Accepted Manuscript

Title: Screening of plant growth-promoting traits inarsenic-resistant bacteria isolated from agricultural soil andtheir potential implication for arsenic bioremediation

Author: Suvendu Das Jiin-Shuh Jean Sandeep Kar Mon-LinChou Chien-Yen Chen

PII: S0304-3894(14)00188-5DOI: http://dx.doi.org/doi:10.1016/j.jhazmat.2014.03.012Reference: HAZMAT 15785

To appear in: Journal of Hazardous Materials

Received date: 24-9-2013Revised date: 5-3-2014Accepted date: 6-3-2014

Please cite this article as: S. Das, J.-S. Jean, S. Kar, M.-L. Chou, C.-Y. Chen, Screeningof plant growth-promoting traits in arsenic-resistant bacteria isolated from agriculturalsoil and their potential implication for arsenic bioremediation, Journal of HazardousMaterials (2014), http://dx.doi.org/10.1016/j.jhazmat.2014.03.012

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Screening of plant growth-promoting traits in arsenic-resistant bacteria

isolated from agricultural soil and their potential implication for arsenic

bioremediation

Suvendu Das a, Jiin-Shuh Jean a,*, Sandeep Kar a, Mon-Lin Chou a, Chien-Yen

Chen b

a Department of Earth Sciences, National Cheng Kung University, Tainan, Taiwan

b Department of Earth and Environmental Sciences, National Chung Cheng University,

Chiayi County, Taiwan

Author for correspondence: jiinshuh@mail.ncku.edu.tw (Jiin-Shuh Jean); Tel.: +886-6-

2757575 Ext. 65426; Fax: +886-6-274-0285

Abstract

Twelve arsenic (As) -resistant bacteria (Minimum inhibitory concentration ranging from

10 to 30 mM and 150 to 320 mM for As(III) and As(V), respectively) were isolated from

the agricultural soil of the Chianan Plain in southwestern Taiwan using enrichment

techniques. Eight isolates capable of oxidizing As(III) (rate of oxidation from 0.029 to

0.059 μM h-1 10-9 cell) and exhibiting As(III)-oxidase enzyme activity belong to

Pseudomonas, Acinetobacter, Klebsiella and Comamonas genera, whereas four isolates

that did not show As(III)-oxidizing activity belong to Geobacillus, Bacillus,

Paenibacillus, and Enterobacter gerera. Assessment of the parameters of plant growth

promotion revealed that Pseudomonas sp. ASR1, ASR2 and ASR3, Geobacillus sp.

ASR4, Bacillus sp. ASR5, Paenibacillus sp. ASR6, Enterobacter sp. ASR10 and

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Comamonas sp. ASR11, and ASR12 possessed some or all of the studied plant growth-

promoting traits, including phosphate-solubilization, siderophore, IAA-like molecules and

ACC deaminase production. In addition, the ability of As-resistant isolates to grow over

wide ranges of pH and temperatures signify their potential application for sustainable

bioremediation of As in the environment.

Keywords: Arsenite oxidation, Chemolithotrophs, bioremediation, Plant growth

promotion, Agricultural soil

1. Introduction

Soil contaminated with arsenic (As) in several agriculturally important areas is an

important current public health issue due to the toxic effects of this metalloid and its

accumulation through the food chain, which poses long term risks to human health [1]. In

the Chianan Plain in southwestern Taiwan, the elevated concentration of As in the soil

and in plants is mainly due to the irrigation of agricultural soil with As-enriched

groundwater [2]. The incidence of high As in the groundwater of the Chianan Plain is

known for generating unique cases of endemic Blackfoot disease (BFD), a peripheral

vascular disease (i.e., gangrene) [3]. At present, even though As-rich groundwater has not

been used for drinking, it is still extensively used for irrigation, aquaculture and industrial

purposes [2]. Agricultural soil acts as a principal sink of As through irrigation of crop

land, and most of the arsenical residues have low solubility and low volatility, generally

accumulating in the topsoil layers [4]. Topsoil thus contaminated with As may have

influence on the entry of As into the food chain [4].

The health hazards associated with As-contaminated soil coupled with the high

remediation cost makes it necessary to search for novel As-resistant bacteria for the

purpose of bioremediation of polluted land. However, bioremediation of heavily As-

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contaminated soil is difficult because this metalloid is very toxic to plants [5]. In addition,

As deposited in the soil may accumulate rapidly since it is only slowly depleted through

plant uptake, leaching, erosion, or methylation [6]. Currently, bioremediation involving

both plant and rhizospheric microorganisms resistant to heavy metals and possessing

plant growth promoting (PGP) traits that contribute to both contaminant degradation and

the promotion of plant growth is gaining in popularity [6, 7, 8]. In metal-polluted sites,

the composition of the indigenous soil microflora adapts to a changing environment, and

these microflora are able to use pollutant substrates as a nutrient source for their growth

and proliferation [4]. In addition, plants grown in metal-contaminated sites harbor unique

metal-resistant microflora in their rhizospheres [9]. These adopted indigenous microbial

communities may have the advantage of secreting PGP substances such as siderophores,

indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase and

solubilize phosphate that alleviate metal toxicity and enhance plant-assisted

bioremediation [9, 10, 11]. A recent review on this topic summarizes this with many more

examples [11]. The hypothesis of this study posits that selection of efficient As-resistant

bacteria and inoculation into the seeds and/or roots of suitable plant species will widen

the perspectives of bioremediation of soils affected by arsenic [12]. The significance of

metal/metalloid-resistant bacteria possessing PGP traits can be enormous in

metal/metalloid-contaminated soils since these bacteria are able to increase the tolerance

of plants against abiotic stress, stimulate plant growth, and contribute in this way to an

accelerated remediation of contaminated soils [9, 10, 11]. Moreover, As-resistant bacteria

capable of oxidizing As and exhibiting PGP traits could not only potentially support plant

growth on As-stressed soils but also lower As-toxicity and uptake, thus minimizing health

risks in crop production [6].

Heavy metal-resistant and plant growth-promoting bacteria (PGPB) have been

reported to increase in the uptake efficiency and enhancement of metal translocation,

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promote growth and increase heavy metal tolerance in plants [9-14]. However, reports on

As-resistant/As-oxidizing bacteria exhibiting PGP traits and their influence on

bioremediation of As remain elusive [6, 8].

The aim of this study was to investigate PGP traits in As-resistant bacterial strains

isolated from the agricultural soil of the Chianan Plain that had been irrigated with As-

enriched groundwater for subsequent studies of plant-microbe interactions and the

development of strategies that minimize health risks in food production and lead to better

and more sustainable agricultural practices.

2. Materials and Methods

2.1. Site description, soil sampling and chemical analysis

Soil samples (0 - 10 cm depth) were collected from eight As-affected areas of the

Chianan Plain, SW Taiwan (23o02″ - 23o38″ N and 120o06″ - 120o21″ E). Individual soil

cores (2 cm diameter, 10 cm depth) were taken with a sample probe from five different

places within each As-enriched site. Each sample was divided into two subsamples (for

soil chemical and microbial analyses). For the soil chemical analysis, the collected

samples were air dried in shade at room temperature, sieved (< 2 mm), and then

refrigerated at 4°C until analysis. Total As in soil was determined as reported by Kar et al.

[2].

