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Accepted Manuscript

Heavy metals in slag affect inorganic N dynamics and soil bacterial community
structure and function

Miyuki Oka, Yoshitaka Uchida

PII: S0269-7491(18)31036-4
DOI: 10.1016/j.envpol.2018.09.024
Reference: ENPO 11566

To appear in: Environmental Pollution

Received Date: 20 March 2018


Revised Date: 3 September 2018
Accepted Date: 4 September 2018

Please cite this article as: Oka, M., Uchida, Y., Heavy metals in slag affect inorganic N dynamics
and soil bacterial community structure and function, Environmental Pollution (2018), doi: 10.1016/
j.envpol.2018.09.024.

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1 Heavy metals in slag affect inorganic N dynamics and soil

2 bacterial community structure and function


3 Miyuki Oka1, Yoshitaka Uchida2,*

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5 School of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo, Hokkaido

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6 060-8589, Japan.
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7 Research Faculty of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo,

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8 Hokkaido 060-8589, Japan.

9 *Corresponding author. Email: uchiday@chem.agr.hokudai.ac.jp

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10 Abstract

11 Heavy metal contamination of soil in the vicinity of mining sites is a serious environmental

12 problem around the world when mining residue (slag) is dispersed as dust. We conducted an

13 incubation experiment to investigate the effect of a slag containing high levels of Pb and Zn

(62.2 and 33.6 g kg−1 slag as PbO and ZnO, respectively, sampled from a site formerly used

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15 as a lead and zinc mine) on the nitrogen cycle when mixed with soil (0–0.048 g slag g−1 soil).

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16 The nitrogen cycle provides many life supporting-functions. To assess the quality of the soil

17 in terms of the nitrogen cycle we focused on the dynamics of nitrate and ammonium, and

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18 bacterial community structure and functions within the soil. After two weeks of pre-

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19 incubation, 15N-labeled urea (500 mg N kg−1) was added to the soil. Changes in soil pH, the
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20 concentration and N ratio of nitrate (NO3−-N) and ammonium, and bacterial relative

21 abundance and community structure were measured. Results indicated that increasing the
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22 ratio of slag to soil had a stronger negative effect on nitrification than ammonification, as

23 suggested by slower nitrate accumulation rates as the slag:soil ratio increased. In the
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24 treatment with the highest amount of slag, the concentration of NO3−-N was 50% of that in
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25 the controls at the end of the incubation. Regarding the bacterial community, Firmicutes had
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26 a positive and Planctomycetes a negative correlation with increasing slag concentration.

27 Bacterial community functional analysis showed the proportion of bacterial DNA sequences
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28 related to nitrogen metabolism was depressed with increasing slag, from 0.68 to 0.65. We
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29 concluded that the slag impacted the soil bacterial community structure, and consequently

30 influenced nitrogen dynamics. This study could form the basis of further investigation into

31 the resistance of the nitrogen cycle to contamination in relation to soil bacterial community.

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33 Keywords: Slag; Heavy metal; Nitrogen cycle; Bacterial community structure; Bacterial

34 community function
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35

36 Capsule: Slag containing heavy metals impacted the soil bacterial community structure,

37 consequently influenced nitrogen dynamics.

38

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39 1. Introduction

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40 Soil contamination caused by the slag produced during mining for metals is one of the most

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41 serious global environmental issues. The slag often contains hazardous components including

42 heavy metals such as Pb and Zn. In contaminated sites, plant biomass and diversity decrease

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43 (Nagajyoti et al., 2010, Hernández and Pastor, 2008). When plant biomass decreases, some of
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44 the benefits provided by the plants to soils (e.g., the formation of aggregates and retention of

45 moisture) may decline or cease. This results in reduced soil stability, and may lead to the
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46 spread of contaminated soils further into the surrounding environment through wind and
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47 water erosion (Zuazo and Pleguezuelo, 2009). In fact, around areas contaminated by heavy
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48 metals the damage caused to human health by ingestion of dust is a significant problem (Zhao

49 et al., 2012, Taylor et al., 2014). To minimize the spread of contaminants into residential
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50 areas, the importance of plant cover has been discussed previously (Wong, 2003).

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52 One of the reasons for the decrease in plant biomass is insufficient nutrient supply, a prime
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53 example being inorganic nitrogen (N) (Bååth, 1989). Nitrogen is a major growth-limiting

54 factor for plants in the natural environment. Available N, including ammonium (NH4+) and

55 nitrate (NO3−), is supplied from the residues of plants, animals, and microorganisms, or

56 dinitrogen gas in the air. For sufficient N to be available to plants, soil microbial activities

57 such as decomposition, ammonification, and nitrification are important. Environmental

58 factors inhibiting soil microbial activity, such as presence of contaminants, can result in
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59 reduced plant growth and biomass production. In soils bearing a high concentration of heavy

60 metals, the diversity and biomass of soil microorganisms is decreased (Bååth, 1989, Giller et

61 al., 1998). Heavy metals affect the growth, morphology, and metabolism of soil

62 microorganisms (Leita et al., 1995). This stagnates N cycling and decreases the abundance

63 and diversity of plant species able to grow in the area (Giller et al., 1998).

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64

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65 Several studies have examined the N cycle and microbial activity in soils contaminated with

66 Pb, Zn or other risk elements. Some of these have shown that contaminants influence

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67 mineralization (Vásquez-Murrieta et al., 2006), while others have shown that heavy metals

68 had only a small effect on ammonification but a larger effect on nitrification (Rother et al.,

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1982; Ueda et al., 1988). Contrastingly, another report suggested that Pb and Cd
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70 contamination of soil had no effect on nitrification (Cela et al., 2002).
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72 Additionally, many studies have been conducted focusing on soil bacterial community
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73 structure and activity in contaminated sites, revealing that these parameters are changed in the
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74 presence of heavy metals (Ellis et al., 2003, Wang et al 2007, Gołębiewski, M et al., 2014).

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76 Although the effects of slag containing high levels of risk elements, including heavy metals,
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77 on the N cycle and soil microbes have been investigated, few studies have shown the
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78 relationship between them. Better understanding of the impacts of slag on soil bacterial

79 community and function related to the N cycle will provide basic information to assist in

80 more efficient recovery of soil N and plants as vegetation cover in the contaminated sites,

81 which in turn will ameliorate the potential environmental risk of mining.

