Transcriptional activity of arsenic-reducing bacteria and genes

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Transcriptional activity of arsenic-reducing bacteria and genes regulated by lactate and biochar during arsenic transformation in flooded paddy soil Jiangtao Qiao, Xiaomin Li, Min Hu, Fangbai Li, Lily Y. Young, Weimin Sun, Weilin Huang, and Jianghu Cui Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03771 • Publication Date (Web): 30 Nov 2017 Downloaded from http://pubs.acs.org on November 30, 2017

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Environmental Science & Technology

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Transcriptional activity of arsenic-reducing bacteria and genes regulated by lactate and

2

biochar during arsenic transformation in flooded paddy soil

3 4 5 6 7 8 9 10 11

Jiang-tao Qiao1, 2, 3†, Xiao-min, Li1†, Min Hu1, Fang-bai Li1*, Lily Y. Young4, Wei-min Sun1, Weilin Huang1, 4, and Jiang-hu Cui1

1

Guangdong Key Laboratory of Integrated Agro-environmental Pollution Control and Management, Guangdong

Institute of Eco-Environmental Science & Technology, Guangzhou 510650, P. R. China

12

2

13

3

14

4

Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, P.R. China University of Chinese Academy of Sciences, Beijing 100049, P. R. China

Department of Environmental Sciences, Rutgers University, New Brunswick, NJ, USA

1 ACS Paragon Plus Environment


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ABSTRACT

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Organic substrates and biochar are important in controlling arsenic release from sediments and soils,

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however, little is known about their impacts on arsenic-reducing bacteria and genes during arsenic

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transformation in flooded paddy soils. In this study, microcosm experiments were established to

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profile transcriptional activity of As(V)-respiring gene (arrA) and arsenic resistance gene (arsC) as

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well

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arsenic-contaminated paddy soils. Chemical analyses revealed that lactate as the organic substrate

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stimulated microbial reduction of As(V) and Fe(III), which was simultaneously promoted by

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lactate+biochar, due to biochar’s electron shuttle function that facilitates electron transfer from

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bacteria to As(V)/Fe(III). Sequencing and phylogenetic analyses demonstrated that both arrA

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closely associated with Geobacter (> 60%, number of identical sequences/number of the total

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sequences) and arsC related to Enterobacteriaceae (> 99%) were selected by lactate and

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lactate+biochar. Compared with the lactate microcosms, transcriptions of the bacterial 16S rRNA

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gene, Geobacter spp., Geobacter-arrA and arsC genes were increased in the lactate+biochar

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microcosms, where transcript abundances of Geobacter and Geobacter-arrA closely tracked with

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dissolved As(V) concentrations. Our findings indicated that lactate and biochar in flooded paddy

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soils can stimulate the active As(V)-respiring bacteria Geobacter species for arsenic reduction and

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release, which probably increases arsenic bioavailability to rice plants.

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Key words: Arsenic-respiring bacteria; Gene transcription; Biochar; Geobacter; Paddy soil

as

the

associated

bacteria

regulated

by

lactate


and/or

biochar

in

anaerobic

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INTRODUCTION

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Arsenic contamination in paddy fields is of worldwide concern because it threatens the health of

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populations consuming rice as a staple food; this is especially acute in South and Southeast Asian

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countries.1,2 During rice growth season, arsenate (As(V)) adsorbed to soil minerals can be reduced

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to arsenite (As(III)) under flooding conditions, and released into aqueous solution, leading to its

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increased bioavailability to rice plants.3,4

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Both cytoplasmic As(V)-reducing and dissimilatory As(V)-reducing microorganisms play a key

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role in As(V) reduction.5 In the cytoplasmic As(V)-reducing bacteria (e.g. Pseudomonas and

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Bacillus spp.), dissolved As(V) is firstly taken up into cytoplasm, and then reduced to As(III) by a

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soluble As(V) reductase (ArsC) encoded by the gene arsC, followed by extrusion out of the cell.6−9

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In the dissimilatory As(V)-respiring bacteria (e.g. Geobacter and Shewanella spp.), As(V) is

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utilized as a terminal electron acceptor for microbial respiration mediated by the gene arrA coupled

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to oxidation of organic or inorganic electron donors.10−14 Analysis of microbial community based on

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arrA gene sequences showed Geobacter spp. were abundant As(V)-respiring bacteria in arsenic-rich

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sediments.15−18 In paddy fields, however, the abundance of arrA gene was found to be lower than

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that of arsC gene19,20 and the arrA sequences were mainly aligned to uncultured bacteria, rather

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than Geobacter spp..19 To date, it remains poorly understood what is the relative contribution of

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each of these pathways to arsenic mobilization under flooded conditions in paddy soils.

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Organic substrates are important in controlling the rate and magnitude of microbially mediated

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arsenic transformation in anoxic sediments and soils.21,22 Prior studies used acetate as the organic

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carbon proxy to stimulate microbial activity for arsenic-rich sediments, and the major results

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indicated relatively high abundance of Geobacter species in the bacterial communities.21,23,24

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Conversely, a recent study reported that incubation of Pleistocene sediment from Cambodia with

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lactate favors the dominance of 16S rRNA-sequences not related to Geobacter spp.18 In paddy soils,

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lactate is not only a fermentation byproduct of anaerobic respiration, but also is one of the important



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organic acids secreted by rice roots.25,26 Its effect on arsenic mobilization and the composition of

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microbial communities in flooded paddy field merits investigation.

