Wen-Wei Li2, Han-Qing Yu2,*. 1School of Life Sciences, 2CAS Key Laboratory of Urban Pollutant Conversion,. Department of Chemistry, University of Science ...
Supporting Information
Rapid Detection and Enumeration of Exoelectrogenic Bacteria in Lake Sediments and Wastewater Treatment Plant Using a Coupled WO3 Nanoclusters and Most Probable Number Method
Zong-Chuang Yang1,2,ǂ, Yuan-Yuan Cheng2, ǂ, Feng Zhang2,*, Bing-Bing Li1, Yang Mu2, Wen-Wei Li2, Han-Qing Yu2,* 1
School of Life Sciences, 2CAS Key Laboratory of Urban Pollutant Conversion,
Department of Chemistry, University of Science & Technology of China, Hefei, 230026, China
This supporting information contains 12-page document, including 3 figures, 3 tables, detailed descriptions about microbial cultures and MPN medium preparation, abiotic, positive and negative control tests for the WO3 nanocluster method, analytical methods of environmental samples, additional experimental results about E. coli as an EEB, references and this cover page.
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Microbial Cultures. Shewanella oneidensis MR-1, Geobacter sulfurreducens DL-1,
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Pseudomonas aeruginosa, Bacillus subtilis 168 and Escherichia coli JM109 were
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cultured as model bacteria in this work. In detail, S. oneidensis MR-1, P. aeruginosa
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and B. subtilis, were cultivated aerobically in LB broth. Cultures were grown at 30 oC
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and agitated at a rate of 150 rpm until the late stationary phase. E. coli was cultivated
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aerobically in LB broth at 37 oC and agitated at a rate of 150 rpm until the late
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stationary phase. G. sulfurreducens DL-1 was cultivated anaerobically at 30 oC in
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sterilized mineral salt medium (described below) with acetate and fumarate as
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electron donor and acceptor respectively until the late stationary phase. The cells were
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collected by centrifugation at 5000 rpm for 5 min, washed three times with sterilized
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mineral salt medium and re-suspended in sterilized mineral salt medium. The bacteria
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concentration was determined by their optical density at 600 nm wavelength (OD600).
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The suspension of S. oneidensis MR-1 or G. sulfurreducens DL-1 was adjusted to an
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OD600 value of 0.56 and 0.13, respectively. The OD600 of P. aeruginosa, B. subtilis
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and E. coli were all adjusted to 1.0. A sterilized mineral salt medium without any
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electron acceptor or donor was used for dilution. Salts used (in g/L) were NH4Cl (1.5),
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KCl (0.1), CaCl2 (0.05), K2HPO4 (0.225), KH2PO4 (0.225), MgSO4·7H2O (0.117),
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NaHCO3 (2.5), HEPES (11.867) and 10 mL trace element solution containing (in g/L)
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NTA (1.5), MnCl2·4H2O (0.1), FeSO4·7H2O (0.3), CoCl2·6H2O (0.17), ZnCl2 (0.1),
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CuSO4·5H2O (0.04), KAl(SO4)2·12H2O (0.005), NaMoO4 (0.009), H3BO3 (0.005),
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NiCl2 (0.12), NaWO4·2H2O (0.02), Na2SeO4 (0.1), which was buffered to pH 7.0 by
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adding NaOH solution (5 mol/L). LB broth was used as bacterial growth medium. 2
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MPN Medium Preparation. Sterilized WO3-containing deionized water (5 g/L)
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was used as electron acceptor and for probing the EEB by showing color changes.
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The WO3 nanoclusters were prepared according to a previous report2. Briefly,
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crystalline WO3 nanoclusters were synthesized using a hydrothermal process with
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Na2WO4·2H2O as a precursor. 0.825 g of Na2WO4·2H2O and 0.290 g of NaCl were
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dissolved in 20 ml of deionized water. Then, 3 M HCl was slowly added under
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stirring until pH reached 2.0. The solution was transferred into a 45-mL Teflon
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autoclave and heated at 180 oC for 16 h in an oven. After cooling down to ambient
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temperature, white powders of WO3 nanoclusters were obtained. The powders were
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washed thoroughly with deionized water, and then filtered through a 0.45-µm
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membrane to collect the solid. All three mediums were purged with high purity N2 for
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20 min and autocalved for 20 min at 121 oC.
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Abiotic, Positive and Negative Controls of the WO3 Nanocluster Method.
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Suspensions of 100 µL cells were transferred to each well that was preloaded with 100
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µL LB broth and 50 µL sterilized WO3 suspension (prepared using deionized water).
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Then, 80 µL petrolatum oil was added into each well immediately to ensure anaerobic
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conditions for the chromogenic reaction. The color changes of the wells in 96-well
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plates were evaluated after 48-h incubation.
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Selected inorganic or organic species (i.e., 50 mM or 50 µM sodium hyposulfite,
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sodium sulfide, mercaptoethanol and glutathione (GSH)), carbon sources or
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fermentation products (glucose, acetate, lactate and ethanol at 10 mM each) were
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individually added into sterilized mineral salt medium. Mineral salt medium of 3
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100-µL was transferred into each well with preloaded LB broth and WO3 and sealed
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as described above. The color changes of the wells were recorded after 48-h
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incubation.
