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Dynamic Effects of Biochar on the Bacterial Community Structure in Soil Contaminated with Polycyclic Aromatic Hydrocarbons Yang Song, Yongrong Bian, Fang Wang, Min Xu, Ni Ni, Xinglun Yang, Chenggang Gu, and Xin Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02887 • Publication Date (Web): 21 Jul 2017 Downloaded from http://pubs.acs.org on July 24, 2017

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Journal of Agricultural and Food Chemistry

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Dynamic Effects of Biochar on the Bacterial Community Structure in Soil

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Contaminated with Polycyclic Aromatic Hydrocarbons

3

Yang Song,*,† Yongrong Bian,† Fang Wang,† Min Xu,† Ni Ni,† Xinglun Yang,† Chenggang

4

Gu,† and Xin Jiang*,†

5

†

6

Chinese Academy of Sciences, 71 East Beijing Road, Nanjing 210008, PR China

7

*(Y.S.) Tel: +86 25 86881193/86881195. Fax: +86 25 86881000. E-mail: [email protected]

8

*(X.J.) E-mail address: [email protected]

9 10 11 12 13

Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science,

TOC/ABSTRACT ART

14

1 ACS Paragon Plus Environment


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ABSTRACT:

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Amending soil with biochar is an effective soil remediation strategy for organic contaminants.

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This study investigated the dynamic effects of wheat straw biochar on the bacterial

18

community structure during remediation by high-throughput sequencing. The wheat straw

19

biochar amended into the soil significantly reduced the bioavailability and toxicity of

20

polycyclic aromatic hydrocarbons (PAHs). Biochar amendment helped to maintain the

21

bacterial diversity in the PAH-contaminated soil. The relationship between the immobilization

22

of PAHs and the soil bacterial diversity fit quadratic model. Before week 12 of the incubation,

23

the incubation time was the main factor contributing to the changes in the soil bacterial

24

community structure. However biochar greatly affected the bacterial community structure

25

after 12 weeks of amendment, and the effects were dependent on the biochar-type.

26

Amendment with biochar mainly facilitated the growth of rare bacterial genera (relative

27

abundance of 0.01%-1%) in the studied soil. Therefore, the application of wheat straw biochar

28

into PAH-contaminated soil can reduce the environmental risks of PAHs and benefit the soil

29

microbial ecology.

30

KEYWORDS: biochar, immobilization, bacterial community structure, PAHs.

31

INTRODUCTION

32

The environmental pollution caused by polycyclic aromatic hydrocarbons (PAHs) is of

33

great concern because of the mutagenicity, ecotoxicity and carcinogenicity of these

34

compounds.1 China made an estimated contribution of approximately 20% to the global PAH

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emissions in 2007.2 According to the soil investigation report, PAHs have been listed as the

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key organic contaminants in the Chinese soils, with approximately 32% of farmland soil 2 ACS Paragon Plus Environment

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heavily polluted.3 Therefore, it is of great importance to reduce the environmental risk posed

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by PAHs in soil and remediate the contaminated soil for safe agricultural production.

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To reduce the bioavailability of contaminants in soil, amending the soil with

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organic/inorganic amendments has been developed to be an efficient in situ soil remediation

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strategy. 4-6 Recently, biochar has been used to immobilize organic contaminants in soil.7,8

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Generally, biochars show an excellent sorption ability and capacity for organic

43

contaminants.9,10

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chlorobenzenes14 and polychlorinated biphenyls15 in soil. However, studies also reported that

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the dissipation of PAHs,16 benzonitrile,17 atrazine18 and clomazone19 in soil can be stimulated

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by biochars, which could be ascribed to the changes of soil microbial community structure

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and activity after biochar amendment.16 Simultaneously, the extracellular polymeric substrates

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exudated by soil microbes may also affect the bioavailability of contaminants,20 and thus the

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immobilization potential of biochar. Generally, contamination by organic and inorganic

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pollutants reduces the soil microbial diversity.21 The resilience of the soil microbial ecology is

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important for contaminated soil.22 A diverse microbial community structure benefits the

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nutrient efficiency23 and the degradation of organic contaminants in soil.24 However, what’s

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the relationship between the immobilization of contaminants and the soil microbial diversity

54

remains unclear.