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Biochar, the solid product from pyrolysis of waste biomass residues from agricultural production,

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is usually returned to the field. As an amendment, it increases soil organic carbon and decreases

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nutrient leaching due to its high specific surface area and large cation exchange capacity.27 Biochar

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contains high quinone functional groups and condensed aromatic structures, and can function as an

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electron shuttle for facilitating biotic and abiotic redox reactions.28,29 Biochar amendment has

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shown negligible or enhanced influences on arsenic release from contaminated soils, with some

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studies reporting that biochar shifted the microbial community and increased the abundance of

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Fe-reducing bacteria.20,30−32 For flooded paddy soils, however, the effect of biochar on the

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transcriptional activity of cytoplasmic and dissimilatory As(V)-respiring genes as well as the

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associated microorganisms remain unclear.

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In this study, anoxic microcosms were set up with an arsenic-contaminated paddy soil and

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amendments of lactate and/or biochar. The objectives were (i) to evaluate the roles of lactate and/or

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biochar on the arsenic transformation in flooded paddy soils, (ii) to investigate the effect of lactate

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and/or biochar on the potentially active microbial community (16S rRNA cDNA based),

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cytoplasmic As(V)-reducing community (arsC gene transcript based), and dissimilatory

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As(V)-reducing community (arrA gene transcript based); and (iii) to quantify changes in the

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transcript abundance of these two arsenic-reducing genes and arsenic-related bacteria over time.

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MATERIALS AND METHODS

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Soil sampling and biochar preparation. The soil sample was collected from the rice paddy field

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in the downstream area of Lianhua mountain tungsten mine, located in Shantou City (23°38'30.4"N,

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116°50'4.7"E), Guangdong Province, China, in November 2010 when it was in the drained season

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with high concentration of solid-phase arsenic oxyanions. The sample was taken from 3-5 sampling

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points of a field randomly and mixed as a composite sample and then transported to the laboratory 4 ACS Paragon Plus Environment

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with ice immediately and stored at 4°C before incubation experiments and soil characterizations

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analysis. The physico-chemical properties of the soil are shown in Table S1, Supporting

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Information (SI). Biochar was made from oil palm fibers, Malaysia. After being air dried, the oil

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palm fibers were charred at 300°C for 4 h in a muffle furnace using N2 as the medium gas. Biochar

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was milled to pass a 2-mm sieve and stored in a drying oven before analysis of elemental

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composition (Table S2), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy

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(FT-IR) (Figure S1). Sodium lactate (≥ 99.0%) was purchased from Sigma-Aldrich (USA).

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Microcosm set up. Anaerobic batch experiments were carried out in triplicate in 120 ml serum

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vials with 7 g paddy soil (wet weight) and 70 ml piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES)

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buffer (30 mM, pH 7.3) (J&K Chemical Ltd). All the vials were incubated at 30°C under an

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atmosphere of N2 in the dark without shaking. PIPES buffer was used to adjust the pH to 7.3, the

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native soil pH, then a trace element solution (1 ml l-1) and a vitamin solution (1 ml l-1) were added.33

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Lactate was used the sole carbon source with an initial concentration of 10 mM according to

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previous study.18 Biochar was amended based on a wet weight basis, thus a 3% biochar treatment

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was equivalent to 0.21 g biochar per culture. A total of eight treatments including four biotic

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treatments and four abiotic treatments (sterilized by 50 KGy of gamma-ray irradiation) were

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conducted with detail shown in Table S3. Triplicate bottles from each treatment were taken out and

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subjected to destructive sampling at various time intervals (day 0, 1, 2, 5, 10 and 20), and 5 ml of

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soil suspension was collected from each bottle were used for analysis of dissolved arsenic and iron

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speciation, and pellets were used for determination of soil arsenic and iron speciation (2 g) and total

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RNA extraction (2 g), respectively. Sampling on day 0 from each treatment was performed 1-2 h

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after the lactate and/or biochar were mixed with soil slurry in the medium thoroughly.

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Arsenic and iron speciation. Dissolved As(III)/As(V) were determined from the supernatant of

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each culture using atomic florescence spectroscopy (SA-20, Jitian Inc., Beijing, China). Briefly,

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each culture was centrifuged at 8000 g for 10 min at room temperature, from which the supernatant



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was filtered through a sterile 0.22 µm filter before arsenic determination. The soil pellets were

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divided into two subsamples: one was immediately frozen in liquid nitrogen and stored at -80°C

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until RNA extraction and the other was used for sequential extraction and determination of arsenic

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fractions in the soils. The soil pellets were sequentially extracted with 1 M KH2PO4 + 0.1 M

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ascorbic acid34 followed by 0.2 M NH4+-oxalate buffer,35 that is, PO4-As(III)/As(V) and

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oxalate-As(III)/As(V), respectively. Dissolved Fe(II) and 0.5 M HCl-extractable Fe(II), i.e.

