Journal of Environmental Radioactivity 139 (2015) 85e90

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Radionuclides distribution, properties, and microbial diversity of soils in uranium mill tailings from southeastern China Xun Yan a, b, Xuegang Luo c, * a

College of Life Science, Northeast Forestry University, Harbin, Heilongjiang 150040, China College of Chemistry and Chemical Engineering, Qiqihar University Qiqihar, Heilongjiang 161006, China c Southwest University of Science and Technology Mianyang, Sichuan 621010, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 January 2014 Received in revised form 5 September 2014 Accepted 30 September 2014 Available online

Objective: To collect the radioactive contamination data for environmental rehabilitation in uranium mill tailings in southeastern China. Method: The sample areas were divided into high, moderate and low concentration areas, according to the uranium concentration. For every area, 3 soil samples were collected at 0e15 cm, 15e30 cm and 30 e45 cm depth respectively, with 5 repetitions for each. Total 45 (3  5  3) soil samples were collected. Physicochemical properties and enzyme activities of soils were determined as described by references. The concentrations of the radionuclides 238U, 232Th, 226Ra and 40K in soils were determined by using HPGe gamma-ray spectrometer. Soil microbial diversity was analyzed via denaturing gradient gel electrophoresis (DGGE). Results: Soil samples were all acidic. Physicochemical properties, like pH, content of total/available N, P and K, as well as enzyme activities were all increased along with decreased uranium concentration. The 232 Th concentration was increased with the decreased uranium concentration and was not influenced by the depth of sample sites. However, uranium concentration and depth of sample showed no significant influence on the concentrations of 226Ra and 40K. The concentration of 232Th was significantly correlated with that of 226Ra and 40K, while the concentrations of 226Ra and 40K were significantly correlated. However, Pearson correlation coefficients between 238U and other radionuclides were not significant. The microbial population in different concentration areas was different with four domain strains in low area, and two for both moderate and high areas. Furthermore, in each sample site, Proteobacteria was the most dominant flora, while environmental samples were the second according to GenBank database. Moreover, Serratia sp. of Proteobacteria was the dominant strain. Conclusion: Radionuclides distribution in the uranium mill tailing showed a profound influence on soil properties and microbial diversity. This primarily study might provide valuable data for further research towards a better understanding of the radioactive contamination in uranium mill tailings in southeast China. © 2014 Published by Elsevier Ltd.

Keywords: Uranium mill tailings Radionuclides Soil properties Soil microbial diversity

1. Introduction With the rapid development of the nuclear industry and nuclear energy, the demand for uranium (U) mining metallurgy products keeps increasing. However, extraction of uranium and ore

* Corresponding author. Engineering Research Center of Biomass Materials, Ministry of Education, Southwest University of Science and Technology, Mianyang, Sichuan 621010, China. Tel.: þ86 816 6089009. E-mail address: [email protected] (X. Luo). http://dx.doi.org/10.1016/j.jenvrad.2014.09.019 0265-931X/© 2014 Published by Elsevier Ltd.

proceeding in milling facilities produce uranium tailings, which result in soil contamination and account for 85% of the total radioactivity originally present in the ore (Jagetiya and Purohit, 2006). The radioactive waste contains a series of long-lived radionuclides, such as uranium, radium (Ra), and thorium (Th) isotopes. Even after the termination of mining activities, uranium tailings would exert significant impact on soil environment for a long time (Mom cilovi c et al., 2013). Investigations on radiation in abandoned uranium mines have received particular attentions worldwide and extensive surveys in many countries have been carried out (Carvalho et al., 2007; Mom cilovi c et al., 2013). In present study, soil

