Pyrosequencing Reveals Soil Enzyme Activities and Bacterial

Subscriber access provided by PEPPERDINE UNIV

Article

Pyrosequencing reveals soil enzyme activities and bacterial communities impacted by graphene and its oxides Yan Rong, Yi Wang, Yina Guan, Jiangtao Ma, Zhiqiang Cai, Guanghua Yang, and Xiyue Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03646 • Publication Date (Web): 26 Sep 2017 Downloaded from http://pubs.acs.org on September 28, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

Journal of Agricultural and Food Chemistry

1

Pyrosequencing reveals soil enzyme activities and bacterial

2

communities impacted by graphene and its oxides

3

Yan Rong1,2, Yi Wang2, Yina Guan2, Jiangtao Ma2, Zhiqiang Cai1,2*, Guanghua

4

Yang1*, Xiyue Zhao1,2

5

1. Advanced Catalysis and Green Manufacturing Collaborative Innovation Center,

6 7 8 9 10

Changzhou University, Changzhou, 213164, China 2. Laboratory of Applied Microbiology, School of Pharmaceutical Engineering & Life Science, Changzhou University, Changzhou, 213164, China * Corresponding author. Tel/Fax: +86 519 86330160 Email address: [email protected]; [email protected]

11 12

ABSTRACT

13

Graphene (GN) and graphene oxides (GOs) are novel carbon nanomaterial, they have

14

been attracted much attention because of their excellent properties and are widely

15

applied in many areas including energy, electronics, biomedicine and environmental

16

science etc. With industrial production and consumption of GN/GO, they will

17

inevitably enter the soil and water environment. GN/GO may directly cause certain

18

harm to microorganisms and lead to ecological and environmental risks. Graphene

19

oxides are graphene derivative with abundant oxygen-containing functional groups in

20

its graphitic backbone. The structure and chemistry of graphene show obvious

21

differences compared with graphene oxide, which lead to the different environmental

22

behaviors. In this study, four different types of soil (S1, S2, S3 and S4) were 1

ACS Paragon Plus Environment


23

employed to investigate the effect of GN and GO on soil enzymatic activity, microbial

24

population and bacterial community through pyrosequencing of 16S rRNA gene

25

amplicons. The results showed that soil enzyme activity (invertase, protease, catalase

26

and urease) and microbial population (bacteria, actinomycete and fungi) changed after

27

GN/GO release into soils. Soil microbial community species are more richness and

28

the diversity also increase after GO/GN application. The phylum of Proteobacteria

29

increased at 90 days after treatment (DAT) after GN/GO application. The phylum of

30

Chloroflexi occurred after GN applying at 90 DAT in S1 and reached 4.6%.

31

Proteobacteria were the most phylum in S2, S3 and S4 soils, it ranged from 43.6% to

32

71.4% in S2, 45.6% to 73.7% in S3, 38.1% to 56.7% in S4, respectively. The most

33

abundant genus were Bacillus (37.5% - 47.0%), Lactococcus (28.0% - 39.0%) in S1,

34

Lysobacter and Flavobacterium in S2, Pedobacter in S3 and Massilia in S4 soil. The

35

effect of GN and GO on soil microbial community is time dependent, and there are no

36

significant differences between the samples at 10 and 90 DAT.

37 38

KEYWORDS: Graphene; Graphene oxides; Pyrosequencing; Bacterial community;

39

Soil enzyme activity

40

2


Page 2 of 28

Page 3 of 28


41

42

Graphene (GN) is a two-dimensional nanomaterial with sp2 hybridization carbon

43

atoms.1,2 And graphene oxides (GOs) are layered graphene sheets with oxygen

44

functional groups, such as carbonyl and hydroxyl group. 3,4 GN and GO have attracted

45

much attention because of their excellent properties (such as low resistivity and rapid

46

electron mobility etc.) and wide application in many areas (including complex

47

material, energy, electronics, biomedicine, and environmental science etc.).1,4 For

48

instance, GN has high-performance properties of adsorbent in water treatment,5 GO

49

are used in removing pollutants due to their excellent adsorbents and photocatalysts.6

50

The GN and GO are now transferred to industrial production, and 1000 ton per annual

51

of GO has been put into operation in Changzhou, China. With industrial production

52

and consumption of GN/GO, they will inevitably enter the environment. GN/GO may

53

directly cause certain harm to animals, plants and microorganisms in the environment,

54

and lead to ecological and environmental risks.7-9 And GN and GO have strong

55

adsorption capacity, the enrichment of poisonous and harmful environment the

56

material, thereby affecting the environmental pollutants transformation and

57

degradation of environmental behavior.7-12

58

INTRODUCTION

Previous studies showed that GO have strong antimicrobial effect on

59

microorganisms, such as Escherichia coli,12 Pseudomonas putida,13 white rot fungus