2.2. Enrichment and isolation of As-resistant bacteria

Aerobic heterotrophic As-resistant bacteria were isolated by inoculation of 1.0 g

of a fresh soil sample into 100 mL of a modified mineral salt (MMS) medium containing

NH4NO3 (1.25 mM), CaSO4 (2 mM), MgCl2 (2 mM), KH2PO4 (10 μM), KOH (1.25 mM)

and FeCl2 (5 μM) [15], supplemented with micronutrients, vitamins [16], 5 mM glucose,

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and either 75 μM As(III) or 250 μM As(V) [15], and incubated at 30°C with shaking (170

rev/min) in the dark for one week. Enrichment cultures showing turbidity after incubation

were subcultured into a fresh MMS medium containing the same concentration of either

As(III) or As(V). After the second subculture, the resulting enriched cultures were serially

diluted and plated onto an R2A nutrient agar (Difco laboratory, MI, USA) medium

containing the same concentration of either As(III) or As(V). R2A nutrient agar medium

was designed to maximize the cultivability of stressed cells, and this plating protocol has

also been used to isolate aerobic heterotrophs [15]. Colonies appearing different in regard

to color, shape and margins on inoculated plates were streak purified at least three times

on the same complex solid medium in the presence of the same concentration of either

As(III) or As(V), and the isolates were stored at -80°C in 30% sterile glycerol.

2.3. Minimum inhibitory concentration (MIC) test

Minimum inhibitory concentration (MIC) has been defined as the lowest

concentration of As(III) or As(V) added that completely inhibits bacterial growth [17]. In

this study, As(III) and As(V) resistance in isolated bacterial strains was evaluated using

MIC tests. Aliquots of 1.0 mL of overnight cultures were incubated in 99.0 mL of

Mueller Hinton broth (Oxoid) supplemented with either As(III) as NaAsO2 (1 - 50 mM)

or As(V) as Na2HAsO4.7H2O (1 - 350 mM) and incubated at 30°C with shaking (170

rev/min) in the dark for 48h. The optical density of the cultures, as a measure of microbial

growth, was detected at a wavelength of 600 nm (OD600) by an UV-vis spectrophotometer

(BIO-RAD SmartSpecTM 3000, USA); a blank with only the medium culture without

bacteria was also analyzed. Experiments were carried out in triplicate.

2.4. Effect of pH and temperature on growth of As-resistant isolates

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For growth at an optimum pH, 10 mL samples of the MMS broth with no added

As were adjusted at different pH values varying from 2 to 11 and equally inoculated with

fresh cultures of As-resistant isolates. After 24h of incubation at room temperature, the

growth of the bacteria was measured through OD600 using a UV-vis spectrophotometer.

Likewise, for growth at optimum temperature, 10 mL samples of the MMS broth with no

added As were equally inoculated with fresh cultures of As-resistant isolates and

incubated at 4, 20, 30, 40 and 70°C. After 24h of incubation, the growth of the bacteria

was tested through OD600 using a UV-vis spectrophotometer.

2.5. Morphological, biochemical and molecular identification of isolated As-resistant

bacteria

The morphological and biochemical characteristics of the bacterial isolates were

examined according to Bergey’s Manual of Determinative Bacteriology. Individual

cultures grown on R2A nutrient agar medium at 30°C were examined for colony

morphology. Motility and morphology were studied using phase contrast microscopy

(Olympus BX-51, Olympus America Inc., USA). Gram staining was performed as per

standard procedures with exponentially growing cultures.

Molecular identifications of the strains were carried out by partial sequencing of

the 16S rRNA gene. The single bacterial colonies were suspended in 50 μL of sterile

deionized water and treated for 5 min at 100°C. Amplification of the 16S rRNA gene was

performed using a heat treated cellular lysate containing ≈ 10 ng of genomic DNA as a

template [18] in 20 μL of 1× AmpliTaq buffer (PerkinElmer) with 25 pmol of each primer

f8 (5’-AGAGTTTGATCCTGGCTCAG-3’) and r1510 (5’-

GGTTACCTTGTTACGACTT-3’) [19], 250 μmol L-1 each of dNTPs and 1 U of

AmpliTaq (PerkinElmer). The PCR cycle used for amplification was as follows: 5 min at

94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 56°C, 1 min at 72°C and a final

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extension of 8 min at 72°C. The amplified PCR products were purified using the

QIAquick PCR purification kit (QIAGEN) and analyzed with an automated DNA

sequencer (Applied Biosystems, Foster City, CA, USA).

Close relative and phylogenetic affiliation of the obtained sequences were

determined by using the BLAST search program at the NCBI website

(www.ncbi.nlm.nih.gov). The 16S rRNA sequences retrieved from the databases were

aligned using ClustalW, version 5.0, which was included in the MEGA software. The

phylogenetic tree was inferred by MEGA 5.0 (the neighbor-joining method). Sequence

divergences between strains were quantified using the Kimura two parameter distance

model [20]. Bootstrap analysis (1,000 replicates) was used to test the topology of the

neighbor-joining method data.

Nucleotide Sequence Accession Numbers

The nucleotide sequences obtained in this study have been submitted to the

GenBank database and assigned accession numbers AB758593 to AB758604.

2.6. Screening of As(III) oxidizing activities

The isolated As-resistant bacterial strains were screened for As(III)-oxidizing

activity using the modified microplate method of Diliana et al. [21] and the method

described by Lett et al. [22]. For the modified microplate method, 12 mL of a 48h

bacterial culture, grown in MMS medium without As (cell suspensions reached optical

density at 600 nm of 0.5 - 0.6), was centrifuged at 6,500 ×g for 10 minutes, and the

pelleted cells were collected, washed twice with sterile deionized water and then

suspended in 1.2 mL deionized water. A 20 μL aliquot of the bacterial suspension was

mixed with 80 μL of 0.2 M Tris-HCl buffer (pH 7.4) supplemented with As(III) to reach a

final concentration of 75 μM As(III). The cell suspensions were incubated at 30°C with

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shaking (170 rev/min) in the dark for 72h, and then silver nitrate (AgNO3) solution was

added to a final concentration of 0.2 M. The changes in color from bright yellow [due to

the reaction between AgNO3 and As(III) in Tris-HCl] to a brownish color [due to the

reaction between AgNO3 and As(V) in Tris-HCl] indicated that As(III)-oxidizing activity

was likely [21]. In the method described by Lett et al. [22], colonies grown on R2A

nutrient agar supplemented with 75 μM As(III) and incubated at 30°C in the dark for 72h

were flooded with AgNO3 solution (0.1 M). Brown precipitates around the colony

indicated a positive As(III) oxidation reaction.

The ability of the As-resistant isolates to grow chemolithoautotrophically with

As(III) as a sole electron donor and CO2 as a primary carbon source was tested as

reported by Macur et al. [15] with only slight modification. The screened As-resistant

bacteria characterized with As(III)-oxidizing activity were inoculated in MMS media (10

mL) supplemented with 50 μM NaH2PO4, 30 mM NaHCO3 and 5 mM As(III) in screw-

capped tubes and incubated at 30°C in the dark for one week. The tubes were properly

capped to maintain the partial pressure of the CO2.