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samples in uranium mill tailings in southeastern China were analyzed, to assess the changes of soil quality and ecological environment in this abandoned uranium mine. The determination of environmental stress sensitive characteristics of soil may help to assess the soil quality. Soil enzyme activities (such as arylsulphatase (ARYL), b-glucosidase (GLU), phosphatase (PHO), urease (UR) and dehydrogenase (DH)) have been suggested as suitable indicators for soil quality, which could be used to measure the degree of soil degradation and the impact of pollution (Gianfreda et al., 2005). DH and GLU are reported to be associated with C-cycle, while UR, PHO and ARYL play important roles in N, P and S compounds. In addition, soil enzymes activities are found to be significantly correlated with soil pH (AcostaMartinez and Tabatabai, 2000). Meanwhile, bacterial communities in soils are one of the most important factors that exert influences on environment rehabilitation (Mondani et al., 2011). Bacteria can modify the redox state of uranium either by reduction or oxidation, and mediate uranium immobilization via adsorption, precipitation and intracellular accumulation (Merroun and Selenska-Pobell, 2008). Additionally, investigation of microbial communities in uranium mill tailings will be beneficial for understanding the indigenous microbial impacts on uranium-mineralization and developing appropriate long-term management strategies for uranium-contaminated areas (Chen et al., 2012). Mondani et al. (Mondani et al., 2011) have suggested that Proteobacteria, the representative of Acidobacteria, was detected in denaturing gradient gel electrophoresis (DGGE) bands of specific uraniferous samples, the iron-reducing bacteria (such as Geobacter and Geothrixand) and iron-oxidizing species (such as Gallionella and Sideroxydans) were also detected. Mumtaz et al. (Mumtaz et al., 2013) applied the bacterial 16S rRNA sequence analysis to identify the dominant cultivable bacteria in samples with different uranium concentrations. Investigation of bacterial community from the contaminated soils collected at different depth revealed the enrichment of Gamma-Pseudomonas (Geissler and Selenska-Pobell, 2005). The aims of this study were: (1) to determine concentrations of radionuclides (238U, 232Th, 226Ra and 4 K) in soils of the investigated area and determine the Pearson correlation coefficient between the radionuclides; (2) to measure the physicochemical soil characteristics and soil enzymes activities; (3) to analyse the diversity of microorganism in the sample sites by DGGE and identify the dominant strains by using phylogenetic relationship analysis. Our study will provide the basis for better understanding of the radioactive contamination in uranium mill tailings in southeastern China, which could guide the environmental rehabilitation in this region. 2. Materials and methods 2.1. Sampling sites The uranium mill tailings are located in southeastern China. The climatic feature is a subtropical humid continental climate. The area receives regular and plentiful rainfall (1329 mm per year) during late spring and early summer. The average annual temperature is 18.1  C with extreme low temperature of 5.9  C and extreme high temperature of 39.9  C. The average sunshine hours per year are 1447 h and the dominant wind direction is northeast. The sampling sites extended over an area of 1000 m2 in uranium mill tailings. In present study, after the preliminary test on the uranium concentrations and retrieving the decommissioning EIA data and other relevant literatures of this uranium mill tailing, the sample areas were divided into 3 regions: high (32e67 mg/g), moderate (15e27 mg/g) and low concentration areas (3.2e12 mg/g), according to the uranium concentrations. Five sample sites were

