Improvement of Soil Ecosystem Multifunctionality by Dissipating

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Improvement of soil ecosystem multifunctionality by dissipating manure-induced antibiotics and resistance genes Yuting Liang, Meng Pei, Dandan Wang, Shengnan Cao, Xian Xiao, and Bo Sun Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b00693 • Publication Date (Web): 10 Apr 2017 Downloaded from http://pubs.acs.org on April 11, 2017

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Environmental Science & Technology

Improvement of soil ecosystem multifunctionality by dissipating manure-induced antibiotics and resistance genes Running title: Improvement of soil EMF by dissipating of antibiotics

Yuting Liang1*, Meng Pei1,2，Dandan Wang1,2, Shengnan Cao2, Xian Xiao1,3, Bo Sun1 1

State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China.

2

School of Environmental and Safety Engineering, Changzhou University, Changzhou, Jiangsu, 213164, China

3

University of the Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China.

*To whom correspondence may be addressed: Yuting Liang Address: No. 71 East Beijing Road, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China Phone: (86-25)86881534 Fax: (86-25)86881000 E-mail address: [email protected]

The authors declare no competing financial interest.

ACS Paragon Plus Environment


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1

ABSTRACT:

2

The application of animal manure containing antibiotic residues to farmlands as an organic fertilizer

3

causes a long-term potential threat to the ecological environment of farmland. This study analyzed

4

the effects of abating typical antibiotics and resistance genes (ARGs) applied with pig manure on

5

farmland soil as well as on soil ecosystem multifunctionality (EMF) and its influencing factor. The

6

results showed that Lolium multiflorum exhibited significantly stronger abatement of typical

7

antibiotics and ARGs when combined with biochar rather than when used alone (p < 0.05). The

8

dissipation of antibiotics significantly enhanced the soil functions (respiratory, ammonification, and

9

nitrification activities) (p < 0.05). A structural equation model was established to explore the effects

10

of abating antibiotics and ARGs in different treatment systems on soil EMF. The treatment of plant

11

roots with ryegrass alone and in combination with biochar exerted direct positive effects on the

12

physical structure and EMF (p < 0.001). The improvement in soil physical structure directly

13

promoted the abatement of antibiotics and ARGs (p < 0.01). Soil pH and trace elements exerted

14

weaker effects on antibiotics and ARGs after the application of biochar. Plant roots were the most

15

important factor in promoting the EMF of soil containing antibiotics and ARGs.

16

Key words: antibiotics, ARGs, soil ecosystem multifunctionality, ryegrass, biochar

17

1. INTRODUCTION

18

Antibiotics are widely used in the treatment of diseases and animal growth

[1]

. In the United

19

States, 227 thousand tons of antibiotics are produced annually, of which 17.8%–70% is used for

20

livestock and poultry breeding [2]. In China, more than 80 thousand tons of veterinary antibiotics are

21

available for livestock and poultry breeding, of which the usage amount of tetracycline is the

22

highest

[3]

. Animals cannot completely adsorb and metabolize the ingested antibiotics.


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23

Approximately 25%–75% of antibiotics are excreted into the environment in the form of parent

24

compounds [1]. The application of animal manure containing antibiotic residues to farmlands as an

25

organic fertilizer causes a potential long-term threat to the farmland ecological environment.

26

Consequently, environmental pollution and its ecotoxicological effects have become a major

27

problem worldwide. Residual antibiotics in farmland soil inhibit the growth of crop roots and soil

28

microorganisms [4-6]. In addition, residual antibiotics in the soil can induce bacterial drug resistance.

29

The antibiotic resistance genes (ARGs) that are generated enter the soil, water, sediment, and other

30

environmental media through migration and transformation, severely destroying the diversity and

31

stability of ecosystems [7]. Therefore, a thorough understanding not only of the dynamic abatement

32

process of soil antibiotics and ARGs but also the underlying mechanism is extremely urgent.

33

It has been reported that plants can absorb and abate antibiotics in the soil and that plant roots

34

show the highest absorption capacity [8]. Vegetable crops such as carrot and lettuce exhibit different

35

degrees of adsorption of antibiotics [9, 10]. At the same time, positive correlations between antibiotics

36

and ARGs were found

37

direct method for abating ARGs in the soil. Recent studies have reported that floating beds formed

38

by water spinach and cress promote the abatement of ARGs in water environments

39

the abatement and mechanism of ARGs in the soil by plants require further investigation because of

40

the complex nature of soil biological and abiotic conditions.

[11, 12]

. Thus, the use of plants to absorb and decrease antibiotics may be a

[13]

. However,

41

The dissipation mechanism of antibiotics and ARGs in soil is mainly affected by the chemical

42

properties of pollutants, soil structure, physical and chemical features, microorganism activities, and

43

many other factors. Du et al. [14] found that abatement of sulfonamides in the soil is slow, whereas

44

that of fluoroquinolones and β-lactam antibiotics is fast, which is mainly related to their type and



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molecular structure. The physical and chemical properties of soil, such as organic matter content,

46

porosity, humidity, temperature, and pH, influence the abatement of antibiotics and ARGs. The

47

abatement rate of multiple antibiotics is faster in sandy loam soils than in sandy soils

48

al. [16] concluded that soil pH is highly correlated with ARGs such as blaCTX and ermB, and that an

49

alkaline environment is conducive to the spread of ARGs. Moreover, Liu et al.

50

microbial activity affects the abatement of antibiotics. Through metabolism, microorganisms can

51

produce enzymes that directly or indirectly modify the structure of antibiotics and inactivate the

52

drugs. At present, whether plants can indirectly abate antibiotics and ARGs by changing the

53

physical, chemical, and biological properties of soil is unclear.

