Pyrosequencing Reveals Soil Enzyme Activities ... - ACS Publications

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Cite This: J. Agric. Food Chem. 2017, 65, 9191-9199

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†,‡ †

Advanced Catalysis and Green Manufacturing Collaborative Innovation Center and ‡Laboratory of Applied Microbiology, School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu 213164, People’s Republic of China S Supporting Information *

ABSTRACT: Graphene (GN) and graphene oxides (GOs) are novel carbon nanomaterial; they have been attracting much attention because of their excellent properties and are widely applied in many areas, including energy, electronics, biomedicine, environmental science, etc. With industrial production and consumption of GN/GO, they will inevitably enter the soil and water environments. GN/GO may directly cause certain harm to microorganisms and lead to ecological and environmental risks. GOs are GN derivatives with abundant oxygen-containing functional groups in their graphitic backbone. The structure and chemistry of GN show obvious differences compared to those of GO, which lead to the different environmental behaviors. In this study, four different types of soil (S1−S4) were employed to investigate the effect of GN and GO on soil enzymatic activity, microbial population, and bacterial community through pyrosequencing of 16S rRNA gene amplicons. The results showed that soil enzyme activity (invertase, protease, catalase, and urease) and microbial population (bacteria, actinomycetes, and fungi) changed after GN/GO release into soils. Soil microbial community species are more rich, and the diversity also increases after GO/GN application. The phylum of Proteobacteria increased at 90 days after treatment (DAT) after GN/GO application. The phylum of Chlorof lexi occurred after GN application at 90 DAT in S1 soil and reached 4.6%. 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 soil, and from 38.1 to 56.7% in S4 soil. The most abundant genera were Bacillus (37.5−47.0%) and Lactococcus (28.0−39.0%) in S1 soil, Lysobacter and Flavobacterium in S2 soil, Pedobacter in S3 soil, and Massilia in S4 soil. The effect of GN and GO on the soil microbial community is timedependent, and there are no significant differences between the samples at 10 and 90 DAT. KEYWORDS: graphene, graphene oxides, pyrosequencing, bacterial community, soil enzyme activity

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INTRODUCTION Graphene (GN) is a two-dimensional nanomaterial with sp2 hybridization carbon atoms,1,2 and graphene oxides (GOs) are layered GN sheets with oxygen functional groups, such as carbonyl and hydroxyl groups.3,4 GN and GOs have attracted much attention because of their excellent properties (such as low resistivity, rapid electron mobility, etc.) and wide application in many areas (including complex material, energy, electronics, biomedicine, environmental science, etc.).1,4 For instance, GN has high-performance properties of an adsorbent in water treatment,5 and GOs are used in removing pollutants as a result of their excellent adsorbents and photocatalysts.6 GN and GOs are now transferred to industrial production, and 1000 tons per year of GO has been put into operation in Changzhou, China. With industrial production and consumption of GN/GO, they will inevitably enter the environment. GN/GO may directly cause certain harm to animals, plants, and microorganisms in the environment and lead to ecological and environmental risks.7−9 GN and GOs have strong adsorption capacity, with the enrichment of poisonous and harmful environmental material, thereby affecting the environmental pollutant transformation and degradation of environmental behavior.7−12 Previous studies showed that GOs have a strong antimicrobial effect on microorganisms, such as Escherichia coli,12 Pseudomonas putida,13 and white rot fungus Phanerochaete chrysosporium.14 © 2017 American Chemical Society

GN also showed toxicity to cells and animals, and the genotoxicity and long-term toxicity caused by GN have been reported.15,16 The large amount of GN and GO application may lead to their residues in the environment, which can affect soil enzyme activity and soil microbial community by changing their number, microbial activity, and diversity.10,13−15,17−20 Soil enzyme activity and microbial community are often used as the key factors in assessing soil quality. Artificial compounds have an effect on soil microbial dominant species and community diversity and richness and also impact soil enzyme activity and microbial population. Until now, only a few reports studied the effects of GO on the microbial community.8,21 GO had a toxic effect on the wastewater microbial communities at concentrations from 0.05 to 0.3 mg mL−1,8 which showed that GOs have potential risk to the soil biochemical characteristics. A better understanding of GN and GO impacts on soil enzyme activity, microbial population, and microbial community diversity and composition in the soils is necessary for their safe use. The present study investigated their effect on the soil microbial community, diversity, and population and soil enzyme activity. Received: Revised: Accepted: Published: 9191