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HCl-Fe(II) were quantified based on the procedures described previously.36

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RNA extraction and reverse transcription. Total RNA was extracted using the PowerSoil Total

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RNA Isolation Kit (Mio Bio Laboratories, Inc., USA) following the manufacturer's instructions. To

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eliminate any possibility of genomic DNA contamination, RNA was reverse transcribed using

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a PrimeScript RT reagent kit with gDNA Eraser (Takara, Shiga, Japan) according to the instructions

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of the manufacturer.

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PCR amplification and Illumina sequencing. Briefly, the cDNA from 3 replicates of each

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sampling time was amplified with primers specific to the bacterial 16S rRNA gene and the arsC

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gene using the primers 515F and 806R37 and amlt-42-F and amlt-376-R,38 respectively. Details are

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in the SI.

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Sequence analysis. Data from 16S rRNA amplicon libraries were processed using the Quantitative

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Insights Into Microbial Ecology (QIIME 1.8.0) toolkit.39 All the 16S rRNA and the arsC gene

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sequences were submitted to the NCBI Sequence Read Archive (SRA) (BioProject accession

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number PRJNA355907 and PRJNA357341).

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Construction of the arrA gene cDNA clone library. Due to the length limitation of

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high-throughput sequencing, a arrA gene-based clone library method was used to characterize

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microbial community of potentially active dissimilatory As(V)-reducing bacteria. The RNA samples

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extracted on day 2 were selected for arrA gene transcript clone library construction. Nested PCR

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amplification of arrA gene (625 bp) was achieved with primers AS1F/AS2R (the first PCR),


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AS2F/AS1R (the nested PCR).40 The GenBank/EMBL/DDBJ accession number of the arrA gene

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sequence is KY780033 to KY780063.

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Abundance of transcripts. Quantification of transcripts of bacterial 16S rRNA gene, Geobacter

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spp., Geobacter arrA gene and arsC gene were performed on an iQ™5 Multicolor Real-Time PCR

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Detection System (Bio-Rad Laboratories, USA) using the SYBR Green I detection method. Each

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real-time PCR reaction (20 µl reaction) contained 10 µl of 2 × IQ™ SYBR Green Supermix

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(Bio-Rad, USA), 0.2 µM of each primer, 1 µl of cDNA (1-10 ng). Real-time PCR amplification and

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primers used for each reaction were described in Table S4. All sample conditions were run in

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triplicate. Plasmid pGEM-T Easy Vetor (Promega, Madison, USA) was used in the cloning of gene

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fragments

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EZNA Plasmid Mini Kit I (Omega Bio-Tek, Doraville, GA, USA) and the concentration was

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determined with a Qubit 3.0 Fluorometer.

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Statistical analysis. Data were presented as mean ± standard deviation (SD). Abundance of

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functional genes were subjected to one-way analysis of variance (ANOVA) followed by Duncan’s

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multiple range tests in SPSS 22.0 with a significance level of P < 0.05. The correlation analysis was

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conducted by Pearson correlation with a significance level at P < 0.05 (two-tailed) using SPSS 22.0.

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RESULTS

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Arsenic and iron transformation. At day 0, PO4-As(V) were the dominant arsenic species in all

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treatments and oxalate-As(V) and PO4-As(III) were also detected, whereas dissolved-As(III) and

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oxalate-As(III) were undetectable (< 0.01 µg l-1) (Figure 1). In the four abiotic controls with the

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sterilized soils, there was no reduction of As(V) to As(III), with dissolved As(III) detected at

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0.14-0.18 mg kg-1 in the Sterilized soil+lactate+biochar treatment on day 10-20 (Figure S2). Thus,

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the majority of arsenic fractions in the abiotic controls were retained in soil.

157 158

to

establish

standard

curves.41

Plasmid

DNA

was

extracted

using

an

In all biotic microcosms, over the 20 day incubation period the concentrations of dissolved As(III) increased to different levels compared to no observed changes in the abiotic controls (Figure 1 and 7 ACS Paragon Plus Environment


159

Figure S2). The slight increase in dissolved As(III) in the Soil+biochar treatment was insignificant

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over Soil alone (Figure 1). In the Soil+lactate treatment, dissolved As(III) quickly increased to 8.8

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mg kg-1 on Day 1-2 without further increase during the rest of incubation (Figure 1). Lactate alone

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appears to promote As(V) reduction, whereas biochar alone does not. In the Soil+lactate+biochar

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treatment, however, significant release of dissolved As(III) was observed and it reached 30.2 mg

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kg-1 on Day 20. This was approximately 3 and 4-fold higher than the levels of As(III) in

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Soil+lactate and Soil+biochar, respectively (Figure 1). A clear decrease in PO4-As(V) concomitant

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with increase of PO4-As(III) was observed in all biotic treatments, with the highest PO4-As(III)

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detected in the Soil+lactate+biochar treatment (Figure 1). Thus, As(V) can be reduced to As(III) by

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indigenous bacteria from soil, and subsequently or simultaneously released from the solid to the

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aqueous phase, which can be facilitated by lactate and biochar. Our results indicated that the

170

presence of biochar has an additive effect over that of lactate alone on the microbial reduction of

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As(V) to As(III). It should be noted that in this study, the total of reduced As(III) only accounts for

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about 30% of the total loss of adsorbed As(V), which suggested that large amounts of As(III) or

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As(V) might be transformed into arsenic fractions in soil that cannot be extracted using phosphate

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and oxalate buffer.

