1

2

Supplementary Figure S1 | Phylogenetic relationship of the nearly-complete 16S rRNA

gene sequences of bacteria recovered from the clone libraries of the original soil sample

(SZS’−0) and the enrichment culture from 15 days (SZS’−1-3) of incubation. The

phylogenetic tree was constructed using the neighbor-joining algorithm, with the Jukes-Cantor

parameter correction factor in the ARB program. GenBank accession numbers of the clones and

reference species are shown in brackets. The tree topology was evaluated to be stable by the

maximum-likelihood and maximum-parsimony algorithms. The scale bar represents substitutions

per nucleotide position.

3

Supplementary Figure S2 | A control experiment where no cells were added in the C-

chamber but it contained the modified m9K medium (i.e., replacement of 41.7 g/L

FeSO4·7H2O by 6.1 g/L FeCl3). The P-chamber contained rutile. This experiment was designed

as a control to calculate the photon-biomass conversion efficiency (see Supplementary Methods).

4

Supplementary Table S1 | Diversities of microbial communities from the natural soil

sample and from those incubated for 5, 10, and 15 days under both light and dark

conditions. The diversities were indicated by Shannon index (H), Simpson index (D), Pielou

evenness (J) and the OTU number defined by terminal restriction fragments (T-RFs) of bacterial

16S rRNA gene in the terminal restriction fragment length polymorphism (T-RFLP)

chromatogram.

Samples Shannon index

(H)

Simpson index

(D)

Pielou evenness

(J) OTU number

SZS’-0 3.7 27.4 0.8 68

SZS’-1-1 1.2 2.1 0.5 9

SZS’-1-2 0.8 1.6 0.5 6

SZS’-1-3 1.0 1.9 0.5 7

SZS’-2-1 3.2 14.8 0.8 42

SZS’-2-2 2.6 7.9 0.7 32

SZS’-2-3 2.9 10.0 0.7 37

SZS’-0 denotes the natural sample at Day 0;

SZS’-1-1 denotes the enrichment sample at day 5 with applied voltage;

SZS’-1-2 denotes the enrichment sample at day 10 with applied voltage;

SZS’-1-3 denotes the enrichment sample at day 15 with applied voltage

SZS’-2-1 denotes the enrichment sample at day 5 without applied voltage;

SZS’-2-2 denotes the enrichment sample at day 10 without applied voltage;

SZS’-2-3 denotes the enrichment sample at day 15 without applied voltage;

5

Supplementary Methods

Procedure for isolating A. ferrooxidans. The pH value of the m9K medium was adjusted to 2.2

(±0.02) using 1 M sulfuric acid. Approximately 5 mL of the original water sample was

inoculated into the m9K medium and the mixture was incubated at 30oC with shaking at 150

rpm. After enrichment, isolation was achieved with serial dilutions using the m9K medium. A

total of 9 dilutions (1:40) were made and the dilution from the 9th dilution was used as a stock

culture for this study.

Culturing procedure for A. faecalis. This bacterium was incubated and activated in a nutrient

broth medium containing 0.75 g/L beef extract, 2.5 g/L peptone, and 5 g/L NaCl for 3 days at

30oC on a shaker (150 rpm). The pH value of the medium was adjusted to 7.0 using 0.1 M

hydrochloric acid.

Acquisition of sterile and non-sterile soil extracts. The freshly collected sample was sealed in

a plastic bag and stored at 4oC until analysis in less than a week. Aqueous soil extract (pH 5.7)

was obtained by mixing the fresh soil sample with five volumes of water (w/w) and centrifuged

for 5 min at 2000 rpm to remove insoluble minerals. One half of the solution was filtered through

0.45 µm membrane and then autoclaved (121oC, 20 min) to remove indigenous cells. The other

half remained non-sterile.

Direct counting and colony-form unit (CFU) procedures. Direct counting was performed

using a Helber Bacteria Z30000 cell counter (Thoma, UK) with 0.02 mm depth and 1 mm2 area

dimensions under a microscope (Olympus BX41, Japan). CFU were determined on LB agar

plates after two days of incubation at 30oC.

