Effects of Various Carbon Nanotubes on Soil Bacterial Community

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Effects of Various Carbon Nanotubes on Soil Bacterial Community Composition and Structure 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


Fan Wu, Yaqi You, Xinyu Zhang, Haiyun Zhang, Weixiao Chen, Yu Yang, David Werner, Shu Tao, and Xilong Wang 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


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Effects of Various Carbon Nanotubes on Soil Bacterial Community Composition

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and Structure

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Fan Wua, Yaqi Youb, Xinyu Zhanga, Haiyun Zhanga, Weixiao Chena, Yu Yangb, David Wernerc, Shu

4

Taoa, Xilong Wanga,

5 6

a

7

University, Beijing 100871, China

8

b Department

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c School

Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, Peking

of Civil and Environmental Engineering, University of Nevada, Reno, NV 89557, USA

of Engineering, Newcastle University, Newcastle upon Tyne, UK

10 11

Corresponding author. Email address: [email protected] (X. Wang)

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ABSTRACT

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Carbon nanotubes (CNTs) have huge industrial potential and their environmental impacts need to be

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evaluated. Knowledge of CNTs impacts on soil microbial communities is still limited. To address this

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knowledge gap, we systematically examined dynamic effects of one type of single-walled carbon

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nanotubes (SWs) and three multi-walled carbon nanotubes (MWs) with different outer diameters on

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the soil bacterial community in an agricultural soil over 56 days. The results showed that SWs

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differently affected soil bacterial abundance, diversity, and composition as compared to MWs. The

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differences could have resulted from the materials’ distinct physical structure and surface composition,

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which in turn affected their bioavailability in soil. For certain treatments, soil bacterial diversity and

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the relative abundance of certain predominant phyla were correlated with their exposure duration.

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However, many phyla recovered to their initial relative abundance within 56 days, reflecting resilience

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of the soil bacterial community in response to CNTs-induced disturbance. Further analysis at the

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genus level showed differential tolerance to MWs, as well as size- and dose-dependent tolerance

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among bacterial genera. Predictive functional profiling showed that while CNTs initially caused

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fluctuations in microbial community function, community function largely converged across all

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treatments by the end of the 56-day exposure.

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INTRODUCTION

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Carbon nanotubes (CNTs) have huge potential in many fields because of their unique mechanical

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properties and thermal and chemical stability.1,2 With global production gradually increasing, an

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increasing amount of CNTs will inevitably be released into the environment from their synthesis, use

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and disposal,2 which may bring about effects on ecosystems and human health.3,4 Soil holds a large

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proportion of the Earth’s biodiversity,5 and soil organisms collectively play a dominant role in the

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processes of soil formation and development. Soil bacteria, are sensitive to environmental changes

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induced by pollutants (such as carbon nanomaterials) and reflect the quality and health status of soil.6

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Hence, they can be used as effective biomarkers for soil quality assessment and indicators of soil

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ecosystem functioning.7 2



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Soil is likely a major sink of the released nanomaterials.8 Consequently, CNTs may accumulate in soil,

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which may affect soil bacterial communities and soil functioning.3,9 Most previous studies regarding

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the impact of CNTs on bacteria mainly focused on the cells level and cytotoxicity mechanisms. It was

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found that highly purified single-walled carbon nanotubes (SWs) and multi-walled carbon nanotubes

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(MWs) significantly affected cellular membrane and metabolic activity of Escherichia coli after direct

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contact with cells.10 Recently, Yang et al.11 documented that MWs with varying diameters exhibited

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different degrees of cell membrane destruction in various bacteria, suggesting a size-dependent

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cytotoxicity of CNTs. Only a few studies focused on the effects of CNTs on soil bacteria at the

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community level. For instance, a study about the influence of MWs on tomato plants and the soil

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microbial community showed that they had no significant effect on bacterial diversity but affected

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bacterial community composition by altering the relative abundance of bacterial groups.12 Another

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study showed that MWs at high concentrations (5000 μg/g) decreased enzymatic activities and

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biomass of soil microorganisms.13 Rodrigues et al.14 demonstrated that SWs affected the soil bacterial

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community and had negative impacts on the soil nutrient cycle. In general, studies concerning the

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influence of CNTs on soil bacterial communities are scarce and there has been no consensus.15

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As a first attempt to systematically address this knowledge gap, we hypothesize that the effect of

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CNTs on soil bacterial community is a function of their physical structure, size, exposure dose and

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duration; different species of bacteria would respond differently. To test these hypotheses, we

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conducted a 56-day incubation experiment and studied the effects of one type of SWs and three

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different MWs with dissimilar physical structure and size on the bacterial community in an

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agricultural soil, including bacterial abundance, diversity, community composition and structure, and

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function, under various exposure doses and incubation durations. By comparing responses of the soil

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bacterial community under various scenarios, this study substantially contributes to a more systematic

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understanding of how CNTs exposure under different conditions would affect the soil bacterial

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community and thus soil functioning.