selected in each of the three areas by using grid (1  1 m) method. For each sample site, 3 soil samples were collected at 0e15 cm, 15e30 cm and 30e45 cm depth, respectively. Totally, 45 (3  5  3) soil samples were collected, and about 1 kg fresh soil samples were collected via soil sampler. Soil samples were dried at 100  C to achieve a constant weight. After homogenization, they were packed in plastic containers (diameter: 7.0 cm; height: 0.0 cm) sealed with a PVC (polyvinyl chloride) tape. 2.2. Radioactivity measurements The concentrations of 238U, 232Th, 226Ra and 4 K in soil samples were determined by using HPGe (high resolution hyper pure germanium) gamma-ray spectrometer. Briefly, dry soil sample (0.5 g) was stored in polyethylene plastic case (diameter, 7 cm; height, 6.5 cm) for more than 4 weeks. Pulses produced during gamma ray interaction in a NaI (Tl) crystal were counted, based on a detection system calibrated in energy and activity concentration through the standard radioactive sources from NBL (New Brunswick Labora-tory), United States Department of Energy. The activity of 238U was evaluated from all the lines in the spectrum, based on gamma-ray lines of 214Bi at 609.3, 1120.3, and 1764.5 keV. The specific activity of 232Ra was evaluated from gamma-ray lines of 214 Bi at 609.3 and 1764.5 keV. The specific activity of 232Th was measured from gamma-ray lines of 214Bi at 609.3. The specific activity of 4 K was determined from its 1460.8 ke V gamma-ray line. 2.3. Physicochemical properties of soil Soil pH was determined via a pH meter (IS139 apparatus). Soil organic matter (OM) was determined by titrimetry with potassium dichromate (Schumacher, 2002). The electrical conductivity of soil was detected via a conductometer (ik67 type). The available P (phosphorus) was determined by MoeSb colorimetry (Lei et al., 2008). After acid dissolution of soil samples, total N (nitrogen) content was analyzed by semi-micro Kjeldahl method (Fawcett, 1954). After pretreatment using reductant, the alkali-hydrolyzable N was measured via alkaline hydrolysis diffusion method (Spargo et al., 2009). The available K (potassium) was determined using AAS (atomic absorption spectrophotometry) after 1 moL/L NH4AC extraction (David, 1960). What's more, enzyme activities of ARYL, GLU, PHO, UR and DH, were determined as described by Gianfreda et al. (Gianfreda et al., 2005). 2.4. DGGE analysis of soil sample For each soil sample, gDNA was extracted using sludge genomic DNA rapid extraction kit (Baisaike Company, Nanjing, China). PCR amplification of bacterial 16S rDNA from gDNA was performed using the universal primers of GC-338F (with 40 GC clamps) and 518R. The PCR reactions were carried out in 50 mL volumes, containing 2 ng gDNA, 1 mL GC-338F (20 mM) and 1 mL 518R (20 mM), 5 mL 10  PCR buffer, 3.2 mL dNTP (2.5 mM), 0.4 mL rTaq (5 U/mL), added to 50 mL with ddH2O. Cycling conditions involved an initial 5 min denaturing step at 94  C, followed by 30 cycles of 1 min at 94  C, 45 s at 55  C, and 1 min at 72  C, and a final extension step of 10 min at 72  C. After verification of PCR product in agarose gel, 10 mL PCR products were analyzed via DGGE. The deformation gradient was from 35% to 55% (100% denaturant corresponds to 7 mol/L urea and 40% (v/v) acrylamide) and the 8% polyacrylamide gel was electrophoresed in 0.5  TAE running buffer for 4 h at 150 V and 60  C. After that, the gel was detected by silver staining and pictured using BioRAD system. The specific bands in DGGE were recycled and amplified as a template. The primer sequences were 5’-

X. Yan, X. Luo / Journal of Environmental Radioactivity 139 (2015) 85e90

ACTCCTACGGGAGGCAGCAG-3’ (338f) and 5’-ATF ACCGCGGCT GC TGG-3’ (518r). PCR products were sequenced. Sequencing reactions for PCR products were optimized using the BigDye Terminator v1.1 sequencing Ready Reaction kit (Applied Biosystems, CA, USA). The reactions system was comprised of 1 ml of purified PCR products, 1 ml of primer (3.2 pM/ml), 8 ml of 2.5  BigDye Terminator enzyme, and 4 ml of 5  sequencing buffer, and the reactions were started with an initial denaturation at 96  C for 1 min, followed by 25 cycles of 96  C/10 s, 50  C/5 s and 64  C/4 min. Next, the sequencing products were precipitated and sequenced in both directions via a Genetic Analyzer 3130xl (Applied Biosystems, CA, USA). Finally, the bands were checked using the BLAST search program at NCBI (National Centre for Biotechnology Information). Sequences showing highest homology with the sequences in GenBank were selected. Furthermore, the phylogenetic characteristics for the soil samples were determined for a mixture of the soil from all depths, briefly, the sequences of the microorganisms were transformed into the FASTA form, and then were checked using the Clustal-X software (Thompson et al., 1997). Next, Neighbor-Joining was used to calculate the evolutionary distance (Gouy et al., 2010), followed by the phylogenetic tree construction via MEGA V4.1, in combination with the Boot-straps test (Balode et al., 2012). The diversity indexes were also calculated from the DGGE banding patterns to indicate the bacterial diversity of soil samples, including ShannoneWiener Index, diversity of communities; Evenness, the degree of uniformity; Richness, the species numbers identified in a sample (De Mesel et al., 2004). 2.5. Statistical analysis Multivariate analysis and analyses of variance (ANOVA) were used to demonstrate differences among the sample using SPSS 19.0. Pearson correlation coefficient (PCC) was also calculated using SPSS. P < 0.05 was considered as significant. 3. Results 3.1. Measurement of radioactivity The radionuclide contents in soil samples were summarized in Table 1. The concentration of 238U in the high concentration area was 6.7 times than that in low concentration area, and the concentrations in samples at different depth exerted no significant differences. The 232Th concentration was increased with the decreased uranium concentration and was not influenced by the depth of sample sites. However, concentrations of 226Ra and 4 K showed no significant changes along with decreased uranium concentration or increased depth of sample site. Correlation analysis of radionuclides contents, as shown in Table 2, suggested that 232Th concentration was significantly correlated with 226Ra (PCC ¼ 0.69, p ¼ 0.04), indicating that 232Th is the direct source of 226Ra in the soil. What's more, 226Ra was significantly correlated with 40K (PCC ¼ 0.88, p < 0.01), which

