Soil Vanadium(V)-Reducing Related Bacteria Drive Community Response to Vanadium Pollution from a Smelting Plant Over Multiple Gradients

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Soil vanadium(V)-reducing related bacteria drive community response to vanadium pollution from a smelting plant over multiple gradients

Wang, Song; Zhang, Baogang; Li, Tingting; Li, Zongyan; Fu, Jie

Published in: Environment International

Link to article, DOI: 10.1016/j.envint.2020.105630

Publication date: 2020

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Citation (APA): Wang, S., Zhang, B., Li, T., Li, Z., & Fu, J. (2020). Soil vanadium(V)-reducing related bacteria drive community response to vanadium pollution from a smelting plant over multiple gradients. Environment International, 138, [105630]. https://doi.org/10.1016/j.envint.2020.105630

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Environment International 138 (2020) 105630

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Environment International

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Soil vanadium(V)-reducing related bacteria drive community response to vanadium pollution from a smelting plant over multiple gradients T ⁎ ⁎ Song Wanga,d, Baogang Zhanga, , Tingting Lia, Zongyan Lia, Jie Fub,c, a School of Water Resources and Environment, MOE Key Laboratory of Groundwater Circulation and Environmental Evolution, China University of Geosciences (Beijing), Beijing 100083, PR China b Department of Environmental Science and Engineering, Fudan University, Shanghai 200438, PR China c School of Environmental Science and Engineering, Huazhong University of Science and Technology, Wuhan 430074, PR China d Department of Environmental Engineering, Technical University of Denmark, DK-2800 Lyngby, Denmark

ARTICLE INFO ABSTRACT

Handling Editor: Zhen (Jason) He The mining and smelting of navajoite has resulted in a serious vanadium pollution in regional geological en- fi fl Keywords: vironments and signi cant in uence on soil microorganisms. However, the core microbiome responsible for Vanadium pollution adjusting community response to vanadium pollution and the driving pattern have been kept unclear. In this Soil microorganisms study, a suite of surface and profile soil samples over multiple gradients were collected in four directions and Vanadium(V)-reducing related bacteria distances of 10–2000 m from a vanadium smelting plant in Panzhihua, China. The indigenous microbial com- Community response munities and vanadium(V)-reducing related bacteria (VRB) were profiled by 16S rRNA gene high-throughput Core microbiome sequencing technique. Five VRB were detected in the original collected soil samples including Bacillus, Geobacter, Clostridium, Pseudomonas and Comamonadaceae based on high-throughput sequencing data analysis, and their abundances were significantly related with the content of vanadium. Low vanadium concentration promoted the growth of VRB, while high vanadium concentration would inhibit VRB multiplication. The Gaussian equation could be used to quantitatively describe the nonlinear relationship between VRB and vanadium. Network analysis demonstrated that the microbial communities were significantly influenced by VRB assemblage, and 1.32–52.77% of microbes in the community showed a close association with VRB. A laboratory incubation experiment also confirmed the core role of VRB to drive community response to vanadium pressure.

1. Introduction Rinklebe et al., 2019). Vanadium has adversely effects on phosphate metabolism (Zhang et al., 2012, 2014). High vanadium ingestion can Vanadium is the 20th most abundant element in the Earth's crust, result in serious diseases, including renal damage and potentially pul- and is beneficial or essential for living organisms due to its unique roles monary tumors (Heinemann et al., 2000; Chen and Liu, 2017). The in biological structures and functions (Crans et al., 2004; Zhang et al., seeding leaves of soybean would become yellow and withered and there 2015a). In modern industry, vanadium is mainly applied to steel to are significantly decreased yields of shoot and roots when the soil va- ameliorate the strength and corrosion resistance, and it is also em- nadium content is high (Wang and Liu,1999). The background value of − − − ployed in many other industries including pigments, catalysts, and different metals in China was 61 mg kg 1, 0.38 g kg 1, 3.0 g kg 1, − − − − pharmaceuticals (Liu et al., 2017; Schlesinger et al., 2017). Anthro- 6.6 g kg 1,23mgkg 1,74mgkg 1 and 82 mg kg 1 for Cr, Ti, Fe, Al, pogenic activities, such as the mining and smelting of navajoite, widely Cu, Zn and V respectively (Wei et al., 1991). The concentration of va- using of vanadium products, and fossil fuel combustion, have resulted nadium in Chinese environmental quality standard for agriculture soil − in substantial vanadium entering into regional geological environments is 130 mg kg 1 (GB15618-2009) (CMEP, 2009). (Imtiaz et al., 2015; Xiao et al., 2017). Severe vanadium contamination Microorganisms play an important role in Earth's biogeochemical in soils has been reported by many researchers (Cao et al., 2017; Imtiaz cycle, which can not only regulate ecosystem processes but also influ- et al., 2015; Larsson et al., 2015). Excessive concentration of vanadium ence biogeochemical dynamics in geological environments (Xu et al., is harmful to both plants and animals and sometimes it even threatens 2017; Das et al., 2019). Microbes are sensitive to environmental pol- human health (Hao et al., 2015; Mandiwana and Panichev, 2010; lution and especially heavy metal pollution, and numerous studies have

