleaching mechanism of heavy metals in the mixture of contaminated soil and slag by using indigenous Penicillium chrysogenum strain F1. J. Hazard. Mater. 248-249, 107–114. Edgar, R.C., 2013. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat. Methods 10, 996–998. EPM, 1995. Environmental quality standard for soils (In Chinese). GB 15618–1995. Office of Technical Standards, Ministry of Environmental Protection, July 1995. Fonti, V., Dell'Anno, A., Beolchini, F., 2013. Influence of biogeochemical interactions on metal bioleaching performance in contaminated marine sediment. Water Res. 47, 5139–5152. Fonti, V., Dell'Anno, A., Beolchini, F., 2016. Does bioleaching represent a biotechnological strategy for remediation of contaminated sediments? Sci. Total Environ. 563-564, 302–319. Gleyzes, C., Tellier, S., Astruc, M., 2002. Fractionation studies of trace elements in contaminated soils and sediments: a review of sequential extraction procedures. Trends Anal. Chem. 21, 451–467. Hao, X.D., Liang, Y.L., Yin, H.Q., Liu, H.W., Zeng, W.M., Liu, X.D., 2017. Thin-layer heap bioleaching of copper flotation tailings containing high levels of fine grains and microbial community succession analysis. Int. J. Miner. Metall. Mater. 24, 360–368. Johnson, D.B., Hallberg, K.B., 2003. The microbiology of acidic mine waters. Res. Microbiol. 154, 466–473. Kaksonen, A.H., Särkijärvi, S., Puhakka, J.A., Peuraniemi, E., Junnikkala, S., Tuovinen, O.H., 2016. Chemical and bacterial leaching of metals from a smelter slag in acid solutions. Hydrometallurgy 159, 46–53. Kang, C.H., Kwon, Y.J., So, J.S., 2016. Bioremediation of heavy metals by using bacterial mixtures. Ecol. Eng. 89, 64–69. Karavaiko, G.I., Bogdanova, T.I., Tourova, T.P., Kondrat'Eva, T.F., Tsaplina, I.A., Egorova, M.A., Krasil'Nikova, E.N., Zakharchuk, L.M., 2005. Reclassification of ‘Sulfobacillus thermosulfidooxidans subsp. thermotolerans’ strain K1 as Alicyclobacillus tolerans sp. nov. and Sulfobacillus disulfidooxidans Dufresne et al. 1996 as Alicyclobacillus disulfidooxidans comb. nov., and emended description of the genus Alicyclobacillus. Int. J. Syst. Evol. Microbiol. 55, 941–947. Kay, C.M., Haanela, A., Johnson, D.B., 2014. Microorganisms in subterranean acidic waters within Europe's deepest metal mine. Res. Microbiol. 165, 705–712. Kelderman, P., Osman, A.A., 2007. Effect of redox potential on heavy metal binding forms in polluted canal sediments in Delft (The Netherlands). Water Res. 41, 4251–4261. Kong, Y., 2011. Btrim: a fast, lightweight adapter and quality trimming program for nextgeneration sequencing technologies. Genomics 98, 152–153. Kumar, R.N., Nagendran, R., 2007. Influence of initial pH on bioleaching of heavy metals from contaminated soil employing indigenous Acidithiobacillus thiooxidans. Chemosphere 66, 1775–1781. Latorre, M., Cortés, M.P., Travisany, D., Di Genova, A., Budinich, M., Reyes-Jara, A., Hödar, C., González, M., Parada, P., Bobadilla-Fazzini, R.A., Cambiazo, V., Maass, A., 2016. The bioleaching potential of a bacterial consortium. Bioresour. Technol. 218, 659–666. Lebeau, T., Braud, A., Jézéquel, K., 2008. Performance of bioaugmentation-assisted phytoextraction applied to metal contaminated soils: a review. Environ. Pollut. 153, 497–522. Li, X.M., Peng, W.H., Jia, Y.Y., Lu, L., Fan, W.H., 2016. Bioremediation of lead contaminated soil with Rhodobacter sphaeroides. Chemosphere 156, 228–235. Li, J.T., Liang, Z.W., Jia, P., Liu, J., Xu, Y.J., Chen, Y.J., Shu, H.Y., Kuang, J.L., Liao, B., Shu, W.S., 2017. Effects of a bacterial consortium from acid mine drainage on cadmium phytoextraction and indigenous soil microbial community. Plant Soil 415, 347–358. Ma, L.Y., Wang, X.J., Feng, X., Liang, Y.L., Xiao, Y.H., Hao, X.D., Yin, H.Q., Liu, H.W., Liu, X.D., 2017. Co-culture microorganisms with different initial proportions reveal the mechanism of chalcopyrite bioleaching coupling with microbial community succession. Bioresour. Technol. 223, 121–130. Mago, T., Salzberg, S.L., 2011. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27, 2957–2963.