60

Phanerochaete chrysosporium.14 GN also showed toxicity to cells and animals, the

61

genotoxicity and long term toxicity caused by GN have been reported.15,16 The large

62

amount of GN and GO application may lead to their residues in environment, which 3



63

can affect soil enzyme activity and soil microbial community by changing their

64

number, microbial activity and diversity.10,13-15,17-20 Soil enzyme activity and microbial

65

community are often used as the key factors in assessing soil quality. Artificial

66

compounds have effect on soil microbial dominant species, community diversity and

67

richness, and also impact soil enzyme activity and microbial population. Until now,

68

only few reports studied the effects of GO on microbial community,8,21 GO had toxic

69

effect on the wastewater microbial communities at concentrations from 0.05 to 0.3 mg

70

ml−1,8

71

characteristics. The better understanding of GN and GO impacts on soil enzyme

72

activity, microbial population, microbial community diversity and composition in the

73

soils are necessary for their safe use. The present study was investigated their effect

74

on soil microbial community, diversity, population and soil enzyme activity.

which showed that GOs have potential risk to the soil biochemical

75 76 77

Materials and Methods Chemicals and Soil Samples. Graphene (GN) and graphene oxides (GOs) were

78

obtained from Jiangnan Graphene Research Institute (Changzhou, China). The

79

morphology of graphene (GN) and graphene oxides (GOs) were investigated by

80

scanning electron microscopy (SEM; Zeiss supra55, Germany, Figure S1) and atomic

81

force microscopy (AFM, JPK Nano Wizard 3, Germany. Figure S1). GN and GO zeta

82

potential and particle sizes were measure by Zetasizer nano potentiometer (Malvern,

83

Zetasizer Nano ZEN 3600, UK. Figure S2). Four types of soil from different

84

agricultural field were sampled and employed in this study, which were Red paddy 4


Page 4 of 28

Page 5 of 28


85

soil (S1, GB/T-H2121315), Yellow loam soil (S2, GB/T-A2111411), Huangshi soil

86

(S3, GB/T-G2511211) and Yellow paddy soil (S4, GB/T-A2111511), respectively. The

87

soil samples were taken from crop fields in Longyou (Zhejiang province), Longquan

88

(Fujian province), Changzhou (Jiangsu province) and Huajiachi, Hangzhou (Zhejiang

89

province, China). All soil samples were air-dried, mixed and passed through a 2-mm

90

sieve. Their basic physicochemical characteristics were listed in Table 1.

91

Experiments Procedure. To study the impact of GN and GO on soil enzyme

92

activities, microbial population and bacterial community, GN and GO were mixed

93

with soils thoroughly to give a final concentration of 100 mg kg-1 soil. Soils were

94

incubated at 25 ± 1o C to allow microorganisms to acclimatize. The control

95

experiment was carried out under the same condition without GN and GO (CK set).

96

All the tests for each soil were conducted with three replicates. The soil moisture

97

content was adjusted to about 60% of the maximum water-holding capacity by adding

98

Milli-Q (MQ) water. At different time intervals (10, 30, 50, 70 and 90 days), 5 g of

99

soil (dry weight equivalent) was sampled from the flask and was used for soil

100

microbial population and soil enzyme activities, soil samples at 10 and 90 days after

101

treatment (DAT) were employed for soil DNA extraction.

102

The activities of soil protease (EC 3.4.21.92), invertase (EC 3.2.1.26), catalase (EC

103

3.5.1.5) and urease (EC 1.11.1.6) were analyzed according to the previous published

104

method.22 Soil microbial population (heterotrophic bacteria, actinomycetes and fungi)

105

counting was assayed through most probable number method (MPN) according to

106

previous reports.22,23 5



Soil DNA Extraction and Pyrosequencing, Bioinformatics Analysis. Soil DNA

107 108

extraction was based on the manufacturer’s protocol (Fast DNA Spin kit, MP

109

Biomedicals, USA). The total DNA was purified by electrophoresis in 1% agarose gel.

110

Pure DNA was extracted using Agarose gel extraction kit (Roche). DNA quality was

111

assessed with a ScanDrop 200 spectrophotometer. The primers 515F (5'- GTGCCAGCMGCCGCGG-3') and 907R (5'-

112 113

CCGTCAATTCMTTTRAGTTT-3') were used for amplifying the fragment of 16S

114

rRNA gene. The PCR reaction program and PCR products purification were was

115

according with previous reported methods.22,23 Totally 865,196 16S rRNA sequence

116

reads were filtered, Trimmomatic was used for denoised and processed. The resulting

117

sequences after quality control were analyzed through QIIME. Operational taxonomic

118

units (OTUs) was used at 97% sequence similarity. The methods used for Rarefaction

119

curves, Shannon Wiener curves, communites composition, Venn and PcoA analysis,

120

and genus heatmap were in accordance with the reported methods.22,23

121 122 123

RESULTS AND DISCUSSION Effect of GN and GOs on Soil Enzyme Activity. Soil environmental change and

124

stress often lead to soil enzyme activities change, which are often used as the soil

125

quality index. GN and GOs release into soils perhaps can impact soil enzyme

126

activities and its effects on enzyme activities (invertase, urease, protease and catalase)

127

are shown in Figure1 and Figure S3.