⁎ Corresponding authors at: China University of Geosciences (Beijing), Beijing 100083, PR China (B. Zhang); Fudan University, Shanghai 200438, PR China (J. Fu). E-mail addresses: [email protected] (B. Zhang), [email protected] (J. Fu). https://doi.org/10.1016/j.envint.2020.105630 Received 24 October 2019; Received in revised form 25 February 2020; Accepted 1 March 2020 0160-4120/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). S. Wang, et al. Environment International 138 (2020) 105630 demonstrated that heavy metal pollution may cause drastic changes in (South Group) at a distance of 10 m, 50 m, 500 m, 800 m, 1000 m and microbial community composition and activity, resulting in a decrease 1800 m (S1, S2, S3, S4, S5 and S6), west direction (West Group) at a of microbial diversity and enrichment of tolerant species via the process distance of 10 m, 50 m, 200 m and 800 m (W1, W2, W3 and W4), north of environmental filtering (Ma et al., 2019; Turpeinen et al., 2004; direction (North Group) at a distance of 10 m, 100 m, 200 m, 500 m, Wang et al., 2018; Wu et al., 2019). On the other hand, microbes may 800 m, 1000 m, 1500 m and 2000 m (N1, N2, N3, N4, N5, N6, N7 and be actively involved in the immobilization, degradation and transfor- N8). Profile samples were collected from five sites: one site was in the mation of the toxic metals (Fierros-Romero et al., 2017; Gong et al., south direction at a distance of 800 m (VS), one site was in the east at a 2016; Luo et al., 2014), and play a crucial role in the in situ remediation distance of 700 m (VE), and the other three sites were in the north at a of heavy metal pollution (Hansen et al., 2017). For example, the highly distance of 200 m (VN1), 1000 m (VN2) and 2000 m (VN3), respec- toxic vanadium(V) (V(V)) could be reduced to less toxic vanadium(IV) tively. Profile samples at VS (VS1-VS6), VN1 (VN1_1-VN1_6), VN2 (V(IV)) by many bacterial species in vanadium polluted environment (VN2_1-VN2_6) and VN3 (VN3_1-VN3_6) were collected at vertical (hereafter referred to as V(V)-reducing related bacteria, VRB) (Hao depths of 0–10, 20–30, 40–50, 60–70, 80–90 and 100–110 cm, and at et al., 2015; Qiu et al., 2017; Rivas-Castillo et al., 2017; Yelton et al., VE site, samples were collected at depths of 0–10, 20–30, 40–50, 60–70, 2013; Zhang et al., 2015a). Similar as sulfate-reducing bacteria, which 80–90, 100–110, 120–130, 140–150 and 160–170 cm (VE1-VE9). All are microorganisms with the function of reducing sulfate to sulfide, collected soil samples were divided into two parts depending on their VRB is a kind of community, which possess a common vanadium re- usages. One part for physicochemical analyses and laboratory incuba- duction function and might play a vital role in vanadium reducing tion experiment was kept in polyethylene bags and stored at 4 °C. The process (Schoeffler et al., 2019). other part for microbial analysis was frozen in liquid nitrogen in the In the last several years, the release of vanadium and the resulting sampling site and stored in −80 °C freezer in the laboratory until use. pollution to geological environments have attracted increasing atten- tion (Gardner et al., 2017; Wright et al., 2014; Yang et al., 2017). A 2.2. Physicochemical analyses considerable amount of researches have been conducted to investigate the environmental pollution of vanadium, including its concentration Soil samples were air-dried at room temperature and sieved through distribution, speciation analysis, mobility, bioaccumulation as well as 2 mm mesh for subsequent physicochemical analyses. pH was de- microbial response (Cao et al., 2017; Teng et al., 2011; Xiao et al., termined by a HI 3221 pH meter (Hanna Instruments Inc., USA). 