High-Throughput Screening of Electrochemically Active Microorganisms Using Bioelectrochromic

1 Supplementary Information for

3 A Photometric High-Throughput Method for Identification of

4 Electrochemically Active Bacteria Using a WO3 Nanocluster Probe

6 Shi-Jie Yuan†, Hui He†, Guo-Ping Sheng*, Jie-Jie Chen, Zhong-Hua Tong, Yuan-

7 Yuan Cheng, Wen-Wei Li, Zhi-Qi Lin, Feng Zhang, Han-Qing Yu*

8 Department of Chemistry, University of Science & Technology of China, Hefei,

9 230026, China

11† These authors contributed equally to this work.

13 *Corresponding authors:

14 Dr. Guo-Ping Sheng, E-mail: gpsheng @ ustc .edu.cn; Prof. Han-Qing Yu, E-mail:

15 [email protected] n

16 Contents

18 Materials and Methods

19 S1. Quantitative assessment of the electricity-producing ability of bacterial

20 strains with the MFC method

21 S2. Isolation of EAB

22 S3. Roles of soluble mediators

23 S4. Relationship between the rate of color development and the population

24density of cells

25 26 Results 27 S5. Isolation of EAB

28 S6. Bioelectrochromic phenomenon induced by the supernatant

29 S7. Correlation between the population density of cells and the chromaticity

31Supporting Figures

32 Figures S3

33 Materials and Methods

35Sections S1. Quantitative assessment of the electricity-producing ability of

36bacterial strains with the MFC method

37 To validate the proposed methods, the electricity-producing abilities of bacterial

38strains were also evaluated using MFC methods. Each MFC had a single-chamber air-

39cathode configuration with carbon paper (GEFC Co., China) as the anode (3 × 7 cm)

40and carbon paper loaded with Pt (2 mg/cm2) as the cathode (2 × 2 cm). The anode was

41connected to the cathode via a 1000 Ω resistor for monitoring the electricity. The

42circuit current was calculated from the voltage across the resistance, which was

43continuously recorded with a data acquisition/switch unit (34970A, Agilent Inc.,

44USA) connected to a computer. The current density was calculated according to the

45projected cathode surface area. The anode chamber was filled with 400 ml of sterile

46sodium lactate minimal salt medium. The medium containing (in per liter): 2.02 g

47sodium lactate, 5.85 g NaCl, 11.91 g Hepes, 0.3 g NaOH, 1.498 g NH4Cl, 0.097 g

48KCl, 0.67 g NaH2PO4·2H2O, plus 0.4 ml of a trace mineral stack solution (containing

49per liter: 1.5 g NTA(C6H9NO6), 30 g MgSO4·7H2O, 5 g MnSO4·H2O, 10 g NaCl, 1 g

50FeSO4·7H2O, 1 g CaCl2·2H2O, 1 g CoCl2·6H2O, 1.3 g ZnCl2, 0.1 g CuSO4·5H2O, 0.1

51g AlK(SO4)2·12H2O, 0.1 g H3BO3, 0.25 g Na2MoO4·2H2O, 0.25 g NiCl2·6H2O, 0.25 g

52Na2WO4·2H2O). 0.4 ml of filter-sterilized amino acid solution (containing per liter: 2

53g L-glutamic acid, 2 g L-arginine, 2 g DL-serine) and 0.4 ml of filter-sterilized

54vitamin solution (containing per liter: 2.0 g biotin, 2.0 g folic acid, 10.0 g pyridoxine

55HCl, 5.0 g riboflavin, 5.0 g thiamine, 5.0 g nicotinic acid, 5.0 g pantothenic acid, 0.1 g

56cyanocobalamin, 5.0 g p-aminobenzoic acid, 5.0 g thioctic acid), were added after

1 57autoclaving . Ultrapure N2 was used to remove trace of oxygen. Cultures for each

58MFC were grown from frozen stocks, then inoculated into liquid LB medium and

59incubated until the late stationary phase was achieved before being transferred into

60MFCs. All solutions were prepared with ultrapure water (Millipore Co., USA). All

61batch tests were conducted in triplicate.