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83 Our study focused on the relationship between the supply of inorganic N, bacterial abundance,

84 and community structure and function. We evaluated the N cycle in soil contaminated with a

85 slag containing Pb and Zn, using the nitrogen-15 (15N) tracing technique. By comparing the N

86 dynamics with the quantitative and qualitative data of microorganism populations, we

87 attempted to link changes in biological properties of a contaminated soil with the N cycle.

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88 2. Materials & methods

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89 2.1. Soil sampling and physicochemical properties of soil and slag

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90 Soil (0‒10 cm depth) was collected from the experimental corn (Zea mays) farm of Hokkaido

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91 University, Sapporo, Japan (43°3′N, 141°20′E) in May 2017. The sampled soil was air dried
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92 and sieved through a 2 mm mesh. The pH of the soil was 5.77 ± 0.33 (s.d., n = 3), and total C

93 and N were 42.3 ± 0.85 g kg−1 and 3.60 ± 0.21 g kg−1, respectively. To measure soil pH, 3 g
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94 of dry soil was mixed with 7.5 ml Milli-Q water and shaken for 30 minutes, followed by pH
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95 measurement using a pH meter (AS800, AS ONE Corporation, Osaka, Japan). Total C and N
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96 content of the soil samples were determined using an elemental analyzer (EA 2400 Series;

97 Perkin Elmer, Foster City, CA, USA).


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99 A Zn-leaching residue (slag) was sampled in February 2017 from the surface of the former
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100 mining site in Kabwe, Zambia (14°27′19″S, 28°26′0″E) and sieved to retain particles ≤7 µm.
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101 The pH of the slag was 6.03 ± 0.04 (s.d., n = 3), and total C and N were 3.55 ± 0.33 g kg−1

102 and 1.68 ± 0.85 g kg−1 (s.d., n = 4), respectively. The same methods and devices as the soil

103 samples (AS800 and EA 2400) were used to measure these slag characteristics. The chemical

104 composition of the sieved slag was analyzed with Cartesian geometry energy dispersive X-

105 ray fluorescence spectrometer (NEX CG; Rigaku Corporation, Tokyo, Japan). As risk

106 elements, the slag contained 6.22% PbO, 3.36% ZnO, 0.07% As2O3 and 0.0003% HgO. The
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107 levels of water-soluble Pb and Zn in the slag were 0.78 and 1.18 mg g−1 slag (measured after

108 3 g of the slag was shaken in 120 ml water for 4 hours). The soil collected from the farm in

109 Hokkaido University contained no detectable Pb or Zn.

110 2.2. Incubation setup

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111 To test the effect of the slag on the sampled soil, 45 g of the soil and varying amounts of the

112 slag were mixed and packed in separate 120 ml plastic cups. Four different contamination

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113 levels for Pb and Zn were created, equating to 0, 100, 500 and 3000 mg Pb and 0, 54, 270,

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114 and 1620 mg Zn kg−1 dry soil, and these were designated as Control (C), and Low (L),

115 Middle (M) and High (H) concentrations of heavy metals (Table 1). The range of slag:soil

116
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ratios was determined based on previously performed field experiments (Ikenaka et al. 2010).
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118 Three replicates for each treatment were prepared on five sampling occasions. In total, 60 (=
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119 4 treatments * 3 replicates * 5 sampling days) cores were arranged. The samples were
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120 compressed to 0.9 g cm−3, and Milli-Q water was added to achieve a water-filled pore space
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121 (WFPS) of 60%. To avoid excess soil moisture loss the cups were closed with plastic lids into

122 which six holes (diameter 1 mm) had been perforated. The moisture content was maintained
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123 at 60% WFPS by adding Milli-Q water every 2 days. The particle density of the soil was

124 taken as 2.65 g cm−3 for the calculation of the WFPS. Thus, the soil porosity was 1 − (0.9 g
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125 cm−3 / 2.65 g cm−3) = 66%.


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126
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127 The soil samples were incubated for two weeks and then three atom% N-labeled urea was

128 applied (500 mg N kg−1 dry soil–slag mixture). The soil was sampled destructively on the day

129 of urea application (immediately after the urea application, day 0), and at 1, 6, 14 and 28 days
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130 after urea application. The samples were divided for the measurement of pH (using the

131 method described previously), inorganic N, and bacterial DNA.

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133 2.3. Measurement of ammonium and nitrate

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134 For the determination of inorganic N (NO3–-N and NH4+-N) concentration, three grams of the

135 sampled fresh soil was extracted with 2M KCl (15 ml). After shaking the mixture for 30

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136 minutes, the suspension was filtered through a filter paper (Grade 5C, <5 mm; Advantec,

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137 Tokyo, Japan). The extracted solution was stored at 4 °C until measurement. For the

138 measurement, a colorimetric method was employed with a flow injection analyzer (AQLA-

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139 700; Aqualab, Tokyo, Japan) as described previously (Hamamoto and Uchida 2015).
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141 In brief the methods used were: for NO3–-N, the cadmium reduction and N-(1-
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142 Naphthyl)ethylenediamine dihydrochloride method; and for NH4+-N, the indophenol blue
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143 method. Results are shown as mg N kg–1 soil.


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144
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145 2.4. Isotopic N measurement

146 To measure the 15


N ratio in the extracted inorganic N, the NO3− in the KCl solution was
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converted into NO2− using spongy cadmium and further reduced to N2O with sodium azide in
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148 acetic acid according to methods previously described (McIlvin and Altabet, 2005). KCl

149 solution (1 ml) was placed in a 20 ml glass vial with 4 ml buffer solution containing NaCl

150 (final concentration 0.5M) and NaHCO3 to make the solution around pH 8. Then, 0.2 g of

151 spongy cadmium was added and the vial was shaken for 18 hours. After shaking, the

152 supernatant was moved to another vial. The azide/acetic acid buffer was prepared on the day
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153 of use. NaN2 (0.65 g) was dissolved in 9 ml Milli-Q water and mixed with 1 ml acetic acid.

154 The buffer was purged with He for 20 minutes to remove N2O in the solution. Using a

155 syringe, 0.2 ml buffer solution was injected and mixed with the sample in the vial closed with

156 a rubber and plastic lid. After 30 minutes shaking, 0.1 ml NaOH (ca. 6M) was added to stop

157 the reaction. The N2O produced was preserved in the vial at room temperature until

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158 measurement.