128

Invertase activity invertase activity increased significantly after GN application in 6


Page 6 of 28

Page 7 of 28


129

soil S1, while it also most kept unchangeable after GO application, except at 70 DAT

130

invertase activity reach 7.10 mg glucose g-1 soil, the control group was only 5.0 mg

131

glucose g-1 soil . In soil S2, invertase activity was inhibited by graphene and GO at 30

132

and 90 DAT, while increased at 50 DAT. Invertase activity remained almost

133

unchangeable in soil S3, and GO inhibit its enzymatic activity during 30-70 DAT in

134

soil S4.

135

Urease activity soil urease plays a key role during the transformation of organic

136

phosphorus. The impact on urease activities by graphene and GO differed in different

137

type of soil. Urease activity in soil S1 inhibited significantly before 50 DAT, it only

138

reached 0.33 and 0.26 mg NH4-N g-1 soil after GN and GO application at 30 DAT,

139

while the activity was 0.58 mg NH4-N g-1 soil in the control soil. However urease

140

activity increased remarkable at 70 and 90 DAT. Graphene inhibited enzyme activity,

141

while GO increased in soil S2. Both GN and GO inhibited urease activity gently in

142

soil S4, and increase significantly in S3 at 90 DAT.

143

Protease activity protease activity presents soil microbial population and fertility.

144

Graphene can inhibit protease gently and GO increase enzyme activity remarkably at

145

90 DAT in soil S1. Protease activity remains unchangeable in soil S3 after GN and

146

GO application. In soil S2 and S4, both GN and GO inhibit enzyme activity. Protease

147

activity was 0.91 mg tyrozine g-1 soil, and 0.79 mg tyrozine g-1 soil after GN and GO

148

application in soil S2 respectively, while in control soil it is 1.52 mg tyrozine g-1 soil

149

90 DAT. In soil S4, enzyme activity is 8.0 mg tyrozine g-1 soil, after GN and GO

150

applying, it declines to 6.24 and 5.21 mg tyrozine g-1 soil, respectively. 7



151

Catalase activity catalase is one of the most important enzyme in microbial growth

152

and can protect organisms from H2O2 toxicity. In this study, catalase activity increased

153

except 30 DAT in soil S1, and it was stimulated to increase after GN and GO

154

application in soils. At 50 DAT, catalase activity reached 3.62 ml 0.02 M KMnO4 / g

155

soil and 2.97 ml 0.02 M KMnO4 / g soil after GN and GO application in S1. The

156

catalase activity increased 47.9% and 21.4% in soil S1 compared with the control

157

group at 50 DAT, respectively. In other 3 type soils, catalase activity both increased

158

with GN and GO application.

159

Enzyme activity in soils is one of the key roles in decomposition of biological

160

residues (animal, plant, microorganism and human), biodegradation and

161

transformation of organic compounds. Therefore the changes in soil microbial

162

population and enzyme activity exposed to GN and GO were investigated in this study,

163

the results were employed to assess the effect of GN and GO on soil quality.9,10,24,25

164

Fig.1 showed that GN and GO could stimulate to increase invertase activities in S1

165

soil, in S2 soil decreased at 30 and 90 DAT, keep almost unchangeable in S3, and

166

decreased at 30 DAT in S4. Urease was inhibited at the initial day and then it

167

increased significantly after 70 DAT. Protease activity decreased in S1 and S2 soils,

168

catalase activity increased in both four different soils. The results indicated that both

169

GN and GO can impact enzyme activity in different soils, and invertase, catalase and

170

protease are sensitive to GN and GO. But these effects were transient, enzyme activity

171

keep with normal level after 50 - 90 DAT. Higher concentration of GO lowered soil

172

enzyme activity.17-19 8


Page 8 of 28

Page 9 of 28


173

Effect of GN and GO on Soil Microbial Population. The effects of GN and GO

174

on the total number of bacteria, actinomycete and fungi in soils were showed in

175

Figure 2 and Figure S4. The bacterial total number in S1 soil increased after GN and

176

GO application at 30 and 70 DAT, and decreased in 10 and 50 DAT. GN was just

177

released in to S2 soil, bacterial population increased compared with the CK obviously

178

especially at 30 DAT, bacterial total number reached 109 CFU g-1 soil, while it was

179

only 108 CFU g-1 soil in the CK soil. It was only 107 CFU g-1 soil after GO application

180

in S2 soil at 30 DAT. In S3 soil, bacteria population inhibited at 10, 30 and 70 DAT,

181

and it increased at 50 and 90 DAT. The total number of bacteria in S4 increased at 30

182

and 70 DAT, and decreased at 10, 50 and 90 DAT.