2.7. As(III)-oxidation by the As-resistant isolates

To test the ability of isolated bacterial strains to oxidize As(III) in growth

medium, the strains were grown overnight in MMS liquid media without As. One percent

of these overnight cultures [initial cell density 106 cells mL-1 based on OD600

measurement of cell suspensions] were incubated in 20 mL of MMS media modified to

include 5 mM MOPS buffer (pH 7.0), 50 μM NaH2PO4, 1 mg L-1 yeast extract and 75 μM

As(III) and were incubated at 30°C with shaking (170 rev/min) in the dark for 50h.

Control flasks without cells were incubated to check for the abiotic transformation of As.

Samples were taken at different time intervals to determine cell growth in terms of optical

density (600 nm), and As(III) and As(V) concentrations were determined using an

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inductively coupled plasma - mass spectrophotometer (Aligent 7500ce ICP-MS, Tokyo,

Japan). Arsenic oxidation rates were calculated from the maximum slopes of As

concentration curves versus incubation time and normalized to cell number (OD

correlating to position of maximum slopes) using the relationship between cell

enumeration with phase contrast microscopy and OD measurement (600 nm) of cell

suspensions, as reported by Macur et al. [15].

2.8. As(III)-oxidase assay

Overnight grown (mid-log to late log growth phase) cultures of As-resistant

bacterial isolates in MMS liquid media supplemented with 75 μM As(III) were harvested

by centrifugation at 10,500 ×g for 2 min. The cell pellets were washed with 50 mM Tris-

HCl (pH 8) buffer followed by resuspension in 2 mL of the same buffer containing 0.5

mM phenylmethylsulfonyl fluoride (PMSF) and 1 mg mL-1 lysozyme and was incubated

for 2h at room temperature with occasional stirring. The cells were lysed using sonication

and centrifuged at 10,500 ×g at 4oC for 30 min. The As(III)-oxidase assay was performed

as described by Anderson et al. [23]. The assay for As(III)-oxidase activity followed the

transfer of reducing equivalents from As(III) to 2,4-dichlorophenolindo-phenol

(DCIP). The reduction of DCIP (60 μM) was monitored at 600 nm in the presence of 200

μM As(III).

2.9. Quantitative determination of potential plant-growth promoting traits of As-resistant

bacteria

The As-resistant isolates were tested for their ability to solubilize phosphate, to

produce siderophores and IAA-like molecules and to grow on ACC as the sole nitrogen

source.

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2.9.1. Screening for phosphate-solubilization

The ability of As-resistant bacterial isolates to solubilize phosphate was tested by

growing the strains in modified Pikovskaya’s medium [24] with 0.5% of tricalcium

phosphate ( TCP) at 30°C for 5 days at 170 rev/min in order to reach a stationary phase

(determined by measuring absorbance at 600 nm). The culture supernatants were

collected by centrifugation at 6,500 ×g for 10 min. The soluble phosphate in the culture

supernatant was estimated according to the method of Zaidi et al. [25].

2.9.2. Screening for siderophore production

The ability of As-resistant isolates to produce siderophores was detected by using

the Chrome Azural S (CAS) method of Schwyn and Neilands [26]. To induce siderophore

production, As-resistant bacteria were grown in a modified M9 liquid medium containing

no Fe [26] and incubated at 30°C for 5 days at 170 rev/min. As a control a flask with

2 μM added iron (freshly prepared, filter sterilized FeSO4·7H2O stock solution) was

also inoculated. After the cultures had grown to the stationary phase, the cells were

pelleted by centrifugation (6,500 ×g for 10 min), and the supernatant was filtered through

a 0.45 μm membrane filter. The concentration of siderophores in the filtrate was

measured by mixing 0.5 mL CAS assay solution [26] with 0.5 mL filtrate, and the

absorbance was measured at 630 nm. The assay was calibrated by generating a standard

curve for samples containing 1 - 100 mM deferoxamine mesylate (DFM).

2.9.3. Screening for IAA-like molecules production

As-resistant bacterial strains were cultured in a minimal medium (KH2PO4 0.4 g

L-1, K2HPO4 2 g L-1, MgSO4.7H2O 0.2 g L-1, FeSO4.7H2O 0.1 g L-1, CaCl2 0.1 g L-1,

NaCl 0.2 g L-1, NaMoO4.2H2O 0.005 g L-1, Glucose 10 g L-1) with 0.5 mg mL-1 L-

tryptophan, a precursor of IAA-like molecules. After 5 days of incubation at 30°C in the

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dark, 2 mL of the cell suspension was transferred into a tube and then mixed vigorously

with 100 μL of 10 mM orthophosphoric acid and 4 mL of Salkowski’s reagent (2% 0.5 M

FeCl3 in 35% perchloric acid) and incubated for 45 min for development of pink color.

Then the absorbance was read at 530 nm. The IAA-like molecule concentration in the

cultures was determined using a calibration curve of pure IAA as a standard following the

linear regression [27].

2.9.4. Screening for utilization of ACC

ACC deaminase activity was determined by monitoring the amount of α-

ketobutyric acid generated from the cleavage of ACC [28]. The ACC deaminase activity

was induced by growing As-resistant bacterial cells in a minimal medium containing

ACC (3 g L-1) as the sole nitrogen source, after growing them in 15 ml MMS medium up

to log phase. α-ketobutyrate produced by the reaction was determined by comparing the

absorbance (540 nm) of the sample to a standard curve of α-ketobutyrate ranging between

0.1 and 1.0 μmol. The ACC deaminase activity was expressed as the amount of α-

ketobutyrate (KB) produced per mg of protein per hour.

3. Results

3.1. Total As content in agricultural soils from the study sites

The total As content in the surface soil (0 – 10 cm depth) from the study sites

ranged from 7.92 ± 3.38 mg kg-1 to 12.7 ± 2.82 mg kg-1 (mean 10.3 ± 3.38 mg kg-1). The

soil from Beimen CN9 had the highest total As content. There was not much variation in

the total As content in the soil among the study sites (Table 1).

3.2. Enrichment and isolation of As-resistant bacteria

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Arsenic-resistant bacteria from agricultural soil in the Chianan Plain were isolated

using enrichment techniques. Several hundred bacterial colonies were able to grow on the

R2A agar plates containing either 75 μM As(III) or 250 μM As(V). Colonies different in

shape, color and margins (192 bacterial colonies) were picked from the most diluted

plates (10-6 – 10-5 dilutions) and screened for their MICs. Arsenic-resistant isolates, which

had MICs ≥ 10 mM and ≥ 150 mM for As(III) and As(V), respectively, were selected for

further characterization and identification. From the twelve As-resistant isolates (MIC

ranged from 10 to 30 mM and 150 to 320 mM for As(III) and As(V), respectively),

isolate ASR1 had the highest MICs of 30 mM and 320 mM for As(III) and As(V),

respectively, whereas isolates ASR11 and ASR12 had the lowest MICs of 10 mM and

150 mM for As(III) and As(V), respectively (Table 1).

3.3. Effect of pH and temperature on growth of As-resistant isolates

All of the twelve As-resistant bacteria were able to grow on wide ranges of pH

(pH 5, 6, 7, 8 and 9) and temperature (20, 30 and 40°C), with the most favorable growth

conditions at pH 7 and a temperature of 30°C. However, none of the bacteria were able to

grow in extreme conditions i.e., pH 2, pH 11 and 70°C (data not presented).