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MATERIALS AND METHODS

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CNTs Characterization. One type of SWs and three MWs with outer diameters of 10-20 nm

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(MW20), 20-30 nm (MW30), and 30-50 nm (MW50) were all purchased from Chengdu Organic

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Chemicals Co. Ltd., Chinese Academy of Sciences. The sizes of these CNTs were characterized using

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transmission electron microscopy (TEM) (FEI Tecnai G2 F30); their outer diameters were calculated

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based on TEM images of 15 tubes, and the values were 3.441 ± 0.143, 19.823 ± 0.216, 24.940 ±

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0.433, and 42.882 ± 0.165 nm for SWs, MW20, MW30 and MW50, respectively (Figure S1; Table

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S1). The specific surface area of individual CNTs was obtained using a surface area analyzer

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(Autosorb-1-MP, Quantachrome Instruments, USA), and the values for SWs, MW20, MW30 and

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MW50 were 495.1, 176.4, 130.4 and 127.5 m2/g, respectively. More details regarding TEM imaging,

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specific surface area and porosity measurements of the CNTs are described in the Supporting

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Information. Methods for determination of the bulk elemental composition and surface oxygen and

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carbon contents are described in SI as well.

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Experimental Design (exposure experiment). Soil was sieved through a 2 mm sieve, after removing

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small stones and plant roots. It was then pre-incubated at 25 °C for one week. After pre-incubation, the

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soil was used for 56-day microcosm experiments. Each sterilized glass bottle was filled with 30 g soil

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and a specific amount of SWs or MWs to create a microcosm. The CNTs were added to soil as dry

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powder to reach a mass fraction of 0.05%, 0.1% or 0.5%, followed by vigorous stirring and agitation

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with a spatula to mix the soil with the CNTs homogeneously. The same method has been used in other

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studies.16,17 Sterilized water was added to microcosms every week to maintain soil moisture content to

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the field level of 15.3%, which was measured after drying for 8 h at 150 °C in an oven prior to the

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microcosm experiments. Details of the soil characterization are described in SI. Soil without CNTs

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added served as the control. All treatments were performed with three replicates and microcosms

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incubated at 25 °C in the dark. During the entire incubation period, soil bottles were sealed with some

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small holes in the lid allowing ventilation. Around 30 g soil was sampled on day 0, 7, 28, and 56

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without prior mixing.

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Quantitative PCR Analysis and High-throughput Sequencing of 16S rRNA Genes. The bacterial 4



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16S rRNA gene abundance was assessed by quantitative PCR (qPCR) with the primer pair

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BACT1369F

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(5’-GGWTACCTTGTTACGACTT-3’)

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(5’-CTTGTACACACCGCCCGTC-3’),18 with details presented in Supporting Information. For

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community analysis, bacterial 16S rRNA genes were amplified using the primer pair 515F

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(GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT).19 The PCR

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products were purified and then the sequencing libraries were established using a TruSeq®DNA

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PCR-Free Sample Preparation Kit (Illumina, USA) following the manufacturer’s protocols. Quality of

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the library was initially determined with [email protected] Fluorometer (Thermo Scientific, USA) and

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Agilent Bio-analyzer 2100 System (Agilent Technologies, USA), and then sequencing analysis was

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performed on an Illumina PE250 platform.20 Raw reads (2250 bp paired-end) obtained from MiSeq

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high-throughput sequencing were analyzed with the pipeline of Quantitative Insights into Microbial

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Ecology (QIIME).21 Chimeras were filtered out using the QIIME’s default program ChimeraSlayer,22

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and reads were clustered into operational taxonomic units (OTUs) using UPARSE,23 with a threshold

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of 97% identity. The representative sequences of OTUs were classified by the Ribosomal Database

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Project (RDP) classifier.24 All representative sequences were also aligned against the latest SILVA

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database.25 All samples were rarefied to the smallest sequencing depth using QIIME before

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downstream analyses to ensure comparisons between samples.