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Table 2 The Pearson correlation matrix of radionuclides. 238 238

U 232 Th 226 Ra 40 K

232

U

226

Th

0.01 1.00

1.00

40

Ra

0.07 0.69** 1.00

K

0.34 0.24 0.88** 1.00

**P < 0.01.

meant that both 226Ra and 40K are dissolved in soil in similar conditions and from same reservoir rocks. However, PCC between 238U and other radionuclides was not significant. 3.2. Basic physicochemical characteristics of soil samples Physicochemical characteristics of soil samples from high, moderate and low concentration areas were examined (Table 3). Soils from 3 regions were all acidic. The contents of OM, total N, total P and total K increased along with the decreasing of uranium concentration. Similarly, the concentration of alkali-hydrolyzable N, available P and available K increased with the decreasing of uranium concentration. In addition, CEC (cation exchange capacity) showed a similar trend as other physicochemical characteristics. Additionally, activities of soil enzymes (PHO, ARYL, GLU, UR and DH) decreased with the increasing of uranium concentration (Table 4). The above changes among different concentration samples were significant (p < 0.05), while the differences of all detected characters were not significant (p > 0.05). 3.3. Analysis of soil microbial diversity (DGGE) Diversity of microorganism in sample site was evaluated by DGGE, indicating the dominant microorganism in different sample sites (Fig. 1). The average of ShannoneWiener Index of low, moderate and high concentration areas were 1.66, 1.44 and 1.55; the average of Evenness were 0.90, 0.84 and 0.89; and the average of Richness were 6.27, 5.47 and 5.87. These results indicated that there were significant differences of microbial diversity in different concentration samples. Total 14 bands, 11 bands and 14 bands were sequenced and further conducted similarity sequence searching via BLAST in GenBank (Table 5) to detect the representative microorganisms. For low concentration area, Proteobacteria, unknown environmental samples (the exact bacteria name could not be identified, as there is no detailed information of similar sequence related with the 'unknown environmental samples' in NCBI), Acidobacteria and Actinobacteria were identified. For moderate and high concentration area, Proteobacteria and unknown environmental samples were screened. In each sample site, Proteobacteria assumed absolute superiority, followed by unknown environmental samples. Additionally, Serratia sp. of Proteobacteria was the dominant strain. Phylogenetic relationship analysis of representative microorganism

Table 1 Measurement of Radioactivity in soil samples (mean ± SD). High concentration area A U (mg/g) 232 Th (mg/g) 238 U/232Th 226 Ra (Bq/g) 40 K (104 mg/g) 238

43.5 15.6 3.3 0.16 2.27

B ± ± ± ± ±

8.85 2.66 0.54 0.03 0.27

41.3 13.8 3.06 0.08 2.13

Moderate concentration area C

± ± ± ± ±

9.16 2.55 1.07 0.02 0.18

48.1 12.7 3.98 0.06 1.90

A ± ± ± ± ±

15.7 2.29 1.12 0.02 0.19

24.7 15.3 1.66 0.17 1.57

B ± ± ± ± ±

3.15 3.52 0.71 0.04 0.11

22.1 14.5 1.50 0.13 1.73

Low concentration area C

± ± ± ± ±

3.06 2.73 0.33 0.02 0.14

Note: A, B and C represented soil samples were collected at 30e45 cm, 15e30 cm and 0e15 cm depth.

23.6 14.5 1.55 0.08 1.62

A ± ± ± ± ±

3.42 2.73 0.51 0.02 0.13

6.7 16.9 0.4 0.14 1.47

B ± ± ± ± ±

2.15 2.50 0.11 0.03 0.23

5.4 16.7 0.36 0.13 1.42

C ± ± ± ± ±

2.3 2.44 0.17 0.02 0.21

5.2 16.0 0.33 0.11 1.29

± ± ± ± ±

1.36 2.09 0.14 0.04 0.28

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Table 3 Physicochemical characteristics of soil samples (mean ± SD).