2015). Our previous study observed a significant influence of vanadium Oxidation-reduction potential (Eh) was analyzed using an Eh meter in shaping microbial community composition, structure and diversity in with a platinum electrode installed at 15 cm depth in each site (Honma surface soil of Panzhihua mining and smelting area, China (Cao et al., et al., 2016). Total nitrogen (TN), organic matter (OM), and available 2017). However, the core microbiome responsible for mediating com- phosphorus (AP) were measured based on reported methods (Cao et al., munity response to vanadium pollution have not been identified and 2017). Total concentrations of vanadium and other metals in soils were potential core role of VRB assemblage in the shift of community under measured by inductively coupled plasma-optical emission spectrometer vanadium oxidation stress has not been well explained. Moreover, (Prodigy XP, Leeman Labs, Inc., USA) after microwave digestion (MARS previous studies on the modification of community by vanadium are 6, CEM Corp., USA) with a mixture of concentrated HNO3, HClO4 and mostly qualitative descriptions, while constructing the relatively HF (1:1:2, v/v/v) (Liu et al., 2015). Precision and accuracy of heavy quantitative relation between vanadium stress and core microbiome metal measurement were verified using standard reference materials response is more meaningful for predicting the effect of vanadium from the National Research Center for Geoanalysis of China. Accepted pollution on microbial community and assessing the impact on the recoveries ranged from 90% to 110%. Each sample was analyzed in ecosystem. triplicate. The relative deviation of the triplicate samples was < 10% In the present study, a comprehensive field survey was conducted to in all batch measurements. explore the profile of microbial community and VRB assemblage in soils around a vanadium smelting plant over horizontal and vertical gra- 2.3. Incubation experiment dients by Illumina high-throughput sequencing of 16S rRNA genes. Combing with the vanadium concentration, the influences of vanadium One low vanadium contaminated soil sample (E6) was selected to pollution on the microbial community and VRB assemblage were conduct the incubation experiment. Serum bottles of 50 mL equipped quantitatively investigated. Furthermore, a laboratory incubation ex- with rubber plugs were used for cultivation. Each bottle was filled with periment was conducted to simulate the community succession and 2 g soils and the residual space was filled with simulated groundwater, validate the responses of community and VRB assemblage to vanadium which contained the following components (per liter): CaCl2 (0.2464 g); observed in the field survey. The objectives of this study are to quan- MgCl2·6H2O (1.0572 g); NH4Cl (0.1557 g); KH2PO4 (0.0299 g); KCl titatively establish the link between VRB assemblage and vanadium (0.0283 g); NaCl (0.4459 g) and NaHCO3 (0.8082 g) (Cao et al., 2017). − oxidation stress and elucidate the core role of VRB to drive the com- At the start of incubation, two gradients of vanadate (1 mg L 1 and − munity response to vanadium pollution. 10 mg L 1 as V(V)) as electron acceptor with corresponding carbon − − source of glucose (10 mg L 1 and 100 mg L 1 as COD) as electron 2. Materials and methods donor were added to soils. During the incubation, the bottles were re- filled with new simulated groundwater every three days, and this mi- 2.1. Site description and sample collection crobial cultivation lasted for three months to achieve a stable evolution. All experiments were carried out in duplicate and at room temperature The studied vanadium smelting plant is located in Panzhihua City (22 ± 2 °C). Incubated soil samples were collected for microbial (South Sichuan Province, China), which is the most famous base of community analysis when incubating for 30 d, 60 d and 90 d, respec- vanadium-bearing titanomagnetite in China. Extensive smelting activ- tively. ities have resulted in severe vanadium contamination in around soils (Cao et al., 2017). In April 2017, representative surface and profile soil 2.4. Microbial community analysis samples over multiple gradients were collected in different directions and various distances from the smelting plant (Fig. 1). A total of 24 All triplicate mixed soil samples and incubated soil samples were surface soil samples were collected in triplicate at a depth of 0–10 cm applied to microbial community analysis by Illumina MiSeq high- from the east direction (East Group) at a distance of 10 m, 50 m, 200 m, throughput sequencing of 16S rRNA gene technique. The genomic DNA 500 m, 800 m and 1000 m (E1, E2, E3, E4, E5 and E6), south direction in 0.5 g soil sample was extracted using a FastDNA Spin Kit for Soil

