Pyrosequencing Reveals Soil Enzyme Activities and Bacterial

Sep 26, 2017 - Proteobacteria was the most abundant phylum in S2, S3, and S4 soils; it ranged from 43.6 to 71.4% in S2 soil, from 45.6 to 73.7% in S3 ...
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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

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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.

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Journal of Agricultural and Food Chemistry

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Pyrosequencing reveals soil enzyme activities and bacterial

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communities impacted by graphene and its oxides

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

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Yang1*, Xiyue Zhao1,2

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1. Advanced Catalysis and Green Manufacturing Collaborative Innovation Center,

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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]

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ABSTRACT

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

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

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

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

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

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

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

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

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

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

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

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population and bacterial community through pyrosequencing of 16S rRNA gene

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

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

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

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

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

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

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

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

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

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

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

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significant differences between the samples at 10 and 90 DAT.

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

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Soil enzyme activity

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

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

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

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

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

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

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

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

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

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

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

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

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

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adsorption capacity, the enrichment of poisonous and harmful environment the

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material, thereby affecting the environmental pollutants transformation and

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degradation of environmental behavior.7-12

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INTRODUCTION

Previous studies showed that GO have strong antimicrobial effect on

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

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

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

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

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

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

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

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

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

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

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

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ml−1,8

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

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

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

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

which showed that GOs have potential risk to the soil biochemical

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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treatment (DAT) were employed for soil DNA extraction.

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

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

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

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

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previous reports.22,23 5

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Soil DNA Extraction and Pyrosequencing, Bioinformatics Analysis. Soil DNA

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

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

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

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

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

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

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

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

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sequences after quality control were analyzed through QIIME. Operational taxonomic

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

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

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

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

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

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

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

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are shown in Figure1 and Figure S3.

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Invertase activity invertase activity increased significantly after GN application in 6

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

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

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

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

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

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soil S4.

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

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

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

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

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

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

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

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

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Protease activity protease activity presents soil microbial population and fertility.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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with GN and GO application.

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

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

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transformation of organic compounds. Therefore the changes in soil microbial

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

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

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

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

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

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

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

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

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

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

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enzyme activity.17-19 8

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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and decreased in S2 and S4 soils.

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

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

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

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

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

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

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

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

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

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

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

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

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

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toxic to Gram-positive bacteria.30

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

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

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

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

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

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

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

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

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

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

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

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

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

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which may weaken GN/GO toxicity to microorganism.

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Microbial Community Species Richness and Diversity. The rarefaction curve

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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90 DAT. 11

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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S3 and S4 soils. 12

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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induction of GN for 1 year.25

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

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

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

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

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

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

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

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Acknowledgments

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

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

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

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TABLE AND FIGURE CAPTIONS

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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.).

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

392

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

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

394

relative read abundance of bacterial phyla within each community)

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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

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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. ).

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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

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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

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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

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30 50 Days

70

90

20 15

70

90

D

S4C S4GN S4GO

10 5 0 10

30

50 Days

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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

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70

90

Journal of Agricultural and Food Chemistry

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Figure 3.

Rarefaction Measure: rarefaction

A

425

Rarefaction Measure: r-shannon

B

426 24

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Figure 4.

Relative abundance (%)

427

428

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Figure 5.

Relative abundance (%)

429

430

431 432

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Figure 6.

434

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Graphical Abstract

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