[17]

[15]

. Knapp et

showed that soil

54

Lolium multiflorum is a high-yielding perennial ryegrass of the Gramineae family. Ryegrass is

55

characterized by rapid growth, large biomass, strong regeneration ability, and easy cultivation [18]; in

56

addition, this species exerts an enrichment effect on organic pollutants

57

dissipate veterinary antibiotics in swine wastewater by up to 89%–99% [21]. However, the abatement

58

effect of ryegrass on antibiotics and ARGs in soil has yet to be evaluated. Previous studies have

59

reported that biochar can effectively improve the physical and chemical properties of soil. For

60

example, biochar can effectively neutralize soil pH; promote soil microbial activity; and increase

61

soil air flux, adsorption capacity, and rhizosphere water retention[22, 23]. However, the application of

62

biochar in the soil environment and its effects on plants and microorganisms are still controversial.

63

It was found that tiny particles of biochar can enter soil pores, thereby increasing soil volume and

64

decreasing water permeability

65

ARGs in the soil has yet to be clarified. In addition, the effect of the combination of biochar and

66

plants on the abatement of antibiotics and ARGs as well as on the soil ecosystem multifunctionality

[19, 20]

. Ryegrass can also

[24, 25]

. Whether biochar promotes the abatement of antibiotics and


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67


(EMF) has yet to be studied.

68

Based on a survey on antibiotic distribution in pig manure and in soil containing pig manure in

69

early stages of our experiment, we selected eight common antibiotics (i.e., tetracycline,

70

chlortetracycline, doxycycline, sulfamethazine, enrofloxacin, lomefloxacin, ciprofloxacin, and

71

norfloxacin) and 10 ARGs (tetM, tetQ, tetO, tetW, tetM, sul1, sul2, gyrA, qnrA, and ermF) as target

72

objects to study the dynamic abatement process of antibiotics and ARGs in soil treated with ryegrass

73

alone or in combination with biochar. This paper aims to determine the following unknowns: (i) the

74

abatement effects of biochar–ryegrass treatment on antibiotics and ARGs in the soil; (ii) the

75

physical, chemical, and biological mechanisms underlying the abatement of antibiotics and ARGs;

76

and (iii) the major factors influencing the abatement of antibiotics and ARGs and improvement of

77

soil EMF.

78

2. MATERIALS AND METHODS

79

2.1 Extraction and determination of antibiotics in soil. Pig manure samples were obtained

80

from a pig farm of a livestock and poultry breeding company in Changzhou, Jiangshu (119.75°E,

81

31.73°N). Soil samples without antibiotics were collected within 10 cm of surface soil from an

82

experimental rice farm of the same company. The pig manure and soil samples were air-dried,

83

crushed with wooden roller, and then sieved through a 2-mm mesh. Homogeneous soil and pig

84

manure were mixed in a proportion of 100:1 (mass ratio). The mixture was allowed to stand for 24 h,

85

stirred, and then allowed to stand again for another 24 h. Finally, the soil was transferred to a plastic

86

pot (17 cm diameter, 15 cm height, and 3 kg of soil per pot) and lined. Ryegrass seeds were obtained

87

from the Forage Research Institute of Jiangsu Academy of Agricultural Sciences. Biochar samples

88

were prepared by pyrolysis and carbonization of wheat stalks at 650 °C under anoxic conditions.



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A total of 81 potted plants were used in this experiment and divided into three groups (n = 27):

90

control, ryegrass, and biochar–ryegrass (biochar was added evenly to ryegrass potted soil equal to 2%

91

of the total amount). Irrigation was performed every 2–3 days with deionized water and soil water

92

contents were maintained at 60% water-holding capacity. Drainage water was captured in a bottom

93

tray and added back to the pots. The pots were placed in a plant growth chamber under the

94

following conditions: 16 h of light (day) and 8 h of darkness (night), and 25 °C temperature. The

95

pots were randomized. At 0, 5, 10, 15, 20, 25, 30, 35, and 40 days after germination, ryegrass was

96

removed from the pots (for the control potted plant, a soil sampler was used to collect soil evenly in

97

multiple spots). Three replicates were removed for per sampling day. Ryegrass was removed gently

98

to separate the root system from the bulk soil. Then, the shake-off method was used to collect

99

root-zone soil, and a small sterile shovel was used to scrape the soil tightly adhering to the roots.

100

Both the root-zone soil and adhering soil were combined and used for further analysis. The collected

101

rhizosphere soil was used to determine antibiotic contents, soil ecosystem functions (respiration,

102

ammonification, and nitrification), and physical and chemical properties. Ryegrass plants collected

103

on days 10, 20, 25, and 35 were maintained to determine the morphological parameters of ryegrass

104

root surfaces.

105

Solid-phase extraction was employed to extract antibiotics from soil

[26]

. Five grams of soil

106

sample and 3 mL of methanol/EDTA (1:1 volume ratio) buffer solution were added to a 50 mL

107

centrifuge tube, followed by oscillation of the tube. The centrifuge tube was subjected to ultrasound

108

extraction for 10 min and then centrifuged at 5000 r/min for 10 min. The supernatant was then

109

collected. The above method was used to extract the residues four times. After combining the four

110

supernatants, the solution was filtered through a 0.75-µm fiber membrane to remove the large


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111

particles in the solution and dried with nitrogen gas in a water bath at 80 °C. The concentrated liquid

112

was filtered through a solid-phase extraction column. The constant volume of the solution was 10

113

mL. The solution was collected into a sample bottle for later tests after filtering through a 0.22-µm

114

membrane. Liquid chromatography (Agilent 1290, USA) coupled with tandem mass spectrometry

115

(MS/MS; TSQ bar, Water TQ Detector, USA) was used to qualitatively and quantitatively detect

116

antibiotics in the soil. The detection column was a ZORBA×RRHD Eclipse Plus C18 (2.1×50 mm,