August 5, 2017 September 23, 2017 September 26, 2017 September 26, 2017 DOI: 10.1021/acs.jafc.7b03646 J. Agric. Food Chem. 2017, 65, 9191−9199

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Journal of Agricultural and Food Chemistry Table 1. Physicochemical Characteristics of the Experimental Soils characteristic location pH (H2O) OMa (%) CECb (cmol kg−1) clay (%) silt (%) sand (%) texture (%) 0.09 mm total N (%) P (mg kg−1) K (g kg−1) a

red paddy soil (S1) Longyou, Zhejiang province 4.20 8.4 6.62 39.0 41.1 19.9 46.20 36.50 17.30 2.03 18.26 21.6

yellow loam soil (S2) Longquan, Fujian province 6.53 2.67 14.09 38.7 50.4 10.9 67.4 28.3 4.3 0.24 21.25 13.47

Huangshi soil (S3) Changzhou, Jiangsu province 5.95 1.52 7.11 33.5 49.8 16.7 60.7 32.6 6.7 0.08 7.65 10.7

yellow paddy soil (S4) Huajiachi and Hangzhou, Zhejiang province 7.02 30.5 10.83 18.2 61.2 20.6 8.0 71.20 20.8 1.12 16.37 15.83

OM = organic matter. bCEC = cation-exchange capacity.

Figure 1. Effects of GN and GOs on soil enzyme activity for different incubation periods in soils (A, S1 invertase activity; B, S2 invertase activity; C, S3 invertase activity; and D, S4 invertase activity).

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Experimental Procedure. To study the impact of GN and GOs on soil enzyme activities, microbial population, and bacterial community, GN and GOs were mixed with soils thoroughly to give a final concentration of 100 mg kg−1 of soil. Soils were incubated at 25 ± 1 °C to allow microorganisms to acclimatize. The control experiment was carried out under the same conditions without GN and GOs (CK set). All of the tests for each soil were conducted with three replicates. The soil moisture content was adjusted to about 60% of the maximum waterholding capacity by adding Milli-Q (MQ) water. At different time intervals (10, 30, 50, 70, and 90 days), 5 g of soil (dry weight equivalent) was sampled from the flask and used for soil microbial population and soil enzyme activities. Soil samples at 10 and 90 days after treatment (DAT) were employed for soil DNA extraction. The activities of soil protease (EC 3.4.21.92), invertase (EC 3.2.1.26), catalase (EC 3.5.1.5), and urease (EC 1.11.1.6) were analyzed according to a previously published method.22 Soil microbial population (heterotrophic bacteria, actinomycetes, and fungi) counting was assayed through the most probable number (MPN) method according to previous reports.22,23 Soil DNA Extraction and Pyrosequencing and Bioinformatics Analysis. Soil DNA extraction was based on the protocol of the

MATERIALS AND METHODS

Chemicals and Soil Samples. GN and GOs were obtained from Jiangnan Graphene Research Institute (Changzhou, China). The morphology of GN and GOs was investigated by scanning electron microscopy (SEM, Zeiss Supra 55, Germany; Figure S1 of the Supporting Information) and atomic force microscopy (AFM, JPK Nano Wizard 3, Germany, Figure S1 of the Supporting Information). GN and GO ζ potential and particle sizes were measure by a Zetasizer nano potentiometer (Malvern, Zetasizer Nano ZEN 3600, U.K., Figure S2 of the Supporting Information). Four types of soil from different agricultural fields were sampled and employed in this study, which were red paddy soil (S1, GB/T-H2121315), yellow loam soil (S2, GB/T-A2111411), Huangshi soil (S3, GB/T-G2511211), and yellow paddy soil (S4, GB/T-A2111511). The soil samples were taken from crop fields in Longyou (Zhejiang province, China), Longquan (Fujian province, China), Changzhou (Jiangsu province, China), and Huajiachi and Hangzhou (Zhejiang province, China). All soil samples were air-dried, mixed, and passed through a 2 mm sieve. Their basic physicochemical characteristics were listed in Table 1. 9192