63Sections S2. Isolation of EAB

64 The nanocluster probe method described in this paper can also be used to isolate

65EAB. In this study, the EAB were isolated from a laboratory-scale bioreactor, which

66had been operated for 6 months with mixed anaerobic sludge as the inoculum and

67acetate as the substrate. The mixed microorganisms were incubated overnight in LB

68medium. Then, they were spread over an LB plate. Twenty minutes later, 25 mL of

69autoclaved WO3 agar suspension was poured as an overlay, which contained WO3

70nanocluster (5 g/L), NaCl (10 g/L) and agar (20 g/L). The entire sandwich-like plate

71was incubated at 30oC. After 36 h, a blue color started to appear and the color

72intensity increased over time. The colored areas of the sandwich plate were chipped

73off, and the colonies between them were selectively isolated from the WO3 plate

74according to the coloration position, and then inoculated into LB liquid medium. The

75strains were incubated overnight with shaking at 125 rpm and 30oC. With these

76colonies as inoculums, the sandwich procedure described above was repeated until the

77appearance of single colony with blue coloration. MFCs were used to validate the

78electricity-producing abilities of these isolates.

79 Genomic DNA was extracted from the isolated bacteria using DNA extraction kit

80(Shanghai Bio-Tech Co., China). PCR amplification was performed using the forward

81primer 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and the reverse primer 1492R

82(5’-GGTTACCTTGTTACGACTT-3’) with an initial denaturation at 98°C for 5 min

83followed by 35 cycles of denaturation at 95°C for 35 s, annealing at 55°C for 35 s,

84and extension at 72°C for 90 s, before the final extension for 8 min. PCR products

85were purified with a UNIQ-10 Column kit (Shanghai Bio-Tech Co., China) and

86sequenced using an ABI 3730XL DNA sequencer (Applied Biosystems Inc., USA).

87The obtained 16S rRNA gene sequences were compared to the GenBank sequences

88using the BLAST search program.

90Sections S3. Roles of soluble mediators

91 To explore the role of soluble mediators in the observed electron transfer process,

92a two-chamber glass reactor (65 mL in volume) separated by a poly(ether sulfones)

93filter membrane (Xiboshi Co, Tianjin, China) with a pore size of 0.45 μm was used A

9460 mL of Shewanella oneidensis MR-1 (in sodium lactate minimal salt medium) was

95added to one chamber and 20 mL of 5 g/L sterile WO3-nanocluster-containing sodium

96lactate minimal salt suspension was dosed to another chamber. Ultrapure N2 was used

97to remove trace of oxygen immediately. The reactor was incubated at 30oC. The color

98development was monitored using a digital camera (SP-600UZ, Olympus Co., Japan).

10 5 11

100Sections S4. Relationship between the rate of color development and the

101population density of cells

102 To validate the relationship between the color development and the

103corresponding population density of cells, a series of diluted solutions of Shewanella

104oneiensis MR-1 suspension were tested in a 96-well plate. The wild type Shewanella

105oneidensis MR-1 was grown from a frozen stock. Each isolated colony was inoculated

106into a flask containing 200 mL of sterile LB broth and grown at 30°C under shaking

107until the exponential phase. The cells were collected by centrifugation at 4000 rpm for

1085 min, washed for three times and re-suspended in sterile sodium lactate minimal salt

109medium, which had the same contents as that used in the MFC anode chamber1. A

110series of the bacterial suspension dilutions were inoculated into a 96-well plate. The

111color development and the Density (mean) of each well were measured.

112 113 Results

115Sections S5. Isolation of EAB

116 Bioelectrochromic phenomenon of the WO3 plate was observed after 36-h

117incubation. The color intensity further increased, and single colonies with blue

118coloration were observed after 48 h. Three single clones, designated as WO-1, WO-2

119and WO-3, were then isolated from the WO3 plate based on their coloration and the

120position of color occurred. Nearly complete 16S rRNA gene sequences (1305

121nucleotides for WO-1; 1406 for WO-2 and 1202 for WO-3) of the isolated strains

12 6 13

122were obtained. Sequence analysis showed that Strain WO-1 was most closely related

123to Kluyvera, whereas WO-2 and WO-3 belonged to Shewanella and Proteus

124respectively (Table S1). The phylogenetic tree was constructed using the neighbor-

125joining method (Fig. S1).