117

1.8 µm). Liquid chromatography conditions were as follows: sample volume of 10 µL, flow speed

118

of 0.2 mL/min, column temperature of 30 °C, and detection time of 30 min. The mobile phase of

119

tetracycline antibiotics and sulfonamide antibiotics was A: water+0.3% formic acid, B:

120

methanol+0.1% formic acid; the mobile phase of quinolones was A: water+0.2% ammonia, B:

121

methanol+0.1% ammonia. The above method was used to measure the content of antibiotics in the

122

soil in preliminary tests. After the antibiotic standard solution was added, the recovery rate was

123

calculated as follows: recovery rate = (measured value − blank value)/additive amount. Preliminary

124

test results showed that the standard recovery rate of antibiotics using this method was high, with a

125

more stable effect for extracting different antibiotics. The standard recovery rates of tetracyclines

126

(doxycycline, tetracycline, and chlortetracycline), sulfanilamides (sulfamethazine), and quinolones

127

(enrofloxacin, lomefloxacin, ciprofloxacin, and norfloxacin) were 73.2%–93.4%, 81.2%–101.1%,

128

and 106.3%–110.8%, respectively. Thus, this method meets the requirements of antibiotic extraction

129

and determination. In addition, concentrations of the heavy metals copper and zinc in the manure

130

and soil were determined using flame atomic absorption spectrometry (Varian Spectra AA 220,

131

USA). The concentrations of copper and zinc in the manure were 771.0 and 1698.3 mg/g dry weight,

132

respectively. The final concentrations of copper and zinc in the soils were 69.9 and 38.5 mg/g soil,



133

respectively.

134

2.2 Quantitative PCR analysis of ARGs. Microbial genomic DNA was extracted from 5 g of

135

well-mixed soil for each sample by combining freeze grinding and sodium dodecyl sulfate for cell

136

lysis as previously described

137

tetM, tetO, tetQ, and tetH; sulfonamide resistance genes: sul1 and sul2; quinolone resistance genes:

138

gyrA and qnrA; and macrolide resistance gene: ermF) were made by ligating PCR products into a

139

pEASY-T3 Cloning Kit (TransGen) and then transformed into a Trans1-T1 Phage-Resistant

140

Chemically Competent Cell (TransGen), as described in the manufacturer’s manual. Plasmids

141

carrying the target genes were extracted using a TIAN Pure Midi Plasmid Kit (Tiangen) and used as

142

standards for quantitative PCR. All qualitative PCR assays were conducted in a 50-µL reaction

143

using an ABI 2720 Thermocycler (Applied Biosystems, USA) to detect ARGs in the soil. The ARG

144

primers and 16S rRNA used in the PCR are shown in Table S1. The PCR mixture consisted of 5 µL

145

of 2× Ex Taq buffer, 3 µL of 25 mM Mg2+, 1 µL of dNTPs (10 mM each), 2 µL of forward/reverse

146

primers (10–20 pmol), 1 µL of Ex Taq DNA Polymerase (5 U/µL), 1 µL of template, and 35 µL of

147

ddH2O. The thermocycler was programmed as follows: denature at 94 °C for 4 min; 35 cycles of 30

148

s at 94 °C, 30 s at different annealing temperatures and 30 s at 72 °C; and a final extension step for 7

149

min at 72 °C. The annealing temperature varied depending on the target gene: 55 °C was used for

150

tetW, tetM, tetO, tetQ, tetH, gyrA, and qnrA, and 60 °C was used for sul1, sul2, and ermF as well as

151

the 16S rRNA. To ensure reproducibility, triplicate PCRs were performed for each sample. Sterile

152

water was used as a negative control in every reaction.

[27]

. Standards of eleven ARGs (tetracycline resistance genes: tetW,

153

The levels of the ARGs and 16S rRNA gene copies were determined by qPCR using a CFX96

154

Real-time PCR System (Bio-Rad). The qPCR primers for ARGs and the 16S rRNA gene were the


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same as those used in the qualitative PCR. One 25-µL qPCR mixture contained 12.5 µL of SYBR®

156

Green Premix Ex Taq (Takara, Japan), 0.5 µL of forward/reverse primers (10–20 pmol), 1 µL of

157

template DNA, and 10.5 µL of ddH2O. The detailed protocol was as follows: 94 °C for 5 min

158

followed by 30 cycles of 94 °C for 30 s, annealing at different temperatures for 30 s, and 72 °C for

159

30 s. Each reaction was run in triplicate. A standard curve was used to calculate the copy number of

160

the genes; the square of the related coefficient of the standard curve was greater than 0.992, and

161

amplification efficiencies ranged from 95% to 110%. Data analysis was carried out using ICycler

162

software (Bio Rad, USA). The specificities of the qPCR products were determined by melting

163

curves and electrophoresis on a 1.8% agarose gel in 5× TAE buffer. All of the qPCR data of ARGs

164

were normalized among samples by dividing the copy numbers by the 16S rRNA gene copy number

165

to minimize variance caused by differential extraction and analytical efficiencies as well as

166

differences in background bacterial abundances. Thus, a time series of relative ARGs for each

167

treatment that was normalized to the background bacterial 16S rRNA signal was created.

168

2.3 Root morphology of ryegrass and analysis of soil physical and chemical properties.

169

Ryegrass was sampled at 10, 20, 25, and 35 days after seed germination. The roots and above

170

ground biomass were washed with distilled water to remove sand and other surface debris, cleansed

171

with deionized water, and then dried. The roots and above ground biomass were then separated. To

172

determine the characteristics of the plant roots, a V700 Epson scanner was used to save a complete

173

root image to a computer [28]. WinRHIZO PRO 2007 root system software (Regent Instruments Inc8,

174

Canada) was used to analyze the total length, average length, average diameter, total surface area,

175

average surface area, total sectional area, average sectional area, and other morphological

176

parameters.