DOI: 10.1021/acs.jafc.7b03646 J. Agric. Food Chem. 2017, 65, 9191−9199

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Journal of Agricultural and Food Chemistry

Figure 2. Effect of GN and GOs on soil microbial population for different incubation times in S1 and S2 soils (A, S1 bacterial population; B, S1 actinomycete population; C, S1 fungal population; D, S2 bacterial population; E, S2 actinomycete population; and F, S2 fungal population). manufacturer (Fast DNA Spin Kit, MP Biomedicals, Solon, OH, U.S.A.). The total DNA was purified by electrophoresis in 1% agarose gel. Pure DNA was extracted using Agarose Gel Extraction Kit (Roche). DNA quality was assessed with a ScanDrop 200 spectrophotometer. The primers 515F (5′-GTGCCAGCMGCCGCGG-3′) and 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) were used for amplifying the fragment of the 16S rRNA gene. The polymerase chain reaction (PCR) reaction program and PCR product purification were used according with previously reported methods.22,23 In total, 865 196 16S rRNA sequence reads were filtered. Trimmomatic was used for denoising and processing. The resulting sequences after quality control were analyzed through QIIME. Operational taxonomic units (OTUs) was used at 97% sequence similarity. The methods used for rarefaction curves, Shannon−Wiener curves, community composition, Venn diagrams and principal coordinate analysis (PCoA), and genera heatmap were in accordance with the reported methods.22,23

released into soils perhaps can impact soil enzyme activities, and their effects on enzyme activities (invertase, urease, protease, and catalase) are shown in Figure 1 and Figure S3 of the Supporting Information. Invertase Activity. Invertase activity increased significantly after GN application in S1 soil, while it was also kept unchangeable after GO application, except at 70 DAT invertase activity, reaching 7.10 mg of glucose g−1 of soil; the control group was only 5.0 mg of glucose g−1 of soil. In S2 soil, invertase activity was inhibited by GN and GOs at 30 and 90 DAT, while it increased at 50 DAT. Invertase activity remained almost unchangeable in S3 soil, and GOs inhibit its enzymatic activity during 30−70 DAT in S4 soil. Urease Activity. Soil urease plays a key role during the transformation of organic phosphorus. The impact on urease activities by GN and GOs differed in different types of soil. Urease activity in S1 soil inhibited significantly before 50 DAT; it only reached 0.33 and 0.26 mg of NH4 N g−1 of soil after GN and GO application at 30 DAT, while the activity was 0.58 mg of NH4 N g−1 of soil in the control soil. However, urease activity increased remarkable at 70 and 90 DAT. GN inhibited enzyme

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RESULTS AND DISCUSSION Effect of GN and GOs on the Soil Enzyme Activity. Soil environmental change and stress often lead to soil enzyme activity change, which is often used as the soil quality index. GN and GOs 9193


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Figure 3. Bacterial diversity comparison with (A) rarefaction curves and (B) Shannon−Wiener curves in different soils at 10 and 90 DAT.

activity, while GO increased in S2 soil. Both GN and GO inhibited urease activity gently in S4 soil and increased significantly in S3 soil at 90 DAT. Protease Activity. Protease activity presents soil microbial population and fertility. GN can inhibit protease gently, and GOs increase enzyme activity remarkably at 90 DAT in S1 soil. Protease activity remains unchangeable in S3 soil after GN and GO

application. In S2 and S4 soils, both GN and GO inhibit enzyme activity. Protease activity was 0.91 and 0.79 mg of tyrozine g−1 of soil after GN and GO application in S2 soil, respectively, while in control soil, it is 1.52 mg of tyrozine g−1 of soil 90 DAT. In S4 soil, enzyme activity is 8.0 mg of tyrozine g−1 of soil; after GN and GO application, it declines to 6.24 and 5.21 mg of tyrozine g−1 of soil, respectively. 9194


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Figure 4. Bacterial phyla composition of the different communities (percentage of relative read abundance of bacterial phyla within each community).