126 The current densities of the MFCs inoculated with the three isolated strains are

127itemized in Supplementary Table S1. Strain WO-2 (with 99% identity to Shewanella

128putrefaciens CN-32) generated a medium-level current density, which was slightly

129higher than that generated by Shewanella oneidensis MR-1, but lower than by Strain

130WO-1 (with 99% identity to Kluyvera cryocrescens TS IW 13). Strain WO-3 (with

13199% identity to Proteus sp. SBP10) produced the lower current density.

14 7 15

132 Table S1. Three isolated EAB.

Seria Closest homologue Homology / Current density of MFC l (accession no.) % /A/m2 numb er WO- Kluyvera cryocrescens TS IW 13 99 0.323±0.006 1 (AM992189) WO- Shewanella putrefaciens CN-32 99 0.275±0.014 2 (CP000681.1) WO- Proteus sp. SBP10 99 0.178±0.015 3 (GU812899.1) 133

16 8 17

136Figure S1. Phylogenetic relationship of the 16S rRNA gene sequences of the three

137isolated strains with other related strains and genera.

138 Note: the dendrogram shows the results of an analysis, in which MEGA 3.1 and

139 Neighbor-Joining were used. Bootstrap values greater than 50%, derived from 1,000

140 replicates, are also shown. Bar: 0.01 sequence divergence.

18 9 19

141 Sections S6. Bioelectrochromic phenomenon induced by the supernatant

144Figure S2. Typical photographs of the cell consist of two chambers separated with a

145 poly(ether sulfones) filter membrane. (a), before incubation. (b), after 24-h

146 incubation.

148 Figure S2 shows the images of the culture before and after 24-h incubation.

149Bioelectrochromic phenomenon was visually observed for the WO3 chamber after

150 12-h incubation and became more evident 24 h later. Since the two chambers were

151separated by filter membrane, the supernatant of Shewanella oneidensis MR-1 in the

152incubated chamber could permeate to the WO3-containing chamber. Such a

153bioelectrochromic phenomenon suggests that there were soluble compounds in the

154 supernatant, which were reduced by Shewanella oneidensis MR-1 in the incubation

155process and then transferred electrons to the WO3 nanocluster. These soluble

156 compounds would be soluble mediators1, and were also involved in the electron

157transfer process from EABs to WO3 nanoclusters.

20 10 21

159Sections S7. Correlation between the population density of cells and the

160 chromaticity

161 Figure S3 illustrates the color changes of samples with different cell densities

162 over time. While only slight blue color could be observed in about 3 min for several

163 wells (Fig. S3a), it became more evident in 15 min (Fig. S3b). Compared with the

164 wells with bacteria that showed different degrees of blue color, no color change was

165observed for the wells in Row A, which were used as the non-bacteria control. The

166color intensity further increased after 30-min incubation (Fig. S3c). Slight and

167 inconspicuous blue color appeared in the wells of Rows B and C with a lower

168 population density, while distinctly higher color intensities were observed for the

169wells of Rows D-G with a higher population density. A close correlation between the

170 population density of cells and the color intensity could be found in Fig. S3d, with a

171value of Spearman's ρ (0.994, P < 0.01) achieved.

22 11 23

172 173Figure S3. Correlation between the population density of cells and the

174chromaticity. (a), typical photographs of the 96-well plate after 3-min incubation.

175 The initial concentration of bacteria inoculated in Rows of A-G was counted to be 0,

176 7.38×106, 3.69×107, 7.38×107, 1.48×108, 2.21×108 and 3.69×108 CFU/well. Seven

177replicates were used. (b), after 15-min incubation. (c), after 30-min incubation. (d),

178Correlation between the population density and the corresponding Density (mean)

179(Spearman's ρ=0.994, P < 0.01) after 30-min incubation.

24 12 25

180 Supporting Figures

185 Figure S4. Hexagonal WO3 cell structures in polyhedral representation (a) 4×4×1

186 supercell: a=b=29.1928 Å, c=3.8992 Å, (b) 2×2×1 supercell: a=b=14.5964 Å,

187 c=3.8992 Å, α=β= 90°, γ=120°, quarter of structure (a).

26 13 27

188 References

1901. Marsili, E. et al. Shewanella secretes flavins that mediate extracellular electron 191 transfer. Proc Natl Acad Sci USA 105, 3968 (2008).