177

The potentiometric method

[29]

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was used to measure the pH of soil samples with FE20-FiveEasy

178

Plus™. An adsorption instrument (Micromeritics Company, II TriStar 3020, USA) was used to

179

perform the nitrogen adsorption and desorption tests for various samples. Nitrogen adsorption was

180

conducted at liquid nitrogen temperature (-196 °C). Before the analysis, samples were degassed at

181

150 °C under vacuum (approximately 10-2 Pa) for 4 h. Afterward, the BET method

182

calculate the specific surface area of samples (SBET), and the t-plot method [31] was used to calculate

183

the specific surface area (Smi) and pore volume (Vmi) of micropores. Contents of the trace elements

184

Si, Al, Fe, K, Ca, Ti, S, Zr, Mn, V, Sr, Cr, Rb, Y, Ni, and Nb were measured with an

185

energy-dispersive X-ray fluorescence spectrometer (EDX-8000, Shimadzu Corporation, Japan).

186

[30]

was used to

2.4 Analysis of soil EMF. Soil respiration was determined by the potential of organic carbon [32]

187

mineralization with aerobic incubation at 28 °C

. A 20-g soil sample was placed in 500 mL

188

wide-mouthed bottle and covered with gauze. Sodium hydroxide (0.1 mol/L) was used to absorb

189

carbon dioxide. Phenolphthalein was used as an indicator for titration with 0.1 mol/L hydrochloric

190

acid. Soil ammonification was determined at 28 °C

191

was measured by chromogenic reaction with Nessler's reagent. A 10-g sample was placed into a

192

1000-mL Erlenmeyer flask, and then 5 mL of sterilized 0.2% peptone and 2 mL of ammonifying

193

bacterial liquid medium were added, with a soil:water ratio of 3:1. To determine soil nitrification

194

potential

195

28 °C with the addition of 200 ppm NH4+-N; concentrations of NO3−-N were measured before and

196

after the incubation to determine the soil nitrification potential.

[33]

. Ammonium ion in the soil water solution

[34]

, 10 g of fresh soil samples were cultured aerobically in 250-mL flasks for 2 weeks at

197

2.5 Data analysis. The raw data of antibiotics are available in the supplementary information file

198

(Fig. S1). The dissipation of antibiotics fit zero-order reaction kinetics. The dissipation rates of


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199

various antibiotics were obtained by calculating the slope of antibiotic residues each day. The least

200

significant difference test was used to calculate the differences among different categories of

201

antibiotics at different stages under different treatments. Statistical significance was considered at p

202

< 0.05. The correlation between antibiotics and ARGs and two-way ANOVA analysis were analyzed

203

using SPSS software. Redundancy analysis and CANOCO software were used to analyze the

204

relationships of antibiotics and ARGs to various environmental factors. Structural equation

205

modeling (SEM) is a multivariate statistical framework that is used to model complex relationships

206

between directly and indirectly observed (latent) variables through multiple regression, factor

207

analysis, path analysis, multivariate analysis of variance, and latent growth curve modeling

208

Recently, SEM has been used to construct complex relationships among soil biotic and abiotic

209

factors

210

environmental factors on antibiotics, ARGs, and soil EMF. Seven indicators were used for the initial

211

model, namely, ryegrass roots, soil pH, soil structure, trace element, residual antibiotics, ARG

212

abundance, and soil ecosystem function. In this model, antibiotics were considered an indicator

213

affecting ARGs. Ryegrass roots, soil pH, soil structure, trace element, residual antibiotics, and ARG

214

abundance were considered independent variables. The “robust” maximum likelihood evaluation

215

program of AMOS 7.0 software was used to analyze the model. The χ2, comparative fit index,

216

goodness-of-fit, and root square mean error of approximation tests were performed to assess model

217

fitness. Non-significant indicators and pathways were eliminated in the final model to obtain the

218

most parsimonious model. Prior to SEM analysis, we examined the normal distributions of data for

219

heteroscedasticity as well as all bivariate relationships for signs of nonlinearities. All statistical

220

analyses were performed using R version 3.0.2 (R Foundation for Statistical Computing, Vienna,

[35]

.

[36, 37]

. In this paper, SEM was used to analyze the effects of plant root systems and various



221

Austria, 2013).

222

3. RESULTS

223

3.1 Dissipation of residual antibiotics in soil. The dissipation rates of the three categories of

224

antibiotics (tetracyclines, sulfonamides, and quinolones) in soil were significantly higher under

225

biochar–ryegrass than ryegrass or in the control (p < 0.05), especially for tetracycline antibiotics

226

(Fig. S2). Antibiotic types, treatment and their interactions significantly affected the dissipation of

227

antibiotics according to two-way ANOVA analysis (p < 0.001) (Fig. S3). Under the control, the

228

natural dissipation rate of chlortetracycline was significantly higher than that of norfloxacin and

229

other antibiotics (p < 0.05) (Fig. 1). The dissipation rates were significantly enhanced under both the

230

ryegrass and biochar–ryegrass treatments (p < 0.05), especially for chlortetracycline, norfloxacin,

231

tetracycline and lomefloxacin. Compared with the ryegrass treatment, the dissipation rates of

232

tetracycline, chlortetracycline, doxycycline, and norfloxacin were higher under the biochar–ryegrass

233

treatment (p < 0.05), whereas the changes in the other four antibiotics were not significant.

234

3.2 Dissipation of ARGs in soil. Under the control, the total ARG abundance showed little

235

attenuation (Fig. 2). No significant difference in ARGs was observed under the three treatments

236

initially (Table S2). However, in the middle and late stages (15–40 days), ARG abundance was

237

significantly lower under ryegrass and biochar–ryegrass treatments than that of control (p < 0.05).