Catalase Activity. Catalase is one of the most important enzymes in microbial growth and can protect organisms from H2O2 toxicity. In this study, catalase activity increased, except 30 DAT in S1 soil, and it was stimulated to increase after GN and GO application in soils. At 50 DAT, catalase activity reached 3.62 and 2.97 mL of 0.02 M KMnO4 g−1 of soil after GN and GO application in S1 soil, respectively. The catalase activity increased 47.9 and 21.4% in S1 soil compared to the control group at 50 DAT, respectively. In the other three types of soils, catalase activity both increased with GN and GO application. Enzyme activity in soils is one of the key roles in decomposition of biological residues (animal, plant, microorganism, and human) and biodegradation and transformation of organic compounds. Therefore, the changes in soil microbial population and enzyme activity exposed to GN and GOs were investigated in this study; the results were employed to assess the effect of GN and GOs on soil quality.9,10,24,25 Figure 1 showed that GN and GOs could stimulate to increase invertase activities in S1 soil, decrease at 30 and 90 DAT in S2 soil, respectively, keep almost unchangeable in S3 soil, and decrease at 30 DAT in S4 soil. Urease was inhibited at the initial day and then increased significantly after 70 DAT. Protease activity decreased in S1 and S2 soils, and catalase activity increased in the four different soils. The results indicated that both GN and GOs can impact enzyme activity in different soils and invertase, catalase, and protease are sensitive to GN and GOs. However, these effects were transient; enzyme activity kept a normal level after 50−90 DAT. A higher concentration of GOs lowered soil enzyme activity.17−19 Effect of GN and GOs on the Soil Microbial Population. The effects of GN and GOs on the total number of bacteria, actinomycetes, and fungi in soils were showed in Figure 2 and Figure S4 of the Supporting Information. The bacterial total number in S1 soil increased after GN and GO application at 30 and 70 DAT and decreased in 10 and 50 DAT. When GN was

just released to S2 soil, bacterial population increased in comparison to the CK soil obviously, especially at 30 DAT, and the bacterial total number reached 109 colony-forming units (CFU) g−1 of soil, while it was only 108 CFU g−1 of soil in the CK soil. It was only 107 CFU g−1 of soil after GO application in S2 soil at 30 DAT. In S3 soil, the bacteria population inhibited at 10, 30, and 70 DAT and increased at 50 and 90 DAT. The total number of bacteria in S4 soil increased at 30 and 70 DAT and decreased at 10, 50, and 90 DAT. GOs inhibit actinomycete growth in S1 and S2 soils, especially at 70 DAT in S1 soil; the actinomycete population is 8 × 103 CFU g−1 of soil, while it was 9 × 104 CFU g−1 of soil in the CK soil. However, GN and GOs were released into S3 and S4 soils after 30 DAT, and the actinomycete population increased compared to the CK soil obviously. In S3 soil, the total number of actinomycetes reached 4 × 106 and 6 × 106 CFU g−1 of soil after GN and GO application, respectively, and it was only 1 × 106 CFU g−1 of soil in the CK soil at 50 DAT. GN and GOs inhibit actinomycetes at 10 DAT and then were stimulated to increase significantly. The fungal population in S1 and S3 soils increased and decreased in S2 and S4 soils. Many studies showed that GOs have antimicrobial activity, which has been investigated through microorganism culture.10,17,26−28 Antimicrobial activity of GOs may also apply to soil as well. However, our findings demonstrate that GOs can promote soil enzyme activity in particular soils and may have a positive effect on the functions of environmental microorganisms in soils. Its negative impact on soil enzyme activity and microbial population may only be transient; with cultural days prolonging, the negative effect will disappear. Especially in S1 soil, both GN and GOs increase invertase, protease, and catalase activities, and the bacterial and fungal populations in S1 soil also increased significantly. The concentration of GN and GOs is 100 mg kg−1 of soil in this study, which is far higher than that in many 9195


