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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.
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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).
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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;
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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.
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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
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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
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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
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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
− −
−
× × × − × ×= =
× × × × ×
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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
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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:
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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
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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.