3.4. Identification and phylogenetic characterization of As-resistant isolates

The morphological and biochemical characteristics of the twelve As-resistant

bacterial isolates are shown in Table 2. Following morphological characterization,

motility and gram staining, the isolates were compared with those of the standard species

using Bergey’s Manual of Determinative Bacteriology. These twelve isolates were further

identified by partial sequencing of 16S rRNA genes. The bacterial isolates ASR1, ASR2

and ASR3 showed the closest GenBank match to Pseudomonas sp. (99% similarity),

Pseudomonas stutzeri (99% similarity) and Pseudomonas putida (99% similarity),

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respectively. Isolates ASR7 and ASR8 showed the closest GenBank match to

Acinetobacter sp. (99% similarity), and isolates ASR11 and ASR12 showed the closest

GenBank match to Comamonas sp. (99% similarity), whereas the closest GenBank

matches to the isoaltes ASR4, ASR5, ASR6, ASR9 and ASR10 were Geobacillus sp.

(99% similarity), Bacillus cereus (99% similarity), Paenibacillus pabuli (99% similarity),

Klebsiella sp. (99% similarity) and Enterobacter sp. (99% similarity), respectively (Table

1). Phylogenetic analysis revealed that from the eight As-resistant bacteria that were

screened positive for As-oxidizing activities, isolates ASR1, ASR2, ASR3, ASR7, ASR8,

and ASR9 belong to Gram-negative γ–Proteobacteria, whereas isolates ASR11 and

ASR12 belong to Gram-negative β-Proteobacteria. From the four As-resistant bacteria

that did not show any As(III)-oxidizing activities, isolates ASR4, ASR5, and ASR6

belong to Gram-positive phylum Firmicutes, whereas isolate ASR10 belongs to Gram-

negative γ–Proteobacteria (Supporting information, SI-Figure 1).

3.5. As(III) oxidation by the isolates

From the twelve As-resistant isolates, Pseudomonas sp. ASR1, ASR2, and ASR3,

Acinetobacter sp. ASR7 and ASR8, Klebsiella sp. ASR9, Comamonas sp. ASR11 and

ASR12 were screened positive for As(III)-oxidizing activity. In contrast, as in the control

(not bacterium-inoculated), negligible As(III) oxidation was observed in isolates

Geobacillus sp. ARS4, Bacillus sp. ASR5, Paenibacillus sp. ASR6 and Enterobacter sp.

ASR10, which could be a result of the strong aeration of the growth medium under 170

rev/min shaking conditions (Fig. 1). The rate of As(III) oxidation during the logarithmic

growth of the isolates Pseudomonas sp. ASR1, ASR2, and ASR3, Acinetobacter sp.

ASR7 and ASR8, Klebsiella sp. ASR9, Comamonas sp. ASR11 and ASR12 varied from

0.029 to 0.059 μM h-1 10-9 cell with corresponding As(III) half-lives ranging from 23.9 to

11.7 h (Table 1, Fig. 1). Pseudomonas sp. ASR1 possessed the highest rate of As(III)

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oxidation (half-life of 11.7 h), whereas Comamonas sp. ASR11 possessed the lowest rate

of As(III) oxidation (half-life of 23.9 h) (Table 1, Fig 1). In addition, none of the As-

resistant isolates which were screened positive for As(III)-oxidizing activity grew in

media designed for chemolithotrophic metabolism using As(III) as the sole electron

donor, and none of the As-resistant bacterial isolates were able to reduce As(V) to As(III)

in the growth medium.

3.6. As(III)-oxidase assay

The specific As(III)-oxidase activity in the cellular lysates of the As(III)-oxidizing

bacterial isolates varied from 2.6 to 10.8 nM min-1 mg-1 of protein (Fig. 2). Pseudomonas

sp. ASR1 exhibited the highest As(III)-oxidase enzyme activity (10.8 nM min-1 mg-1

protein), followed by Pseudomonas sp. ASR2 (6.8 nM min-1 mg-1 protein), Acinetobacter

sp. ASR7 (6.4 nM min-1 mg-1 protein), Acinetobacter sp. ASR8 (5.2 nM min-1 mg-1

protein), Pseudomonas sp. ASR3 (4.8 nM min-1 mg-1 protein), Klebsiella sp. ASR9 (3.3

nM min-1 mg-1 protein), Comamonas sp. ASR12 (3.0 nM min-1 mg-1 protein), and

Comamonas sp. ASR11 (2.6 nM min-1 mg-1 protein) (Fig. 2). It was noted that isolates

having higher As(III) oxidation capacity also exhibited higher levels of As(III)-oxidase

enzyme activity. As expected, isolates screened negative for As(III)-oxidizing activities

did not exhibit specific As(III)-oxidase enzyme activity.

3.7. Screening of potential plant growth promoting As-resistant bacteria

From the twelve As-resistant isolates, nine isolates were assayed for one or more

characteristics considered to be important for PGP activity (Table 3). From the twelve As-

resistant bacterial isolates, Pseudomonas sp. ASR1, Bacillus sp. ASR5 and Enterobacter

sp. ASR10 were able to solubilize phosphate, produce siderophores and IAA-like

molecules and to grow on ACC as the sole nitrogen source (Table 3). Isolates

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Pseudomonas sp. ASR2, Pseudomonas sp. ASR3, Geobacillus sp. ARS4, Paenibacillus

sp. ASR6, Enterobacter sp. ASR10, Comamonas sp. ASR11 and Comamonas sp. ASR12

possessed one or more than one PGP trait (Table 3). However isolates Acinetobacter sp.

ASR7, Acinetobacter sp. ASR8 and Klebsiella sp. ASR9 did not possess any of the PGP

characteristics studied. Notably, the maximum phosphate-solubilization and production of

siderophore, IAA-like molecules and ACC diaminase were observed in Pseudomonas sp.

ASR1 compared to the other As-resistant potential PGPB (Table 3).

4. Discussion

4.1. As-resistant bacteria found in less As-enriched agricultural soils

Even though the total As content of agricultural soils from the study sites was

around the background level (5-10 mg kg-1 is considered the background level for As [4,

8, 29]), the As concentration in groundwater from the same study sites was found to be

higher than the WHO’s permissible limit of 10 μg L-1, and in some of the study sites,

(e.g., Yenshuei and Budai) it was found to exceed 800 μg L-1 [2]. Irrigation of cropland

with As-enriched groundwater from the study sites has been shown to substantially

enhance As content in the plants in such locations [2]. Recent research also suggests that

several crops and vegetables can accumulate As in substantial quantities where

groundwater with elevated As concentrations is used for irrigation purposes [30, 31].

Although the As content of the soils from which the As-resistant bacteria were

isolated is very close to the background level of As in soil, these isolates showed very

high resistance to both As(III) and As(V) (MIC ranging from 10 to 30 mM and 150 to

320 mM for As(III) and As(V), respectively), indicating that the level of As resistance to

bacteria is independent of environmental As content [17]. Resistance to As(III) and As(V)

concentrations higher than 30 mM and 100 mM, respectively, is considered to be very

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high [6]. In most cases, As-resistant microorganisms have been obtained from As-

contaminated environments such as As-contaminated soil and sediment [4, 18], gold

mines [32], geothermal areas [33], aquatic areas and groundwater [34]. However, there

have also been reports of highly As-resistant microorganisms in As-free environments

[17, 35]. These suggest a wide distribution of As-resistant bacteria in the environment.