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160 The isotopic N ratio of NH4+-N was measured by oxidizing to N2O using NaOBr according to

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161 the method of Laughlin et al. (1997). To prepare the NaOBr, first 50 g NaOH was dissolved

162 in 400 ml Milli-Q water and preserved at 4 °C overnight. Then, 16 ml of Br2 was added to the

163
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prepared NaOH solution on ice over a period of 30 minutes. The solution was then diluted to
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164 500 ml total and stored at 4 °C until use. An aliquot (1 ml) of the extracted KCl solution was
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165 placed in a 20 ml vial, the vial was sealed with a rubber and plastic lid, and its headspace was

166 replaced with He. NaOBr purged with He was then injected into the vial using a syringe and
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167 in another syringe, the produced N2O was collected. The N2O was preserved in a new vial at
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168 a room temperature until measurement.

169
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170 The N2O was diluted with zero air to increase the gas volume and to reduce the N2O

concentration to about 100 µl l−1 for NH4+-derived N2O, and to between 300 and 5000 µl l-1
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for NO3−-derived N2O. An Isotopic Nitrous Oxide Analyzer (Model 914-0027, Los Gatos
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173 Research) was used for the measurement. The 15


N ratio of NO3−-N was calculated by

174 dividing 14N15NO by total N2O since the NO3−-N in the original solution bonds in the middle

175 position (α site) of the N2O. The 15N ratio of NH4+-N was calculated by dividing total isotopic

176 N2O (14N15NO plus 15N14NO) by total N2O.

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178 2.5. Measurement of bacterial abundance

179 On each sampling day, DNA was extracted from the fresh soil with PowerSoil DNA Isolation

180 Kit (MoBio Laboratories, Carlsbad, CA, USA) according to the manufacturer’s instructions.

181 The extracted DNA was purified with Agencourt AMPure XP (Beckman Coulter) according

182 to a predetermined protocol. The concentration of the purified DNA was measured with

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183 Qubit dsDNA HS Assay Kit (Invitrogen, USA). The purified DNA was then diluted 100

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184 times with nuclease-free water for qPCR. A standard curve was prepared with a DNA sample

185 from the control treatment on day 0 to determine the relative bacterial abundance among

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186 different treatments at different timings. We used the QuantiTect SYBR Green PCR kit

187 (Qiagen, Mississauga, ON, Canada) and Mx3000P/Mx3005P QPCR Systems (Agilent). For

188
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primers, 27F/519R (Abujabhah et al. 2017) were chosen for bacterial (16S rRNA)
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189 quantitative analysis. The initial denaturation temperature was 95 °C with an annealing
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190 temperature of 95 °C, and the extension was conducted for 1 min at 58 °C for 35 cycles. The

191 final extension was done at 72 °C for 30 s. The relative abundance was expressed based on
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192 the abundance of the 16S rRNA genes of the control treatment at day 0.
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193
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194 2.6. Amplification and sequencing of 16S rRNA in soil

195 Bacterial community analysis was performed on all DNA samples extracted from soil on day
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196 0 and 28. The first polymerase chain reaction (PCR) was conducted using primers to amplify
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197 the V4 region of 16S rRNA (amplicon size ~250 bp, forward primer = 515F: 5′-

198 GTGCCAGCMGCCGCGGTAA-3′, reverse primer = 806R: 5′-

199 GGACTACHVGGGTWTCTAAT-3′). For PCR, samples were prepared with 10 µL of

200 AmpliTaq Gold® 360 Master Mix (Applied BiosystemsTM, Foster City, U.S.A.), 0.4 µL of

201 forward primer, 0.4 µL of reverse primer and 1–2 µL of DNA extract. Nuclease-free water
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202 was added to make up to a final volume of 20 µL. The first PCR cycle was 95 °C for 10 min,

203 then 20 cycles at 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 1 min, followed by 72 °C for 7

204 min. The first PCR products were then purified with Agencourt AMPure XP (Beckman

205 Coulter) according to the given protocol.

206

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207 Using the amplicon obtained from the first PCR procedure, another PCR was performed to

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208 make it Ion Torrent sequencing sample-specific. To achieve this, a forward primer of 515F

209 attached with the sequence of Ion Xpress Barcode Adapters 19–42 Kit (Life Technologies),

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210 and reverse primer of 806F attached with the sequence of Ion P1 adaptor (Ion Torrent; Life

211 Technologies) were used (Table S1). The PCR sample contained 10 µL of AmpliTaq Gold®

212
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360 Master Mix (Applied BiosystemsTM, Foster City, U.S.A.), 0.4 µL of forward primer, 0.4
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213 µL of reverse primer and 4 µL of purified first PCR product. Nuclease-free water was added
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214 to make up to a final volume of 20 µL. The second PCR cycle was 95 °C for 10 min, then 5

215 cycles at 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 1 min, followed by 72 °C for 7 min.
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216 The second PCR products were purified with the same method as above. The concentration
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217 of purified DNA was measured using Qubit dsDNA HS Assay Kit (Invitrogen, USA). The

218 final length and concentration of the amplicons were confirmed using a Bioanalyzer High
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219 Sensitivity DNA Kit (Agilent Technologies, Palo Alto, CA, USA).
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220
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221 The library was diluted to 50 pM using Low TE (Tris-EDTA; 10 mM Tris base, 0.1 mM

222 EDTA, Ion Torrent; Life Technologies). Ion Chef Instruments (Ion Torrent Life

223 Technologies, USA) with the Ion PGM Hi-Q Chef kit was used for the loading of the library

224 into the Ion 318 chip (Ion Torrent Life Technologies, USA).

225
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226 2.7. Analyses of the 16S rRNA based bacterial community structures

227 The obtained sequences were analyzed through Metagenomics 16S w1.1 in Ion Reporter.

228 Then, to analyze the changes in microbial community structure and their interactions with

229 soil environments, Detrended Correspondence Analysis (DCA) was conducted using the

230 diversity data at phylum or family level and environmental factors, including slag

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231 concentration (g slag g−1 soil), pH, concentration of NH4+-N and NO3−-N, and bacterial

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232 abundance. The importance of phyla or families that constituted <5% of each sample was

233 down-weighted. The value of DCA1 was less than 2.0 in each case, indicating that linear

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234 relationships existed between microbial abundance and environmental factors. Based on this

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235 result, Redundancy Analysis (RDA) was conducted using the square root-converted data of
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236 species proportions. The result of RDA is shown in a tri-plot.

