Supplementary Information

As(V) resistance and reduction by bacteria and their performances in As removal from As-contaminated soils

Fig S1. The locations of 8 soil samples in the realgar mine areas in Shimen County of Huanan

Province of China. The red and blue triangles mean the realgar mine location and the slag heap site, respectively.

Table S1. The selected physicochemical properties of the experimental soils. Items Experimental soils Soil-1 Soil-2 Soil stype Alluvial soils Alluvial soils Total N g kg-1 1.85 1.53 Total P g kg-1 0.82 0.71 Total K g kg-1 12.3 18.1 Organic Matter g kg-1 22.1 19.3 Total As mg·kg-1 38.7 65.9 Available Fe mg·kg-1 21.5 33.2 Available Mn mg·kg-1 16.3 24.8 Available Al g·kg-1 0.96 1.33 pH 7.62 7.55 CEC cmol(+) kg-1 9.7 12.1 Fig S2. The growth situations of 28 bacterial isolates after being inoculated onto the solid LB culture media containing As(V) of 500, 1000, or 2000 mg L-1 for 48h. The labels in red indicated these isolates were selected out according to their growth situations in response to the corresponding As(V) levels.

500 mg L-1

3-2 8-3 8-4 1-2 1-3 3-1 5-3 6-1

2-1 3-3 6-2 8-2 8-5 1-1 2-6 5-2

2-2 5-1 4-1 6-3 2-5 8-1

2-4 2-3 4-3 4-2 7-2 7-1

1000 mg L-1

8-3 8-4 1-2 1-3 3-1 3-2 5-3 6-1

3-3 5-2 6-2 8-2 8-5 1-1 2-1 2-6

2-2 5-1 4-1 6-3 2-5 8-1

2-4 2-3 4-3 4-2 7-2 7-1

2000 mg L-1 6-1 1-2 1-3 3-1 3-2 5-3 8-3 8-4

2-1 8-5 1-1 2-6 3-3 5-2 6-2 8-2

2-2 4-1 6-3 2-5 5-1 8-1

2-4 2-3 4-3 4-2 7-2 7-1 Fig S3. The optical density of 9 bacterial isolates after exposed to different contents of As(V) in LB culture media.

4.0

3.5 2-2 4-3 4-2 4-1 7-2 7-1 8-5 8-4 8-3 3.0 m n 2.5 0 0 6

y t

i 2.0 s n e d

1.5 l a c i t

p 1.0 O

0.5

0.0 0 100 200 300 400 500 600 700 800 As content in culture medium mg L-1 Fig S4. The linear relationship between the decreased As calculated via the change of As fractionations before and after bio-extraction and the decreased As calculated via the change of total

As contents before and after bio- extraction.