183

GO inhibit actinomycete growth in S1 and S2 soils, especially at 70 DAT in S1 soil,

184

actinomycete population is 8 × 103 CFU g-1 soil, while it was 9 × 104 CFU g-1 soil in

185

the CK soil. However, GN and GO were released into S3 and S4 soils after 30 DAT,

186

actinomycete population increased compared with the CK obviously. In S3 soil, the

187

total number of actinomycete reached 4 × 106 CFU g-1 soil and 6 × 106 CFU g-1 soil

188

after GN and GO application, respectively, and it was only 1 × 106 CFU g-1 soil in the

189

CK S3 soil at 50 DAT. GN and GO inhibit actinomycete at 10 DAT, and then it was

190

stimulated to increased significantly. Fungal population in S1 and S3 soils increased

191

and decreased in S2 and S4 soils.

192

Many studies showed that GO has antimicrobial activity, which have been

193

investigated through microorganisms culture.10,17,26,27,28 Antimicrobial activity of GO

194

may also apply to soil as well. However, our findings demonstrate that GO can 9



195

promote soil enzyme activity in particular soils, and may have positive effect on the

196

functions of environmental microorganisms in soils. And its negative impact on soil

197

enzyme activity and microbial population may be only transient, with cultural days

198

prolonging, negative effect will disappear. Especially in S1 soil, both GN and GO

199

increase invertase, protease and catalase activities, and the bacterial and fungal

200

population in S1 soil also increased significantly, and the concentration of GN and

201

GO is 100 mg kg-1 soil in this study, which is far higher than that in many

202

studies.7,8,17,19,20,29 Lots of studies showed that the potential toxicological impact of

203

GN and GO on many microorganism, GO has strong cytotoxicity toward bacteria,30,31

204

fungi32 and algae29 etc. Compared with Gram-negative bacteria, GO and GN are more

205

toxic to Gram-positive bacteria.30

206

Figure 1 indicated that the catalase activity reached maximum at 50 DAT, and then

207

it was higher than that in control soil, lower catalase activity at the beginning of

208

GN/GO application. This showed catalase activity are sensitive to GN/GO.10,11 The

209

activity of catalase was related to microbial quantity, but also microbial environmental

210

stress, including ROS, superoxide anions (*O2-), hydrogen peroxide (H2O2), and

211

hydroxyl radicals (*OH-).33-34 Microorganism is often induced to generate ROS and

212

its subsequent oxidative stress after they are exposed to GN and GO. The results in

213

this study GN and GO can inhibit or increase soil enzyme activities and microbial

214

population in different soils, which showed GN and GO have different toxicity to

215

bacterial in different environment, these are in accordance with the results of

216

microbial population in different soils. The organic matter (OM) can provide effective 10


Page 10 of 28

Page 11 of 28


217

carbon and energy sources and its content leads to different microbial population and

218

growth rate in soils. Higher OM content brought about higher microbial population,

219

which may weaken GN/GO toxicity to microorganism.

220

Microbial Community Species Richness and Diversity. The rarefaction curve

221

(Figure 3A) can assess environmental microbial species richness, Shannon -Wiener

222

curve (Figure 3B) reflect the index of microbial species diversity and Venn diagram

223

(Figure S5) shows all possible logical relations in the sample. Microbial species are

224

more richness after GO and GN application in S4 soil than that in control at 90 DAT.

225

The species decrease after GO applying at 10 DAT, while it increases after GN

226

application in S4. And microbial species are relatively poor after GN and GO

227

application in S2 and S3 soil than that in control soil at 90 DAT. The sequence of

228

species richness from high to low is S4 > S3 > S2 > S1. OTUs in S4 are above 1500,

229

in S3 and S2 are higher than 1000, however in S1 soil, OTUs are below 300 at 90

230

DAT. The microbial species are higher at 90 DAT than that at 10 DAT in S2, S3 and

231

S4 soils. Species diversity was showed in Fig. 3B. With the increase of inocubation

232

time, the microbial species diversity in soils with GN/GO application and control

233

group increased. GO can inhibit species diversity and GN can increase microbial

234

species diversity at 10 DAT in S4 soil, and the microbial species diversity are almost

235

unchangeable compared with the control group at 90 DAT after GN and GO

236

application. GN/GO both can inhibit gently species diversity in S3 soil at 90 DAT,

237

however, GO can increase diversity and GN also can inhibit species diversity in S2 at

238

90 DAT. 11



239

Community Species Composition and Structure. The results showed in Figure 4

240

all the reads were assigned to bacteria. Firmicutes were the most phylum in S1 soil

241

(87.2%-95.3%), the second phylum is Proteobacteria (3.6%-9.7%). The ratio of

242

Proteobacteria increased at 90 DAT after GN/GO application. The phylum of

243

Chloroflexi occurred after GN applying at 90 DAT in S1 and reached 4.6%.