X. Hao et al. / Science of the Total Environment 655 (2019) 347–355 Peng, J.F., Song, Y.H., Yuan, P., Cui, X.Y., Qiu, G.L., 2009. The remediation of heavy metals contaminated sediment. J. Hazard. Mater. 161, 633–640. Ren, W.X., Li, P.J., Geng, Y., Li, X.J., 2009. Biological leaching of heavy metals from a contaminated soil by Aspergillus niger. J. Hazard. Mater. 167, 164–169. Robinson, B.H., Anderson, C.W.N., Dickinson, N.M., 2015. Phytoextraction: Where's the action? J. Geochem. Explor. 151, 34–40. Sauvé, S., Hendershot, W., Allen, H.E., 2000. Critical review solid-solution partitioning of metals in contaminated soils: dependence on pH, total metal burden, and organic matter. Environ. Sci. Technol. 34, 1125–1131. Sessitsch, A., Kuffner, M., Kidd, P., Vangronsveld, J., Wenzel, W.W., Fallmann, K., Puschenreiter, M., 2013. The role of plant-associated bacteria in the mobilization and phytoextraction of trace elements in contaminated soils. Soil Biol. Biochem. 60, 182–194. Tang, H.L., Shuai, W.T., Wang, X.J., Liu, Y.S., 2017. Extraction of rare earth elements from a contaminated cropland soil using nitric acid, citric acid, and EDTA. Environ. Technol. 38, 1980–1986. Vakilchap, F., Mousavi, S.M., Shojaosadati, S.A., 2016. Role of Aspergillus niger in recovery enhancement of valuable metals from produced red mud in Bayer process. Bioresour. Technol. 218, 991–998. Wang, S.H., Zheng, Y., Yan, W.F., Chen, L.X., Mahadevan, G.D., Zhao, F., 2016. Enhanced bioleaching efficiency of metals from E-wastes driven by biochar. J. Hazard. Mater. 320, 393–400. WHO, 1992. Cadmium. Environmental Health Criteria. Vol. 134. WHO, Geneva.

355

Williams, S.T., Robinson, C.S., 1981. Role of streptomycetes in decomposition of chitin in acidic soils. Microbiology 127, 55–63. Wuana, R.A., Okieimen, F.E., Imborvungu, J.A., 2010. Removal of heavy metals from a contaminated soil using organic chelating acids. Int. J. Environ. Sci. Technol. 7, 485–496. Xin, B.P., Jiang, W.F., Aslam, H., Zhang, K., Liu, C.H., Wang, R.Q., Wang, Y.T., 2012. Bioleaching of zinc and manganese from spent Zn–Mn batteries and mechanism exploration. Bioresour. Technol. 106, 147–153. Yahya, A., Hallberg, K., Johnson, D., 2008. Iron and carbon metabolism by a mineraloxidizing Alicyclobacillus-like bacterium. Arch. Microbiol. 189, 305–312. Yang, Z.H., Zhang, Z., Chai, L.Y., Wang, Y., Liu, Y., Xiao, R.Y., 2016. Bioleaching remediation of heavy metal-contaminated soils using Burkholderia sp. Z-90. J. Hazard. Mater. 301, 145–152. Zeng, X.F., Twardowska, I., Wei, S.H., Sun, L.N., Wang, J., Zhu, J.Y., Cai, J.C., 2015. Removal of trace metals and improvement of dredged sediment dewaterability by bioleaching combined with Fenton-like reaction. J. Hazard. Mater. 288, 51–59. Zhang, H.J., Gao, Y.T., Xiong, H.B., 2017. Removal of heavy metals from polluted soil using the citric acid fermentation broth: a promising washing agent. Environ. Sci. Pollut. Res. 24, 9506–9514. Ziegler, S., Waidner, B., Itoh, T., Schumann, P., Spring, S., Gescher, J., 2013. Metallibacterium scheffleri gen. nov., sp. nov., an alkalinizing gammaproteobacterium isolated from an acidic biofilm. Int. J. Syst. Evol. Microbiol. 63, 1499–1504.