Rapid Detection and Enumeration of Exoelectrogenic Bacteria in Lake

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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 Yang, Yuan-Yuan Cheng, Feng Zhang, Bing-Bing Li, Yang Mu, Wen-Wei Li, and Han-Qing Yu Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.6b00112 • Publication Date (Web): 31 Mar 2016 Downloaded from http://pubs.acs.org on April 3, 2016

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Environmental Science & Technology Letters

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

1

ACS Paragon Plus Environment


1

ABSTRACT

2 3

Exoelectrogenic bacteria (EEB) play important roles in biogeochemical cycling,

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environmental remediation, wastewater treatment and bioenergy recovery. Methods to

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effectively and rapidly probe the abundance of EEB in environments are highly

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desirable. In this work, a novel approach that couples WO3 nanoclusters and most

7

probable number (MPN) method for rapid detection and enumeration of EEB was

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developed. This WO3-MPN approach enabled rapid and reliable estimation of the

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population size of two typical EEB, Shewanella oneidensis MR-1 and Geobacter

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sulfurreducens DL-1. In addition, it was successfully applied to detect and count EEB

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in environmental samples from the sediments of a freshwater lake (9.9×104 to 4.1×106

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cells/g dry sediment), and engineered samples of a municipal wastewater treatment

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plant (1.0×103 to 7.5×105 cells/mL). This work may facilitate better identification and

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practical applications of EEB in natural and engineered environments.

15

2


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INTRODUCTION

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Exoelectrogenic bacteria (EEB) are capable of exocellular electron transfer to

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extracellular electron acceptors1-3 and inhabit in a wide range of natural and

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engineered environments.4-6 They play important roles in biogeochemical cycling,7

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environmental remediation, waste treatment, bioenergy recovery and microbial

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electrosynthesis processes.1 Various EEB, such as Geobacter sulfurreducens,2

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Shewanella oneidensis8 and Ochrobactrum anthropic,9 have been isolated from the

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environments and characterized. However, in contrast with the environmental

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ubiquity of EEB, the overall number of identified EEB species is still very small so far.

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Furthermore, information about the population size of EEB in natural and engineered

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environments remains sparse. Thus, effective methods to measure the abundance of

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EEB in the environment are highly desirable.

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Recently, a WO3 probe method was developed to provide rapid detection of

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EEB.10 With unique electric conductivity, selectivity and biocompatibility, WO3

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nanoclusters can rapidly receive electrons from EEB, which immediately lead to color

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change of WO3 from white to blue, and the color intensity increases with more

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electrons received. The behavior of extracellular electron transfer from microbes to

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WO3 nanoclusters is very similar to that from microbes to a solid electrode. In

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addition, it possesses high detection selectivity for EEB, as has been validated by the

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use of non-EEB as a negative control, which caused no distinct color change of WO3

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nanoclusters.10 Thus, the WO3 method offers an ideal tool to identify EEB, although 3



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its validity for detecting complex environmental samples is to be confirmed. In

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addition, the WO3 method alone cannot reveal the EEB population size information,

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thus effective methods for quantifying EEB abundance in environmental samples are

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still needed.

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The most-probable-number (MPN) methods have been widely applied in

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combination with molecular biology techniques or other qualitative detection methods

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to reveal the population size of bacteria with specific functions or physiological

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features.11-13 Given the good reliability of the MPN method for measuring bacteria

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population size, in this work we integrated the WO3 probe with MPN for the detection

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and enumeration of EEB. To validate the detection selectivity and reliability of the

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WO3 nanocluster method for EEB in environmental sample, several controls with

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different bacteria and reductive chemicals were tested. The effectiveness of this

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WO3-MPN approach was evaluated by using the pure cultures of Shewanella

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oneidensis MR-1, Geobacter sulfurreducens DL-1 (two main typical EEB), and the

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results were compared with those of 4',6-diamidino-2-phenylindole (DAPI) and

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colony-forming unit (CFU) counting methods. Furthermore, the population sizes of

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EEB in environmental samples from lake sediments and a municipal wastewater

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treatment plant (WWTP) were evaluated using such a new approach.

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MATERIALS AND METHODS

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Microbial Cultures and Medium. S. oneidensis MR-1 and G. sulfurreducens DL-1 4


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were provided by Prof. K. H. Nealson at the University of Southern California and

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Prof. D. R. Lovley at the University of Massachusetts, respectively. Pseudomonas

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aeruginosa (ATCC27853), Escherichia coli JM109 (ATCC53323) and Bacillus

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subtilis 168 (ATCC23857) were purchased from American type culture collection

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(ATCC, American Type Culture Collection). The culture details of these bacteria are

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shown in the Supporting Information (SI). The preparation of the medium for WO3

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test and MPN measurement is described in details in SI.

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Cell Counting and Color Intensity Determination. The suspensions of S.