High area Moderate area Low area

PH

OM (g/kg)

Total N (g/kg)

Total P (g/kg)

Total K (g/kg)

CEC cmol(þ)/kg

Alkali-hydrolyzable N (mg/kg)

Available P (mg/kg)

Available K (mg/kg)

3.62 ± 0.31 4.15 ± 0.20 4.51 ± 0.28

6.35 ± 1.89a 12.80 ± 2.08b 18.95 ± 1.96c

0.298 ± 0.02a 0.529 ± 0.08b 0.818 ± 0.19c

0.416 ± 0.1a 0.544 ± 0.13 ab 0.706 ± 0.16b

9.83 ± 0.47a 14.7 ± 1.43b 24.2 ± 2.02c

9.01 ± 0.35a 15.8 ± 0.42b 20.8 ± 0.51c

69.6 ± 5.62a 66.9 ± 5.33a 111 ± 7.15b

1.9 ± 0.12a 5.3 ± 0.31b 9.7 ± 0.42c

4.65 ± 0.18a 7.45 ± 0.26b 9.1 ± 0.31c

Note: organic matter: OM; cation exchange capacity: CEC. a There was significant difference compared with the Lower area samples. b There was significant difference compared with the High area samples. c There was significant difference compared with the Moderate area samples.

Table 4 Analysis of enzyme activities in soil samples (mean ± SD). High concentration area A PHO ARYL GLU UR DH

0.95 0.07 0.64 6.54 0.33

Moderate concentration area

B ± ± ± ± ±

a

0.43 0.02a 0.04a 0.2a 0.04a

0.93 0.08 0.68 6.24 0.35

C ± ± ± ± ±

a

0.41 0.02a 0.03a 0.11a 0.06a

0.92 0.08 0.67 6.89 0.40

A ± ± ± ± ±

a

0.09 0.02a 0.08a 0.17a 0.05a

1.35 0.17 1.11 15.73 1.10

Low concentration area

B ± ± ± ± ±

b

0.11 0.04b 0.06b 0.56b 0.62b

1.39 0.18 1.14 16.86 1.11

C ± ± ± ± ±

b

0.09 0.03b 0.07b 0.77b 0.37b

1.43 0.19 1.20 19.29 1.05

A ± ± ± ± ±

b

0.09 0.02b 0.08b 0.97b 0.44b

2.09 0.28 2.00 34.56 1.7

B ± ± ± ± ±

c

0.26 0.02c 0.04c 0.95c 0.15c

2.11 0.27 2.01 35.83 1.78

C ± ± ± ± ±

c

0.30 0.04c 0.05c 1.30c 0.16c

2.10 0.30 2.06 36.6 1. 9

± ± ± ± ±

0.29c 0.04c 0.04c 1.65c 0.15c

Note: arylsulphatase: ARYL; b-glucosidase: GLU; phosphatase: PHO; Urease: UR and dehydrogenase: DH. A, B and C represented soil samples were collected at 30e45 cm, 15e30 cm and 0e15 cm depth. a There was a significant difference compared with the Lower area samples. b There was a significant difference compared with the High area samples. c There was a significant difference compared with the Moderate area samples.

showed two main clusters and most microorganisms showed high similarity with Serratia sp. (Fig. 2). 4. Discussion In present study, the average concentrations of 238U and 232Th were significantly higher than the background value suggested as 238 U (2.72 mg/g) and 232Th (12.4 mg/g) for soils in China (GB15618, 1995). However, 238U concentration and depth of sampling sites showed no significant influence on the concentrations of 226Ra and 40 K. The increased concentration of radionuclides might be due to the industrial extraction process of ore, the abandoned waste of uranium mill tailings and the damages of soil-covers (Gavrilescu et al., 2009). To identify on what extent these radionuclides can exert influence together on this mill tailing, correlation analyses were carried between radionuclides pairs. It turned out that the concentration differences between 238U and other radionuclides