Reportedly,

biochar

immobilizes

PAHs,11

atrazine,12

diuron,13

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Recent studies have reported that amending soil with biochars resulted in varying effects

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on the soil microbial community structure.8 Biochar can increase the microbial community

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diversity: the bacterial diversity of terra preta increased by 25% compared to a similar soil 3 ACS Paragon Plus Environment


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without biochar.25 Amendment with rice or wheat straw biochar significantly increased the

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α-diversity in dry land soil and paddy soils in China.26,27 Addition of biochar increased the

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bacterial community richness and diversity in a chlorpyrifos contaminated soil.28 In contrast,

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decreased soil microbial diversity resulting from biochar amendment was also reported,29,30

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and other studies reported that biochar amendment into soil did not change the soil microbial

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community structures.31,32 The reporting of the different results above was because that these

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studies were conducted not only with different biochars, but also with different incubation

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periods after biochar amendment. The soil microbial community structure could also be

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affected by the factor of time.33 Whether the dynamic effect of biochar on the microbial

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community structure is changeable over time, specifically in PAH-contaminated soil, remains

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unclear. If biochar can immobilize PAHs or stimulate the dissipation of PAHs in soil on the

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one hand and enhance the soil microbial diversity on the other hand, amending contaminated

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soil with biochar will greatly reduce the environmental risk of PAHs and benefit the soil

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microbial ecology. Furthermore, elucidating the dynamic effect of biochar on the microbial

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community structure in PAH-contaminated soil will guide the use of biochar in soil

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remediation.

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Therefore, the objectives of this study were to study the relationship between the

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bacterial diversity and the potential immobilization of PAHs in soil caused by biochar

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addition and to elucidate the dynamic effects of biochar on the bacterial community structure

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of PAH-contaminated soil during remediation. In this study, a PAH-contaminated soil was

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amended with wheat straw biochars, which were produced at different temperatures and 4 ACS Paragon Plus Environment

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added to the soil with different application levels. The dissipation and bioavailability of PAHs

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in the soil were measured to estimate the immobilization potential of the biochars. The

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bacterial community structures were analyzed by high-throughput sequencing to elucidate the

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dynamic effects of biochar on the soil microbial ecology.

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MATERIALS AND METHODS

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Soil Sampling. The PAH-contaminated soil was collected from the top 20 cm in an

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arable field near a steel mill in a suburb of Nanjing, China. The soil was air-dried and passed

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through a 2 mm sieve. The pH of the soil was 7.4. The soil had an organic matter content of

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2.0%, a composition of 8.9% clay, 62.5% silt, and 27.6% sand. The total soil concentration of

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16 PAHs in the US EPA priority pollutant list was 7581.61 ng g−1. Biochar

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Characterization.

Wheat

straw

biochars

were

pyrolyzed

under

90

oxygen-limited conditions at 300 °C or 600 °C 34 and labeled BC300 or BC600, respectively.

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The elemental C, N and H compositions of the biochar were determined on a CNH analyzer

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(Vario MICRO, Germany elementar, Germany). The specific surface areas, pore volume and

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size

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Barrett-Joyner-Halenda analysis, respectively. Table S1 shows the detailed physicochemical

95

properties of the biochars. Fourier transform infrared (FTIR) spectrometry (NEXUS 870,

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Thermo Nicolet, USA) was used for analyzing functional groups of biochar (Figure S1).

of the biochar were measured

using

Brunauer-Emmett-Teller method and

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Soil Amendment with Biochar. Briefly, 495 g of PAH-contaminated soil was

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amended and mixed thoroughly with 5 g of BC300 or BC600 (1% application level) in a glass

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beaker. The biochar-amended soil was transferred to a 1000-mL flask. Deionized water was 5 ACS Paragon Plus Environment


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added into the flask to maintain a soil water content of 28%. The soil was compacted using a

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glass stick to a volume equivalent to 1.3 g cm−3 of soil density. The flask was then closed with

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a glass cap and incubated at 25 °C in the dark. The 2% biochar application level was prepared

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via the same procedure. Soil unamended with biochar was used as the control. There were

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therefore five treatments, i.e., control, 1%BC300, 1%BC600, 2%BC300, and 2%BC600, all

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analyzed in triplicate.

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The flasks were aerated once per week for 20 min over the incubation period. After

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aeration, 10 g of the soil was sampled for total and bioavailable PAH concentrations analysis.