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oneidensis MR-1, G. sulfurreducens DL-1 and environmental samples were

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homogenized by votexing and 10-fold serially diluted in the sterilized mineral

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medium to 10-11. Aliquots of 100-µL dilution of the homogenate each were transferred

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to the wells of a 96-well plate. Each well was preloaded with 100 µL Luria-Bertani

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(LB) broth and 50 µL sterilized WO3 suspension (the total amount of WO3 was 250

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µg). After adding the bacteria solution, 80 µL petrolatum oil was added into each well

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immediately to ensure anaerobic conditions for the chromogenic process. For each

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sample, three replicate MPN assays were set up with five replicate wells for each

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dilution step. The plates were incubated at 30 oC in an incubator (Sheldon Inc., USA).

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The color changes of WO3 in the wells were evaluated after 48-h incubation.

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The wells with changed color were considered positive for EEB growth. MPN

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tables were constructed according to the standard method.14 To evaluate the accuracy

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of the WO3-MPN method, the cell number of S. oneidensis MR-1 or G.

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sulfurreducens DL-1 suspension was validated by direct counting using an 5



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epifluorescence microscope (BX51, Olympus Co., Japan) after dying with DAPI, and

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CFU counting on LB-agar plates at 30 °C for 24 h.

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In the MPN assay of S. oneidensis MR-1 and G. sulfurreducens DL-1, the 96-well

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plates were scanned using a scanner at given time intervals. The color change degrees

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of the wells were recorded according to a previously established protocol.10

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Environmental Samples. Sediment samples were collected from six different

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locations (designated as NFH, NFHK, AB, XHX, HX and DHX, Figure S1a) of Chao

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Lake (117°16′- 117°51′E and 31°43′- 31°25′N), one of the five largest freshwater lakes in

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China with severe eutrophication.15 In each location, at least three sediment cores

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(5-cm diameter and 5-cm depth) were collected and the samples were then mixed in

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an anaerobic glove box. The sediment organic content was determined by the K2Cr2O7

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oxidation method. Phosphate was extracted using 0.5 M NaHCO3, whereas NH3-N

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and nitrate were extracted using 2 M KCl. Sediment slurries were prepared by mixing

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the sediment samples and deionized water at a ratio about 1:3 (w/v). The water

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content of these slurries was estimated using a freeze dryer (Labconco Co., USA).

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Influent, effluent and mixed liquor samples were collected from the Wangtang

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Municipal WWTP in Hefei, China (Figure S1b). This plant adopts Carrousel

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oxidation ditch process and has a treatment capacity of 100,000 m3/day and a

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hydraulic retention time of 15 h.16 The samples were analyzed immediately after

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being collected. The analytical methods are detailed in SI.

102 103

RESULTS AND DISCUSSION 6


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Validation of WO3 Nanoclusters as an EEB Probe. We first validated whether the

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WO3 nanoclusters could distinguish EEB from non-EEB by setting various abiotic,

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positive and negative controls (SI). When B. subtilis was used as a non-EEB inoculum,

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the color of the WO3 probe remained unchanged, indicating that there were no

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extracellular electrons produced from this bacterium (Figure S2a). The substrate,

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fermentation products, and some reductive metabolic products of non-EEB, e.g.,

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mercaptoethanol and glutathione, or reductive inorganic salts, e.g., sodium hyposulfite,

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sodium sulfide, at environmental concentrations also failed to reduce WO3 (Figure

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S2). Notably, although some specific metabolites such as reductive-state riboflavin did

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cause color change of WO3, they could take electrons from extracellular proteins of

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EEB only, indicating an indirect reduction of WO3 by EEB in this case. In all, these

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results indicate that WO3 color change occurred only in the presence of EEB, whether

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in a direct or indirect way, and the interference of non-EEB and abiotic factors could

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be excluded.

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To find out whether the WO3 method is applicable for various types of EEB,

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rather than just dissimilatory Fe(III) reducing bacteria (DIRB), we also tested a

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non-DIRB EEB, P. aeruginosa. Color change of WO3 occurred after 48-h inoculation

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(Figure S2a). Another model bacterium, E. coli, caused slight color change of WO3

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(Figure S2a), indicating a week exoelectrogenic ability of this strain. While there has

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been controversy about the current production ability of E. coli,17-20 our current

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generation results (Figure S3) confirm its identity as an EEB, which is consistent with 7



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the above WO3 detection result. Thus, this method offers a general and useful tool for

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detecting various EEB.

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Evaluation of the WO3-MPN Method. The effectiveness of the WO3-MPN

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method was verified by comparing with the DAPI and CFU counting methods. The

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cell number of S. oneidensis MR-1 counted by the WO3-MPN method was

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1.5×109/mL and remained constant after 48-h incubation (Figure 1a). This value was

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comparable to the results of the CFU method (9.2×108/mL) and DAPI method

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(8.4×108/mL) (Table S1). A good consistency between the different methods was also

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obtained for counting G. sulfurreducens DL-1 (Table S2).