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The production of Fe(II) was observed in the biotic microcosms with lactate and/or biochar

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during the whole reaction time (Figures S3 and S4), in which dissolved Fe(II) accounted for ≥ 90%

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of the HCl-extractable Fe(II) (Figure S4). Compared to the respective abiotic control, the biotic

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microcosms had high dissolved Fe(II) with lactate and/or biochar, suggesting that Fe(III) reduction

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was predominantly driven by microorganisms in the anoxic paddy soil (Figure S3). Among all

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biotic microcosms, the extents of Fe(II) production were ranked as follow: Soil+lactate+biochar >

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Soil+lactate > Soil+biochar > Soil (Figure S3), indicating that amendment of biochar and/or lactate

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promoted microbial Fe(III) reduction. The dissolved Fe(II) reached 6.2 mg l-1 on day 20 in the

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Soil+lactate+biochar treatment, which was 2.5 and 4-fold higher levels of Fe(II) than Soil+lactate


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(2.3 mg l-1) and Soil+biochar (1.4 mg l-1), respectively (Figure S3). Therefore, biochar can

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significantly promote microbial Fe(III) reduction when lactate is present. It should be noted that

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concentrations of As(III) and Fe(II) increased coincidently over time, demonstrating that arsenic

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release and Fe(III) reduction may occur simultaneously in the tested paddy soil, and both were

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significantly promoted in presence of biochar and lactate.

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Composition of active bacterial communities. Overview of the active bacterial communities is in

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Table S5 and Figure S5. Within the domain Bacteria, 16 distinct phyla/Class (Figure S6), and more

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than 21 genera (Figure 2) were detected with a relative abundance > 1%. Generally,

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β-Proteobacteria (19-46%), δ-Proteobacteria (8-38%), Actinobacteria (2-14%), Acidobacteria

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(2-11%), α-Proteobacteria (1-9%) and Bacteroidetes (1-7%) represented the most dominant active

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bacterial phyla in all biotic treatments. Higher abundance of Firmicutes (2-41%), δ-Proteobacteria

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(13-38%) and γ-Proteobacteria (3-19%) were detected in Soil+lactate+biochar and Soil+lactate

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relative to Soil+biochar and Soil (< 12%).

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Figure 2 and Table S6 illustrate the diversity and relative abundance of active microbes at the

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genus level. Azoarcus (9-22%) and Anaeromyxobacter (4-11%) were dominant genera in all biotic

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microcosms throughout the incubation. There was a big bloom of Clostridium in the treatments of

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Soil+lactate+biochar (24%) and Soil+lactate (40%) on day 1, while its abundance was < 0.03% in

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Soil+biochar and in Soil treatment. A significant increase in Geobacter was clearly observed in

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Soil+lactate+biochar (5-14%) over Soil+lactate, Soil+biochar and Soil (< 6%). The relative

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abundances of Pseudomonas and Desulfobulbus were 1-5% in Soil+lactate+biochar, whereas their

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relative abundances were under 1% or undetectable in the other biotic microcosms. Thus, the active

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members of Geobacter, Pseudomonas and Desulfobulbus can be stimulated by biochar in anoxic

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arsenic-contaminated paddy soil when lactate is presented.

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Active members of arrA and arsC gene transcripts. Overview of the active dissimilatory

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As(V)-respiring bacterial community is in Table S7. Phylogenetic analysis of the retrieved arrA



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gene sequences with the known As(V)-respiring bacteria indicated four main clades (Figure 3). The

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21 OTUs affiliated within Cluster A (70% of the arrA sequences, 61 and 65 arrA sequences for

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Soil+lactate+biochar and Soil+lactate treatments, respectively), are closely related to the arrA gene

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sequences in Geobacter uraniireducens Rf4, and Geobacter sp. OR-1. G. uraniireducens Rf4 is an

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anaerobic bacterium isolated from subsurface sediment containing uranium 42 and the strain OR-1 is

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a known anaerobic As(V)-respiring bacteria isolated from a paddy field.

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(8 OTUs, 26.6% of the arrA sequences) are not closely related to any cultivated organism. In

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addition, the 1 arrA gene sequence within Cluster C and the 5 arrA gene sequences in Cluster D

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were phylogenetically closed to the known arsenic respiring bacteria within the Desulfosporosinus

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and Shewanella, respectively. Details of the retrieved arrA gene sequences and their

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phylogenetically relatives are shown in Table S8. These results suggest that Geobacter species are

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the most abundant and active As(V)-respiring bacteria in the presence of lactate and/or biochar.

14

Sequences in Cluster B

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Regarding arsC gene sequences, the most abundant and active cytoplasmic As(V)-reducing

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bacteria are represented in the Enterobacteriaceae family. In both Soil+lactate and

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Soil+lactate+biochar treatments, about 10 families were detected, in which > 99% of arsC gene

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reads were affiliated with Enterobacteriaceae family of γ-Proteobacteria (Table S9). The PCoA

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analysis showed that symbols representing the Soil+lactate+biochar and Soil+lactate did not

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separate from each other (Figure S7).