244

Proteobacteria were the most phylum in S2, S3 and S4 soils, it ranged from 43.6% to

245

71.4% in S2, 45.6% to 73.7% in S3, 38.1% to 56.7% in S4, respectively. GO can

246

inhibit Proteobacteria in S2, which decreased from 53.7% to 43.6% at 90 DAT. The

247

phylum of Chloroflexi increased, which was 10.3% after GO application in S2 and

248

was only 7.7% in control soil. Firmicutes were also stimulated to increase by GN and

249

GO. In S3 soil, the phylum of Bacteroidetes increased by 3.4% after GN application,

250

and 3.1% after GO application compared with that in control soil. Firmicutes

251

decreased from 14.4% to 11.2%. In S4 soil, Acidobacteria was inhibited at 90 DAT

252

after GN/GO application. The most abundant genus were Bacillus (37.5% - 47.0%),

253

Lactococcus (28.0% - 39.0%) in S1 soil. GN inhibit Lactococcus and Bacillus growth

254

at 90 DAT, GO only inhibit Bacillus gently (Figure 5). Microbial genus is more

255

richness in S2, S3 and S4 soils than that in S1, and bacterial community composition

256

was strongly affected by GN/GO in S2. after application of GO at 90 DAT in S2,

257

Bacillus, Nocardioides, Roseiflexus, increased obviously from 3.8% to 6.4%, 0.4% to

258

3.0%, 1.1% to 2.8%, respectively, and the genus of Arenimonas decreased obviously

259

from 6.8% to 1.5%. The community composition was gently affected by GN/GO in

260

S3 and S4 soils. 12


Page 12 of 28

Page 13 of 28


261

From Fig. 4, 5 and 6, it can be concluded that the richness and diversity of soil

262

bacterial communities increased after GN and GO application. Especially GN can

263

selectively enrich some bacterial phylum (such as Chloroflexi and Firmicutes etc. )

264

and genus (such as Lactococcus and Baccillus etc.), and GO also can inhibit some

265

bacterial genus. Previous reports showed that the change in soil microbial biomass in

266

response to the GO exposure was not obvious, which indicated that the GO toxicity is

267

transient in the short-term response.17,18 The results in this study are in accordance

268

with the previous reports. The effect of GN and GO on soil microbial community is

269

time dependent.20 And no significant differences existed compared with the samples at

270

10 DAT and 90 DAT. The bacterial community structure and composition analysis

271

showed a significant shift after introduction of GN and GO after 10 DAT, and then

272

weaken at 90 DAT. Previous studies also showed that GN and GO impact on

273

microbial community is transient and time dependent. Ge et al reported that GN could

274

not affect fungal communities, whereas it did alter the bacterial community after

275

induction of GN for 1 year.25

276

The results in this study showed that effects of GN and GO on soil enzyme activity,

277

population and microbial communities varied at different incubation time, GN/GO

278

can make some of phyla and genus increase or decrease in soils. The heat map

279

analysis revealed clearly that the bacterial communities after GN/GO introduction and

280

without GN/GO application were different in soils (Fig. 6). Soil microbial community

281

composition and population are not only the simple reflection of the microorganisms

282

in soils, also result from the specific environmental pressures. 13



283 284

Acknowledgments

285

The research was financially funded by the grants “National Natural Science

286

Foundation of China” (Project No. 11275033) and “Natural Science Foundation of

287

Jiangsu Province, China” (Project No. BK20151185).

288 289

References

290

(1) Geim, A.K.; Novoselov, K.S. The rise of grapheme, Nat. Mater. 2007, 6, 183-191.

291

(2) Rao, C.N.; Sood, A.K.; Subrahmanyam, K.S.; Govindaraj, A. Graphene: the new

292

two-dimensional nanomaterial, Angew. Chem. Int. Ed. 2009, 48, 7752–7777.

293

(3) Lerf, A.; He, H.; Forster, M.; Klinowski, J. Structure of graphite oxide revisited. J.

294

Phys. Chem. B. 1998, 102, 4477–4482.