was not significant, while 232Th concentration was significantly correlated with 226Ra, and 226Ra connected with 40K. Similar relationships have been reported in previous studies that a significant correlation is observed between 232Th and 226Ra, 226Ra and 40K (Prasad et al., 2008) (Mehra et al., 2007). In this case, the individual concentration of a certain radionuclide could be a good predictor for the concentration of another radionuclide (Jibiri et al., 2011). Collectively, the study on the radioactive contamination data in uranium mill tailings will help in the environmental rehabilitation. All soil samples were acidic and soil acidity was increased along with the increasing 238U concentrations, which might be associated with a lot of acidic ions produced during the extraction process of uranium, such as nitrate and sulfate (Gavrilescu et al., 2009). Soil PH has great effects on the generation of soil colloid, radionuclide hydrolysis and ion exchange reaction, which in turn affect the adsorption of nuclide (Van Bergeijk et al., 1992). Meanwhile, nutrients in soils (such as total N, the alkali-hydrolyzable N, available P

Fig. 1. The denaturing gradient gel electrophoresis (DGGE) profiles of amplified 16S rDNA fragments from soil samples used to for sequencing. A, B and C represented soil samples were collected at low, moderate and high concentration areas.

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Table 5 Sequence comparative analysis of DGGE bands (A, B and C represented soil samples were collected at low, moderate and high concentration areas; the numbers represent the sample numbers). Samples

The most similar strains

Accession number

Similarity

Diversity

A1 A2

JF496532 EF417700

100% 100%

Proteobacteria Acidobacteria

A3 A4 A5 A6 A7 A8 A9 A10

Serratia proteamaculans Uncultured Acidobacteria bacterium Uncultured Serratia sp. Uncultured Serratia sp. Uncultured Serratia sp. Uncultured Serratia sp. Hafnia sp. Kocuria marina strain Serratia sp. Uncultured bacterium

JQ799000 JQ799000 JQ799000 JQ799000 JF312960 JX007973 JQ665444 HQ120313

99% 99% 99% 100% 100% 100% 100% 100%

A11

Uncultured bacterium

EU714780

99%

A12

JN630882

100%

A13 A14

Serratia proteamaculans strain Uncultured Serratia sp. Uncultured bacterium

Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Actinobacteria Proteobacteria Unknown environmental samples Unknown environmental samples Proteobacteria

JF833667 JN199327

100% 100%

B1 B2 B3 B4 B5 B6 B7

Serratia proteamaculans Hafnia sp. Hafnia sp. Uncultured Serratia sp. Uncultured Serratia sp. Hafnia sp. Uncultured bacterium

JF496532 JF312960 JF312960 JQ799000 JQ799000 JF312960 HQ120313

100% 100% 99% 99% 99% 99% 100%

B8

Uncultured bacterium

EU714780

99%

B9

JN630882

100%

B10 B11 C1 C2 C3 C4 C5 C6 C7 C8 C9

Serratia proteamaculans strain Uncultured Serratia sp. Uncultured Serratia sp. Serratia proteamaculans Hafnia sp. Hafnia sp. Uncultured Serratia sp. Uncultured Serratia sp. Uncultured Serratia sp. Uncultured Serratia sp. Hafnia sp. Uncultured bacterium

JF833667 JQ799000 JF496532 JF312960 JF312960 JQ799000 JQ799000 JQ799000 JQ799000 JF312960 HQ120313

100% 99% 100% 100% 99% 99% 99% 99% 100% 100% 100%

C10

Uncultured bacterium

EU714780

99%

C11

Serratia proteamaculans strain Serratia sp. Hafnia sp. Uncultured bacterium

JN630882

100%

HM242268 JF312960 JN199327

100% 100% 100%

C12 C13 C14

Proteobacteria Unknown environmental samples Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Unknown environmental samples Unknown environmental samples Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Unknown environmental samples Unknown environmental samples Proteobacteria Proteobacteria Proteobacteria Unknown environmental samples

and K) all decreased along with the increasing of 238U concentration, which was due to the increasing of proportion of tailing in soil. Soil nutrients, especially OM, are important links among soil, microorganism, plant and animal (Baumgardner et al., 2013; Miransari, 2013). Additionally, soil enzyme activities (ARYL, GLU, PHO, UR and DH) decreased with the increased 238U concentration and the increased depth of sample site. Soil enzyme activity has been used to assess the influence of various pollutions on soil quality (Badiane et al., 2001). Radionuclides inhibit soil enzymes

Fig. 2. Phylogenetic analysis of 16S rDNA gene sequences from gDNA isolated from soil samples. Bootstrap values (percentages) are given at the nodes.