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Transcript abundance of bacterial 16S rRNA cDNA, Geobacter spp. and arrA and arsC genes.

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The time-course transcription behavior of total bacterial 16S rRNA gene, Geobacter spp., as well as

229

the arsC and Geobacter-related arrA genes in the Soil+lactate+biochar and Soil+lactate treatments

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were compared by quantitative reverse transcription-PCR (RT-qPCR). Transcriptions of bacterial

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16S rRNA gene were 1010-1011 gene copies ml-1 soil slurry in lactate+biochar amendment, which

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were significantly (P < 0.05) higher than those (109-1010 gene copies ml-1) in lactate amendment

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during the course of the incubation (Figure 4a). The biochar+lactate amendment also favored the


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transcription of Geobacter species and Geobacter arrA gene when compared to those of the lactate

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amendment (Figure 4b and 4c). After normalized to the absolute gene copy numbers of ambient

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16S rRNA gene, a positive correlation was observed between the transcripts of Geobacter spp. and

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dissolved As(V) (Pearson’s correlation r = 0.71, P > 0.05) as well as between the transcripts of

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Geobacter arrA gene and dissolved As(V) (r = 0.82, P < 0.05) in Soil+lactate+biochar treatment

239

(Figure S8a). In Soil+lactate treatment, however, the dissolved As(V) did not correspond well with

240

the transcripts of Geobacter spp. (r = -0.23, P > 0.05) or of Geobacter arrA (r = 0.20, P > 0.05)

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(Figure S8b). However, no significant correlation was found between the relative transcript

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abundance of Geobacter or arrA gene and dissolved As(III) or Fe(II) concentration in

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Soil+lactate+biochar and Soil+lactate (Table S10).

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Transcript numbers of arsC gene (102-103 gene copies ml-1) were much lower than those of

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Geobacter arrA gene (106-107 gene copies ml-1) in both lactate and lactate+biochar amendments

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(Figure 4c and 4d). The transcript abundance of arsC did not correspond well with dissolved As(V)

247

either in lactate+biochar or in lactate amendments (r = 0.029 and -0.083, P > 0.05, respectively)

248

(Figure S9). Therefore, ArsC mediated As(V) reduction, i.e. cytoplasmic As(V) reduction, was not

249

the major process determining arsenic release and reduction in anoxic paddy soil.

250

DISCUSSION

251

Effect of lactate. Arsenic dissolution and reduction in anoxic soil are mainly due to microbial

252

reductive

253

reduction.3,14,43Although lactate is thought to facilitate iron dissolution abiotically under anoxic

254

conditions,44 negligible aqueous arsenic and Fe(II) were found in the abiotic treatment with lactate

255

in arsenic-rich aquifers and sediments in previous studies.18,45 Similarly, no detectable As(V) release

256

or Fe(III) reduction was observed in the Sterilized soil+lactate treatment (Figure S2), underscoring

257

that the abiotic effect of lactate on iron reduction and arsenic release was very limited.

258

dissolution

of

Fe(III)

(hydr)oxide

and

microbially

mediated

As(V)

Our results indicated that lactate principally stimulated indigenous microorganisms and promoted 11 ACS Paragon Plus Environment


259

arsenic release and reduction in the anoxic paddy soil (Figure 1 and 2). Incubations of arsenic-rich

260

sediments have been conducted primarily with addition of acetate as organic proxy to promote

261

microbial activity.17,18,21,23,32 The majority of these studies have demonstrated enrichment of

262

bacterial population closely related to Geobacter species. Lactate can be secreted from the rice roots

263

particularly under low oxygen stress, and can be produced via anaerobic fermentation of

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large-molecular organic substrates under anoxic conditions.25,26 In this study, incubation of anoxic

265

paddy soil with lactate favored enrichment of Clostridium and Geobacter, Azoarcus and

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Anaeromyxobacter (Figure 2), with Geobacter identified as the most active bacteria responsible for

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As(V) reduction (Figure 3). Besides acetate, Geobacter spp. have been also shown to oxidize lactate

268

during iron reduction.42,46 Therefore, when supplied with different organic substrates (e.g. lactate or

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acetate), Geobacter species can be readily stimulated for iron and arsenic reduction under anoxic

270

conditions.

271

When using a DNA-based sequencing method to profile community composition of arrA gene in

272

the paddy fields, most of the sequences were aligned to the arrA genes of uncultured bacteria with

273

only a few related to Geobacter sp..19 Such a low amount of Geobacter is probably attributed to the

274

difference in primers used to target arrA gene, in which the nested PCR amplification employed in

275

this study can generate more positive arrA gene products,40 relative to the common PCR

276

amplification that was used by Zhang et al.19 Previous study reported the discovery of highly

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diverse communities of As(V)-respiring bacteria in unamended near-surface sediments, with highest

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sequence similarity to known Geobacter species using the same nested primers in our study.47 In

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addition, the high abundance of Geobacter in our arrA gene cDNA clone library is also associated

280

with the application of lactate as a simple organic substrate to stimulate bacterial growth.