12 s t n e t

1 y = 0.8913x + 2.2465 n - g o

c k R² = 0.4592

10 s g A

m l

a n t o o i t t

c

g 8 a n r i t r x a e p - o i m y = 0.5994x + 3.0702 b o

c 6 r

e R² = 0.4559 y t b f

a s

A d

n d a e 4 s e a r e o r f c e e b d

e 2 Soil-1 Soil-2 h T

0 0 5 10 15 The decreased As by comparing As fractionations before and after bio-extraction mg kg-1 Table S2. The morphological and biochemical characteristics of three bacterial isolates Characteristics Isolate 2-2 Isolate 4-3 Isolate 8-5 Cell morphology rod rod rod Gram stain - - - Oxidase + + + Catalase + + + Starch hydrolysis - - - Casein hydrolysis - - - nitrate reduction + + + Temperature (℃) 40 - - + Urease + + - V-P reaction - - + Citrate utilization + + + (Note：- negative；+ Positive) Table S3. Recently (2010-now) reported bacterial isolates capable of As(V) reduction. Bacterial isolates Initial concentration of As(V) Abilities of As(V) reduction Reference Pseudomonas taiwanensis SSMGP 2-2 10 mg L-1 (0.13 mM) After 6 h, all As(V) was completely reduced into As(III). This study Pseudomonas Monteilii SSMGP 4-3 Pseudomonas sp. SSMGP 8-5 10 mg L-1 (0.13 mM) After 24 h, almost 32% of initial As(V) was reduced into As(III). This study Shewanella sp. OM1 187.5 mg L-1 (2.5 mM) In 96 h, all As(V) was anaerobically reduced into As(III). Lukasz et al. 2014 Pseudomonas sp. OM2 187.5 mg L-1 (2.5 mM) In 72 h, all As(V) was anaerobically reduced into As(III). Lukasz et al. 2014 Serratia sp. OM17 Aeromonas sp. OM4 187.5 mg L-1 (2.5 mM) In 120 h, all As(V) was anaerobically reduced into As(III). Lukasz et al. 2014 Pseudomonas sp. PAHAs-1 112.5 mg L-1 (1.5 mM) In 48 h, all As(V) was reduced into As(III). Feng et al. 2014 Anaeromyxobacter sp. PSR-1 375 mg L-1 (5 mM) In 96 h, all As(V) was completely reduced into As(III). Kudo et al. 2013 Geobacter sp. OR-1 450 mg L-1 (6 mM) In 72 h, all As(V) was completely reduced into As(III). Ohtsuka et al. 2013 Citrobacter sp. NC-1 1500 mg L-1 (20 mM) In 24 h, all As(V) was reduced into As(III). Chang et al. 2012 Pantoea agglomerans sp. DSM 3493 75 mg L-1 (1 mM) In 60 h, 97% of initial As(V) was reduced into As(III). Wu et al. 2013 Bacillus thuringiensis sp.IAM 12077 75 mg L-1 (1 mM) In 22 h, 95% of initial As(V) was reduced into As(III). Wu et al. 2013 Bacillus cereus OSBH5 0.75 mg L-1 (0.01 mM) In about 140 h, all As(V) was reduced into As(III). Maity et al. 2011 Pseudomonas stutzeri OSBH2 0.75 mg L-1 (0.01 mM) In 160 h, all As(V) was reduced into As(III). Maity et al. 2011 Pseudomonas sp. AR-3 18.75 mg L-1 (0.25 mM) In 25 h, all As(V) was aerobically reduced into As(III). Liao et al. 2011 Citrobacter sp. AR-7 18.75 mg L-1 (0.25 mM) In 10 h, all As(V) was aerobically reduced into As(III). Liao et al. 2011 Geobacillus kaustophilus A1 1500 mg L-1 (20 mM) In 20 h, about 10% of initial As(V) was reduced into As(III). Cuebas et al. 2011 Pseudomonas sp. MP1400 150 mg L-1 (2.0 mM) In 24 h, all As(V) was completely reduced into As(III). Pepi et al. 2011 Pseudomonas sp. APSLA3 150 mg L-1 (2.0 mM) In about 100 h, all As(V) was completely reduced into As(III). Pepi et al. 2011 Pseudomonas sp. BPSLA3 150 mg L-1 (2.0 mM) In about 70 h, all As(V) was completely reduced into As(III). Pepi et al. 2011 Pseudomonas sp. MP1400d Pseudomonas sp. MP1400a References: Lukasz D, Liwia R, Aleksandra M, Aleksandra S (2014) Dissolution of arsenic minerals mediated by dissimilatory arsenate reducing bacteria: estimation of the physiological potential for arsenic mobilization. BioMed Res Int, 2014(1): 841892. Feng TC, Lin HP, Tang JZ, Feng YY (2014) Characterization of polycyclic aromatic hydrocarbons degradation and arsenate reduction by a versatile Pseudomonas isolate. Int Biodete Biodegr 90: 79-87. Kudo K, Yamaguchi N, Makino T, Ohtsuka T, Kimura K, Dong DT, Amachia S (2013) Release of arsenic from soil by a novel dissimilatory arsenate-reducing bacterium, Anaeromyxobacter sp. strain PSR-1. Appl Environ Microbiol 79: 4635-4642 Ohtsuka T, Yamaguchi N, Makino T, Sakurai K, Kimura K, Kudo K, Homma E, Dong DT, Amachi S (2013) Arsenic dissolution from Japanese paddy soil by a dissimilatory arsenate-reducing bacterium Geobacter sp. OR-1. Environ Sci Technol 47: 6263-6271 Chang YC, Nawata A, Jung K, Kikuchi S (2012) Isolation and characterization of an arsenate-reducing bacterium and its application for arsenic extraction from contaminated soil. J Ind Microbiol Biot 39: 37-44 Wu Q, Du J, Zhuang G, Jing C (2013) Bacillus sp SXB and Pantoea sp IMH, aerobic As(V)-reducing bacteria isolated from arsenic-contaminated soil. J Appl Microbiol 114: 713- 721 Maity JP, Kar S, Liu JH, Jean JS, Chen CY, Bundschuh J, Santra SC, Liu CC (2011) The potential for reductive mobilization of arsenic [As(V) to As(III)] by OSBH2 (Pseudomonas stutzeri) and OSBH5 (Bacillus cereus) in an oil-contaminated site. J Environ Sci Health A Tox/Hazard Subst Environ Eng 46: 1239-1246 Liao VHC, Chu YJ, Su YC, Hsiao SY, Wei CC, Liu CW, Liao CM, Shen WC, Chang FJ (2011) Arsenite-oxidizing and arsenate-reducing bacteria associated with arsenic-rich groundwater in Taiwan. J Contam Hydrol 123: 20-29 Cuebas M, Villafane A, McBride M, Yee N, Bini E (2011) Arsenate reduction and expression of multiple chromosomal ars operons in Geobacillus kaustophilus A1. Microbiology 157: 2004-2011 Pepi M, Protano G, Ruta M, Nicolardi V, Bernardini E, Focardi SE, Gaggi C (2011) Arsenic-resistant Pseudomonas sp. and Bacillus sp. bacterial strains reducing As(V) to As(III), isolated from Alps soils, Italy. Folia Microbiol 56: 29-35