Jackson and Dugas [36] suggested that the genetic and cellular mechanisms of As

resistance evolved millions of years ago when higher levels of As were present in the

natural environment, and thus the traits may be evolutionarily widespread. Our results

support the idea that As resistance might not be confined to bacteria inhabiting As-

contaminated environments and also suggest that even less As-contaminated or As-free

environments might also harbor As-resistant bacteria.

4.2. Phylogenetic relatedness of As-resistant isolates

The isolates Pseudomonas, Acinetobacter, Enterobacter, Geobacillus, Bacillus

and Comamonas are broadly represented among the As-resistant bacterial strains from

As-contaminated environments [33, 35, 37]. The other adjoining genera of the

phylogenetic tree of 16S rRNA gene sequences of As-resistant bacterial isolates (SI-Fig.

1) have been reported to have As-resistant and/or plant growth promoting capabilities.

For instance, Pseudomonas sp. (GQ497248), Acinetobacter sp. (GQ497238 and

GQ497249), Klebsiella sp. (GQ497245), Enterobacter sp. (GQ497247) and Comamonas

sp. (GQ497244) were isolated from As-enriched tannery wastes and agricultural soils in

Thailand [35]. The As-resistant bacteria Geobacillus sp. (HM776457) was reported in the

geothermal soils of Tuscany, Italy [33]. The As-oxidizing bacteria Acinetobacter sp.

(FJ607348) and Paenibacillus sp. (FR874235) were isolated from As-contaminated

abandoned mine areas and adjacent coastal sediments in Korea and acid mine drainage

located in Carnoules, Gard (France), respectively [37, 38]. Comamonas sp. (JF345176), a

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novel As-resistant bacteria exhibiting siderophores production, was isolated from the

rhizosphere of As-hyperaccumulator Pteris vittata [39]. In addition, Pseudomonas sp.

(JX514407), Acinetobacter sp. (JX514424) and Paenibacillus sp. (JN899577) were

reported as potential PGPB [40, 41]. In our study, Pseudomonas sp. ASR1 was the most

As-resistant bacterial strain isolated from the comparatively high As-contaminated

agricultural soil of Beimen CN9 (Table 1). The bacterium Pseudomonas has been broadly

represented among the As-resistant strains isolated from As-contaminated and/or As-free

environments because of their ubiquitous nature and remarkable adaptability to diverse

environments [18].

4.3. As(III)-oxidation by the isolates: a detoxification mechanism as opposed to energy

generation

The oxidation of As(III) leads to the formation of the less bioavailable and less

toxic As(V). Thus, bacteria capable of As(III) oxidation can contribute to natural

remediation processes, as observed in different As-contaminated environments [42, 43].

In our study, we found that the As-resistant isolates Pseudomonas, Acinetobacter,

Klebsiella and Comamonas genera were capable of oxidizing As(III) during their

logarithmic growth phase. The As(III) oxidation capability of these isolates were further

proved by the exhibition of specific As(III)-oxidase enzyme activities in these isolates.

Bacteria oxidize As both for detoxification and energy generation purposes [42].

Interestingly, in our study, none of the As(III)-oxidizing isolates grew in media designed

for chemolithotrophic metabolism using As(III) as the sole electron donor. This suggests

that the mechanism of As(III) oxidation by these isolates is better explained as a

detoxification mechanism rather than as an energy generation mechanism. Macur et al.

[15] reported that As-contamination in oxic environments may not select for

microorganisms capable of utilizing As in energy generation but rather may instead favor

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bacteria capable of detoxification via As(III) oxidation. It is noteworthy that the

metal/metalloid oxidation capability of metal/metalloid-resistant bacteria can potentially

be exploited for phytoextraction [10]. For instance, Fe/S oxidizing bacteria have been

reported to enhance metal bioavailability in soils through acidification reaction [44].

4.4. Implication of As-resistant bacteria possessing potential PGP traits for

bioremediation

Concerning metal resistant PGPB, it has been reported that bacteria belonging to

diverse families possessing PGP traits enhance metal translocation and uptake efficiency,

the promotion of growth in trial plants and increases in the heavy metal tolerance of

plants in the presence of metals/metalloids at a high level (SI-Table 1). Reports on As-

resistant bacteria possessing PGP traits and their influence on bioremediation are,

however, far and few between (SI-Table 1). For instance, Srivastava et al. [6] reported

that addition of As-resistant PGPB Staphylococus arlettae to Brassica juncea (L.) grown

in As-spiked (15 mg kg-1) soil increased biomass as well as the protein, chlorophyll and

carotenoid content of trial plants and accumulated As maximally in the plant roots. In

contrast, Wang et al. [5] reported that inoculation of Agrobacterium radiobacter, an As-

resistant PGPB, to Populus deltoids grown in As-spiked (300 mg kg-1) soil could remove

54% of the As in the soil resulting in significant increases in As concentrations in roots,

stems and leaves by 229, 113 and 291%, respectively. However, such high As

concentrations inhibited the growth of the trial plants under consideration. de-Bashan et

al. [45, 46] reported that PGPB isolates Azospirillum brasilense strain Sp6 and Cd and

Bacillus pumilus strains ES4 and RHIZO1 enhanced the growth and development of

Atriplex lentiformis growing in As-contaminated tailings. In addition, As-resistant

bacteria belonging to Alphaproteobacteria, Betaproteobacteria, and

Gammaproteobacteria have been reported to exhibit potential PGP traits [39, 47]. In this

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study, we revealed that the As-resistant isolates Pseudomonas sp. ASR1, Bacillus sp.

ASR5 and Enterobacter sp. ASR10 were able to solubilize phosphate, produce

siderophores, IAA-like molecules and ACC deaminase. Hence, these strains could have a

great potential for application in bioremediation of As. Moreover, the ability of the

isolated bacterial genera Pseudomonas and Comamonas to oxidize As(III) and to exhibit

potential PGP traits could lower As(III) toxicity and support plant growth in As-

contaminated soil [47]. In this study, Pseudomonas sp. ASR1, which was comparatively

more resistant to As, possessed greater As(III)-oxidizing activity and also exhibited

higher phosphate-solubilization, as well as higher production of siderophores, IAA-like

molecules and ACC deaminase as compared to the other isolates, could be the better

choice for potential application in As bioremediation processes.

In aerobic soils, As(V) is present as HAsO42- and H2AsO4

- , and is often bound to

Fe and/or Al minerals, making it insoluble. However bacterial solubilization of Fe

releases As from these minerals, making it available to plants and soil microorganisms

[39]. Thus As-resistant microorganisms might have a selective advantage with regard to

survival under As-stressed conditions. In addition to siderophore production, phosphate-

solubilization by As-resistant bacteria has also been reported to play an important role in

plant growth and survival of bacteria under As-stressed conditions [6]. The growth of As-

sensitive plants in As-contaminated soil could be adversely affected because As(V) in

soils reduces the amount of phosphorous in plants [48]. This deficiency can be

compensated for by the phosphate-solubilization ability of As-resistant strains, reducing

the pH of the inoculated soil [6, 48]. Recent studies by Bashan et al. [49, 50] urged that a

combination of two or three metal phosphate compounds (according to the end use of the

bacteria) rather than TCP is appropriate as a universal selection factor for the isolation of

a true phosphate-solubilizing bacteria (PSB) because the TCP yields thousands of PSB

per study, but when tested in field conditions, only a few have proven to be successful.

