Electronic Supplementary Material s26

Electronic Supplementary Material

Characterization of the arsenite oxidizer Aliihoeflea sp. strain 2WW and its potential application in the removal of arsenic from groundwater in combination with Pf-ferritin

Anna Corsini1, Milena Colombo1, Gerard Muyzer1,2, Lucia Cavalca1*

1DeFENS - Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente, Università degli Studi di Milano, via

Celoria 2, Milano, Italy, I-20133

2Microbial Systems Ecology, Department of Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics,

University of Amsterdam, 1090 GE Amsterdam, The Netherlands

*Corresponding author. Tel.: +390250319116; fax: +390250319238.

E-mail address: [email protected] (L. Cavalca) Material and methods

Chemicals

All chemicals used in this study exceeded 98% purity. All buffers and solutions were prepared with Milli-Q water, and sterilized by membrane filtration (0.22 µm pore size, Millipore, MA, USA). As(III) and As(V) solutions were prepared from stock solutions of As(III) (NaAsO2) and As(V) (Na2HAsO4 7H2O) (Sigma) respectively. Stock solutions were stored at 4°C.

Growth medium preparation

-1 The growth medium used, hereafter referred as BBWM, consisted as follows: solution A (g L ): KH2PO4 0.04; K2HPO4

-1 0.04; NaCl 1.0; (NH4)2SO4 0.4; trace element solution 2 mL. The pH of solution A was adjusted to 6.5. Solution B (g L ):

CaCl2 0.2; MgSO4 0.2. Solutions A and B were sterilized separately by autoclaving. Equal volumes of solutions A and B were mixed after cooling and then supplemented with 10 ml of solution C 0.95 M, and with 10 ml of a vitamin solution 1%

(v/v) vitamin solution. Vitamin solution was filter sterilized and contained (mg L-1): p-aminobenzoic acid 5.0; biotin 5.0; folic acid 2.0; pyridoxine-HCl 1.0; riboflavin 5.0; thiamine 5.0; nicotinic acid 5.0; panthotenic acid 5.0; vitamin B12 0.1.

The final pH was adjusted to 8.0. Appropriate carbon sources, previously sterilized, were added to BBWM.

Resting cells preparation

The strain 2WW was grown for 48 h in BBWM supplemented with yeast extract (0.2% w/v) at 30 °C under shaking conditions at 150 rpm. After growth, cells were centrifuged at 10,000 rpm at 10 °C for 30 min. The cell pellet was washed three times with TRIS-HCl (5 mM, pH 7.2) and resuspended in the same medium. This cell suspension served as an inoculum in order to obtain a final cell density of 107 cell mL-1.

PCR conditions

PCR reactions of 16S rRNA and of arsenic genes were performed in a final volume of 25 L containing: 10 ng of

genomic DNA, 1.5 U of Taq polymerase, 0.2 M of each primer, 0.2 mM of dNTPs, 1.75 mM MgCl2, and 1x PCR buffer. DNA amplification conditions for 16S rRNA were: initial denaturation at 95°C for 5 min, 35 cycles of 95°C for 1 min,

55°C for 40 sec, 72°C for 1 min 40 sec, and then a final extension step at 72°C for 10 min. DNA amplification conditions for aioA, arsC, arsB, ACR3(1) and ACR3(2) were the same as reported by Quéméneur et al (2008), Bachate et al. (2009) and Achour et al. (2007).

All the reagents were from Invitrogen. PCR reactions were carried out using the T-Gradient apparatus (Biometra,

Germany). PCR products were checked on a 2% (w/v) agarose gel containing GelRed™ stain (Biotium) at 0.01% (v/v) and visualized using the Gel Doc image analyzer system (Bio-Rad). Fig. S1 Effect of temperature on growth and As(III) oxidation rate of growing cells of Aliihoeflea sp. 2WW strain in

-1 BBWM-Y (YE 0.2%) and 75 mg L As(III); ■, OD600nm in presence of As(III); □, OD600nm in absence of As(III); ♦,

As(III);▲, As(V). (Mean ± SD; n = 3. SD bars were smaller than symbols)

30 °C a 1.2 75 1.0 60 0.8 45 0.6 30 0.4 0.2 15 0.0 0 0 8 16 24 32 40

15 °C b 1.2 75

m 1.0 n 60 0

O 30 0.4 0.2 15 0.0 0 0 16 32 48 64 80 96 112 128 144

5 °C c 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 50 100 150 200 250 300 350 400 450 Time (h) Fig. S2 Effect of pH on growth and As(III) oxidation rate of growing cells of Aliihoeflea sp. 2WW strain in BBWM-Y (YE

-1 0.2%) and 75 mg L As(III); ■, OD600nm in presence of As(III); □, OD600nm in absence of As(III); ♦, As(III);▲, As(V). (Mean

± SD; n = 3)

pH 5.0 pH 6.0 1.2 90 1.2 90

1 75 1 75

0.8 60 0.8 60

0.6 45 0.6 45

0.4 30 0.4 30

0.2 15 0.2 15

0 0 0 0

m 0 24 48 72 m 0 24 48 72 n n 0 0

0 Time (h) 0 Time (h) 6 6 D D O O pH 7.0 pH 8.0 1.2 90 1.2 90

1 75 1 75

0.8 60 0.8 60

0.6 45 0.6 45

0.4 30 0.4 30

0.2 15 0.2 15

0 0 0 0 0 24 48 72 0 24 48 72 Time (h) Time (h)