281

It should be noted that the presence of lactate stimulated the transcription activity of Geobacter

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spp., Geobacter arrA gene (Figure 4b) and arsC gene (Figure 4d), all of which reached their highest

283

values on Day 5 (Figures S8 and S9). This suggested that microbial As(V) reduction regulated by


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lactate should have been mainly taking place during the early stage of incubation (day 0−5), which

285

was consistent with the arsenic transformation (Figure 1). Hence, the arsenic-reducing bacteria and

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genes could response quickly to the lactate amendment at the beginning of incubation, resulting in

287

enhancement of arsenic release and reduction in the anoxic paddy soils. In addition, lactate can also

288

stimulate microbial Fe(III) reduction (Figure S3) followed by an increased release of As(V) (Figure

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1), which can be subsequently reduced to As(III) by arsenic-reducing bacteria. The addition of

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lactate has caused an increase in arsenic release and reduction in the anaerobic microcosm

291

experiment of the present study. It should be noted that many organic substances can be found in the

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rice plant exudates which may show a different response to the arsenic transformation in the paddy

293

field. Investigation about the effect of complex organic mixture extracted from soil (such as

294

dissolved organic matter or humic substances) instead of single organic proxy should be considered

295

in the future.

296

Effect of biochar. It has been widely reported that biochar can influence the fate of heavy metals by

297

sorption/desorption due to its large surface area, strong adsorption capacity and high cation

298

exchange capacity.27,48 Biochar can also change soil chemical properties such as pH to influence

299

heavy metal transformation and bioavailability.48 In this study, the pH values of all treatments did

300

not change greatly throughout the incubation (pH 7.3-7.6), suggesting that the effect of pH on

301

arsenic desorption is negligible in our study.

302

In addition, microbial reductive dissolution of Fe(III) facilitated by the biochar can increase

303

arsenic mobility by arsenic release from the Fe(III)-bearing minerals.49,50 This can explain why the

304

extent of As(V) release from soil to aqueous phase in the Soil+biochar treatment was higher than

305

the Sterilized soil+biochar treatment (Figures 1 and S2). The biochar alone had minimal effect on

306

the composition and diversity of the microbial community (Figure 2). Therefore, the majority of

307

arsenic in soil with biochar alone is retained in the soil, while arsenic release as a consequence of

308

microbial iron reductive dissolution is limited in the absence of an organic substrate.



309

When supplied with lactate, the biochar amendment modestly enhanced the release of As(V) to

310

the aqueous phase (Figure 1), while the microbial reduction of As(V) and Fe(III) was significant

311

(Figure 1 and Figure S3). This can be due to several factors. Firstly, biochar can create new habitats

312

for microorganisms by adsorbing organic substrates and nutrients on its surface, leading to shifts in

313

microbial abundance, community composition and activities.51 In this study, biochar coupled with

314

lactate elevated the initial relative abundances of Geobacter, Pseudomonas and Desulfobulus, as

315

compared with lactate only (Figure 2). The transcripts of Geobacter species and Geobacter arrA in

316

the lactate+biochar amendment were higher than those of lactate alone (Figure 4). Hence, in

317

presence of lactate, biochar can stimulate the dissimilatory As(V)-respiring bacteria (mainly

318

Geobacter spp.), which play a more important role in the As(V) reduction than the cytoplasmic

319

As(V)-reducing bacteria in flooded paddy soil. In addition, the enhanced microbial Fe(III) reduction

320

by biochar in the presence of lactate (Figures S3 and S4) could also increase arsenic release and

321

provide more available As(V) for microbial As(V) reduction (Figure 1).

322

Secondly, biochar is reported to facilitate electron transfer from microbes to terminal electron

323

acceptor by functioning as an electron shuttle due to its condensed aromatic structures and

324

quinone/hydroquinone moieties.28,52 These functional moieties also can be found in dissolved and

325

particulate soil organic matters, such as humic substances.53−55 In the amendment of biochar+lactate,

326

the transcript numbers of arrA gene were significantly higher over that of arsC gene (Figure 4).

327

Electron transfer from the cytoplasmic ArsC reductase to the biochar outside the cell is unlikely,

328

while the biochar at the cell surface can serve as electron shuttles between the ourter-membrane

329

reductases from dissimilatory Fe(III)-/As(V)-reducing bacteria and the terminal electron acceptor

330

Fe(III)/As(V).56 Supplementary experiment for As(V) reduction with biochar and Geobacter

331

sulfurreducens strain PCA that cannot reduce As(V) directly57 confirmed that microbially reduced

332

biochar can abiotically transfer electron to As(V), resulting in the reduction of As(V) to As(III)

333

(Figure S10). In summary, the enhancement of As(V) reduction by biochar in the presence of lactate


Page 14 of 32

Page 15 of 32


334

is mainly due to: (i) facilitation of As(V) release into soil solution following enhanced microbial

335

Fe(III) reduction; (ii) stimulation on the transcripts of arrA gene and dissimilatory As(V)-respiring

336

bacteria; (iii) function as electron shuttle between metal-reducing bacteria and As(V) by a two-step

337

process, i.e., the biotic reduction of biochar by Fe(III)/As(V)-reducing bacteria, followed by an

338

abiotic As(V) reduction by the microbially reduced biochar.