activities through interaction with their SH group and/or imidazole ligand, which was competitive inhibition (Francis et al., 2012; Gao et al., 2010). However, soil enzyme activities in uranium mill tailings soils in different countries were not always coincide with each other (Gianfreda et al., 2005; Klose and Tabatabai, 2000). Additionally, the genetic fingerprinting technique of DGGE, which enables the separation of the double-stranded DNA fragments with length up to 500bp, was used in this study to analyze the diversity of microorganisms in different sample sites (Kocherginskaya et al., 2005). DGGE can screen the soil bacterial communities by detecting community changes. Many bacteria and

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X. Yan, X. Luo / Journal of Environmental Radioactivity 139 (2015) 85e90

fungi have been detected in uranium mill tailings around the world (Landa, 2004), and Proteobacteria has been reported as the dominant stain by previous studies (Gillow et al., 2000). The results in this study showed that Proteobacteria was the most dominant flora, while unknown environmental samples were the second. Moreover, Serratia sp. of Proteobacteria was the dominant strain. The microbial structures in all the three concentration areas were simple with limited strains of microbial population, with less diversity in high concentration areas suggesting an impact of the radionuclides. .The decrease of uranium concentration could increase the physicochemical properties and enzyme activities of soil from uranium mill tailings, whereas the concentration changes of 238U exhibited significant correlations with the changes of other radionuclides. Furthermore, Proteobacteria was identified as the flora with absolute superiority in the soils. The current analysis concerning the influence of radionuclide distributions on soil properties and soil microbial diversity might provide a better understanding for radioactive contamination and guide the reuse of this area in future. Acknowledgments This study was supported by the National Defense key projects of China (NO. B3120110001), Qiqihar university research project (No. 2012155). We wish to express our warm thanks to Engineering Research Center of Biomass Materials, Ministry of Education, Southwest University of Science and Technology. References Acosta-Martinez, V., Tabatabai, M., 2000. Enzyme activities in a limed agricultural soil. Biol. Fert. Soils 31, 85e91. Badiane, N.N.Y., Chotte, J.-L., Pate, E., Masse, D., Rouland, C., 2001. Use of soil enzyme activities to monitor soil quality in natural and improved fallows in semi-arid tropical regions. Appl. Oil Ecol. 18, 229e238. Balode, D., Skar, H., Mild, M., Kolupajeva, T., Ferdats, A., Rozentale, B., Leitner, T., Albert, J., 2012. Phylogenetic analysis of the Latvian HIV-1 epidemic. AIDS Res. Hum. Retrov. 28, 928e932. Baumgardner, M., Kristof, S., Johannsen, C., Zachary, A., 2013. Effects of organic matter on the multispectral properties of soils. Proc. Indiana Acad. Sci. 413e422. Carvalho, F., Madruga, M., Reis, M., Alves, J., Oliveira, J., Gouveia, J., Silva, L., 2007. Radioactivity in the environment around past radium and uranium mining sites of Portugal. J. Environ. Radioact. 96, 39e46. Chen, Z., Cheng, Y., Pan, D., Wu, Z., Li, B., Pan, X., Huang, Z., Lin, Z., Guan, X., 2012. Diversity of microbial community in Shihongtan sandstone-type uranium deposits, Xinjiang, China. Geomicrobiol. J. 29, 255e263. David, D., 1960. The determination of exchangeable sodium, potassium, calcium and magnesium in soils by atomic-absorption spectrophotometry. Analyst 85, 495e503. De Mesel, I., Derycke, S., Moens, T., Van der Gucht, K., Vincx, M., Swings, J., 2004. Top-down impact of bacterivorous nematodes on the bacterial community structure: a microcosm study. Environ. Microbiol. 6, 733e744. Fawcett, J., 1954. The semi-micro Kjeldahl method for the determination of nitrogen. J. Med. Lab. Technol. 12, 1. Francis, A., Poinssot, C., Geckeis, H., 2012. Impacts of microorganisms on radionuclides in contaminated environments and waste materials. Radionucl. Behav. Nat. Environ. Sci. Implic. Lessons Nucl. Ind. 161e225.

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Radionuclides distribution, properties, and microbial diversity of soils in uranium mill tailings from southeastern China.

To collect the radioactive contamination data for environmental rehabilitation in uranium mill tailings in southeastern China...
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