238

Ryegrass dissipates ARGs in soil, and biochar helps accelerate this process. The dissipation rates of

239

ARGs under the different treatments are shown in Table S3. The two types of sulfonamide ARGs

240

exhibited the highest dissipation rates under the ryegrass and biochar–ryegrass treatments. The

241

dissipation rates of quinolone ARGs were relatively low, and that of qnrA was consistently the

242

lowest.


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243

Based on the correlation analysis between antibiotics and ARGs (Table 1), the tetracycline

244

resistance gene tetW showed a significant positive correlation with chlortetracycline and

245

doxycycline (p < 0.01), and tetQ and tetH showed a significant positive correlation with

246

doxycycline (p < 0.01). For sulfonamide resistance genes, only qnrA showed a significant negative

247

correlation with lomefloxacin (p < 0.05). In general, ARG levels did not correlate with changes in

248

respective antibiotics in manure-fertilized soils.

249

3.3 Changes in the physical structure and physical and chemical properties of soil and plant

250

root systems. A nitrogen adsorption–desorption experiment was conducted to characterize the

251

physical structure of biochar and soil samples at different periods (Table S4). The isothermal

252

adsorption–desorption curve of biochar shows that its adsorption pattern follows that of the

253

Langmuir isotherm (Fig. S4). Pores smaller 10 nm in diameter of biochar were abundant, resulting in

254

a larger specific surface area. At the start of the experiment, the soil isothermal adsorption–

255

desorption curves and pore size distribution curves under different treatments were similar (Fig. 3).

256

In the middle stage of the experiment, the isothermal adsorption–desorption curves under different

257

treatments showed the same adsorption type, that is, a type II S-pattern isotherm. The soil under

258

ryegrass and biochar–ryegrass treatments had a stronger nitrogen adsorption capacity than that under

259

the control (p < 0.05). The pore size distribution curves for the three treatments were still similar. In

260

the late stage of the experiment, the specific surface area under the biochar–ryegrass treatment was

261

the largest, and that under the control was the smallest. The pore size distribution curve shows that

262

the soil under the biochar–ryegrass treatment contained more porous structures than that under

263

ryegrass treatment. This result indicates that the addition of biochar increased the soil porosity at this

264

stage.



265

The contents of trace elements in the soil samples under the three treatments at different stages

266

are shown in Table S5. The trace elements show a certain difference under different treatments, and

267

this difference may be a factor affecting the dissipation mechanism of antibiotics and ARGs. We

268

further determined the surface characteristics of the plant root system at different stages (Fig. S5).

269

The indicators of plant surface characteristics under the ryegrass and biological carbon–ryegrass

270

treatments showed no significant difference. Moreover, the total surface area of the ryegrass root

271

system demonstrated a significant negative correlation with the total residual antibiotics in the soil

272

under the two treatments (p < 0.05) (Table S6).

273

3.4 Relationship among antibiotics, ARGs, and environmental factors. Redundancy analysis

274

was used to analyze the effect of environmental factors on the dissipation of antibiotics and ARGs in

275

soil (Fig. 4). The environmental factors were constrained to the first two sorting axes, completely

276

explaining 59.04% and 95.46% of variation in the dissipation of antibiotics and ARGs, respectively.

277

Samples were significantly distinguished according to the different treatments for both antibiotics

278

and ARGs. Soil specific surface area and total root system length were the main factors influencing

279

the dissipation of both antibiotics and ARGs.

280

3.5 Effect of antibiotic and ARG dissipation on soil EMF. Soil functions including respiration,

281

ammonification, and nitrification were determined during the experiment (Fig. 5). Soil respiration

282

under ryegrass and biochar–ryegrass treatments was significantly higher than that under the control

283

(p < 0.05). The difference between the ryegrass and biochar–ryegrass treatments was not significant.

284

Soil ammonification under the ryegrass and biochar–ryegrass treatments was significantly higher

285

than that under the control (p < 0.05) in the middle stage of the experiment; at the late stage, the soil

286

ammonification under the biochar–ryegrass treatment was significantly higher than that under


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287

ryegrass treatment (p < 0.05). Soil nitrification was significantly inhibited under the three treatments

288

initially (p < 0.05). Later, soil nitrification under the biochar–ryegrass treatment was significantly

289

higher than that under the ryegrass treatment and the control (p < 0.05). Correlation analysis

290

between soil ecosystem function and residual antibiotics at the different stages revealed that in the

291

late stage of the experiment, soil ammonification and nitrification were significantly negatively

292

correlated with norfloxacin residues (p < 0.05) (Table S7). This result indicates that ryegrass and

293

biochar actively improve the soil ecosystem functions and that the effect of ryegrass is stronger

294

when combined with biochar than when used alone.

295

Structural equation models (SEM) were fitted to infer the direct and indirect effects of ryegrass

296

roots, soil structure, soil pH, trace element antibiotics and ARGs on EMF (Fig. 6). In the ryegrass

297

treatment (Fig. 6-a), plant roots directly (r = 0.782, p < 0.001), and indirectly through trace element

298

(r = -0.384, p < 0.001), impacted EMF. In addition, ryegrass roots showed a marginally positive

299

effect on ARGs (r = -0.13, p = 0.071). For total effects, the most important factor influencing EMF

300

was ryegrass roots. The SEM demonstrated that the influence of the biochar–ryegrass treatment on

301

EMF was mediated by the roots and trace elements (Fig. 6-b). In this system, plant roots directly (r

302

= 0.544, p < 0.001) impacted EMF and was the most important parameter influencing EMF. The

303

strongest relationship observed in the SEM analysis was between the root and soil structure (r =

304

0.860, p < 0.001).