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238 Heatmaps were constructed with the metagenomics data at phylum and family levels. Phyla

239 or families whose relative abundance was <1% on average are not shown on the heatmaps.
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240
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241 For the functional analysis of microbiota from 16S rRNA data, Phylogenetic Investigation of

242 Communities by Reconstruction of Unobserved States (PICRUSt) software was used


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243 (Langille et al., 2013). The analysis was conducted following the tutorial. Operational
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244 Taxonomic Units (OTUs) were prepared by eliminating all the OTUs that matched the
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245 GreenGenes 13_5 reference sequence with 97% similarity. The functions of each sample

246 were predicted from the normalized OTU tables referencing KEGG orthology values level 3.

247
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248 2.8. Statistical Analysis

249 For the concentrations and isotopic ratios of inorganic N, and the relative amounts of soil

250 bacterial DNA, two-way analysis of variance (ANOVA) was used to determine the effects of

251 the amount of heavy metal and the sampling date. One-way ANOVA and Tukey’s test was

252 performed for the analysis of significant differences among samples on each date or for each

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253 treatment. For microbial diversity analysis, the Shannon–Wiener index was calculated, and

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254 the significant effect of day and treatment was also investigated in the same way. All data are

255 presented as mean ± s.d. Statistical analysis was performed using R ver. 3.1.2 (R Foundation

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256 for Statistical Computing, Vienna, Austria).

257

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258 3. Results
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259 3.1. Dynamics of ammonium, nitrate and pH

As shown in Fig. 1a, NH4+-N concentration peaked on day 6 in the control treatment and on
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day 14 in the slag-treated variants. By day 28, NH4+-N concentration had decreased to 220
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262 mg N kg−1 dry soil in C and L and to around 310 mg N kg−1 dry soil in M and H. Statistically
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263 significant effects (p < 0.05) of the slag application rates were seen on all sampling days

264 except for day 0 (Table 2). The isotopic ratios of NH4+-N remained constant at about 3.0 ±
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265 0.7% in all samples and there were no significant differences throughout the incubation
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266 period (Fig. 1b).

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268 In all treatments, the extracted NO3−-N concentrations increased throughout the incubation

269 period (Fig. 1c). On all sampling days, treatment C contained the highest and the treatment H

270 contained the lowest level of NO3−-N. In contrast, there were no clear differences in the
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271 isotopic ratios in NO3−-N among the four treatments (Fig. 1d). However, the treatments with

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272 a higher concentration of slag (M and H) tended to have a lower N ratio compared to the

273 control.

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275 No significant differences were seen in total inorganic N concentration among the four

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276 treatments on all sampling days except on days 1 and 6 (Fig. S1, Table S2). On day 14, total

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277 inorganic N concentration reached a peak at about 600 mg N kg−1 in all treatments. The total

278 concentration decreased to about 440 mg N kg−1 on day 28. While concentrations were

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279 similar, the NO3−-N to NH4+-N ratio showed significant differences on days 14 and 28.

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281 There were no significant effects on pH from the treatments or the soil sampling times (Fig.

282 S2), although soil pH tended to decrease with increasing slag concentration. At the beginning
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283 of the incubation, soil pH ranged from 4.1 to 4.4 in all treatments. On day 6, the effect of the

284 slag was clearest; pH was about 5.2 in treatments C and L and 4.7 in M and H. In all
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285 treatments, pH rose to its highest on day 6 and decreased to around 4.2 by the end of the
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286 incubation.

287
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288 3.2. Soil bacterial abundance


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289 The relative abundance of soil bacteria tended to decrease with increasing slag concentration.
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290 However, this trend was only seen in the first 2 weeks and there was no difference among the

291 treatments by day 28 (Fig. S3).

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293 In the control treatment, on day 1, the relative abundance of bacteria was the highest among

294 all samples, after which abundance decreased continuously until the end of the experiment. In
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295 treatment L, bacterial DNA was highest on day 0 and then decreased over time. There were

296 peaks for treatments M and H on day 6. By day 28, the relative quantity of DNA was almost

297 the same in all treatments.

298

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299 3.3. Bacterial community structures

300 On average, 67,267 reads were mapped per sample for 16S rRNA. The following diversity

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301 and community analyses were conducted at phylum and family levels.

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302

303 The average Shannon–Wiener diversity index of each sample is shown in Table 3. No

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304 interaction effects of sampling day and treatment were seen (two-way ANOVA). There were
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305 no significant effects of slag application on diversity at either phylum level (p > 0.05) or

306 family level (p > 0.1). Treatment H had the lowest, and L the highest, diversity indexes while
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307 treatments C and M had similar moderate levels of bacterial diversity.


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309 3.3.1. Community structure analysis

310 Fig. S4 shows the percentage of each phylum in all DNA samples. Proteobacteria or
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311 Firmicutes were either the first or second largest phylum in all samples.
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312
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313 The result of RDA is shown in Fig. 2. The first component (RDA1) accounted for 62.7% of

314 the variation in the data and the second (RDA2) and third component (RDA3) accounted for

315 15.9% and 2.5%, respectively. Overall, RDA1 was positively correlated with the amount of

316 NH4+-N, and RDA2 was negatively correlated with the contamination level of slag containing

317 heavy metals. Using RDA with stepwise regression, slag concentration, pH, and NH4+-N

318 concentration were selected as significant explanatory variables.


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319

320 The bacterial communities in soil contaminated with >8.0 g slag kg−1 dry soil (M and H)

321 tended to have similar community structures, with relatively higher proportions of Firmicutes

322 (28% to 42%) and Actinobacteria (5.6% to 9.3%), and lower percentages of Planctomycetes

323 (<5.9%, except 28M2 (7.36%)). When sampling days were compared, the abundance of

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324 Bacteroidetes was relatively higher in samples taken on day 28. The samples lacking or with

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325 low amounts of slag (C and L) measured on day 0 were clustered into the same group (based

326 on pH) with a dominance of Proteobacteria.

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327

328 Heatmaps were constructed based on the relative abundance of each phylum and family (Fig.

329
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S5). Regarding community structure at phylum level, samples were clustered into three major
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330 groups; (1) C and L on day 0, (2) M and H on day 0, and (3) all groups on day 28. The
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331 patterns for each phylum indicated four major groups, with Firmicutes and Proteobacteria

332 clustered alone. Firmicutes appeared more frequently in treatments M and H than in C and L.
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333 The relative abundance of Proteobacteria was higher in samples on day 0. Other phyla were
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334 better represented on day 28, except for Armatimonadetes and Verrucomicrobia.