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Another 5 g of soil was sampled and stored at -80 °C before soil DNA extraction. After

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sampling, the flasks were closed and incubated further.

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Total and Bioaccessible PAH Concentrations. Accelerated solvent extraction

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(ASE 200, Dionex) was used for extracting total PAHs from soil.35 One g of the soil was

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homogenized with 5 g of diatomaceous earth and subjected to extraction with hexane/acetone

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(4:1, v/v) at 100 °C and 1500 psi in ASE. The extracts were rotary concentrated to 1 mL at

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50 °C and then were cleaned up with silica gel/anhydrous sodium sulfate column, eluted with

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15 ml hexane/dichloromethane (9:1, v/v). Then the eluate was concentrated to 1 mL for PAH

116

detection

117

7890A/5975C).

by

gas

chromatography−mass

spectrometer

analysis

(GC−MS,

Agilent

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The bioaccessible PAHs in the soil were extracted with hydroxypropyl-β-cyclodextrin

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(HPCD).36 Briefly, 1 g soil was extracted with 20 mL of HPCD (50 mM) by shaking at 200

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rpm for 20 h with an orbital shaker. After shaking, the soil suspension was centrifuged for 30 6 ACS Paragon Plus Environment

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min and then the supernatant was discarded. The residue soil was shaking-washed with

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deionized water for 2 min. Then total PAH concentrations in the residue soil were measured

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again by the method described above. The bioaccessible PAH concentration in the soil was

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calculated as the total PAH concentration in initial soil minus the total PAH concentration in

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residue soil after extraction with HPCD. The analysis of variance (ANOVA) and the least

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significant difference (LSD) post hoc comparison tests were applied for data analysis with

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SPSS 17.0. The significant level was at p < 0.05.

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Bacterial Community Structures in Soils. The biochar-amended soils with

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different incubation period were subjected to microbial analyses. DNA was extracted from

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soil samples using the E.Z.N.A.

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manufacture’s protocols and assessed by agarose gel electrophoresis. The V4−V5 regions of

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bacterial 16S rRNA gene were amplified using 338F and 806R primer set.37 PCR reactions

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were performed using standard and established method (Text S1). Amplicons were purified

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using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA)

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following the manufacturer’s instructions and quantified using Quanti Fluor™-ST (Promega,

136

USA). The Illumina MiSeq platform by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai,

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China) was used for sequencing (Text S1). The raw reads were deposited into the NCBI

138

Sequence Read Archive (SRA) database (Accession Number: SRP080209). Raw fastq files

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were demultiplexed, quality-filtered using QIIME (version 1.17) to remove the low-quality

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sequences (Text S1). Operational Taxonomic Units (OTUs) were clustered with a 97%

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similarity cutoff, using UPARSE (version 7.1 http://drive5.com/uparse/). Chao and Shannon

®

Soil DNA Kit (OMEGA, USA) according to the



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indexes were calculated in MOTHUR v.1.30.1 (http://www.mothur.org/). The principal

143

coordinates analysis (PCoA) and clustering analysis were conducted in R v.3.2.1.38 (Text S1)

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The differences in the bacterial abundances among treatments were analyzed through

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statistical analysis of the metagenomic profiles (STAMP).39

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Phytotoxicity Assays. At the end of the incubation, a root elongation experiment was

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performed to test the phytotoxicity of the biochar-amended soil. Briefly, 50 g of the soil was

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sampled from the incubation flask, transferred to petri dishes and watered to saturation for 6 h.

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Then 20 ryegrass seeds (Lolium perenne L.) were placed over the soil surface. The petri

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dishes were incubated in a growth chamber at 28 °C and 60% relative humidity for 96 h under

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darkness. Then, the root lengths of the successfully emerged ryegrass were counted and

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statistically analyzed through ANOVA and LSD post hoc comparison tests.