305

In the two SEM models, plant roots exerted a direct positive impact on soil EMF but a negative

306

impact on trace elements (p < 0.001). Soil physical structure positively affected residual antibiotics

307

and dissipation of ARG abundance (p < 0.01). At the same time, antibiotics demonstrated a

308

significant positive impact on ARGs (p < 0.001). The positive impact of the root system on EMF



309

and its negative impact on trace elements were weaker under the biochar–ryegrass treatment than

310

under the ryegrass treatment, but the positive impact on soil physical structure was enhanced.

311

Moreover, the impact of soil structure on antibiotics increased, but the impact on ARGs was

312

decreased. At the same time, the positive impact of antibiotics on ARGs increased. The negative

313

impact of plant root growth on soil pH under the biochar–ryegrass treatment was no longer

314

significant, and the positive impacts of pH on antibiotics and of trace elements on ARGs under the

315

biochar–ryegrass treatment were no longer significant. Furthermore, the comprehensive influence of

316

trace elements on ARGs and EMF was significantly weaker under the biochar–ryegrass treatment

317

than under the ryegrass treatment, but that of soil physical structure on EMF was significantly

318

higher under the former than the latter (Fig. 6a-2; Fig. 6b-2). In addition, the comprehensive

319

influence of soil pH on antibiotics/ARGs and EMF significantly decreased, and the comprehensive

320

influence of antibiotics on ARGs increased under the biochar–ryegrass treatment.

321

4. DISCUSSION

322

4.1 Dissipation of antibiotics and ARGs by ryegrass and biochar. Our results revealed that

323

ryegrass treatment can dissipate several antibiotics common in soil and that its combination with

324

biochar enhances this dissipation effect. Different types of antibiotics in soil exhibit different

325

dissipation effects. For example, ryegrass demonstrates strong dissipation effects on tetracycline,

326

aureomycin, and norfloxacin in the soil but weaker effects on sulfamethazine. This result may be

327

related to the chemical properties of the antibiotics and their molecular structure stability. Compared

328

with sulfamethazine, tetracycline antibiotics have more unstable chemical properties and structure

329

under acidic conditions and are easily broken down by bacteria and enzymes around plant roots into

330

inorganic substances and carbohydrates

[38]

. Moreover, this result may be closely related to the


Page 16 of 33

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331

adsorption performance of different antibiotics. The soil–water balance coefficients of sulfonamides,

332

tetracyclines, and quinolones are 0.9–10, 100–3910, and 496–61000 L/kg, respectively, which are

333

several thousand times higher than that of sulfanilamides

334

quinolones are more easily adsorbed and fixed by the soil or other media than are sulfanilamides. In

335

addition, natural dissipation was observed, especially for chlortetracycline and norfloxacin under the

336

control conditions. The increased dissipation rate of antibiotics by plant roots and biochar may

337

largely reduce the risk of antibiotics spreading to other environmental compartments, such as

338

surface water. Further field-scale mass balance is required to understand the attenuation rates and

339

spread of antibiotics. In the present study, the addition of biochar promoted antibiotic dissipation in

340

the soil, possibly through the adsorption effect. Some studies have shown that biochar exerts a

341

strong direct adsorption effect on sulfapirazinmetossina

342

fixation effect on ciprofloxacin in the soil [41]. Another possible reason is the effect of biochar on the

343

physical and chemical properties of the soil. For example, biochar can effectively alter the pH and

344

increase the soil cation exchange capacity and mineral nitrogen contents

345

soil environment conductive to ryegrass and soil microbes and indirectly promoting the dissipation

346

of antibiotics. The pure effect of biochar on the dissipation of antibiotics and ARGs is important to

347

get a full understanding of the underlying mechanisms. Additionally, our laboratory-based

348

experiments may not necessarily reflect what would occur in field conditions. Further

349

field-condition experiments are required to test the effects of ryegrass, biochar, and the combination

350

and dynamic changes of antibiotics and ARGs under a gradient of biochar dosages, which is of

351

critical importance for applying ryegrass and biochar to dissipate manure-induced antibiotics and

352

improving soil ecosystem functions.

[39]

. This comparison directly shows that

[40]

and an even better adsorption and


[22, 23]

, thereby providing a


353

Some studies have shown that a certain relationship exists between ARGs and antibiotics and that

354

selection pressure is closely related to the concentration of residual antibiotics [11]. The present study

355

found no significant correlation between ARGs and their corresponding antibiotics. This may be

356

because the antibiotics can be rapidly dissipated, adsorbed, or chelated, and the residual antibiotics

357

in the soil can rapidly decrease. The selection of ARG abundance was always short lived; therefore,

358

the correlation between antibiotics and ARG abundance is weak

359

contribute to the inconsistency between antibiotics and ARGs. For example, the cross-selection of

360

heavy metals or other antibiotics can weaken the correlation between ARGs of mobile genetic

361

elements and corresponding antibiotics [43-45].

[42]

. In addition, other factors can

362

4.2 Mechanism of physical structure and chemical properties of soil and plant roots

363

affecting the dissipation of antibiotics and ARGs. Soil treated with biochar–ryegrass had higher

364

specific surface area and porosity than did soil treated with ryegrass (Fig. 3). The porous structure of

365

biochar increased the specific surface area and pore structure of the soil. In addition, the biochar–

366

ryegrass treatment elicited stronger dissipation effects on antibiotics and ARGs than did ryegrass.

367

This result indicates that the specific surface area, pore structure, and other physical properties of

368

the soil are the important factors influencing the dissipation of antibiotics, which is in accordance

369

with results of a study by Accinelli

370

surface area and pH of soil are the main factors affecting antibiotic dissipation in soil and that the

371

specific surface area of soil, total root length, and average surface area of roots are the main factors

372

affecting for the dissipation for ARGs. In this study, the pH of ryegrass soil decreased with time,

373

and biochar alleviated this effect (Table S8). The change in pH may promote the dissipation of

374

antibiotics [46].