339

Role of Geobacter species and other active bacteria. Our results identified Geobeacter spp.,

340

which are well-known dissimilatory Fe(III)-reducing bacteria,58,59 as the major As(V) reducers in

341

our microcosms (Figure 3). Geobacter species have been frequently detected in arsenic-rich South

342

and Southeast Asia sediments and groundwater through 16S rRNA and arrA gene analyses.15,17,21,23

343

Geobacter uraniireducens was also detected in paddy soils from South China.19 Genome

344

sequencing of G. uraniireducens Rf4 and G. lovleyi SZ indicate that they possess the necessary

345

genes for As(V) respiration (arr operon), but direct As(V) respiration by these bacteria has not yet

346

been reported.56,59,60 Direct evidence for arsenic reduction by Geobacter isolate was only reported

347

with Geobacter sp. OR-1 isolated from paddy soil,14 while its genome sequencing of OR-1 revealed

348

a similar arsenic respiration operon that had been previously described.61 In this study, we reported

349

that the transcript abundances of Geobacter species and Geobacter arrA gene closely tracked with

350

dissolved As(V) concentrations in the Soil+lactate+biochar treatment (Figure S8). These results

351

imply that Geobacter spp. could rapidly respond to dissolved As(V) concentrations by regulating

352

the transcription of arsenic respiration gene (arrA) in anoxic paddy soil, particularly when amended

353

with lactate and biochar simultaneously. From the results in microbial community (Figure 2),

354

phylogenetic analysis of arrA gene sequences (Figure 3), quantification of transcripts (Figure 4),

355

and correlation analysis (Figure S8), and its known ability to carry out dissimilatory Fe(III) and

356

As(V) reduction, Geobacter appears to be the key member of the soil-biochar-lactate community.

357

Another active genus is Clostridium, members of which are commonly known as fermentative

358

bacteria and are also able to respire As(V) and Fe(III),62 such as arsenic-respiring Clostridium sp.



Page 16 of 32

359

OhiLAs63 and Fe(III)-reducing C. beijerinckii,64 C. butyricum65 and C. saccarobutylicum BS2.66 The

360

relative abundance of Clostridium was dramatically high on day 1 in both Soil+lactate and

361

Soil+lactate+biochar, indicating that Clostridium can be stimulated promptly by the presence of

362

lactate and biochar, and likely affects arsenic and iron transformation in flooded paddy soils.

363

Among the other active members of microbial community, Anaeromyxobacter is also able to

364

oxidize

365

Anaeromyxobacter has been frequently detected in arsenic-contaminated sites and a dissimilatory

366

As(V)-respiring bacteria affiliated with Anaeromyxobacter have been isolated,69 suggesting that this

367

genus can be associated with arsenic mobility and transformation.69,70 Members of Azoarcus,

368

Pseudomonas and Desulfobulbus have not previously been known to have associated with arsenic

369

reduction,71 but genera of Azoarcus and Pseudomonas can be resistant to high As(III).72 It has been

370

verified that a putative ars operon is present in the genomes of many members of Azoarcus revealed

371

by whole-genome analyses.73−76 This may explain why the Azoarcus genus can survive in the

372

arsenic-rich environments. In this study, the abundance of Azoarcus is high in all biotic treatments

373

throughout the incubation, indicating that members of Azoarcus may be the indigenous soil bacteria

374

that could play a role in arsenic transformation in flooded paddy soils. Whether there are more

375

specific interactions of this community with arsenic or iron transformations remains to be

376

determined.

377

Perspectives. According to previous reports, biochar amendment can have positive,77,78

378

negative20,30,32 or negligible31 impacts on arsenic release. So far, knowledge of the potential

379

influences of biochar on arsenic transformation in anoxic arsenic-contaminated paddy soils is still

380

very limited. While the microbial community and the copy numbers of functional genes in previous

381

study were only characterized at the end of incubation (day 60) using DNA-based analysis,20 our

382

study focused on the transcriptional activity of As(V)-reducing genes and identification of

383

As(V)-reducing bacteria, as well as the potential correlation between arsenic transformation and

organic

compounds

coupled

to

dissimilatory


Fe(III)

reduction.67,68

In

fact,

Page 17 of 32


384

transcriptional activity of As(V)-reducing bacteria/genes with time dependence. When compared

385

with molecular techniques targeting rRNA genes (the present microbial community), analyses of

386

rRNA cDNA and gene transcripts employed in this study have been proved to be a better indicator

387

of physiological activity of microbial population.79−81 In addition to the common sense that biochar

388

can function as electron shuttle to facilitate electron transfer from bacteria to As(V) and Fe(III),20

389

our findings that biochar mainly up-regulated the active expression of As(V)-respiring arrA gene

390

and stimulated the active As(V)-respiring bacteria Geobacter species for arsenic reduction and

391

release provide a better understanding of the microbial mechanisms that drive arsenic mobility in

392

flooded paddy soil amended with biochar.

393

However, the amendment of biochar may raise a serious environmental issue, such as elevating

394

arsenic accumulation in rice plant due to an increase in arsenic release and bioavailability. It has

395

been reported that dissolved As(III) can readily be taken up by plants from soil pore water.82 In fact,

396

biochar amendment in soil has resulted in higher total arsenic in porewater in previous rice

397

cultivation experiment in pot trial, leading to an increased arsenic accumulation in rice.83 Therefore,

398

biochar amendment in arsenic-contaminated paddy soil should be considered very carefully. For

399

example, other factors should be taken into consideration including content of organic matter in

400

contaminated sites and dosage of biochar to be used when using biochar for heavy metal

401

remediation.