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RESULTS AND DISCUSSION

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Dissipation of PAHs in Soils. The dynamic changes in the residue percentages of

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total PAHs in soil are shown in Figure S2. The dissipation of PAHs from the control soil was

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fast in the first 12 weeks of incubation and stabilized after that period. After 24 weeks of

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incubation, the residue percentage of total PAH concentration in the control was 30%. There

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were no significant differences in the dissipation of PAHs in 2%BC300 and 1%BC600

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compared with that in the control throughout the incubation period (p > 0.05). Amendment

160

with 2%BC600 significantly inhibited the dissipation of PAHs (p < 0.05). For BC300, more

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biochar amendment resulted in lower PAH residues in the soil. For BC600, more biochar

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amendment resulted in higher PAH residues in soil. This difference may be ascribed to the 8 ACS Paragon Plus Environment

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two roles played by biochar: nutrient stimulation for potential PAH- degraders and sorption

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inhibition of PAHs in soils. Biochars pyrolyzed at low temperature contain more nutrients,

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such as total and available N and P (Table S1 and S2), than those at higher temperatures,8 thus

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may facilitate the growth of potential degrading microbes. Biochars pyrolyzed at high

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temperatures exhibit a stronger sorption capacity for organic contaminants than those at low

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temperatures.7 The wheat straw biochars used in this study mainly inhibited the dissipation of

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PAHs in soil.

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Bioavailability and Toxicity of PAHs in Soils. After 24 weeks of incubation, the

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HPCD extraction was conducted to elucidating the changes of the bioavailability of PAHs in

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soil (Figure 1). Biochar amendment of the soil significantly reduced the HPCD extraction

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efficiency (p < 0.05), thus reducing the bioavailability of the PAHs in soil. The 2%BC600

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treatment resulted in the lowest HPCD extraction efficiency, while there was no significant

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difference in the HPCD extraction among the 1%BC300, 1%BC600 and 2%BC300 treatments

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(p > 0.05). Simultaneously, the root elongation of ryegrass in the biochar-amended treatments

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was significantly greater than that in the control (p < 0.05) (Figure 1), which was consistent

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with the HPCD extraction efficiency. BC600 performed better than BC300 in enhancing root

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elongation. These results indicate that the amending soil with biochar resulted in reduced

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bioavailability and toxicity of PAHs in soil, i.e., immobilization of PAHs occurred in the

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biochar-amended soil.40 These results explained the inhibitory effect of biochar on the

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dissipation of PAHs in soil. Such immobilizing effect of biochar on PAHs was also reported in

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studies on hardwood-, sewage sludge-, peanut shell- and rice straw-derived biochars.11,41 9 ACS Paragon Plus Environment


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Bacterial Abundance and Diversity. After high-throughput sequencing process,

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there were an average of 34032 reads per sample, with an average length of 442 bp. The

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number of OTUs (97% similarity) ranged from 2307 to 3193 per sample, and the coverage

187

estimates ranged from 96.8% to 98.0%, indicating that almost all of the bacterial species were

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included. A total of 1983 OTUs co-existed in the 5 treatments after 1 week of incubation.

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With increasing incubation time, the co-existing OTU number increased, while the number of

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the specific OTUs in each treatment decreased (Figure S3). This indicates that the bacterial

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communities were becoming increasingly similar over time. As shown in Figure 2a, the

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bacterial abundances, as expressed by the Chao index, increased before 4 weeks of incubation

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in each treatment, significantly in the control, 1%BC300 and 2%BC300 treatments (p < 0.05).

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The abundances stabilized over weeks 4 to 24 of incubation in all treatments (p > 0.05).

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Compared to BC600, BC300 was more conducive to bacterial growth in this study, in

196

agreement with another report.16 This is potentially because the pH of BC300 was more

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neutral than that of BC600 (Table S1). Bacteria are sensitive to changes in pH, and their

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abundances have been proved to be highest in pH neutral soils.42 Moreover, amendment of

199

BC300 resulted in higher available N and P contents than amendment with BC600 did (Table

200

S2).

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Figure 2b shows the soil bacterial diversity (Shannon index). Generally, the bacterial

202

diversity in each treatment first increased before 4 weeks of incubation and then decreased

203

with the incubation time. It is not surprising that the soil bacterial abundance and diversity

204

increased under the ideal moisture and temperature conditions during the first 4 weeks of 10 ACS Paragon Plus Environment

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incubation. A significant decrease in bacterial diversity in the control was observed after 12

206

weeks of incubation (p < 0.05). However, biochar amendment mediated the decrease in soil

207

bacterial diversity. After 24 weeks of incubation, the 1%BC300 treatment contained the

208

highest bacterial diversity, followed by 2%BC300 and 2%BC600, which were all significantly

209

higher than that in the control (p < 0.05). These differences could also be ascribed to the

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multiple roles of biochar played in soil, e.g. sorption of toxic compounds, providing nutrients

211

and habitats for microbes and improving soil physical properties.7,8,23 However, more biochar

212

addition did not further enhance the abundance or the diversity of bacteria in soil (Figure 2).