6

Calculation of the total number of electrons and current density. Current-time (I-t) curves

were obtained by continuously recording the voltage across a 1000 Ω external resistor by a data

logger (ADC-16, Pico Technologies Limited, UK). Current was calculated, integrated over time,

and converted to the number of electrons recovered by using the following conversions: 1C = 1A

× 1s = 6.24 × 1018

electrons and 1 mol = 6.02×1023

electrons. Current density was calculated by

dividing the current by the total area of the graphite electrode in the C-chamber).

Light absorption by minerals. Mineral absorption spectra were scanned using a Lambda 950

UV–vis spectrophotometer with an integrating sphere from 350 to 780 nm. The slit width was

2.00 nm.

DNA extraction and polymerase chain reaction (PCR) amplification. Genomic DNA was

extracted with commercially available kits (MP Biomedicals, USA) from seven samples: the

natural soil sample (SZS’-0) and the enrichment samples at days 5, 10, and 15 under light (SZS’-

1-1, SZS’-1-2, SZS’1-3) and dark conditions (SZS’-2-1, SZS’-2-2, SZS’2-3). The bacterial 16S

rRNA gene fragment was PCR-amplified in a 50 µL reaction volume containing 5 µL of 10×

PCR-buffer with 15 mM MgCl2 (Takara, Dalian, China), 200 µM dNTP (Takara), 10 pmol of

each primer (Applied Biosystems, Shanghai, China) 8F:5’-AGAGTTTGATCCTGGCTCAG-3’;

1492R:5’-GGTTACCTTGTTACGACTT-3’ 49,50

, 1.5 U Taq DNA-polymerase (Takara) and

approximately 10 ng template DNA. PCR was performed in a thermocycler (PTC-200, MJ

Research Inc., Watertown, USA) using the following thermal profile: initial denaturing for 5 min

at 94oC followed by 30 cycles of denaturing (94

oC for 1 min), annealing (51

oC for 45 sec), and

elongation (72oC for 2 min). Both unlabeled and 6-carboxyfluorescein (6-FAM) labeled 8F

primers were used to amplify the DNA fragment for the clone library and terminal restriction

7

fragment length polymorphisms (T-RFLP) analysis, respectively. The DNA amplicons were

purified with the QIA quick PCR Purification Kit (Qiagen China, Shanghai, China).

T-RFLP analysis. Purified DNA amplicons from all four samples were digested in a 20-µl

reaction volume for 6 h at 37°C with 20 U of RsaI (New England Biolabs, Beverly, MA, USA)

according to the manufacturer’s instruction, followed by a desalting step51

. The purified and

digested DNA was mixed with 12 µL Hi-Di formamide and 0.5 µL of DNA fragment length

internal standard (GeneScan Liz-500, Applied Biosystem, IL, USA) followed by a denaturation

step at 95 °C for 5 min and immediately snap-cooling on ice. The “Genescan” analysis was then

conducted in a capillary electrophoresis system (ABI 3130 Genetic Analyzer, Applied

Biosystems) according to the manufacturer’s instructions. Fragment separation data were

collected with ABI 3130 Collection (version 2.7) and GeneMapper Analysis Software (version

3.7). The peaks with a terminal restriction fragment (T-RF) of 50-800 bp in length were treated

as effective peaks and the relative peak area of every single T-RF was calculated by dividing the

individual T-RF peak area by the total area of all the peaks.

Cloning, screening and sequencing. Two bacterial 16S rRNA gene clone libraries were

constructed: one for the natural soil sample (SZS’-0) and the other for the 15-day soil sample

from the C-chamber with a bias voltage (SZS’-1). One hundred eighty (180) and 190 positive

clones were picked from SZS’-0 and SZS’-1 clone libraries, respectively and sequenced. The

clones were screened by both restriction fragment length polymorphism (RFLP) and T-RFLP

analyses with RsaI and HhaI (New England Biolabs, Beverly, MA, USA) restriction enzymes.