295

(4) Bianco, A. Graphene: Safe or toxic? The two faces of the medal. Angew. Chem. Int.

296

Ed. 2013, 52, 2-14.

297

(5) Xu, J.; Lv, H.; Yang, S.T.; Luo, J. Preparation of graphene adsorbents and their

298

applications in water purification. Rev. Inorg. Chem. 2013, 2-3, 139-160.

299

(6) Zhao, G.; Wen, T.; Chen, C.; Wang, X. Synthesis of graphene-based nanomaterials

300

and their application in energy related and environmental-related areas. RCS Adv.

301

2012, 2, 9286–9303.

302

(7) He, K.; Chen, G.; Zeng, G.; Peng, M.; Huang, Z.; Shi J. Stability, transport and

303

ecosystem effects of graphene in water and soil environments. Nanoscale. 2017, 9,

304

5370. 14


Page 14 of 28

Page 15 of 28


305

(8) Ahmed, F.; Rodrigues, D.F. Investigation of acute effects of graphene oxide on

306

wastewater microbial community: A case study. J. Hazard. Mater. 2013, 256-257,

307

33-39.

308

(9) Zhang, P.; Zhang, R.; Fang, X.; Song, T.; Cai, X.; Liu, H; Du, S. Toxic Effects of

309

Graphene on the Growth and Nutritional Levels of Wheat (Triticum Aestivum L.):

310

Shortand Long-Term Exposure Studies. J. Hazard. Mater. 2016, 317, 543–551.

311

(10) Huang, G.; Guo, H.; Zhao, J.; Liu, Y.; Xing B. Effect of Co-Existing Kaolinite

312

and Goethite on the Aggregation of Graphene Oxide in the Aquatic Environment.

313

Water Res. 2016, 102, 313–320.

314

(11) Lin, D.; Tian, X.; Wu, F.; Xing, B. Fate and Transport of Engineered

315

Nanomaterials in the Environment. J. Environ. Qual. 2010, 39, 1896–1908.

316

(12) Liu, S.; Zeng, T.; Hofmann, M.; Burcombe, E.; Wei, J.; Jiang, R.; Kong, J.; Chen,

317

Y. Antibacterial activity of graphite, graphite oxide, graphene oxide, and reduced

318

graphene oxide: membrane and oxidative stress. ACS Nano. 2011, 9, 6971–6980.

319

(13) Combarros, R.G.; Collado, S.; Diaz, M. Toxicity of graphene oxide on growth

320

and metabolism of Pseudomonas putida. J. Hazard. Mater. 2016, 310, 246-252.

321

(14) Xie, J.; Ming, Z.; Li, H.; Yang, H.; Yu, B.; Wu, R. Toxicity of graphene oxide to

322

white rot fungus Phanerochaete chrysosporium. Chemosphere. 2016, 151, 324-331.

323

(15) Ma, Y.; Shen, H.; Tu, X.; Zhang, Z. Assessing in vivo toxicity of graphene

324

materials: current methods and future outlook. Nanomedicine. 2014, 9(10),

325

1565-1580.

326

(16) Akhavan, O.; Ghaderi, E.; Hashemi, E.; Akbari, E. Dose-dependent effects of 15



327

nanoscale graphene oxide on reproduction capability of mammals. Carbon. 2015, 95,

328

309-317.

329

(17) Chung, H.; Kim, M.J.; Ko, K.; Kim, J.H.; Kwon, H.; Hong, I. Effects of

330

graphene oxides on soil enzyme activity and microbial biomass. Sci. Total Environ.

331

2015, 514: 307-313.

332

(18) Du, J.; Hu, X.; Zhou, Q. Graphene oxide regulates the bacterial community and

333

exhibits property changes in soil. RSC Adv. 2015, 5, 27009.

334

(19) Oyelami, A.O.; Semple, K.T. Impact of carbon nanomaterials on microbial

335

activity in soil. Soil Biol. Biochem. 2015, 86, 172-180.

336

(20) Ren, W.; Ren, G.; Teng, Y.; Li, Z.; Li, L. Time-dependent effect of graphene on

337

the structure, abundance, and function of the soil bacterial community. J. Hazard.

338

Mater. 2015, 297, 286-294.

339

(21) Wang, J.; Chen, J.; Zhu, W.; Ma, J.; Rong, Y.; Cai, Z. Isolation of the Novel

340

Chiral Insecticide Paichongding (IPP) Degrading Strains and Biodegradation

341

Pathways of RR/SS-IPP and SR/RS-IPP in an Aqueous System. J. Agric. Food Chem.

342

2016, 64, 7431−7437.

343

(22) Cai, Z.; Ma, J.; Wang, J.; Cai, J.; Yang, G.; Zhao, X. Impact of the novel

344

neonicotinoid insecticide Paichongding on bacterial communities in yellow loam and

345

Huangshi soils. Environ Sci Pollut Res. 2016, 23, 5134–5142.