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Similar results were also reported in a study conducted with Brassica rapa-derived biochar43

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and wheat straw gasified biochar,44 and these results may be related to the increased C/N ratio

215

of soils amended with greater amounts of biochars,23 which suppresses the growth of specific

216

microbes in soil.

217

Relationships between Bacterial Diversity and PAH Immobilization. The

218

reduced bioavailability and toxicity of PAHs in soil (Figure 1) reflected the immobilizing

219

effect of biochar in soils. Immobilization may also be affected by microbes since some strains

220

exudate the extracellular polymeric substrates, which mediate the bioavailability of PAHs in

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soil.20 As shown in Figure 3., the relationships between bacterial diversity and the

222

immobilization of PAHs (i.e., reduced HPCD extraction and enhanced root length of ryegrass)

223

were fitted into quadratic models. The lowest HPCD extraction efficiency and the highest

224

ryegrass root length were both reached at a Shannon index of 6.0, indicating that an

225

appropriate level of bacterial diversity facilitates the immobilization of PAHs in soil. More 11 ACS Paragon Plus Environment


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biochar addition (within 2%) did not further significantly enhance the soil bacterial diversity

227

(Figure 2) or significantly reduce the bioavailability of PAHs in soil (Figure 1). Therefore,

228

reasonable levels of biochar should be applied to soil when using biochar as an amendment in

229

PAH-contaminated soils. It is not clear of the relationship between bacterial diversity and the

230

immobilization of PAHs at lower extent of bacterial diversity in this study with only one soil

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and without sterilization. Therefore, further studies with the sterile control and more

232

soil/biochar types should be conducted to elucidate the effects of microbes on the

233

immobilization of organic contaminants in biochar-amended soil.

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Bacterial Community Structures. Based on PCoA analysis (Figure 4), the first two

235

PCs explained a 86.7% of the variance in the bacterial communities. The PC1 explained

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76.0% of the variance. The bacterial communities in all treatments incubated for 1 week, 4

237

weeks and 12 weeks were tightly clustered in three separate groups, indicating that incubation

238

time was the main factor affecting the changes in the bacterial community structure during the

239

initial 12 weeks of incubation. The data for the control at 24 weeks was far away from the

240

cluster containing all of the 12-week data. However, the biochar amendment resulted in an

241

overlap of the clusters of the samples incubated for 12 weeks and 24 weeks, indicating similar

242

changes in bacterial communities due to biochar amendment in the soils incubated for 12 and

243

24 weeks. The analysis of the cluster trees also confirmed that the similarity of the soil

244

bacterial community structures was dependent on the incubation time in the initial 12 weeks

245

of incubation, but on the type of biochar after 12 weeks of incubation (Figure S4). Others

246

have reported that rice straw biochar shifted the soil bacterial community structure after 6 12 ACS Paragon Plus Environment

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weeks of incubation27 and that biochar had a negative effect on the soil bacterial diversity at

248

12 or 26 weeks after amendment.29,30 There are also reports of biochar not affecting the

249

bacterial community structure after 7 weeks and 6 years.31,32 However, all these studies were

250

conducted using different biochars and soils from those used in the present study. In general,

251

the microbial community structures are affected by the soil properties that are changed by

252

biochar addition, rather than by the direct utilization of biochar by microbes.8,27,29 Therefore,

253

the present study reveals that the long-term lasting effect of biochar on soil properties is

254

important in shifting soil bacterial communities.

255

The weighted UniFrac distances were determined to elucidate the differences in the soil

256

bacterial community among treatments (Figure 5). Obviously, the differences in the bacterial

257

community structure between biochar-amended treatments and the control were dependent on

258

the biochar- type (Figure 5a). Similar changes were observed in the treatments amended with

259

the same type of biochar. More interestingly, the inflection points appeared at a time point of

260

12 weeks for the BC600-amended treatments (Figure 5a). The inflection or decreasing of this

261

distance was also existed among different biochar-amended treatments at 12 weeks (Figure

262

5b). A greater amount of BC300 amended into the soil resulted in a greater difference in the

263

soil bacterial community structure than did a greater amount of BC600. Moreover, the

264

difference among BC300 and BC600 was greater at the 1% application level than that at the

265

2% application level, indicating that lower amounts of biochar result in greater differences in

266

the soil bacterial community structure among different biochar-amended treatments.