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Fig. 1. The sampling locations around the vanadium smelting plant in Panzhihua, China.

(Oiagen, CA, USA) according to the manufacturer's protocol. Bacterial 2.5. Data processing and statistical analyses 16S rRNA genes were amplified with PCR primers 338F (ACTCCTAC GGGAGGCAGCAG) and 806R (GGACTACHVGGGTWTCTAAT) tar- The potential ecological risk index (EI) was introduce to assess the geting the V4-V5 hyper variable regions (Liu et al., 2017). Each PCR degree of vanadium pollution in soils, which can be calculated using the reaction was performed in 20 μL reaction mixture, which consisted of following equation (Hakanson, 1980): 4 μL of 5 × FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer TRF× C (5 μM), 0.4 μL of FastPfu polymerase, and 10 ng of template DNA, using EI = obs C (1) the following thermal cycling conditions: an initial denaturation at 0 95 °C for 3 min, 25 cycles of 95 °C for 30 s, 57 °C for 30 s and 72 °C for where TRF (=2) is a given toxic factor response to vanadium (Pan 45 s, and a final extension at 72 °C for 10 min. The 16S rRNA gene −1 et al., 2016), Cobs (mg kg ) is the measured concentration of vana- amplicon sequencing was conducted on the Illumina MiSeq platform by −1 dium in the soil samples and C0 (mg kg ) is the corresponding back- Majorbio (Shanghai, China). Sequencing data including all field and ground value of China (CNEMC, 1990). This ecological risk assessment incubated samples have been submitted to the NCBI Sequence Read method has been widely used in heavy metal polluted soil (Pan et al., Archive (accession No. SRP188735, SRP224622). 2016; Yang et al., 2017; Sun et al., 2010). Based on obtained EI value, The obtained sequence data were processed and analyzed using the ecological risk of vanadium can be categorized as low (< 40), FLASH and Trimmomatic (Sun et al., 2017; Wang et al., 2016). The low moderate (40–80), considerable (80–160), high (160–320) and very quality and chimeric sequences were filtered to get high quality se- high (> 320) levels (Hakanson, 1980; Pan et al., 2016). quences, which were then clustered into operational taxonomic units Multivariate statistical methods were used to account for the colli- (OTUs) by UPARSE (version 7.0 http://drive5.com/uparse/) at the 97% nearity between VRB assemblage and vanadium, other possible physi- similarity level. Taxonomic classification of the OTUs was determined cochemical properties or gradient factor. Redundancy analysis (RDA) using RDP Classifier (http://rdp.cme.msu.edu/) against the silva was applied using the R software package (vegan 2.4.4) to present the (SSU115) 16S rRNA database using a minimum confidence of 70% relations between VRB assemblage and the significant variables and (Amato et al., 2013). The rarefaction curve was constructed by random explain the effect of vanadium gradient on the VRB assemblage and the sampling for all the sequences (Zhang et al., 2019a). Relative abun- related bacteria. To prevent disproportionate effects of highly abundant dance (%) of individual taxa within each community was estimated by taxa, all the species abundance values were standardized using corre- comparing the number of sequences assigned to a specific taxon versus lation coefficients, and the inertial correlations were obtained. The the number of total sequences obtained for that sample. Alpha diversity hierarchical cluster analysis based on the relative abundance of top 30 analysis (i.e., Chao1/Ace richness estimator, and Simpson/Shannon abundant genera was conducted using average linkage between groups diversity index) was conducted using the Mothur software (Zhang et al., and Pearson correlation as measure interval by using Origin 9.0. 2020). Network analysis was carried out to illustrate the significant