[15]

. Redundancy analysis further revealed that the specific


Page 18 of 33

Page 19 of 33


375

The growth of the plant root system directly affects the number of microorganisms around the

376

root system. Plant exudations benefit the growth and enrichment of rhizosphere microorganisms

377

such that microorganisms around the root system are several dozen times more abundant than

378

non-root microorganisms

379

antibiotics

380

negative correlation with the total residual antibiotics in the soil (p < 0.05). Furthermore, the growth

381

of the root system directly affected the absorption and dissipation of ryegrass to antibiotics, in

382

accordance with the findings of Boxall [9].

[17]

[46]

. These microorganisms are important in the dissipation of soil

. In the present study, the total surface area of ryegrass roots showed a significant

383

4.3 Mechanism underlying soil EMF improvement during the dissipation of soil antibiotics.

384

At the early stage of this experiment, treatments showed slight differences, and the overall soil

385

ecosystem function was weak and nearly suppressed. Weak soil ecosystem function in the early

386

stage may be related to residual antibiotics and heavy metals induced by manure. Antibiotics and

387

heavy metals inhibit the growth of microorganisms in the soil, decreasing soil ecosystem function [6].

388

In the middle and late stages of this experiment, ryegrass and biochar–ryegrass treatments

389

significantly enhanced the soil ecosystem function (p < 0.05). Smith et al.

390

addition of biochar to soil accelerates the decomposition of organic matter and thus promotes soil

391

respiration. The porous and loose characteristics of biochar provide more space for the growth of

392

soil bacteria. It has been reported that the nutrient elements introduced from biochar to the soil

393

promote the growth of ammonifying bacteria, significantly improving the ammonification function

394

of the soil [49]. Nelissen et al.

395

in the soil and accelerate the transformation of NH4+-N into NO3--N, thus improving soil

396

nitrification.

[50]

[48]

reported that the

reported that biochar can promote the growth of nitrifying bacteria



397

In the present study, SEM for ryegrass showed that plant roots can improve EMF directly and

398

indirectly through trace elements. In addition, the plant root system under the biochar–ryegrass

399

treatment can directly accelerate the EMF improvement. The improvement of soil EMF may be due

400

to the growth of microorganisms in the rhizosphere soil [49]. The addition of biological carbon into

401

the soil weakened the direct positive effect of the root system on EMF. This result may be because

402

biochar and its chemical reactions in the soil environment generate substances that are harmful to

403

certain microorganisms [51]. Moreover, under the ryegrass and biochar–ryegrass treatments, the plant

404

root system positively affected soil physical structure (p < 0.001), proving a relationship between

405

the root system and soil physical structure

406

physical structure exerted a significant negative impact on antibiotic residues and ARG abundance

407

(p < 0.01). This finding may be related to the migration, transformation, and adsorption of

408

antibiotics and ARGs

409

the soil physical and chemical properties. Compared with the ryegrass model, the biochar–ryegrass

410

treatment removed both the significant negative impact of the plant root system growth on soil pH

411

and the significant positive impact of soil pH on antibiotics. These results may be attributed to the

412

improvement of soil pH by biochar [23].

413

Supporting Information Available: Concentrations and dissipation rate of antibiotics in different

414

treatments, adsorption-desorption and pore-size of biochar, plant roots index, primer information of

415

ARGs, dissipation rate of ARGs and statistical analysis, structure parameters of samples, contents of

416

trace elements, pH values and correlation analysis between root index, multifunctionality and

417

antibiotics.

[39]

[22, 23]

. At the same time, under the two treatments, soil

. Thus, antibiotics and ARGs can be dissipated and blocked by adjusting


Page 20 of 33

Page 21 of 33

418


ACKNOWLEDGEMENTS

419

This study was supported by National Key R&D Program of China (2016YFD0200309), National

420

Natural Scientific Foundation of China (No. 41622104, 41371256), Distinguished Young Scholar

421

Program of the Jiangsu Province (BK20160050), Foundation for Distinguished Young Talents in

422

State Key Laboratory of Soil and Sustainable Agriculture (Y412010008), and Youth Innovation

423

Promotion Association of Chinese Academy of Sciences (2016284).

424

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425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453