267

Abundances of Bacterial Phyla. The composition of the soil bacterial phyla is 13 ACS Paragon Plus Environment


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shown in Figure 6. Thirteen phyla had relative abundances greater than 1% in each treatment,

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and these phyla accounted for 96.3%-98.9% of the total bacterial abundances. Another 13

270

phyla had relative abundances between 1% and 0.1% and accounted for 1.0%-3.0% of the

271

total abundance (Figure S5). Of the main phyla, Proteobacteria (23.0%-44.5%), Firmicutes

272

(10.5%-37.5%), Gemmatimonadetes (4.5%-10.3%), Acidobacteria (5.7%-13.7%), Chloroflexi

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(4.1%-10.4%), Actinobacteria (3.2%-11.4%) and Bacteroidetes (2.7%-9.1%) were more

274

abundant, accounting for a total of 83.2%-95.2% of the whole bacterial community (Figure 6).

275

Proteobacteria was the main bacterial phylum in this PAH-contaminated soil, in accordance

276

with another report in which Proteobacteria dominated the bacterial community.33 As shown

277

in Figures S6 and S7c, the relative abundances of Proteobacteria, Bacteroidetes,

278

Actinobacteria and Gemmatimonadetes significantly decreased in each treatment (p < 0.05),

279

while Firmicutes, Chloroflexi, Acidobacteria and Parcubacteria significantly increased after

280

24 weeks of incubation (p < 0.05) regardless of the treatment. Moreover, the relative

281

abundances of most of the phyla increased (positive data in Figure S7ab) or decreased

282

(negative data in Figure S7ab) regardless of the different treatments with the exception of

283

Saccharibacteria, Verrucomicrobia (Figure S7a) and Amatimonadetes (Figure S7b). These

284

results indicate that biochar had a limited effect on the main changes in the bacterial phyla

285

over time.

286

The dynamic effects of biochar on the relative abundances of the bacterial phyla with

287

relative abundance higher than 1% were changeable with the incubation period (Figure S8).

288

For most of these phyla, such as Proteobacteria, Firmicutes, Acidobacteria, Nitrospirae, 14 ACS Paragon Plus Environment

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Verrucomicrobia, Planctomycetes and Parcubacteria, dramatic changes in the differences in

290

relative abundance in the biochar-amended treatments relative to that in the control occurred

291

after 12 weeks of incubation. This result partly explains the changes in the bacterial

292

community structures over time (Figures 3 and 4). Firmicutes showed the greatest differences

293

in relative abundance in the biochar-amended treatments relative to that in the control at 12

294

weeks (Figure S8) and was most abundant phylum in the control after 24 weeks of incubation

295

(Figure 6). Amendment with biochar, especially BC600, shortened the period required for

296

Firmicutes to become abundant to 12 weeks. After 24 weeks of incubation, the abundances of

297

Bacteroidetes, Actinobacteria and Gemmatimonadetes in the biochar-amended treatments

298

were significantly increased (p 1%), intermediate (0.1%-1%) and low (0.01%-0.1%) abundances.

330

For highly abundant bacteria, amendment with biochar resulted in negative tending after 24 16 ACS Paragon Plus Environment

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weeks of incubation. However, for bacteria of intermediate and low abundance, amendment

332

with biochar mainly resulted in increased abundance after 24 weeks of incubation. This

333

explains why biochar amendment enhanced the bacterial diversity in the soil. Another study

334

also reported that biochar mainly increased the relative abundance of the rare members in

335

microbial communities.44

336

In summary, the above results showed that amending the PAH-contaminated soil with

337

wheat straw-derived biochar reduced the bioavailability and toxicity of the PAHs in soil and

338

helped to maintain the high bacterial diversity in the soil. The effects of biochar on the

339

bacterial community structure were considerable after 12 weeks of soil amendment and were

340

dependent on the biochar type. BC600 performed better than BC300 in immobilizing the

341

PAHs in soil, while BC300 performed better than BC600 in enhancing the bacterial diversity

342

in the soil. Therefore, a quadratic mode relationship was observed between the bacterial

343

diversity and PAHs immobilization in soil affected by biochar. Amendment with biochar

344

mainly facilitated the growth of the rare members of the soil bacterial communities, which is

345

helpful

346

PAH-contaminated soil with biochar can reduce the environmental risk of PAHs in soil and

347

benefit the soil microbial ecology.