335
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336 At the family level, the effect of the slag was clearer than at phylum level. Treatments C and
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337 L, and M and H, were clustered together regardless of sampling day.


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338 Thermoanaerobacteraceae (Firmicutes) was the only family that showed a positive

339 association with the M and H treatments. Ectothiorhodospiraceae and Sphingomonadaceae

340 appeared more often on day 0 (although the proportion of the former decreased in treatments

341 M and H on day 28). Planctomycetaceae was clustered as the family that showed the clearest

342 negative correlation to slag concentration.

343
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344 3.3.2. Community functional analysis

345 Fig. S6 shows bacterial functional features with significant differences (p < 0.05) between

346 control and slag-containing treatments (those with sequence proportions <0.5% are omitted).

347 On days 0 and 28, the functions related to bacterial motility (“Bacterial motility,” “Flagellar

348 assembly”) were decreased in the slag-containing treatments. In contrast, “General functions,”

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349 “Pyruvate metabolism,” “Valine, leucine and isoleucine biosynthesis” and “Protein export”

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350 were increased in the slag-containing treatments. Some functions only featured in the results

351 on days 0 and 28. For example, on day 0, “Bacterial chemotaxis” and “Two-component

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352 system” were considerably lower in the contaminated samples, while the metabolism of some

353 amino acids and sugars (“Histidine metabolism,” “Starch and sucrose metabolism,” “Cysteine

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and methionine metabolism,” and “Galactose metabolism”) was higher in the slag treatments.
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355 On day 28, functions related to the metabolism of carboxylic acids (“Fatty acid metabolism,”
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356 “Propanoate metabolism,” and “Butanoate metabolism”) and carboxylates (“Glyoxylate and

357 dicarboxylate metabolism”) were high in slag-containing samples.


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358
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359 The proportions of sequences of N metabolism are shown in Fig. 3. Nitrogen metabolism

360 includes dissimilatory NO3−-N reduction, assimilatory NO3−-N reduction, denitrification, N


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361 fixation, nitrification, and anaerobic ammonium oxidation (anammox). Both on day 0 and 28,
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362 the relative abundance of bacteria that have genes related to N metabolism tended to decrease
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363 in the slag-contaminated soil. The impact was clearer on day 0 and there was a negative

364 correlation of relative abundance with slag concentration. On day 28, the differences between

365 the four treatments were reduced.

366
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367

368 4. Discussion

369 4.1. Slag effects on N dynamics

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370 The appearance of the peak concentration of NH4+-N was delayed in the slag-containing

371 treatments (L, M, and H) compared to the control treatment (Fig. 1a). This might be the result

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372 of the slag depressing urease activity through which NH4+ is released from urea added to the

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373 soil. Yang et al. (2006) investigated the effects of Cd, Zn, and Pb (in concentrations of 0–50,

374 0–800, and 0–1000 mg kg−1, respectively) on soil urease activity and reported that these

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375 heavy metals decreased urease activity. Heavy metal ions are known to react with the
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376 sulfhydryl groups on the active site of urease (Shaw, 1954) and metal ions including Mn2+,

377 Pb2+, Co2+, Cd2+, Ni2+, and Cu2+ have been reported to be urease inhibitors (Upadhyay, 2012).
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378 There is a possibility that urea was adsorbed by the slag, but the slag used in the current
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379 experiment contained very low levels of C (0.36%), so the urea adsorption capacity of the
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380 slag could be considered low. It has been reported for a variety of soils that C levels

381 positively influence urea adsorption capacity (Chin and Kroontje, 1962).
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382

383 We were unable to identify the substance or element within the slag that had impacted the N
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384 cycle in the current experiment. The slag contained various metals (Section 2.1). Several
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385 preliminary experiments were performed to test the impact of pure heavy metal compounds,

386 such as PbNO3 and Pb(CH3COO)2, on N cycle activities in soils. However, we concluded that

387 the presence of C and N in the test compounds influenced N cycle activities. Thus, the pure

388 heavy metals were not used in the current study and this study aimed to identify the potential

389 effect of the slag accumulating in the open air, and the mixing ratios of slag to soil were

390 determined from these field data (Ikenaka et al., 2010).


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391

392 In the current study, most of the extracted NH4+-N was derived from the applied urea since
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393 the isotopic ratio was constant at around 3 atom%, the same value as the N levels of the

394 added urea (Fig. 1b). Thus, soil N was not contributing to the soil NH4+-N pool, and it was

395 urea hydrolysis that was likely controlling the increase of NH4+-N. If so, urea hydrolysis

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396 might have been delayed due to inhibition of urease activity by the slag treatments in this

experiment. The amount of NH4+-N was 200–300 mg N kg−1 soil even at the end of the

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397

398 current experiment, whereas the original concentration of soil NH4+-N was <20 mg N kg−1

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399 soil (Table 2, day 0). The relatively constant N ratio of NH4+-N in the current experiment

400 may simply have been due to the relative excess of NH4+-N from the applied urea compared

401
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to NH4+-N from soil N. Also, the soil was pre-incubated for two weeks, and the active
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402 mineralization of soil N might have ceased before the commencement of the experiment.
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403 Previous studies have reported soil N mineralization during urea hydrolysis due to increasing

404 soil pH (e.g., Tong and Xu, 2012) but we did not observe an increase in soil pH during the
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405 current experiment, probably due to the buffering capacity of the soil.
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406

407
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408 Increasing amounts of slag negatively influenced the accumulation of NO3−-N in the soils
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409 (Fig. 1c, Table 2). Compared to the effects of slag on NH4+-N concentration, the effect on
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410 NO3−-N was more significant, suggesting that the slag influenced nitrification more than

411 ammonification. This inference is in agreement with the study by Rother et al (1982) who

412 reported that nitrification was considerably more sensitive than ammonification to Cd, Zn and

413 Pb. They studied the effects on soil of Cd, Zn and Pb at concentrations of 1000‒5000, 1000‒

414 5000 and 10,000‒20,000 mg kg−1, respectively. Ueda et al (1988) investigated the effects of

415 anionic heavy metals, such as CrO42−, MoO42−, WO42−, and VO3−, on ammonification and
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416 nitrification and found similar tendencies. This study suggested that the different sensitivities

417 of the two processes to heavy metals may be due to the difference in the number of reaction

418 steps between nitrification and ammonification. One chemical reaction step is needed to

419 convert urea to NH4+-N whereas two steps are required to nitrify NH4+-N to NO3−-N (NH4+

420 → NO2− → NO3−). Heavy metal inhibition of nitrification in wastewater treatment plants has

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421 been well studied and it is reported that the oxidation of NH4+ to NO2− by ammonia oxidizing

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422 bacteria is thought to be the most sensitive step in the process (EPA, 1993). Stephen et al

423 (1999) showed ammonia and NH4+ accumulated in the presence of toxic metals (Cd, Co, Cs

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424 and Sr) in soil and referred to the negative influence of these metals on ammonia oxidation.