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Figure 5. Bacterial genera composition of the different communities (percentage of relative read abundance of bacterial genera within each community).

studies.7,8,17,19,20,29 A lot of studies showed the potential toxicological impact of GN and GOs on many microorganisms. GOs have strong cytotoxicity toward bacteria,30,31 fungi,32 algae,29 etc. In comparison to Gram-negative bacteria, GN and GOs are more toxic to Gram-positive bacteria.30 Figure 1 indicated that the catalase activity reached a maximum at 50 DAT and then was higher than that in the control soil, with lower catalase activity at the beginning of GN/GO application. This showed that the catalase activity is sensitive to GN/GO.10,11 The activity of catalase was related to microbial quantity but also microbial environmental stress, including reactive oxygen species (ROS), superoxide anions (O2• −), hydrogen peroxide (H2O2), and hydroxyl radicals (•OH).33,34 The microorganism is often induced to generate ROS and its subsequent oxidative stress after it is exposed to GN and GOs. The results in this study of GN and GOs can inhibit or increase soil enzyme activities and microbial population in different soils, which showed that GN and GOs have different toxicities to bacterial in different environments;

these are in accordance with the results of the microbial population in different soils. The organic matter (OM) can provide effective carbon and energy sources, and its content leads to different microbial populations and growth rates in soils. A higher OM content brought about a higher microbial population, which may weaken GN/GO toxicity to the microorganism. Microbial Community Species Richness and Diversity. The rarefaction curve (Figure 3A) can assess environmental microbial species richness. The Shannon−Wiener curve (Figure 3B) reflects the index of microbial species diversity. The Venn diagrams (Figure S5 of the Supporting Information) show all possible logical relations in the sample. Microbial species are more rich after GN and GO application in S4 soil than that in the control at 90 DAT. The species decrease after GO application at 10 DAT, while it increases after GN application in S4 soil. Microbial species are relatively poor after GN and GO application in S2 and S3 soils than in control soil at 90 DAT. The sequence of species richness from high to low is S4 soil > S3 9196


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Figure 6. Distribution heatmap of microbial genera arranged by hierarchical clustering of soils with different treatments.

inhibit species diversity in S3 soil at 90 DAT; however, GOs can increase diversity while GN also can inhibit species diversity in S2 soil at 90 DAT. Community Species Composition and Structure. The results shown in Figure 4 were all relative read abundances assigned to bacteria. Firmicutes were the most abundant phylum in S1 soil (87.2−95.3%), and the second most abundant phylum is Proteobacteria (3.6−9.7%). The ratio of Proteobacteria increased at 90 DAT after GN/GO application. The phylum of Chloroflexi occurred after GN application at 90 DAT in S1 soil and reached 4.6%. Proteobacteria was the most abundant phylum

soil > S2 soil > S1 soil. OTUs in S4 soil are above 1500, and OTUs in S3 and S2 soils are higher than 1000; however, in S1 soil, OTUs are below 300 at 90 DAT. The microbial species are higher at 90 DAT than at 10 DAT in S2, S3, and S4 soils. Species diversity was shown in Figure 3B. With the increase of the inocubation time, the microbial species diversity in soils with GN/GO application and the control group increased. GOs can inhibit species diversity while GN can increase microbial species diversity at 10 DAT in S4 soil, and the microbial species diversity is almost unchangeable compared to the control group at 90 DAT after GN and GO application. GN/GO can both gently 9197