3 S. Wang, et al. Environment International 138 (2020) 105630 correlations between core microbiome and soil microbial community, samples are shown in Fig. 2. The average vanadium concentration in all − which was performed using software Cytoscape with input correlations collected surface soil samples was 1616.91 ± 12.35 mg kg 1, which calculated in the software local similarity analysis (LSA) using default was much higher than the background level of vanadium in China soils − settings (Sun et al., 2013). All the correlations between any two genera (82 mg kg 1)(Chen et al., 1991). This indicated that the soils around in this microbiome were calculated and then we chose the genera to this area had been severely contaminated by vanadium. Remarkably, a structure network based on their significant relations to VRB decreasing tendency of vanadium with the distance away from the (|r| > 0.8). smelting plant had been observed (Fig. 2), which demonstrated the vanadium pollution in soils was mostly resulted from the pathway of atmospheric deposition of vanadium released from the vanadium 3. Results and discussion smelter. Moreover, the vanadium concentrations in the soil profiles decreased over vertical gradient (Fig. 2). Vanadium concentration 3.1. Physiochemical properties and vanadium contents changes in different orientation were small, among which the soils in South Group were polluted more heavily than other directions rela- The physiochemical properties of soil samples over horizontal and tively. There was more vanadium related industry in south direction vertical gradients are summarized in Table S1. Overall, the surface soils comparing the mountain in north group, so soils in the south were in this area were alkaline with an average pH value of 8.18 ± 0.32, contaminated more seriously. Even when soil depth reached to 170 cm, − which is consistent with the previous observation by Cao et al. (2017). the vanadium concentration (109.65 ± 3.58 mg kg 1) still exceeded The average content of OM in the surface soils was the background value of soil vanadium in Sichuan Province − − 22.18 ± 0.53 g kg 1, which is lower than the average value for soils in (96 mg kg 1)(CNEMC, 1994), indicating the vertical migration of − China (43.75 g kg 1)(Wu and Cai, 2006). For the profile samples, there vanadium contamination to the deeper soil. The ecological risk index was a decreasing tendency of OM along the soil depth, which was also indicated the vanadium in the soils near the smelting plant would in- reported by Liang et al. (2019). The average contents of TN and AP in duce considerable or moderate ecological risk. − − the surface soils were 0.66 ± 0.08 g kg 1 and 12.01 ± 0.54 g kg 1, − respectively. The average contents of Cr (510.81 ± 0.21 mg kg 1), Ti − − (15.05 ± 0.57 g kg 1), Fe (85.83 ± 0.13 g kg 1), Al 3.2. Microbial community composition and diversity − − (26.71 ± 1.58 g kg 1), Cu (63.58 ± 0.11 mg kg 1) and Zn − (228.36 ± 0.61 mg kg 1) in the surface soils were 3–40 times higher The high-throughput sequencing generated total 763,340 quality than the background values of China soils (Wei et al., 1991), suggesting sequences across 33 soil samples including all the surface samples and a severe heavy metal contamination. An evident decreasing trend of the VE profile samples. A total of 7569 OTUs were identified, with a concentrations of these heavy metals along the distance to vanadium range of 1178 to 3341 for individual samples. The number of OTUs, smelter and soil depth was also observed. Chao1/Ace richness estimator and Simpson/Shannon diversity index Contents of vanadium as well as the ecological risk levels in soil are shown in Table S2. Rarefaction curves of microbial communities in

Fig. 2. Vanadium distributions and potential ecological risk in surface and profile soils in Panzhihua, China. Different size circles show different vanadium concentrations, and various colors stand for different ecological risks.