The clones were classified into different groups according to the T-RFLP and RFLP patterns.

Representative clones from each T-RF group were randomly chosen and sequenced. After the

Chimera Check by the Ribosomal Database Project (http://rdp.cme.msu.edu/), the clone

8

sequences were aligned and phylogenetic trees were constructed by the neighbor-joining method

with the Molecular Evolutionary Genetics Analysis (MEGA) software52.

Calculation of the photoelectric efficiency. The photoelectric efficiency was calculated by

dividing the number of photoelectrons transferred from the P-chamber to the C-chamber by the

number of photons received on the mineral surface over the logarithmic growth period of A.

ferrooxidans. The number of photons was calculated by dividing the total light energy by the

energy of each photon. The total light energy (J) was equal to light intensity (8 mW/cm2) x the

total surface area of the mineral-coated electrode (35 cm2) x irradiation time (2 or 3 days

depending on wavelength). The energy of a photon (J) = Planck constant (J·s) x speed of light

(m/s) / light wavelength (nm).

Example calculation of the photon-biomass conversion efficiency-the first method. The first

method calculates the ratio of the amount of energy derived from Fe2+

oxidation in the C-

chamber (i.e., energy output to support the growth of A. ferrooxidans) to the amount of light

energy received in the P-chamber (i.e., energy input) over the logarithmic growth period (1.5 to 4

days, Fig. 2) of A. ferrooxidans cells. Under acidic conditions, the amount of energy derived

from Fe2+

oxidation is 8.1 kcal/mol (or 33.9 kJ/mol)53

. The number of moles of Fe2+

oxidized by

A. ferrooxidans was calculated by determining the number moles of photoelectrons used to

oxidize Fe2+

in the C-chamber (Eeffective) assuming 1:1 stoichiometric ratio (i.e., 1 mole of

electrons is used to oxidize 1 mole of Fe2+

) according to the following equation:

Eeffective = Etotal - Eoxygen

Eeffective is the number of moles of photoelectrons used to reduce Fe3+

to Fe2+

during the log

growth phase period of A. ferrooxidans (1.5 to 4 days, Fig. 2), Etotal is the total number of moles

9

of photoelectrons transferred from the P-chamber to the C-chamber over the same period

(integrated from current-time curves), and Eoxygen is the number of moles of photoelectrons

consumed by O2 in the C-chamber (i.e., not used to recycle Fe3+). This parameter was measured

in a control experiment, where no cells were present (Supplementary Fig. S2) but Fe3+

(along

with O2) was present at the same concentration as that in Fig. 2. So the amount of electrons

consumed by Fe3+

was subtracted from the measured values. An integration of electric current

between the two chambers over the 1.5 to 4 days period resulted in 4.56×10-4

mol for Etotal. An

integration of electric current over the same period in the absence of cells (control) resulted in

1.04×10-6

mol/hour for Eoxygen.

The photon-biomass energy conversion efficiency of the rutile - A. ferrooxidans system

was then calculated by the following equation:

Energy conversion efficiency (ECEphoton)=33.9 kJ/mol× Eeffective /(P×S×time)

Where P is the power of the lamp (13.5 mW/cm2 for Fig. 2), S is the electrode surface

area (35 cm2 in the anode). So the photon-biomass energy conversion efficiency was calculated

as:

3 4 6

3 2 2

33.9 10 / (4.56 10 1.04 10 / 60 )0.013%

13.5 10 / 35 (60 60 60)photon

J mol mol mol h hECE

W cm cm s

− −

−

× × × − × ×= =

× × × × ×

10

Example calculation of the photon-biomass conversion efficiency-the second method. In the

second method, the efficiency was calculated by dividing the amount of energy required to result

in the observed cell growth by the amount of light energy received in the P-chamber. In this

experiment (Fig. 2), during the log growth phase of A. ferrooxidans (1.5 to 4 days) the total

amount of cell growth was:

4.51×107 cell/mL×350 mL (chamber volume) =1.58×10

10 cells

However, a fraction of this growth was due to consumption of Fe2+ initially present in the m9K

medium (~3500 mg/L over the 1.5 to 4 days) and must be subtracted to obtain the amount of net

growth due to photoelectrons. The amount of cell growth driven by the medium Fe2+

was

estimated from the rutile-free control (orange squares in Fig. 2) assuming that cell growth was

proportional to Fe2+

concentration. In the rutile-free control, consumption of 3727 mg/L Fe2+

(from day 1.5 to day 2.5) produced 3.48×107 cells/mL. For the 36-96 hour period in the presence

of rutile and visible light, the amount of the medium-Fe2+

consumed was 2531 mg/L, which

should have resulted in cell growth of 2.37×107 cells/mL (or 8.30×109 cells). This amount of cell

growth due to oxidation of Fe2+ initially present in the m9K medium was subtracted from the

measured total growth to obtain a net growth amount of 7.5×109 cells due to photoelectrons.

According to the average carbon to volume ratio of 0.12 pg/µm3 for both natural and cultured

bacteria54

, the total amount of carbon was:

7.5×109 cell×1 µm

3/cell×0.12 pg/µm

3 = 9.0×10

-4 g carbon

The cell volume of 1 µm3/cell was based on SEM observations: cell length 1~2 µm, cell diameter

~0.5 µm). A previous study reported that one mole of biomass synthesis (in the form of

11

CH1.8O0.5N0.2) requires 0.855 to 9.8 mol ATP55

. Taking an intermediate value of (5.33), the total

amount of ATP required to support the measured amount of cell growth was:

(9.0×10-4

g/12.0 g/mol)×5.33 = 4.0×10-4

mol ATP

A previous study reported that at pH 7.0 and 25 oC, ATP-AMP transformation releases 45.6

kJ/mol energy56

. The total amount of ATP energy was then calculated

4.0×10-4

mol×45.6 kJ/mol = 18.24 J

Finally, the photon-biomass energy conversion efficiency was calculated as follows:

3 2 2

18.240.018%

13.5 10 / 35 (60 60 60)ATP

JECE

W cm cm s−

= =× × × × ×

These two methods of calculating the photon-biomass conversion efficiency matches fairly well.

This close match clearly demonstrates the reliability of the data and the validity of assumptions.

Supplementary References:

49. Dojka MA, Hugenholtz P, Haack SK, Pace NR (1998) Microbial diversity in a hydrocarbon-

and chlorinated-solvent-contaminated aquifer undergoing intrinsic bioremediation. Appl.

Environ. Microbiol. 64:3869-3877.

50. Park S, et al. (2006) The characterization of bacterial community structure in the rhizosphere

of watermelon (Citrullus vulgaris SCHARD.) using culture-based approaches and terminal

fragment length polymorphism (T-RFLP). Appl. Soil Ecol. 33:79-86.

51. Fedi S, et al. (2005) T-RFLP analysis of bacterial communities in cyclodextrin-amended

12

bioreactors developed for biodegradation of polychlorinated biphenyls. Res Microbiol.

156:201-210.

52. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular evolutionary genetics

analysis (MEGA) software version 4.0. Mol Biol Evol. 24, 1596-1599.

53. Ingledew W (1982) Thiobacillus ferrooxidans the bioenergetics of an acidophilic

chemolithotroph. Biochimica et Biophysica Acta, 683:89-117.

54. Nagata T, Watanabe Y (1990) Carbon- and nitrogen-to-volume ratios of bacterioplankton

grown under different nutritional conditions. Appl Environ Microb, 56:1303-1309.

55. Mignone C, Donati E (2004) ATP requirements for growth and maintenance of iron-

oxidizing bacteria. Biochem Eng J, 18:211-216.

56. Nelson D, Cox M (2004) in Lehninger Principles of Biochemistry 4ed, (W. H. Freeman), pp

493.