346

(23) Cai, Z.; Wang, J.; Ma, J.; Zhu, X.; Cai, J.; Yang, G. Anaerobic degradation

347

pathway of the novel chiral insecticide Paichongding and its impact on bacterial

348

communities in soils. J. Agr. Food Chem. 2015, 63, 7151-7160. 16


Page 16 of 28

Page 17 of 28


349

(24) Asad, M.A.; Lavoie, M.; Song, H.; Jin, Y.; Fu, Z.; Qian. H. Interaction of chiral

350

herbicides with soil microorganisms, algae and vascular plants. Sci. Total Environ.

351

2017, 15, 1287-1299.

352

(25) Ge, Y.; Priester, J.H.; Mortimer, M.; Chong, H.C.; Ji, Z.; Schimel, J.P.; Holden,

353

P.A. Long-term effects of multi-walled carbon nanotubes and graphene on microbial

354

communities in dry soil. Environ. Sci. Technol. 2016, 50, 3965–3974.

355

(26) Chen, J.; Wang, X.; Han, H.; A new function of graphene oxide emerges:

356

inactivating phytopathogenic bacterium Xanthomonas oryzae pv. Oryzae. J. Nanopart.

357

Res. 2013, 15, 1658.

358

(27) Pretti, C.; Oliva, M.; Pietro, B.D.; Monni, G.; Cevasco, G.; Chiellini, F.; Pomelli,

359

C.; Chiappe, C. Ecotoxicity of pristine graphene to marine organisms. Ecotoxicol.

360

Environ. Saf. 2014, 101, 138–145.

361

(28) Wang, X.; Liu, X.; Chen, J.; Han, H.; Yuan, Z. Evaluation andmechanism of

362

antifungal effects of carbon nanomaterials in controlling plant fungal pathogen.

363

Carbon. 2014, 68, 798–806.

364

(29) Du, S.; Zhang, P.; Zhang, R.; Lu, Q.; Liu, L.; Bao, X.; Liu, H. Reduced graphene

365

oxide induces cytotoxicity and inhibits photosynthetic performance of the green Alga

366

Scenedesmus Obliquus. Chemosphere. 2016, 164, 499– 507.

367

(30) Akhavan, O.; Ghaderi, E. Toxicity of graphene and graphene oxide nano walls

368

against bacteria. ACS Nano, 2010, 4, 5731–5736.

369

(31) Liu, S.; Zeng, T.H.; Hofmann, M.; Burcombe, E.; Wei, J.; Jiang, R.; Kong, J.;

370

Chen, Y. Antibacterial activity of graphite, graphite oxide, graphene oxide, and 17



371

reduced graphene oxide: membrane and oxidative stress. ACS Nano. 2011, 5, 6971–

372

6980.

373

(32) Chen, M.; Cao, F.; Li, F.; Liu, C.; Tong, H.; Wu, W.; Hu, M. Anaerobic

374

Transformation of DDT Related to Iron(III) Reduction and Microbial Community

375

Structure in Paddy Soils. J. Agric. Food Chem. 2013, 61, 2224−2233.

376

(33) Seabra, A.B.; Paula, A.J.; de Lima, R.; Alves, O.L.; Duran, N. Nanotoxicity of

377

graphene and graphene oxide. Chem. Res. Toxicol. 2014, 27, 159–168.

378

(34) Nadres, E.T.; Fan, J.; Rodrigues, D.F. Toxicity and Environmental Applications

379

of Graphene-Based Nanomaterials. Graphene-based Materials in Health and

380

Environment, Springer, Cham, Switzerland. 2016, 323–356.

381

18


Page 18 of 28

Page 19 of 28


382

TABLE AND FIGURE CAPTIONS

383

Table 1 Physico-chemical characteristics of the experimental soils.

384

Figure 1. Effects of GN and GO on soil enzyme activity for different incubation

385

periods in soils (A: S1 invertase activity. B: S2 invertase activity. C: S3 invertase

386

activity. D: S4 invertase activity)

387

Figure 2. Effect of GN and GO on soil microbial population for different incubation

388

time in S1 and S2 soils (A: S1 bacterial population. B: S1 actinomyces population. C:

389

S1 fungal population. D: S2 bacterial population. E: S2 actinomyces population. F: S2

390

fungal population.).

391

Figure 3. Bacterial diversity comparison with rarefaction curves (A) and Shannon

392

-Wiener curves (B) in different soils at 10 Day and 90 DAT

393

Figure 4. Bacterial phyla composition of the different communities (Percentage of

394

relative read abundance of bacterial phyla within each community)

395

Figure 5. Bacterial genus composition of the different communities (Percentage of

396

relative read abundance of bacterial genus within each community).