4 S. Wang, et al. Environment International 138 (2020) 105630 different groups of soils are shown in Fig. S1. High microbial diversity (2.64–12.90%), Unclassified-f-Anaerolineaceae (0.58–10.03%), Un- and richness was found in the samples with a moderate vanadium level classified-o-JG30-KF-CM45 (0.61–7.32%), Sphingomonas (0.65–7.23%) (e.g., E2, S2, W3 and N2), whereas a low microbial diversity was found and RB41 (0.19–8.14%), while the dominant genera in profile soils in the samples with either very high vanadium content or low vana- included Bacillus (4.55–37.13%), Unclassified-o-Gaiellales dium content. It seems that appropriate vanadium level was beneficial (1.39–16.67%), Gaiella (1.26–7.21%), Unclassified-c-Actinobacteria for enhancement on the microbial diversity and richness, but excessive (0.08–8.03%) and Unclassified-c-KD4-96 (1.14–5.88%). vanadium level would definitely pose an adverse impact. fi The identi ed OTUs of bacterial microbiome across all soil samples 3.3. VRB assemblage and influence of vanadium pollution can be assigned to 24 phyla, 56 classes, 117 orders, 230 families, and – 421 genera. The dominant phyla were Actinobacteria (16.27 50.57%), Many bacteria, some archaea and fungi have been reported to be – fl – Proteobacteria (4.29 41.85%), Chloro exi (7.9 33.38%), Acidobacteria able to reduce vanadium (Table S3). In the present study, five genera – – (0.39 23.43%) and Firmicutes (0.19 49.76%) (Fig. S2). Cao et al. from phyla of Proteobacteria and Firmicutes, which had been reported (2017) has reported that Actinobacteria, Proteobacteria, Acidobacteria having the ability to reduce vanadium and possessing the potential and Firmicutes were widely found in the soils around a vanadium vanadium reduction activity in this local environment, were detected in fl smelting plant. In addition, Chloro exi was also detected in a microbial the collected soil samples, including Bacillus, Clostridium, fuel cell with V(V) as the electron acceptor (Zhang et al., 2015a). Re- Comamonadaceae, Geobacter, and Pseudomonas. There must be more markably, the richness of individual phylum changed a lot along the unidenti fied bacteria having the similar vanadium reducing functions ff distance to the smelting plant and di erent directons, which may be and features belong to VRB, which have relationship to vanadium re- ff a ected by the disparity of vanadium concentration or other physico- ducing process. However, due to limitation of microbial culture, their chemical properties. With the increase of depth, Actinobacteria was vanadium reducing functions have not been explored yet. In this study, fi fi observed to signi cantly increase in the pro le soils, while Firmicutes in order to explore the distribution VRB assemblage and its interaction showed a declining trend. with the environment, the five detected known VRB genera were con- The dominant genera in all soil samples were Bacillus sidered as the surrogate of VRB assemblage. When VRB was discussed fi (6.61 ± 9.82%), Unclassi ed-c-Acidobacteria (5.58 ± 3.32%), as assemblage, VRB abundance was calculated by summing the relative fi fi Unclassi ed-f-Anaerolineaceae (3.73 ± 2.30%), Unclassi ed-o- abundance of these five genera. This kind of assemblage abundance has fi Gaiellales (3.68 ± 4.65%) and Unclassi ed-o-JG30-KF-CM45 also been used in analyzing sulfate-reducing bacteria (Jia et al., 2018). (3.41 ± 1.61%) (Fig. 3). The microbial compositions of surface soils The distribution of these VRB genera in different spatial gradients are ff fi were di erent from those of pro le soils. Hierarchical cluster analysis shown in Fig. 4. Notably, Bacillus occupied the overwhelming majority based on the relative abundances of the top 30 genera showed that 33 of the VRB assemblage, which suggested this genus might play a critical fi samples could be organized into two groups: the rst group contained role in V(V)-reducing process. Rivas-Castillo et al. (2017) have also fi surface soils and the other contained pro le samples (Fig. 3). The reported that some strains of Bacillus megaterium present not only high fi dominant genera in surface soils included Unclassi ed-c-Acidobacteria vanadium resistance, but also excellent removal capability of

Fig. 3. Hierarchical cluster analysis of microbial communities in all soil samples based on the top 30 abudant genera. The relative abundance of genera was indicated by color intensity.

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Fig. 4. Relative abundance of VRB assemblage (Bacillus, Geobacter, Clostridium, Pseudomonas and Comamonadaceae) across all analyzed soils: (a) East surface soil, (b) south surface soil, (c) west surface soil, (d) north surface soils, (e) profile soil and (f) cultivated soil. vanadium. important explanatory soil properties for VRB assembly. To explore the Considering the severe vanadium contamination in local area, va- relations between VRB assemblage and environmental factors, RDA was nadium is undoubtedly a main factor influencing the soil microbial conducted by taking vanadium as the main factor and Eh, TN, AP, Al community. Besides, the Monte Carlo permutation test (n = 499) and and Cu as the co-factors. The ordination diagram of RDA illustrated correlation analysis (Table S4) identified Eh, TN, AP, Al and Cu as the genus Bacillus had extremely similar distribution with total VRB