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Further study using two or three metal phosphate compounds instead of TCP for

screening potential As-resistant PSB and its implementation in As and nutrient stress

conditions will provide a clearer picture. The ability of As-resistant bacteria to produce

IAA has been reported to induce higher shoot length and higher numbers of leaves and

total chlorophyll content in inoculated plants [6]. Plant growth-promoting bacteria

synthesize IAA utilizing tryptophan excreted by roots in the rhizosphere. The synthesized

IAA is then secreted and transported into the plant cells, (i) participates in plant cell

growth and (ii) promotes ACC synthase activity to increase the ethylene titer [9]. The

bacteria possessing ACC deaminase metabolize the ethylene precursor, ACC, and lower

the stress ethylene production in plants, thus facilitating the formation of longer roots in

plants growing in heavy metals/metalloids-contaminated soil [47]. Recent studies have

revealed that plants inoculated with bacteria containing ACC deaminase are better able to

thrive in As-spiked soils [6].

5. Conclusions

The isolated bacterial strains Pseudomonas, Geobacillus, Bacillus, Paenibacillus,

Enterobacter and Comamonas genera are of particular interest because they offer great

potential in regard to novel crop production strategies due to their resistance to As and the

presence of several potential PGP traits. Geobacillus sp. has never been reported as metal

resistant potential PGPB. The isolate Pseudomonas sp. ASR1, which is comparatively

more resistant to As, possesses greater As(III)-oxidizing activity and exhibits higher

phosphate-solubilization, production of siderophore, IAA-like molecules and ACC

deaminase as compared to other As-resistant isolates. It could therefore be a better choice

for potential application in As remediation as well as for sustainable agronomic

production programs in As-contaminated soils.

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Acknowledgements

This work was supported by the National Science Council of Taiwan (NSC 100–2116–

M–006-009).

References

[1] F.-J. Zhao, S.P. McGrath, A.A. Meharg, Arsenic as a food chain contaminant:

mechanisms of plant uptake and metabolism and mitigation strategies, Annu.

Rev. Plant Biol. 61 (2010) 535–559.

[2] S. Kar, S. Das, J.-S. Jean, S. Chakraborty, C.-C. Liu, Arsenic in the water-soil-plant

system and the potential health risks in the coastal part of Chianan Plain,

Southwestern Taiwan, J. Asian Earth Sci. 77 (2013) 295-302.

[3] W.-P. Tseng, Effects and dose-response relationships of skin cancer and blackfoot

disease with arsenic, Environ. Health Perspect. 19 (1997) 109-119.

[4] S. Das, J.-S. Jean, S. Kar, C.-C. Liu, Changes in bacterial community structure and

abundance in agricultural soils under varying levels of arsenic contamination,

Geomicrob. J. 30(2013) 635-644.

[5] Q. Wang, D. Xiong, P. Zhao, X. Yu, B. Tu, G. Wang, Effect of applying an arsenic-

resistant and plant growth-promoting rhizobacterium to enhance soil arsenic

phytoremediation by Populus deltoides LH05-1, J. Appl. Microb. 111(2011) 1065-

1074.

[6] S. Srivastava, P.C. Verma, V. Chaudhry, N. Singh, P.C. Abhilash, K.V. Kumar, N.

Sharma, N. Singh, Influence of inoculation of arsenic-resistant Staphylococcus

arlettae on growth and arsenic uptake in Brassica juncea (L.) Czern. Var. R-46, J.

Hazard. Mater. (2012), http://dx.doi.org/10.1016/j.jhazmat.2012.08.019.

Page 22 of 33

Accep

ted

Man

uscr

ipt

  22

[7] K. Li, W. Ramakrishna, Effect of multiple metal resistant bacteria from

contaminated lake sediments on metal accumulation and plant growth, J. Hazard.

Mater. 189 (2011) 531-539.

[8] A.L. Wevar Oller, M.A. Talano, E. Agostini, Screening of plant growth-promoting

traits in arsenic-resistant bacteria isolated from the rhizosphere of soybean plants

from Argentinean agricultural soil, Plant Soil 369 (2013) 93-102.

[9] Y. Ma, M.N.V. Prasad, M. Rajkumar, H. Freitas, Plant growth promoting

rhizobacteria and endophytes accelerate phytoremediation of metalliferous soils,

Biotech. Adv. 29 (2011) 248-258.

[10] M. Rajkumar, S. Sandhya, M.N.V. Prasad, H. Freitas, Perspectives of plant-

associated microbes in heavy metal phytoremediation, Biotech. Adv. 30 (2012)

1562-1574.

[11] L.E. de-Bashan, J.-P. Hernandez, Y. Bashan, The potential contribution of plant

growth-promoting bacteria to reduce environmental degradation - A comprehensive

evaluation, Appl. Soil Ecol. 61 (2012) 171-189.

[12] W.W. Wenzel, Rhizosphere processes and management in plant-assisted

bioremediation (phytoremediation) of soils, Plant Soil 321 (2009) 385-408.

[13] B.R. Glick, Using soil bacteria to facilitate phytoremediation, Biotech. Adv. 28

(2010) 367-374.

[14] A. Sessitsch, M. Kuffner, P. Kidd, J. Vangronsveld, W.W. Wenzel, K. Fallmann, M.

Puschenreiter, The role of plant-associated bacteria in the mobilization and

phytoextraction of trace elements in contaminated soils, Soil Biol. Biochem. 60

(2013) 182-194.

[15] R.E. Macur, C.R. Jackson, L.M. Botero, T.R. Mcdermott, W.P. Inskeep, Bacterial

populations associated with the oxidation and reduction of arsenic in an unsaturated

soil, Environ. Sci. Tech. 38 (2004) 104-111.

Page 23 of 33

Accep

ted

Man

uscr

ipt

  23

[16] D.K. Newman, T.J. Beveridge, F.M.M. Morel, Precipitation of arsenic trisulfide by

Desulfotomaculum auripigmentum, Appl. Environ. Microbiol. 63(1997) 2022-2028.

[17] M.M. Bahar, M. Megharaj, R. Naidu, Kinetics of arsenite oxidation by Variovorax

sp. MM-1 isolated from a soil and identification of arsenite oxidase gene, J. Hazard.

Mater. 262 (2013) 997-1003.

[18] M. Pepi, M. Volterrani, M. Renzi, M. Marvasi, S. Gasperini, E. Franchi, S.E.

Focardi, Arsenic- resistant bacteria isolated from contaminated sediment of the

Orbetello Lagoon, Italy, and their characterization, Appl. Microbiol. 103(2007)

2299-2308.

[19] D.A. Lipson, S.K. Schmidt, Seasonal changes in an Alpine soil bacterial community

in the Colorado Rocky Mountains, Appl. Environ. Microbiol. 70 (2004) 2867-2879.

[20] M. Kimura, A simple method for estimating evolutionary rates of base substitutions

through comparative studies of nucleotide sequences, J. Mol. Evol. 16 (1980) 111-

120.

[21] D.S. Diliana, L. Didier, L. Florence, A.E. Daniel, I.G. Veneta, L. Marie-Claire,

Microplate screening assay for the detection of arsenite-oxidizing and arsenate-

reducing bacteria, FEMS Microb. Lett. 237 (2004) 249-253.