402

ASSOCIATED CONTENT

403

Supporting Information

404

Table S1: Chemical properties of the contaminated soil. Table S2: The elemental composition of the

405

biochar. Table S3: The experimental treatments conducted in this study. Table S4: Primers used for

406

the real-time RT-qPCR. Table S5: Microbial diversity metrics (α-diversity). Table S6: Relative

407

abundances of abundant genera by 16S rRNA cDNA sequencing. Table S7: Comparison of

408

α-diversity indices of the arrA gene cDNA clone library. Table S8: Summary of the arrA gene 17 ACS Paragon Plus Environment


Page 18 of 32

409

cDNA clone library. Table S9: The relative abundance of the potentially active arsenic-resistant

410

bacteria at the family level. Table S10: Relationships of concentrations of dissolved As(III) and

411

dissolved Fe(II), and transcripts levels of Geobacter and arrA gene normalized to 16S rRNA gene.

412

Figure S1: X-ray diffractogram and FTIR spectrogram of the biochar. Figure S2: Time-dependent

413

concentrations of dissolved As(III)/As(V), PO4-As(III)/As(V), and oxalate-As(III)/As(V) arsenic

414

fractions in the abiotic controls. Figures S3-S4: Time-dependent concentrations of dissolved Fe(II),

415

HCl-Fe(II). Figure S5: Unweighted and Weighted UniFrac principal coordinate analysis (PCoA) of

416

the potentially active bacterial community. Figures S6: The relative abundances of the potentially

417

active bacterial community at the phylum and class levels. Figure S7: Weighted UniFrac principal

418

coordinate analysis of the active arsenic-resistant bacteria. Figure S8: Dissolved As(V)

419

concentrations and transcripts levels of Geobacter species and Geobacter arrA gene normalized to

420

16S rRNA gene. Figure S9: Dissolved As(V) concentrations and transcripts levels of arsC

421

normalized to 16S rRNA gene. Figure S10: The concentration of As(III) and As(V) during the

422

incubation

423

sulfurreducens+biochar+As(V).

424

AUTHOR INFORMATION

425

Corresponding Author

426

Phone: +86 20 37021396 ; Fax: +86 20 87024123; e-mail: [email protected].

427

Author Contributions

428

†

429

Notes

430

The authors declare no competing financial interest.

431

ACKNOWLEDGMENTS

432

This work was supported by the National Science Foundation of China (41330857 and 41471216),

of

the

biochar+As(V),

Geobacter

sulfurreducens+As(V),

and

Geobacter

These authors contributed equally to this work.


Page 19 of 32


433

the national key Basic Research Program (2016YFD0800701), the National Key Technology R & G

434

Program of China (2015BAD05B05 and 2015A030313752), the Guangdong Natural Science Funds

435

for Distinguished Young Scholars (2017A030306010), the SPICC Program (Scientific Platform and

436

Innovation Capability Construction Program of GDAS), the High-Level Leading Talent

437

Introduction Program of GDAS (2016GDASRC-0103), and Guangdong Key Technologies R & D

438

Program (2015B020207001).

439

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666

FIGURE CAPTIONS

667

Figure

668

phosphate-extractable (PO4-As(III)/As(V)), and oxalate-extractable (oxalate-As(III)/As(V)) arsenic

669

fractions in the soil samples of Soil+lactate+biochar, Soil+lactate, Soil+biochar and Soil over the

670

course of the incubation. The small inserted graphs show a magnified view of the data for

671

PO4-As(III) and oxalate-As(III) over the course of the incubation, respectively. Bars represent

672

standard errors (n = 3).

673

Figure 2 Relative abundance of the potentially active bacterial community at the genus level

674

detected in the treatments of Soil+lactate+biochar (a), Soil+lactate (b), Soil+biochar (c) and Soil (d)

675

during the incubation by 16S rRNA cDNA high-throughput sequencing. Only the genera with

676

relative abundance higher than 1% in at least two treatments were selected for further analysis. The

677

abundance is presented as the average percentage of three replicates, classified by RDP classifier at

678

a confidence threshold of 97%.

679

Figure 3 Phylogenetic tree of putative ArrA sequences retrieved from the Soil+lactate+biochar and

680

Soil+lactate treatments on day 2, and ArrA from other known arsenate-respiring bacteria based on

681

neighbor-joining analysis of approximately 203 amino acid residues of the alpha subunit

682

dissimilatory arsenate reductase. Bars following each OTU indicate the frequency of appearance of

683

the completely matched sequences (clones) in the total clones (number of identical clones/number

684

of the total clones; red, Soil+lactate+biochar; blue, Soil+lactate).

685

Figure 4 Transcript copy numbers of bacterial 16S rRNA cDNA (a), Geobaccter spp. (b),

686

Geobacter arrA gene (c), and arsC gene (d) in the Soil+lactate+biochar and Soil+lactate treatments

687

during the course of the incubation. Significant differences are indicated by different letters (P

Transcriptional activity of arsenic-reducing bacteria and genes

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