425 Radniecki and Ely (2008) investigated both ammonia-dependent and hydrazine-dependent

426
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specific oxygen uptake rate in cell suspensions of an ammonia oxidizing bacteria species and
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427 reported that an increase in Zn2+ inhibited ammonia oxidation, the first step of nitrification.
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428 On this basis we concluded that the slow accumulation of NH4+-N and the reduced

429 concentration of NO3−-N in the slag-containing treatments seen in the current study were due
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430 to relatively stronger inhibition of ammonia oxidation by heavy metals compared to that of
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431 the urease activity, which converts urea to NH4+-N.

432
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433 In the slag-contaminated soils, the ratio of NH4+-N to NO3−-N rose compared to the control.
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434 However, the total amount of inorganic N decreased in all of the treatments to a similar level
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435 from day 14 to 28 (Fig. S1). Since the total amount of inorganic N (NO3−-N plus NH4+-N)

436 was the same in all treatments for each day (day 14 and 28), the mechanism behind the loss

437 of inorganic N during this period was apparently different between the less contaminated

438 soils (soil C and L) and the heavily contaminated soils (soil M and H). Processes such as

439 denitrification and immobilization, related to the loss of inorganic N from soils, is a topic

440 recommended for future study.


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441

442 The concentration of NH4+-N can decrease as a result of immobilization. Yet, as described

443 below, bacterial relative abundance decreased in the treatments with higher slag

444 concentrations, indicating that the NH4+-N might not have been actively immobilized in the

445 presence of the slag. The loss of NH4+-N in the slag-contaminated treatments in the current

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446 experiment was more likely due to NH3 volatilization rather than immobilization. Kim et al.

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447 (2012) reported that nitrification inhibitor application increased the NH3 loss from soil. It is

448 probable that the heavy metals in slag inhibited nitrification and that accumulated NH4+-N

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449 volatilized as NH3 rather than becoming NO3−-N and remaining in the soil. From this, we

450 conclude that the mechanism for the loss of inorganic N was different in contaminated and

451
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uncontaminated soils although further studies are needed to confirm this.
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452
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453 There was a tendency for the isotopic ratio of NO3−-N to be lower in slag-treated variants

454 (especially H), though the differences were not statistically significant (Fig. 1d). The decrease
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455 in isotopic ratio was correlated to the decrease in concentration of NO3−-N in general. Dai et
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456 al (2004) reported that net N mineralization was lower in the presence of elevated levels of

457 risk elements in a pasture soil. In the current study, the contribution made by mineralized soil
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458 organic N to increase the inorganic N pool was small, even in the control soil. Thus, a
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459 negative effect of slag on soil N mineralization was not clearly demonstrated in the current
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460 study.

461

462 4.2. Slag effects on the abundance of bacterial DNA

463 A marked increase in the relative abundance of bacterial DNA was observed from day 0 to 1

464 only in the control treatment (Fig. S3). Thus, in the absence of slag, soil bacteria responded
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465 positively to the urea application over a short period. Previous reports have stated that the soil

466 microbial population tends to decrease with an increase in heavy metal concentration (Bååth,

467 1989). This is due to a decrease in substrate utilization efficiency as a result of a high energy

468 cost metabolism against metal stress (Chander and Joergensen, 2001). The relatively lower

469 abundance of bacteria in the control treatment on day 0 might be due to nutrient deficiency

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470 caused by higher microbial activity during the pre-incubation period.

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471

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472 4.3. The explanation from the change in bacterial community structure

473 4.3.1. Diversity analysis

474
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Slag application did not change bacterial diversity significantly at either phylum or family
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475 level (Table 3). Microbial diversity in the sample with a low amount of slag (L, about 0.2%
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476 by weight) was the highest among all treatments; as the amount of slag increased in

477 treatments M and H, the bacterial diversity tended to decrease. This result is consistent with a
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478 previous study in which a low application rate of biosolids (containing heavy metals)
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479 increased soil microbial diversity while at high rates diversity decreased (Mossa et al., 2017).

480 This increase in microbial diversity with a low level of biosolid application was due to the
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481 high organic matter content of the biosolid. In the current study, in treatment L, the bacteria
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482 present in the applied slag might have grown in the soil to produce the relatively higher
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483 diversity compared to the control. The presence of a low level of slag might also act as a

484 moderate environmental stress, decreasing competitive exclusion, and leading to the observed

485 increase in diversity (Giller et al., 1998). As the concentration of heavy metals in the soil

486 increased (treatments M and H), more bacterial species might have died due to greater stress.

487 In the presence of an environmental stress, bacterial diversity is considered to decrease as a

488 result of the death of some intolerant species and the prospering of more tolerant species
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489 (Atlas, 1984). This is consistent with many previous studies reporting that bacterial diversity

490 tends to be lower in heavy metal-contaminated soil (Sandaa et al., 1999, Xie et al., 2016).

491

492 Bacterial diversity tended to be higher on day 28 than on day 0 in all treatments. The

493 application of urea, from which NH4+-N is easily released, might have improved the growth

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494 of bacterial species that were depressed due to substrate depletion.

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495

496

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4.3.2. Community structure analysis

497 RDA showed that pH, the amount of NH4+-N, and slag concentration were the environmental

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498 factors that most influenced bacterial community structure. However, the pH fluctuation
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499 observed during the incubation period ranged from 4.1–5.3, thus other potential factors

500 influenced by the addition of the slag have to be investigated in future, such as changes in
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501 physical properties of the soil. Also, it is possible that microbes were introduced with the slag,

thereby influencing the microbial communities in the soil. The high levels of NH4+-N
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502

503 released from urea on day 28 might explain why all samples (except 28C1) taken on day 28
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504 had high RDA1 values (Fig. 2). The relative increase of Crenarchaeota, Chloroflexi,
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505 Gemmatimonadetes, and Bacteroidetes on this day was due to this NH4+-N abundance.