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Characterization of Soil Bacterial Communities. To understand effects of CNTs on the soil

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bacterial community, the bacterial abundance, diversity and community composition were

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characterized.26,27 The total bacterial abundance was calculated by copy numbers of the bacterial 16S

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rRNA gene measured using qPCR. Alpha diversity was used to measure the amount of species in the

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community and the relative abundance among species. In this study, we used the Shannon index to

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describe the alpha diversity of the bacterial community, as it evaluates both evenness and richness.28

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Bray-Curtis distance was calculated as a measure of control-to-treatment dissimilarity in bacterial

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community composition (beta diversity). Weighted UniFrac distance was measured to assess pairwise

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dissimilarity in bacterial community composition (beta diversity). To describe the bacterial

(5’-CGGTGAATACGTTCYCGG-3’) using

and the

probe

PROK1492R TM1389F

5


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community composition change more quantitatively, we calculated the percentage change in the

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relative abundance of individual phyla relative to the control using the following formula:

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Tx - C0 C0

× 100%

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Tx: relative abundance of a certain phylum exposed to X (CNT treatment, including CNT type, dose,

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and duration); C0: relative abundance of a certain phylum in the control; + represents a positive shift; -

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represents a negative shift.

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Principal coordinate analysis (PCoA) was employed on weighted UniFrac distances with QIIME to

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visualize the differences in microbial community composition. Moreover, Adonis permutational

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multivariate

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(compare_categories.py) to verify the results of PCoA. Linear discriminant analysis coupled with

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effect size (LEfSe) was used to determine species with significant differences in abundance among

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different treatments.29 Phylogenetic investigation of communities by reconstruction of unobserved

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states (PICRUSt) was used to infer metagenome functional contents from 16S metagenomics data for

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all samples.30

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Statistical Analyses. The CNTs treatment effects on soil bacterial abundance and alpha diversity were

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determined using one-way analysis of variance (ANOVA) or Kruskal-Wallis analysis, depending on

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the results from a Levene's test of homogeneity of variances. Here, p < 0.05 indicates significant

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differences. Pearson correlation analysis was used to test for significant correlations between changes

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in the soil bacterial community and the CNT exposure doses and durations. All statistical analyses

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were performed using SPSS 19.0 (IBM Co., Armonk, New York, USA).

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RESULTS

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Effects of CNTs on Soil Bacterial Abundance. The copy number of bacterial 16S rRNA genes

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detected by qPCR is a general index of the total bacterial abundance in a soil microbial community

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(Figure 1). Considering different CNT types, SWs at varying doses generally led to a small increase in

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soil bacterial abundance relative to the control, and this effect was most significant for the highest

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dose (p < 0.05). This indicated that SWs generally slightly promoted the bacterial abundance in soil.

analysis

of

variance

(PERMANOVA)

was

conducted

with

QIIME

6



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Among the three MWs, MW50 with the largest diameter generally reduced soil bacterial abundance,

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whereas MW20 and MW30 with a smaller diameter promoted it in some cases. These observations

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suggested that different CNTs exerted dissimilar effects on soil bacterial abundance. Likewise, either

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positive or negative effect of CNTs on soil bacterial abundance was reported in previous studies.15, 31

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Figure 1. Changes in copy numbers of bacterial 16S rRNA genes in soil treated by various CNTs with varying doses. Significance (p < 0.05 indicated by different letters) was tested based on a comparison of the treatments with different CNTs at a certain dose and that with the control on a specific incubation day.

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Figure 2. Temporal trends in within-community (alpha) diversity, measured as the Shannon index, of soil bacterial communities exposed to CNTs at various doses.

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Effects of CNTs on Soil Bacterial Community Alpha Diversity. Based on high-throughput

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sequencing, the alpha diversity (Shannon index) of the control remained largely stable over the 56-day

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experimental period (p = 0.688) (Figure 2). Exposure to various CNTs had differentiating effects on

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soil bacterial alpha diversity. SWs at higher doses increased the soil bacterial alpha diversity (p

Effects of Various Carbon Nanotubes on Soil Bacterial Community

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