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Fig. 5. Redundancy analysis of the effect of environmental factors on VRB assemblage. The red arrow is the main factor, the grey arrows are the co-factors, Fig. 6. The relationships between the log value of vanadium content and the and the blue arrows are VRB. (For interpretation of the references to color in abundance of VRB assemblage in soils. The red line is the fitting curve using this figure legend, the reader is referred to the web version of this article.) Gaussian equation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (Fig. 5), which was resulted from the dominating of Bacillus in VRB assemblage. The distribution of Clostridium also resembled with that of abundance of VRB assemblage and the richness and diversity of the Bacillus or total VRB, while the arrows of Comamonadaceae and Pseu- whole community. Ace/Chao1 richness estimator had slightly negative domonas lay on the opposite direction of Bacillus and Clostridium.It effect with VRB (Fig. S3a and b) (Ace: r = −0.458, p < 0.05; Chao1: implied that the different bacteria of VRB would compete with each r = −0.457, p < 0.05), which meant the richness of microbial com- other for the limited living space and resources to support growth (Xu munity would decrease with the increase of VRB abundance. Notice- et al., 2017). For the relation between VRB and vanadium concentra- ably, Shannon/Simpson diversity index significantly correlated with tion, a negative and weak correlation was observed (r = −0.307, VRB abundance (Fig. S3c and d) (Ace: r = −0.772, p < 0.05; Chao1: p = 0.082, n = 33), indicating there was no linear relation between r = 0.915, p < 0.05), indicating the enhancement of VRB assemblage vanadium and VRB. Furtherly, it was found the sample distribution would remarkably impact the diversity and evenness of the whole mi- presented a unimodal pattern along with the vanadium (Fig. 5), which crobial community. might be described by the Gaussian distribution model (discussed To furtherly explore how VRB drove the community shift, a network below). Other co-factors generally exhibited linear relations to VRB analysis was conducted (Fig. 7). All the correlations between any two (Fig. 5). Eh, TN, AP and Al showed significantly positive correlations genera in this microbiome were calculated and then we chose the (r = 0.445–0.623, p < 0.01, n = 33), while Cu had a significantly genera to structure network based on their significant relations to VRB negative correlation (r = −0.541, p < 0.01, n = 33), resulting in (|r| > 0.8) (Banerjee et al., 2016). This network consisted of 49 genera relatively longer lengths of their arrows. (first level interaction) that had direct relationship with VRB and 29 As discussed above, vanadium was a main factor influencing the soil genera (second level interaction) that were closely associated with the microbial community, and the relation between vanadium and VRB was first level interaction genera and could be influenced indirectly by VRB. nonlinear. To empirically and mathematically model the relation, we In the first level, many genera were identified to be metal-resistant or constructed the function of VRB abundance versus log concentration of metal-reducing genera, such as Acidobacterium (Stroud et al., 2014), vanadium, and found the relation could be well described by Gaussian Alcanivorax (Baltar et al., 2018), Rhodoferax (Ma et al., 2015), Thauera equation (R2 = 0.653, p < 0.001) as follows (Fig. 6): (Zhang et al., 2019b), Rhodanobacter (Hemme et al., 2016), Fictibacillus

2 (Zheng et al., 2017), Janibacter (Vetrovsky and Baldrian, 2015), Leu- ye=+3.43 43.41 −−61.73(x 2.61) (2) cobacter (Sturm et al., 2018), Dyella (McGee et al., 2018), and Granu- − Here, x (mg kg 1) is the vanadium concentration in soils and y (%) is licella (Falagan et al., 2017). These genera positively correlated to VRB, abundance of VRB in the microbial community. The abundance of VRB revealing the existence of co-occurring patterns between VRB and the increased with the vanadium content when the vanadium concentration first level interaction genera. In the second level, the genera could was at a low level. However, when the vanadium was aggrandized connect with VRB indirectly through the first level interaction genera. constantly, the richness of VRB significantly decreased. Thus, our re- Totally, the bacteria of the first and second levels as well as VRB ac- sults indicated that low level of vanadium could promote the growth of counted for 1.32–52.77% of the whole community. Therefore, VRB VRB and the high content of vanadium would inhibit the multiplication might drive the change of such a large group of bacteria under the of VRB. vanadium pressure, resulting in shift at the community level. Moreover, the network analysis was only based on the five genera of VRB surro- 3.4. Role of VRB assemblage to drive the community shift gate, more unidentified VRB were likely to exist in community, and the influence coverage of VRB assemblage might be even larger. According to ecological theory, the microbial community is shaped by deterministic factors such as species competition and niche differ- 3.5. Validation from the laboratory incubation test entiation (e.g., environmental filtering) (Wu et al., 2019). The present study has demonstrated vanadium pollution was a critical factor in- The laboratory incubation test indicated the exposure of vanadium fluencing the soil microbial community, and the VRB was the re- could significantly change the soil microbial community. Principal co- sponsive microbial assemblage. To evaluate the contribution of VRB ordinates analysis (PCoA) on the community structures of the original change on the shift of soil microbial communities, we forward selected and cultivated soil samples shown that bacteria converged to similar regression model to analyze the relation between the relative structure at lower vanadium loading, while the structure of microbial