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and nitrification rates in organic and conventional cropping systems. Soil Biol. Biochem. 2003, 35, 29-36. 34. Nannipieri, P.; Ascher, J.; Ceccherini, M.; Landi, L.; Pietramellara, G.; Renella, G. Microbial diversity and soil functions. Eur. J. Soil Sci. 2003, 54 (4), 655-670. 35. Grace, J.; Schoolmaster JR., D.; Guntenspergen, G.; Little, A.; Mitchell, B.; Miller, K.; Schweiger, W. Guidelines for a graph-theoretic implementation of structural equation modeling. Ecosphere 2012, 3(8), art 73. 36. Zhang, S.; Li, Q.; Lü, Y.; Zhang, X.; Liang, W. Contributions of soil biota to C sequestration varied with aggregate fractions under different tillage systems. Soil Biol. Biochem. 2013, 62 (5), 147-156. 37. Flores-Rentería, D.; Rincón, A.; Valladares, F.; Yuste, J. C. Agricultural matrix affects differently the alpha and beta structural and functional diversity of soil microbial communities in a fragmented Mediterranean holm oak forest. Soil Biol. Biochem. 2016, 92, 79-90. 38. Homem, V.; Santos, L. Degradation and removal methods of antibiotics from aqueous matrices: A review. J. Environ. Manage. 2011, 92 (10), 2304-2347. 39. Shi, Y.; Gao, L.; Li, W.; Liu, J.; Cai, Y. Investigation of fluoroquinolones, sulfonamides and macrolides in long-term wastewater irrigation soil in Tianjin, China. B. Environ. Contam. Tox. 2012, 89 (4), 857-861. 40. Zheng, H.; Wang, Z.; Zhao, J.; Herbert, S.; Xing, B. Sorption of antibiotic sulfamethoxazole varies with biochars produced at different temperatures. Environ. Pollut. 2013, 181, 60-67. 41. Li, F.; Feng, D.; Deng, H.; Yu, H.; Ge, C. Effects of biochars prepared from cassava dregs on sorption behavior of ciprofloxacin. Procedia Environ. Sci. 2016, 31, 795-803. 42. Ghosh, S.; Lapara, T. The effects of subtherapeutic antibiotic use in farm animals on the proliferation and persistence of antibiotic resistance among soil bacteria. ISME J. 2007, 1, 191-203. 43. Hall, A.; Colegrave, N. Decay of unused characters by selection and drift. J. Evolution. Biol. 2008, 21 (2), 610-617. 44. Binh, C.; Heuer, H.; Gomes, N.; Kotzerke, A.; Fulle, M.; Wilke, B.; Schloter, M.; Smalla, K. Short-term effects of amoxicillin on bacterial communities in manured soil. FEMS Microbiol. Ecol. 2007, 62 (3), 290–302. 45. McKinney, C. W.; Loftin, K. A.; Meyer, M. T.; Davis, J. G.; Pruden, A. tet and sul antibiotic resistance genes in livestock lagoons of various operation type, configuration, and antibiotic occurrence. Environ. Sci. Technol. 2010, 44 (16), 6102-6109. 46. Loftin, K. A.; Adams, C. D.; Meyer, M. T.; Surampalli, R. Effects of ionic strength, temperature, and pH on degradation of selected antibiotics. J. Environ. Qual. 2008, 37 (2), 378-386. 47. Wu, H. W.; Haig, T.; Pratley, J.; Lemerle, D.; An, M. Allelochemicals in wheat (Triticum aestivum L.): Cultivar difference in the exudation of phenolic acids. J. Agr. Food Chem. 2001, 49 (8), 3742-3745. 48. Smith, J. L.; Collins, H. P.; Bailey, V. L. The effect of young biochar on soil respiration. Soil Biol. Biochem. 2010, 42 (12), 2345-2347. 49. Han, G.; Meng, J.; Zhang, W.; Chen, W. Effect of biochar on microorganisms quantity and soil physicochemical property in rhizosphere of spinach (Spinacia oleracea L.). Appl. Mech. Mater. 2013, 295-298 (6), 210-219. 50. Nelissen, V.; Rütting, T.; Huygens, D.; Staelens, J.; Ruysschaert, G.; Boeckx, P. Maize biochars accelerate short-term soil nitrogen dynamics in a loamy sand soil. Soil Biol. Biochem. 2012, 55, 20–27. 51. Mašek, O. Biochar and carbon sequestration. In Fire Phenomena and the Earth System: An Interdisciplinary Guide to Fire Science; Belcher, C., Ed.; John Wiley: Oxford, 2013; pp 309-322.

538

Figure legends

539

Figure 1 Dissipation rate of antibiotics in different treatments: control, ryegrass and biochar–



540

ryegrass. Lowercase letters indicate significant differences of the same antibiotic in different

541

treatments (p < 0.05); different uppercase letters indicate significant differences among different

542

antibiotics in the same treatments (p < 0.05). TC, tetracycline; CTC, chlortetracycline; DC,

543

doxycycline; SM2, sulfonamides; ENR, enrofloxacin; LOM, lomefloxacin; CIP, ciprofloxacin; NOR,

544

norfloxacin.

545

Figure 2 Abundance of ARGs in samples. Different lowercase letters indicate significant differences

546

of samples in different treatments at the same period (p < 0.05); different uppercase letters indicate

547

significant differences among different periods in the same treatments (p < 0.05). ARGs of

548

tetracycline: tetH, tetQ, tetO, tetM, tetM, tetM; ARGs of sulfonamides: sul1 and sul2; ARGs of

549

quinolones: gyrA and qnrA; ARG of macrolide: ermF.

550

Figure 3 Adsorption-desorption N2 curve and pore-size distribution curves of samples. (a) Early

551

stage (1st day); (b) Middle stage (25th day); (c) Later stage (40th day).

552

Figure 4 Relationship between antibiotics, ARGs and environmental factors. Redundancy analysis

553

of (a) antibiotics and environmental factors and (b) ARGs and environmental factors. Color from

554

dark to light represents the dissipation of antibiotics from the beginning to the end of the experiment

555

(40 days). Environmental variables were chosen based on the significance calculated from

556

individual redundancy analysis results. ASA, root average surface area; TL, root total length; SA,

557

soil surface area; MR, soil respiration; MA, soil ammonification; MN, soil nitrification.

558

Figure 5 Changes of soil ecosystem multifunctionality (EMF), (a) Soil respiration; (b) Soil

559

ammonification; (c) Soil nitrification. Different lowercase letters indicate significant differences of

560

samples in different treatments at same period (p < 0.05). Early stage: 0-10 days; middle stage:

561

10-25 days; later stage: 25-40 days.

562

Figure 6 Structural equation models of the treatments (a) ryegrass and (b) biochar–ryegrass,

563

depicting the direct and indirect influences of ryegrass, soil pH, soil structure, soil trace element,

564

antibiotics, ARGs on EMF. Arrows depict casual relationships: red lines indicate positive effects,

565

and black lines indicate negative effects. Arrow widths are proportional to r values. Paths with

566

coefficients non-significant different from 0 (p > 0.05) are presented with dotted gray lines. *p

Improvement of Soil Ecosystem Multifunctionality by Dissipating

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