397

Figure 6. Distribution heatmap of microbial genus arranged by hierarchical clustering

398

of soils with different treatment.

399

Supporting Information Available: [Figure S1. SEM and AFM photo of graphene

400

and graphene oxide (a: GN-SEM; b: GO-SEM; c: GN-AFM; d: GO-AFM). Figure S2.

401

Graphene and graphene oxide zeta potential and particle sizes (a: GN sizes; b: GN

402

zeta potential; c: GO sizes; d: GN zeta potential). Figure S3. Effects of GN and GO

403

on soil enzyme activity for different incubation periods in soils.(a: S1 urease activity.

404

b: S2 urease activity. c: S3 urease activity. d: S4 urease activity. e: S1 protease activity. 19



405

f: S2 protease activity. g: S3 protease activity. h: S4 protease activity. i: S1 catalase

406

activity. j: S2 catalase activity. k: S3 catalase activity. l: S1 catalase activity. ).

407

Figure S4. Effect of GN and GO on soil microbial population for different incubation

408

time in S3 and S4 soils (a: S3 bacterial population. b: S3 actinomyces population. c:

409

S3 fungal population. d: S4 bacterial population. e: S4 actinomyces population. f: S4

410

fungal population.). Figure S5. Venn diagram in different soils at 10 Day and 90

411

DAT.]

412

20


Page 20 of 28

Page 21 of 28


413

Table 1

414 Red paddy soil

Yellow loam soil

Huangshi soil (S3)

Yellow paddy soil

(S1)

(S2)

Longyou, Zhejiang

Longquan,

Changzhou, Jiangsu

Huajiachi, Hangzhou,

Province

Fujian Province

Province

Zhejiang Province

pH (H2O)

4.20

6.53

5.95

7.02

OMa /%

8.4

2.67

1.52

30.5

CEC b / cmol kg-1

6.62

14.09

7.11

10.83

Clay / %

39.0

38.7

33.5

18.2

Silt / %

41.1

50.4

49.8

61.2

Sand / %

19.9

10.9

16.7

20.6

0.09mm

17.30

4.3

6.7

20.8

Total N / %

2.03

0.24

0.08

1.12

P /mg kg-1

18.26

21.25

7.65

16.37

K / g kg-1

21.6

13.47

10.7

15.83

Characteristics

Location

Texture/%

415

a

Organic matter.

416

b

Cation Exchange Capacity.

(S4)

417

21



Figure 1. 12 10 8

20

A

Invertase (mg glucose /g soil）

Invertase (mg glucose /g soil）

418

S1C S1GN S1GO

6 4 2 0 10

30

50

70

90

B

S2C S2GN S2GO

15 10 5 0 10

30

Days S3C

14

S3GN

12

S3GO

10 8 6 4 2 0 10

50 Days

C

16

Invertase (mg Glucose /g soil）

Invertase (mg Glucose /g soil）

419

420

Page 22 of 28

30 50 Days

70

90

20 15

70

90

D

S4C S4GN S4GO

10 5 0 10

30

50 Days

22


70

90

Page 23 of 28


Figure 2.

7 6 5 4 3 2 1 0

6 5

30

50 Days

70

S1GN S1GO

4 3 2 1 0 10

30

50

S2GN

8.5

S2GO

8 7.5 7 6.5 6 5.5 5 10

30

70

7.5 7 6 5.5 5 4.5 4 3.5 3

90

10

30

6 5

8

S1G N S1G O

70

90

F

S2C

7 S2G N

6 5

4

4

3

3

2

2 1

1

0

0 10

422

50 Days

C

S1C

90

6.5

Fungal population (lg CFU/g soil)

Fungal population (lg CFU/g soil)

7

70

E

S2C S2GN S2GO

Days 8

50 Days

B

S1C

S2C

D

9

90

Actinomyces population (lgCFU/g soil)

10

Actinomyces population (lgCFU/g soil)

A

S1C S1GN S1GO

Bacterial population (lg CFU/g soil)

8

Bacterial population (lg CFU/g soil)

421

30

50

70

90

10

Days

423

23


30

50 Days

70

90


424

Figure 3.

Rarefaction Measure: rarefaction

A

425

Rarefaction Measure: r-shannon

B

426 24


Page 24 of 28

Page 25 of 28


Figure 4.

Relative abundance (%)

427

428

25



Figure 5.

Relative abundance (%)

429

430

431 432

26


Page 26 of 28

Page 27 of 28


433

Figure 6.

434

27



Graphical Abstract


Page 28 of 28

Pyrosequencing Reveals Soil Enzyme Activities and Bacterial

Recommend Documents