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Journal of Agricultural and Food Chemistry in S2, S3, and S4 soils, and it ranged from 43.6 to 71.4% in S2 soil, from 45.6 to 73.7% in S3 soil, and 38.1 to 56.7% in S4 soil. GOs can inhibit Proteobacteria in S2 soil, which decreased from 53.7 to 43.6% at 90 DAT. The phylum of Chloroflexi increased, which was 10.3% after GO application in S2 soil and was only 7.7% in the control soil. Firmicutes were also stimulated to increase by GN and GOs. In S3 soil, the phylum of Bacteroidetes increased by 3.4% after GN application and 3.1% after GO application compared to the control soil. Firmicutes decreased from 14.4 to 11.2%. In S4 soil, Acidobacteria was inhibited at 90 DAT after GN/GO application. The most abundant genera were Bacillus (37.5−47.0%) and Lactococcus (28.0−39.0%) in S1 soil. GN inhibits Lactococcus and Bacillus growth at 90 DAT, while GOs only gently inhibit Bacillus (Figure 5). Microbial genera are more rich in S2, S3, and S4 soils than in S1 soil, and bacterial community composition was strongly affected by GN/GO in S2 soil. After application of GOs at 90 DAT in S2 soil, Bacillus, Nocardioides, and Roseiflexus increased obviously from 3.8 to 6.4%, from 0.4 to 3.0%, and from 1.1 to 2.8%, respectively, and the genus of Arenimonas decreased obviously from 6.8 to 1.5%. The community composition was gently affected by GN/GO in S3 and S4 soils. From Figures 4−6, it can be concluded that the richness and diversity of soil bacterial communities increased after GN and GO application. Especially, GN can selectively enrich some bacterial phyla (such as Chloroflexi, Firmicutes, etc.) and genera (such as Lactococcus, Baccillus, etc.), and GOs also can inhibit some bacterial genera. Previous reports showed that the change in soil microbial biomass in response to the GO exposure was not obvious, which indicated that the GO toxicity is transient in the short-term response.17,18 The results in this study are in accordance with the previous reports. The effect of GN and GOs on the soil microbial community is time-dependent.20 No significant differences existed in comparison to the samples at 10 and 90 DAT. The bacterial community structure and composition analyses showed a significant shift after introduction of GN and GOs after 10 DAT and then weaken at 90 DAT. Previous studies also showed that the GN and GO impacts on the microbial community are transient and time-dependent. Ge et al. reported that GN could not affect fungal communities, whereas it did alter the bacterial community after induction of GN for 1 year.25 The results in this study showed that the effects of GN and GOs on soil enzyme activity and microbial community composition and population varied at different incubation times. GN/GO can make some phyla and genera increase or decrease in soils. The heatmap analysis clearly revealed that the bacterial communities after GN/GO introduction and without GN/GO application were different in soils (Figure 6). The soil microbial community composition and population are not only the simple reflection of the microorganisms in soils but also result from specific environmental pressures.

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for different incubation periods in soils (a, S1 urease activity; b, S2 urease activity; c, S3 urease activity; d, S4 urease activity; e, S1 protease activity; f, S2 protease activity; g, S3 protease activity; h, S4 protease activity; i, S1 catalase activity; j, S2 catalase activity; k, S3 catalase activity; and l, S1 catalase activity) (Figure S3), effect of GN and GOs on the soil microbial population for different incubation times in S3 and S4 soils (a, S3 bacterial population; b, S3 actinomycete population; c, S3 fungal population; d, S4 bacterial population; e, S4 actinomycete population; and f, S4 fungal population) (Figure S4), and Venn diagrams in different soils at 10 and 90 DAT (Figure S5) (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Telephone/Fax: +86-519-86330160. E-mail: [email protected]. *Telephone/Fax: +86-519-86330160. E-mail: yguanghua@cczu. edu.cn. ORCID

Zhiqiang Cai: 0000-0002-9180-675X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research was financially funded by grants from the National Natural Science Foundation of China (Project 11275033) and the Natural Science Foundation of Jiangsu Province, China (Project BK20151185).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b03646. SEM and AFM photo of GN and GOs (a, GN-SEM; b, GO-SEM; c, GN-AFM; and d, GO-AFM) (Figure S1), GN and GO ζ potential and particle sizes (a, GN sizes; b, GN ζ potential; c, GO sizes; and d, GN ζ potential) (Figure S2), effects of GN and GOs on soil enzyme activity 9198


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