506 Among these phyla there were some containing species known to conduct ammonium
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507 oxidation (e.g., Crenarchaeota) (Nicol et al., 2006) or nitrite oxidation (e.g., Chloroflexi)
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508 (Udert et al., 2005). The abundance of NH4+-N in the environment might have favored those

509 species that oxidize NH4+ and NO2−.

510

511 The abundance of Firmicutes was positively correlated with slag concentration while that of

512 Planctomycetes was negatively correlated (Fig. 2). Ellis et al (2003) reported that Firmicutes

513 (mainly Bacillus spp.) exhibit high relative abundance in slag-contaminated sites. Liu et al
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514 (2018) also found that this phylum is among the five most abundant phyla in sediment

515 polluted with heavy metals. In the current study, the high proportion of

516 Thermoanaerobacteraceae (Firmicutes, Clostridia) with increasing slag concentration

517 explains the increase in abundance of Firmicutes (Fig. S7a). This family is mainly isolated

518 from hot springs and is known for its strictly anaerobic, rod-shaped, spore-forming character

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519 (Stackebrandt, 2014). There are few other studies published to date about the environments

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520 occupied by this bacterial family. The ability to form spores might be an advantage for these

521 microbes, enabling them to survive in environments with heavy metal stress. A study by

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522 Šmejkalová et al (2003) showing an increase in spore-forming bacteria in heavy metal-

523 contaminated soil supports this hypothesis. Another factor potentially influencing bacterial

524
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survival under heavy metal stress is sequestration ability. Previous studies suggest that in
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525 heavy metal-contaminated sites, gram-negative bacteria are more widespread than gram-
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526 positive bacteria because the cell walls of gram-positive bacteria are better able to bind metal

527 cations (Evans and Furlong, 2003, Çolak et al., 2011). Also, Bacillus spp seen in metal-
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528 stressed environments can act as a biosorbent (Volesky and Holan, 1995). The mechanism of
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529 heavy metal resistance of members of the Thermoanaerobacteraceae requires further

530 investigation.
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531
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532 The major proportion of extracted 16S rRNA originated from Planctomycetes, and RDA
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533 results showed that they were negatively affected by the heavy metals. Fuerst and Sagulenko

534 (2011) reported several unique features of this phylum, such as peptidoglycan-lacking

535 proteinaceous cell walls, and intracellular membranes that form separate compartments

536 within the cell cytoplasm. These unique features might make this phylum especially sensitive

537 to heavy metals. Bacterial resistance mechanisms, such as the excretion of metals out of the
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538 cell (Silver et al., 1996), have been previously reported, but further study is needed to

539 understand the susceptibility of this phylum to heavy metals.

540

541 In Planctomycetes, there are families that conduct anaerobic NH4+-N oxidation (anammox)

542 and which possess distinctive cell structure, for example Candidatus. Anammox is known to

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543 be inhibited in the presence of heavy metals (Jin et al., 2012). Hu et al. (2011) and Xi et al.

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544 (2016) investigated anammox bacteria in agricultural soil and forest soil, respectively, and

545 reported Candidatus Brocadia fulgida and Candidatus Jettenia asiatica as being the

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546 dominant anammox phylotypes. In the current study, it was found that Candidatus

547 Brocadiaceae tended to decrease as the proportion of slag in the soil increased (Fig. S7b).

548
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Thus, it could be expected that the presence of heavy metals decreased the proportion of this
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549 phylum and family and, although the total amount of N removed by their anammox activity
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550 would not have been large, this could have had an influence in the slag-contaminated soil.

551
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552 Both environmental factors and species relationships may change the proportion of a species
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553 in the community (Perez-Garcia et al., 2016), therefore it is important to further investigate

554 the reasons behind these changes, and to ask whether they are the direct result of the effects
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555 of the slag or are driven by microbial metabolism interactions.


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556
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557 4.3.3. Community function

558 There were influences of both the treatments and the sampling days on the bacterial

559 community functions. Some previous studies reported that the motility of specific bacterial

560 species was inhibited in the presence of heavy metals (Adler and Templeton, 1967, Singh et

561 al., 2014). In their study, Singh et al concluded that the presence of Cd2+ and Pb2+ ions

562 prevented flagella development of a metal-resistant bacterium (strain CM100B). This agrees
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563 with the current results that showed a decrease in DNA sequences related to bacterial motility

564 in the more slag-containing treatments (Fig. S6).

565

566 The current study demonstrated that some general metabolic functions, such as pyruvate

567 metabolism, were relatively high in the slag-containing samples both on day 0 and day 28

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568 (Fig. S6). Bacteria resistant to heavy metals have various detoxification strategies, such as

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569 intra- or extracellular sequestration, export and permeability barriers (Rouch et al., 1995).

570 These mechanisms involve special biochemical processes seen only under metal stress, for

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571 example membrane transport protein expression and increased ATP consumption (Nies,

572 1999). The relatively active functions seen in the contaminated treatments in this study may

573 be related to these mechanisms.


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574
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575 On day 0, very significant differences between the control and the slag-treated samples were

576 found especially in bacterial functions related to motility (such as "Bacterial chemotaxis" and
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577 "Bacterial motility proteins") and repair ("DNA repair and recombination protein"). In
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578 contrast, on day 28, the functions showing significant differences between the control and the

579 slag-treated samples were related to fatty acid metabolism ("Fatty acid metabolism,"
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580 "Butanoate metabolism," "Propanoate metabolism") (Fig S6). One reason for the difference
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581 might be due to the application of urea. On day 28, with a high level of inorganic N, bacterial
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582 groups favoring high N conditions might have become active and the effect of the slag on

583 these groups was different from that on microbes present at the beginning of the experiment.

584

585 The functional analysis also suggested negative impacts of the slag on N metabolism by

586 bacteria (Fig. 3). From the results, we cannot determine which particular reaction(s) within

587 the N cycle were more sensitive than others. Yet, the proportion of N metabolizing bacteria
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588 tended to decrease in the slag-containing treatment, even in the presence of a high level of

589 inorganic N (day 28).

590

591 Kandeler et al (2000) examined the impact of heavy metals on soil microbial biomass,

592 enzyme activity (urease, alkaline phosphatase, and xylanase), and microbial community

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593 structure, concluding that microbial community structure was the least affected of the three.