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Analysis of Environmental Samples. The dissolved oxygen (DO), pH,
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temperature (T) and oxidation-reduction potential (ORP) of the samples were
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determined in-situ in WMWTP. Briefly, the DO was determined using an HQ30d
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analyzer (HACH Inc., USA), the ORP and pH were determined using a PHS-3C
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analyzer (INESA Inc., China). The total organic carbon (TOC), NH3-N, nitrate and
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phosphate were measured after samples were transported to our laboratory. The
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samples were centrifuged at 6,000g for 5 min to collect the supernatant. The TOC was
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determined using a multi N/C 2100 analyzer (Analytic Jena AG Inc., Germany).
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Suspended solids (SS) and volatile suspended solids (VSS) were measured according
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to the Standard Methods (APHA)1. The determination of phosphate, ammonium and
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nitrate of water and environmental samples was conducted using a water quality
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analyzer (Aquakem, Thermo Fisher Scientific Inc., USA).
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Additional experimental results about E. coli as an EEB. MEC tests were
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carried out by using a three-electrode cell system with an Ag/AgCl (3 M KCl)
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reference electrode, a Pt wire counter electrode and a pyrolytic graphite sheet working
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electrode. The concentrated E. coli culture was inoculated into the reactor containing
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60 ml medium to an initial OD600 of 1.0. The culture medium (pH=7) contained:
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glucose 10 g/L, NH4Cl 0.46 g/L, K2HPO4 0.225 g/L, KH2PO4 0.225 g/L,
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MgSO4·7H2O 0.117 g/L, (NH4)2SO4 0.225 g/L, HEPES 11.91 g/L, CaCl2 0.01 g/L, 4
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trace element 10 mL/L. The redox potential of the working electrode was kept at 0.4
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V (vs. Ag/AgCl) using a very sensitive PARSTAT MC Potentiostat (Princeton Applied
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Research, USA). The running temperature was 37 °C. A continuous and rapid rising
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current was observed for the MEC inoculated with E. coli, while the abiotic control
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showed no current generation (Figure S3a).
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For further validation, the E. coli culture was also tested in microbial fuel cells
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(MFCs). An H-shaped double-chamber MFC with potassium ferricyanide catholyte
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(50 mM, 100 mM PBS, pH=7.0) was used. The working volume of both electrode
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chambers was 80 mL. Carbon felt with a uniform dimension of 2 × 1.5 cm2 was used
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as the anode and the cathode. The electrodes were separated by a proton exchange
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membrane, and connected through external circuit with a fixed external resistance of
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100 kΩ to allow current measurement. Before experiments, the MFCs components
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were sterilized in autoclave at 121 oC for 20 min. The E. coli culture was inoculated
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into the anodic chamber containing 80 mL of glucose minimal salt medium to an
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initial OD600 of 1. The running temperature was 37 oC. Again, the MFC with E. coli
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showed obvious current generation relative to the abiotic control, although the current
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was weak (Figure S3b). We can thus conclude that E. coli is an EEB with a weak
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exoelectrogenic activity.
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Figure S1. Sampling sites in Chao Lake, Hefei, China (a) and Wangtang Municipal Wastewater Treatment Plant (WWTP), Hefei, China (b).
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Figure S2. Color changes of WO3 nanoclusters in wells of a 96-well plate with different bacteria, carbon sources or fermentation products (a) and different concentrations of organic or inorganic compounds (b).
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Figure S3. Current densities of the MECs with and without E. coli inoculation (a); current densities of two parallel MFCs inoculated with E. coli (b).
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Table S1 Cell Count of S. oneidensis MR-1 obtained by the CFU, DAPI and MPN Methodsa. cell/mL average
a
P
p1
p2
p3
MPN
1.7×109
4.9×108
2.2×109 (1.5±0.9)×109
CFU
9.2×108
7.8×108
1.1×109 (9.2±1.4)×108
0.36
DAPI
1.0×109
6.8×108
8.4×108 (8.4±1.7)×108
0.29
p1-p3: three replicates; P: significant difference between the results from the MPN
method with the CFU and DAPI method, OD600 = 0.56
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Table S2 Cell Count of G. sulfurreducens DL-1 obtained by the DAPI and MPN Methodsa. cell/mL average
a
p1
p2
MPN
1.4×107
7.0×106
9.4×106 (1.0±0.4)×107
DAPI
5.4×107
3.8×107
5.2×107 (4.8±0.9)×107
P
p3
0.0022
p1-p3: three replicates; P: significant difference between the results from the MPN
method with the CFU and DAPI method. OD600 = 0.13
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Table S3 Comparison between the WO3-MPN Method and Other Methods Reported in Literature. process
bottlenecks
an MPN method based on bio-PCR
no
results
available for EEB
Fe(III)
an MPN method based on the color
time-consuming
reduction-MPN12
change
specificity
PCR-MPN11
of
ferrihydrite
from
common
primers
are
and
low
red-brown to nearly black WO3 probe10
a WO3-nanoclusters probe designed to
detect
electrons
can not count EEB
transferred
outside cells this method
an MPN method based on the color change of WO3-nanocluster from whit to blue
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high specificity and time-saving
REFERENCES
1 American Public Health Association. Standard Methods for the Examination of Water and Wastewater, 18th ed. American Public Health Association, Washington, D. C. 1995. 2 Yuan, S. J.; He, H.; Sheng, G. P.; Chen, J. J.; Tong, Z. H.; Cheng, Y. Y.; Li, W. W.; Lin, Z. Q.; Zhang, F.; Yu, H. Q., A Photometric High-throughput Method for Identification of Electrochemically Active Bacteria Using a WO3 Nanocluster Probe. Sci. Rep. 2013, 3, 1315.
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