[22] M.C. Lett, K. Paknikar, D. Lihvremont, A simple and rapid method for arsenite and

arsenate speciation, in: V. Ciminelli, O. Jr. Jr. Garcia (Eds), Biohydrometallurgy-

fundamentals, technology and sustainable development, Part B, 2001, Elsevier,

Amsterdam, pp. 541-546.

[23] G.L. Anderson, J. Williams, R. Hille, The purification and characterization of As(III)

oxidase from Alcaligenes faecalis, a molybdenum containing hydroxylase, J. Biol.

Chem. 267 (1992) 23674-23682.

[24] W.V.B. Sundara-Rao, M.K. Sinha, Phosphate dissolving microorganisms in the soil

and rhizosphere, Indian J. Agric. Sci. 33 (1963) 272-278.

Page 24 of 33

Accep

ted

Man

uscr

ipt

  24

[25] S. Zaidi, S. Usmani, B.R. Singh, J. Musarrat, Significance of Bacillus subtilis strain

SJ-101 as a bioinoculant for concurrent plant growth promotion and nickel

accumulation in Brassica juncea, Chemosphere 64 (2006) 991-997.

[26] B. Schwyn, J.B. Neilands, Universal chemical assay for detection and determination

of siderophores, Anal. Biochem. 160 (1987) 47-56.

[27] J.M. Bric, R.M. Bostock, S.E. Silversone, Rapid in situ assay for indole acetic acid

production by bacteria immobilization on a nitrocellulose membrane, Appl. Environ.

Microbiol. 57(1991) 535-538.

[28] D.M. Penrose, B.R. Glick, Methods for isolating and characterizing ACC

deaminase-containing plant growth-promoting rhizobacteria. Physiol. Plant 118

(2003) 10-15.

[29] A. Heikens, Arsenic contamination of irrigation water, soil and crops in Bangladesh:

risk implications for sustainable agriculture and food safety in Asia. FAO

Publication, 2006, ISBN, 974-9746-88-2.

[30] M.A. Rahman, H. Hasegawa, M.M. Rahman, M.A. Rahman, M.A.M. Miah,

Accumulation of arsenic in tissues of rice plant (Oryza sativa L.) and its distribution

in fractions of rice grain, Chemosphere 69 (2006) 942-948.

[31] D. Chatterjee, D. Halder, S. Majumder, A. Biswas, B. Nath, P. Bhattacharya, S.

Bhowmick, A. Mukherjee-Goswami, D. Saha, R. Hazra, P.B. Maity, D. Chatterjee,

A. Mukherjee, J. Bundschuh, Assessment of arsenic exposure from groundwater and

rice in Bengal Delta Region, West Bengal. India. Water Res. 44 (2010) 5803-5812.

[32] L. Drewniak, A. Styczek, M. Majder-Lopatka, A. Sklodowska, Bacteria

hypertolerant to arsenic in the rocks of an ancient gold mine, and their potential role

in dissemination of arsenic pollution, Environ Pollut 156 (2008) 1069-1074.

Page 25 of 33

Accep

ted

Man

uscr

ipt

  25

[33] M. Cuebas, A. Villafane, M. McBride, N. Yee, E. Bini, As(V) reduction and

expression of multiple chromosomal ars operons in Geobacillus kaustophilus A1,

Microbiology 157 (2011) 2004-2011.

[34] D. Davolos, B. Pietrangeli, A molecular study on bacterial resistance to arsenic-

toxicity in surface and underground waters of Latium (Italy), Ecoxicol. Environ.

Safety 96 (2013) 1-9.

[35] K. Chitpirom, A. Akaracharanya, S. Tanasupawat, N. Leepipatpiboon, K.W. Kim,

Isolation and characterization of arsenic resistant bacteria from tannery wastes and

agricultural soils in Thailand, Ann. Microbial. 59 (2009) 649-656.

[36] C.R. Jackson, S.L. Dugas, Phylogenetic analysis of bacterial and archaeal arsC gene

sequences suggests an ancient, common origin for arsenate reductase, BMC Evol.

Biol. 3(2003) 18

[37] J.-S. Chang, J.-H. Lee, I.-S. Kim, Bacterial aox genotype from arsenic contaminated

mine to adjacent coastal sediment: Evidences for potential biogeochemical arsenic

oxidation, J. Hazard. Mater. 193 (2011) 233-242.

[38] F. Delavat, M.C. Lett, D. Lievremont, Novel and unexpected bacterial diversity in an

arsenic-rich ecosystem revealed by culture-dependent approaches, Biol. Direct 7:28

(2012) 1-14.

[39] P. Ghosh, B. Rathinasabapathi, L.Q. Ma, Arsenic-resistant bacteria solubilized

arsenic in the growth media and increased growth of arsenic hyperaccumulator

Pteris vittata L, Biores. Technol. 102 (2011) 8756-8761.

[40] R. Dey, K.K. Pal, D.M. Bhatt, S.M. Chauhan, Growth promotion and yield

enhancement of peanut (Arachis hypogaea L.) by application of plant growth-

promoting rhizobacteria, Microbiol. Res. 159 (2004) 371-394.

[41] J. Zhang, J. Liu, L. Meng, Z. Ma, X. Tang, Y. Cao, L. Sun, Isolation and

characterization of plant growth-promoting rhizobacteria from wheat roots by wheat

Page 26 of 33

Accep

ted

Man

uscr

ipt

  26

germ agglutinin labeled with fluorescein isothiocyanate, J. Microbiol. 50 (2012)

191-198.

[42] R.S. Oremland, J.F. Stolz, Arsenic, microbes and contaminated aquifers, Trends

Microbiol. 13(2005) 45-49.

[43] S. Das, S. Kar, J.-S. Jean, J. Rathod, S. Chakraborty, H.S. Liu, J. Bundschuh, Depth-

resolved abundance and diversity of arsenite-oxidizing bacteria in the groundwater

of Beimen, a blackfoot disease endemic area of southwestern Taiwan, Water Res. 47

(2013) 6983-6991.

[44] S.Y. Chen, J.G. Lin, Effect of substrate concentration on bioleaching of metal-

contaminated sediment, J. Hazard. Mater. 82 (2001) 77-89.

[45] L.E. de-Bashan, J.P. Hernandez, K.N. Nelson, Y. Bashan, R.M. Maier, Growth of

quailbush in acidic, metalliferous desert mine tailings: effect of Azospirillum

brasilense Sp6 on Biomass production and rhizosphere community structure, Microb

Ecol 60 (2010) 915-927.

[46] L.E. de-Bashan, J.P. Hernandez, Y. Bashan, R.M. Maier, Bacillus pumilus ES4:

Candidate plant growth-promoting bacterium to enhance establishment of plants in

mine tailings, Environ. Exp. Bot. 69 (2010) 343-352.

[47] L. Cavalca, R. Zanchi, A. Corsini, M. Colombo, C. Romagnoli, E. Canzi, V.

Andreoni, Arsenic-resistant bacteria associated with roots of the wild Cirsium

arvense (L.) plant from an arsenic polluted soil, and screening of potential plant

growth-promoting characteristics, Syst. Appl. Microbiol. 33 (2010) 154-164.