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594 In our study, we found that the slag containing heavy metals influenced the bacterial

595 community structure, as well as the N dynamics and the relative abundance of bacteria. The

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596 slag did not affect the bacterial diversity significantly, but it influenced the bacterial

597 community structure and functions. This is consistent with the study by Li et al (2017) that

598
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concluded long-term heavy metal (Cd, As, Pb, and Zn) contamination had a greater effect on
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599 microbial composition than on diversity, although in our study, no significant tolerance by
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600 archaea (ex. Crenarchaeota) was seen. Changes in bacterial community structure may

601 influence bacterial functions in N cycling. Nutrient cycling in slag-contaminated sites may be
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602 influenced by a combination of changes in bacterial community structure and abundance.


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603
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604 5. Conclusion

605 This study suggests that slag from a mining site containing heavy metals (Pb and Zn) had a
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606 strong negative impact on nitrification, more so than on ammonification. Due to the
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607 disturbance of the nitrification process, the accumulation rate of NO3−-N was slower in slag-

608 contaminated soils. Slag applied to soil at 4.9% by weight caused a ~50% reduction in the

609 accumulation of NO3−-N compared to the control four weeks after the application of urea, an

610 outcome that could limit the growth of plants dependent on NO3−-N. Soil bacterial abundance

611 tended to be lower with increasing slag concentration. Soil bacterial community structure was
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612 significantly influenced by the slag; as the slag to soil ratio increased, the relative abundance

613 of Firmicutes increased and that of Planctomycetes decreased. Families within

614 Planctomycetes that conduct anammox also considerably decreased. The change in microbial

615 community structure affected community functions including N metabolism. Our study

616 suggests that slag mixed with soil influences soil microbial community structure, and

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617 consequently the N cycle-related soil functions. Future research on the effect of slag on

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618 microbial community structure, the N cycle in the soil, and their relationships and

619 mechanisms is recommended.

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620

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621 Acknowledgement
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622 This work was supported by Science and Technology Research Partnership for Sustainable

623 Development (SATREPS) “Visualization of Impact of Chronic/Latent Chemical Hazard and


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624 Geo-Ecological Remediation.”


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625
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Table 1

Incubation setup C L M H
Dry soil (g) 45.0 45.0 45.0 45.0
Slag (g) 0.0 0.1 0.4 2.2
Pb (mg/kg) 0.0 100.0 500.0 3000.0
Zn (mg/kg) 0.0 54.0 270.1 1620.6

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Volume (cm3) 50.0 50.1 50.4 52.4
WFPS (%) 60.0 60.0 60.0 60.0
Urea (mg) 48.2 48.3 48.6 50.5

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Table 2

Property Treatment Date


0 1 6 14 28
NH4+-N C 10.4 (0.3) a 113.2 (7.8) a 518.4 (12.3) a 446.9 (16.3) a 225.9 (0.6) a
L 14.1 (5.5) a 95.9 (2.9) ab 462.4 (17.3) ab 522.0 (22.1) ab 222.2 (8.1) a
M 13.4 (1.8) a 92.3 (5.4) ab 426.5 (12.5) b 467.4 (14.4) ab 328.6 (16.0) b

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H 15.6 (1.0) a 86.1 (2.4) b 446.9 (16.3) b 520.2 (9.5) b 305.8 (3.7) b
ANOVA n.s * * * ***

NO3 -N C 16.4 (0.4) a 17.6 (0.5) a 72.1 (3.4) a 140.7 (2.0) a 221.0 (7.0) a

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L 16.0 (0.1) ab 17.6 (1.0) a 57.3 (4.6) ab 96.3 (2.5) b 208.7 (1.6) a
M 14.6 (0.7) ab 15.4 (0.4) a 47.2 (3.8) b 124.8 (1.8) c 146.3 (0.7) b

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H 13.3 (0.5) b 15.1 (0.4) a 44.4 (0.5) b 67.3 (2.4) d 124.6 (5.4) c
ANOVA * n.s ** *** ***
Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05

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Table 3

Shannon-Wiener diversity index (H')


Treatment Phylum Family
0 28 0 28
C 1.63 (0.04) 1.80 (0.08) 3.10 (0.04) 3.21 (0.08) ab
L 1.70 (0.03) 1.87 (0.04) 3.24 (0.02) 3.33 (0.10) a

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M 1.63 (0.02) 1.80 (0.02) 3.24 (0.02) 3.23 (0.07) ab
H 1.56 (0.02) 1.78 (0.03) 2.97 (0.05) 3.20 (0.10) b

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Day *** *
Treatment *** *

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Day:Treatment *** *
Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05

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(a) (b)
6.0

5.0

4.0
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3.0 L
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Day *** Day ***

ratio in NH4+-N (%)


C Treatment n.s 1.0 Treatment n.s

15N
Day:Treatment *** Day:Treatment n.s
0.0
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Day *** Day n.s
Treatment *** Treatment n.s
Day:Treatment *** Day:Treatment n.s
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Figure 1. The time course changes of the concentration and 15N ratio of ammonium (a and b) and nitrate (c and d). The x axis represents days
after the application of slag on the soil. C, L, M and H stand for the control, low, medium, high amount of slag mixed into the soils,
respectively. The results from two-way ANOVA were shown on the right bottom of each figure. “***” and “n.s.” stand for the significance
level of p<0.001 and not significant, respectively.
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Figure 2. RDA plot of the bacterial community structures. Blue arrows are influential environmental factors
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and the red letters were phylum names. The black letters were the sample names. The sample names represent
sampling days, treatment, and the replicate number. For example, "0L2" is the sample taken from the soil of
replicate number 2 with low Pb concentration (100 mg-Pb/kg dry soil) on day 0. Similarly, C, M and H
represent the soil samples received control, medium and high levels of the slag, respectively.
(a) (b)

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Figure 3. The proportion of the sequences (%) of N metabolism (dissimilatory NO3--N reduction, assimilatory NO3
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--N reduction, denitrification, N fixation, nitrification, and anammox) within the soil bacterial community, on day 0
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(a) and 28 (b). The treatment (x axis) C, L, M and H stand for the control, low, medium and high level of slag
application rates to the soils, respectively.
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1 Highlight

2 ● Nitrification was inhibited in the soil contaminated with slag containing Pb and Zn.

3 ● Slag did not alter bacterial diversity but changed community structure and function.

4 ● Slag increased the proportion of Firmicutes and decreased that of Planctomycetes.

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5 ● Bacterial DNA related to nitrogen metabolism was decreased in the presence of slag.

6 ● Bacterial community structure may influence the N cycle in the sites with slag.

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