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Fig. 7. Network analysis of VRB assemblage and closely associated bacteria in the microbial communities in soils. The first level interaction is the genera influenced by VRB directly, containing 49 genera. The second level, containing 29 genera, could be influenced by the first level microbiome, and influenced by VRB indirectly. community held distinct alteration during the cultivation process at a incubation environment. But as the cultivation proceeded, VRB seemed higher vanadium level (Fig. S4). This implied the soil microorganisms to accommodate the high vanadium content and incubation environ- could adapt to the environment containing low level of vanadium and ment, and the total abundance dramatically increased to 47.20% evolve a stable structure; however, the high concentration of vanadium (E6_3M10) 90 days later. The species of VRB had the intrinsic capacity would dramatically impact the balance of microbial community, which to remove vanadium by reducing the highly toxic V(V) to less toxic V was difficult to restore stability. The rarefaction curves showed the (IV) and possessed a wide range of resistance mechanisms to metals, diversity of soil microbial communities was significantly reduced after which could counteract the toxic effect of vanadium under the oxida- exposing vanadate and in a dose-dependent pattern (Fig. S5), which tive stress (Ding et al., 2014; Rivas-Castillo et al., 2017). Moreover, in was also reflected by the alpha diversity estimator/index (Table S5). some cases, vanadium could be involved in nitrogen fixation as a − Higher vanadate concentration (10 mg L 1) induced a greater impact growth factor and promote the growth of VRB, e.g., Bacillus (Hao et al., on the community diversity. This is consistent with the observation of 2018). Vanadium(V) reduction is also an energy-conserving process, field survey (Figs. 2 and S1; Table S2). which could result in proton translocation and promote the growth of Furthermore, the evolutions of various phylum and VRB were in- microorganisms in vanadium reduction process (Carpentier et al., vestigated during the incubation process (Fig. S2f, Fig. 4f). The domi- 2005). In addition, we calculated the bacteria closely related to VRB nant phylum Acidobacteria decreased from 10.49% in E6 to 3.78% in (|r| > 0.8), and found the abundance of VRB and the associated bac- E6_3M1 and to 2.83% in E6_3M10. Firmicutes had an increase tendency teria increased from 10.26% in E6 to 15.13% in E6_3M1 and 47.66% in in the process of incubation (Fig. S2f). Like the observation of field E6_3M10 during the cultivation process, suggesting a predominant survey, Bacillus spp. was still predominant in VRB during the whole drive of community change caused by VRB under vanadium pressure. In cultivation process. Abundance of total VRB increased consecutively general, the laboratory incubation experiment validated the conclusion from 5.88% (E6) to 14.81% (E6_3M1) during the 90-d cultivation at a of field survey that the abundance of VRB increased at low vanadium − lower vanadium concentration (1 mg L 1). However, at the higher concentration but decreased at high vanadium level; VRB played the − vanadium concentration (10 mg L 1), the abundance of total VRB core role to drive community response to vanadium pressure. sharply decreased from 5.88% (E6) to 2.45% (E6_1M10) after culti- vating for 30 days, which is probably due to the strong toxicity of high level of vanadium to microbes (Zhang et al., 2015b) and the new

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4. Implications and limitations Shewanella oneidensis MR-1 using vanadate as the sole electron acceptor. J. Bacteriol. 187, 3293–3301. Chen, G.D., Liu, H.Z., 2017. Understanding reduction kinetics of aqueous vanadium (V) This study showed that the smelting plant caused heavy vanadium and transformation products using rotating ring-disk electrode. Environ. Sci. Technol. pollution in local area, which posed a serious impact on soil micro- 51, 11643–11651. organisms over horizontal and vertical gradients. Combing field survey Chen, J.S., Wei, F.S., Zheng, C.J., Wu, Y.Y., Adriano, D.C., 1991. Background concentrations of elements in soils of China. Water Air Soil Pollut. 57–58, 699–712. and laboratory incubation experiment, we found the VRB assemblage CMEP (Ministry of Environmental Protection of the People’s Republic of China), 2009. played the core role to drive community response to vanadium pollu- Environmental Quality Standard for Soils (GB 15618-2009) (in Chinese). tion. 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