[48] N. Singh, L.Q. Ma, Arsenic speciation and arsenic and phosphate distribution

in arsenic hyperaccumulator Pteris vittata L. and non hyperaccumulator Pteris

ensiformis L, Environ. Pollut. 141 (2006) 238-246.

Page 27 of 33

Accep

ted

Man

uscr

ipt

  27

[49] Y. Bashan, A.A. Kamnev, L.E. de-Bashan, A proposal for isolating and testing

phosphate-solubilizing bacteria that enhance plant growth, Biol. Fertil. Soils 49

(2013) 1-2.

[50] Y. Bashan, A.A. Kamnev, L.E. de-Bashan, Tricalcium phosphate is inappropriate as

a universal selection factor for isolating and testing phosphate-solubilizing bacteria

that enhance plant growth: a proposal for an alternative procedure, Biol. Fertil. Soils

49 (2013) 465-479.

Figure captions:

Fig. 1. Arsenic oxidation and cell density (optical density measurement at 600 nm) as a

function of time during aerobic culturing of As-resistant bacteria. The total concentration

of As in solution ([Total As] = [As(III) + As(V)]) remained nearly constant during the

experiment. Isolates are named after their nearest match found in the GenBank database.

Vertical bars represent standard deviation.

Fig. 2. Specific As(III)-oxidase enzyme activity of isolates. Mean of three replicated

observations. Vertical bars represent standard deviation. Columns denoted by a different

letter in each subfigure differ significantly by one-way ANOVA at p < 0.05.

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Table 1. Closest GenBank neighbors, sequence similarities, total As content of soil and As(III)-oxidizing activities of As-resistant bacteria

isolated from agricultural soil of Chianan Plain, SW Taiwan

Strains with

accession no.

16S rRNA

fragment

length (bp)

Closest GenBank neighbor

(% similarity)

Sampling sites

(Total As in mg kg-1

)

MIC (mM) As(III) oxidation activities

As(III) As(V) rate

(μM h-1

10-9

cell)

half-life

(h)

ASR1 (AB758593) 1459 Pseudomonas sp. (99) Beimen CN9 (12.7 ± 2.82) 30 320 0.059 11.7

ASR2 (AB758594) 1443 Pseudomonas stutzeri (99) Beimen 2A (10.7 ± 3.14) 20 250 0.043 16.1

ASR3 (AB758595) 1443 Pseudomonas putida (99) Yenshuei (11.9 ± 2.64) 20 280 0.035 19.8

ASR4 (AB758596) 1425 Geobacillus sp. (99) Yichu (7.92 ± 3.38) 10 300 ND ND

ASR5 (AB758597) 1409 Bacillus cereus (99) Budai (11.5 ± 4.92) 10 300 ND ND

ASR6 (AB758598) 1428 Paenibacillus pabuli (99) Beimen CN9 (12.7 ± 2.82) 10 250 ND ND

ASR7 (AB758599) 1491 Acinetobacter sp. (99) Yichu (7.92 ± 3.38) 15 250 0.042 16.5

ASR8 (AB758600) 1499 Acinetobacter sp. (99) Liujiao (8.12 ± 2.74) 15 180 0.038 18.2

ASR9 (AB758601) 1526 Klebsiella sp. (99) Budai (11.5 ± 4.92) 15 180 0.033 21.2

ASR10 (AB758602) 1492 Enterobacter sp. (99) Lucao (10.2 ± 4.29) 15 300 ND ND

ASR11 (AB758603) 1481 Comamonas sp. (99) Jianjiun (9.42 ± 3.05) 10 150 0.029 23.9

ASR12 (AB758604) 1487 Comamonas sp. (99) Liujiao (8.12 ± 2.74) 10 150 0.032 21.7

ND: not detected; MIC: Minimum inhibitory concentration

Total As contents are mean of three replicates ± standard deviation

Table

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Table 2. Morphological and biochemical characterization of isolated As-resistant bacterial strains

Characters Isolates

ASR1 ASR2 ASR3 ASR4 ASR5 ASR6 ASR7 ASR8 ASR9 ASR10 ASR11 ASR12

Morphological

Gram stain - - - + + - - - - - - -

Cell shape Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod

Cell length (μ) 2.5±0.1 2.0±0.1 2.0±0.2 2.2±0.1 3.4±0.1 2.8±0.1 2.0±0.1 2.0±0.1 2.5±0.2 2.0±0.1 2.2±0.1 2.0±0.1

Colony color White White White White White Yellow White White White White White White

Motility + + + + + + - - - + + +

Biochemical

MR - - - + - + - - - + - -

MRVP - + - - + - - - + + - -

Oxidase + + + + + + - - - - + +

Catalase + + + + + + + - + - - -

Urease - - + - - + - + + - - -

Citrate utilization + + + - + + + - + + - -

Nitrate reduction - + - - - + - - + + + +

Starch hydrolysis - + - - - + - - - - - -

Tween 80 hydrolysis - - - - - - + - - - - -

Aesculin hydolysis - - - - + + - - + + - -

Utilization of

Manitol + - + + + + - - + + - -

D-Xylose - - - - + + - + + + - -

L-Arabinose - - - + + + - + + - - -

D-malonate + + + + + + + - + + - -

L-Citrulline - - - - + + + - + - - -

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Table 3. Quantitative assessment of plant growth promoting traits of As-resistant bacterial isolates

As-resistant bacterial isolates Phosphate-solubilization

(μg mL-1

)

Siderophore production

(μM DFM mL-1

)

IAA production

(μM IAA mL-1

)

ACC deaminase production

(μM α-KB mg−1

protein h−1

)

Pseudomonas sp. ASR1 222.5 ± 3.2 32.6 ± 1.8 58.4 ± 4.2 6.2 ± 0.5

Pseudomonas sp. ASR2 126.2 ± 2.4 24.4 ± 1.2 - -

Pseudomonas sp. ASR3 187.6 ± 2.7 26.5 ± 1.3 1.4 ± 0.2 -

Geobacillus sp. ARS4 - 11.6 ± 0.6 18.7 ± 1.2 1.6 ± 0.1

Bacillus sp. ASR5 98.4 ± 1.8 22.8 ± 1.5 34.6 ± 1.4 3.5 ± 0.4

Paenibacillus sp. ASR6 - 8.6 ± 0.8 28.7 ± 1.1 2.4 ± 0.2

Acinetobacter sp. ASR7 - - - -

Acinetobacter sp. ASR8 - - - -

Klebsiella sp. ASR9 - - - -

Enterobacter sp. ASR10 202.8 ± 3.6 13.1 ± 0.6 42.8 ± 1.6 4.8 ± 0.3

Comamonas sp. ASR11 - 28.3 ± 1.2 - -

Comamonas sp. ASR12 - 30.0 ± 1.6 - -

All values are mean of three replicates ± standard deviation (SD); IAA: Indole-3-acetic acid; ACC: 1-aminocyclopropane-1-carboxylate; α-KB: α-

Ketobutyrate

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Accep

ted

Man

uscr

ipt

Figure 1.

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Accep

ted

Man

uscr

ipt

Figure 2.

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Accep

ted

Man

uscr

ipt

Highlights

As-resistant bacteria possessing plant growth promoting (PGP) traits isolated.

Pseudomonas and Comamonas genera oxidized As(III) and possessed PGP traits.

As-free environments might also harbor As-resistant bacteria.

*Highlights (for review)