348

ASSOCIATED CONTENT

349

Supporting Information

350

The Supporting Information is availabile free of charge on the ACS Publications website

351

at…..

for

maintaining

the

soil

bacterial

diversity.

Therefore,

amendment

of



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Supplmental results including properties of biochars, FTIR spectra, dissipation of PAHs,

353

Venn of OTUs, UPGMA cluster analysis, changes of phylum and genus abundances.

354

AUTHOR INFORMATION

355

Corresponding Authors

356

*(Y.S.) Tel: +86 25 86881193/86881195. Fax: +86 25 86881000. E-mail: [email protected]

357

*(X.J.) E-mail address: [email protected]

358

Funding

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This study was financially supported by the National Key Basic Research Program of China

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(2014CB441105), the National Natural Science Foundation of China (41671236), the “135”

361

Plan and Frontiers Program of Institute of Soil Science, Chinese Academy of Sciences

362

(ISSASIP1614), and the Outstanding Youth Fund of Natural Science Foundation of Jiangsu,

363

China (BK20150050).

364

Notes

365

The authors declare no competing financial interest.

366

REFERENCES

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Figure 1. The hydroxypropyl-β-cyclodextrin (HPCD) extraction efficiency and root

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elongation based toxicity test of total PAHs in soils amended with and without biochars

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pyrolyzed at 300 °C (BC300) and 600 °C (BC600) after 24 weeks of incubation.


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4200

a

4000

Chao index

3800 3600 3400 3200 3000 2800

Control

1%BC300

1%BC600

2%BC300

2%BC600

2600 0

4

8

12

16

20

24

7.0

b

Shannon index

6.5

6.0

5.5

5.0

Control 1%BC600 2%BC600

4.5 0

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4

8

12 16 Time (week)

1%BC300 2%BC300

20

24

510

Figure 2. Time course of the bacterial abundance expressed as the Chao index (a) and

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diversity expressed as the Shannon index (b) in soils amended with and without biochars

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pyrolyzed at 300 °C (BC300) and 600 °C (BC600).



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Figure 3. Relationships between the soil bacterial diversity with the

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hydroxypropyl-β-cyclodextrin (HPCD) extractions of PAHs and root elongations.

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Figure 4. Principal coordinate analysis (PCoA) of the bacterial communities in soils amended

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with and without biochars pyrolyzed at 300 °C (BC300) and 600 °C (BC600).



0.30

0.30

a Weighted UniFrac distance

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b

0.25

0.25

0.20

0.20

0.15

0.15

0.10

0.10

0.05

1%BC300-Control

0.05 1%BC600-Control

2%BC300-1%BC300

2%BC600-1%BC600

2%BC300-Control

2%BC600-Control

1%BC600-1%BC300

2%BC600-2%BC300

0.00

0.00 0

520

4

8

12

16

20

0 4 24 Time (week)

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12

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20

24

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Figure 5. Weighted UniFrac distances of soil bacterial communities between different soils

522

amended with and without biochars pyrolyzed at 300 °C (BC300) and 600 °C (BC600). a:

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between biochar-amended treatments and the control; b: between different biochar-amended

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treatments.


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100

Relative abundance (%)

80

60

40

20

0 1w 4w 12w 24w 1w 4w 12w 24w 1w 4w 12w 24w 1w 4w 12w 24w 1w 4w 12w 24w

Control

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1%BC300

Proteobacteria Gemmatimonadetes Nitrospirae Elusimicrobia

Firmicutes Actinobacteria Parcubacteria Others

1%BC600

2%BC300

Acidobacteria Bacteroidetes Verrucomicrobia

2%BC600 Chloroflexi Saccharibacteria Planctomycetes

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Figure 6. Composition of the bacterial phyla in soils amended with and without biochars

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pyrolyzed at 300 °C (BC300) and 600 °C (BC600).


1 Dynamic Effects of Biochar on the Bacterial ... - ACS Publications

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