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For S. oneidensis MR-1, color change of WO3-nanoclusters occurred within 30

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min (Figure 1b). When this culture was diluted by 10, 104, 107-fold, the visible color

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change occurred after incubation of 2, 7, and 12 h, respectively (Figure 1b). In

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contrast, the negative controls showed no color change even after 48-h incubation.

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Wells incubated with the sterilized cell suspension (as a sterilized control) and

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deionized water (as a blank control) also exhibited no color change. These results

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clearly indicate that the WO3-MPN method was effective for quantifying the

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abundance of EEB.

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Enumeration of EEB in Environmental Samples. The WO3-MPN method was

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also applied for samples from sediments of Chao Lake and the WWTP. The NFH

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sample from Chao Lake sediments showed the highest abundance of EEB, about

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4.1×106 cells/g dry sediment, while the XHX samples had the lowest counts, about

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9.9×104 cells/g dry sediment (Table 1). The abundance of EEB at different sampling 8


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sites of the WWTP varied substantially, from 1.0×103 cells/mL (effluent) to 7.5×105

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cells/mL (influent) (Table 2). The corresponding total bacteria counts by DAPI

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counting were 1.6×106 cells/mL in effluent and 1.3×108 cells/mL in influent. The

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estimated EEB abundance here was close to those of the iron reducers counted by

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FeOOH-MPN method21 and the sulfate-reducing bacteria (SRB) counted by

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35

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total bacteria.22 The sterilized samples exhibited no color change, excluding the

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interference of abiotic factors.

S-radiotracer-MPN method in an activated sludge sample with a similar level of

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The uneven distribution of EEB in environmental samples is associated with the

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different environmental properties. Pearson correlation analysis shows that the

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population sizes of EEB in the samples were highly correlated with their ammonium

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and phosphate contents (Tables 1 and 2). This finding of higher EEB abundance in

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nitrogen and phosphorous-rich samples is consistent with previous studies. Pinhassi &

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Berman reported that addition of nitrogen and phosphorous caused a fastest growth of

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Shewanella putrefacien.23 Also, the occurrence and abundance of Shewanella tend to

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decline when environments become more oligotrophic.24 Similarly, the abundance of

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Geobacter spp. was also strongly affected by phosphorus level.25 This result implies

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that abundant EEB might inhabit in eutrophic freshwater lakes.

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Features of the WO3-MPN Approach. Various bacteria species including EEB

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in environmental samples were detected and enumerated. Several EEB detection

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methods and their features are listed in Table S3. The PCR-MPN method has been

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previously used to detect and enumerate microorganisms in environmental samples,11 9



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but specific primes have to be designed for different EEB.26 Until now no common

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primers for EEB are available because of the diversity of EEB species and their

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complexity in extracellular electron transfer pathways. The Fe(III) reduction-MPN

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method can also be used to detect and enumerate EEB, but needs a long time,12, 24 and

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may give false positive results because iron oxides cannot truly distinguish EEB (e.g.,

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Pelobacter carbinolicus).27

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The WO3 probe method showed consistent results with the current generation test,

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indicating a good selectivity of this method for EEB detection.10 Its reliability was

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further validated by the results of the several controls (Figure S2). Overall, the

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WO3-MPN approach allows a simple, broad-spectrum and rapid detection and

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quantification of EEB. However, the WO3 probe method alone cannot provide a

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quantitative estimation of EEB population size in environmental samples. This

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drawback was solved by its combination with the MPN method here. Notably, the

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WO3-MPN method is ineffective for uncultured microbes, which is a common

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limitation for all culture-dependent methods used for complex microbial ecosystems.

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Overcoming this restriction may rely on the development of more sensitive

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nano-probes for single-cell scale detection, which warrants further studies.

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Distribution of EEB in Fresh Lake Sediments and WWTP. EEB in anoxic

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environments, especially in marine sediments, are heavily involved in the anaerobic

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degradation of organic matters and reduction of iron and manganese oxides.6 However,

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so far information about their roles in fresh lake sediments is very limited.25 A recent

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work revealed that EEB were largely involved in the biogeochemical Fe cycling in 10


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hypersaline sediments of a salt lake,12 but their abundance in hypersaline sediments

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was much lower than that in fresh lake sediments obtained in this study. Such a

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difference implies that EEB might be involved in the degradation of organic matters

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and possibly biogeochemical cycling of other elements in fresh environments.

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Information about the abundance of EEB in WWTP is also lacking. Our results,

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for the first time, clearly show the existence of EEB at a considerable level in the

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influent and mixed liquor in WWTP. Their presence in the influent might be attributed

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to the anoxic environments and steel material in sewage pipelines,28 which provide

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favorable habitats for EEB. For the mixed liquor samples, the EEB abundance in the

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anaerobic tank was similar to that in the influent because of the anaerobic conditions.

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The EEB abundance in the anoxic and aerobic tanks decreased because of the

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unfavorable aeration environments. It should be noticed that the EEB in the plant

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were at a similar level to SRB, another class of bacteria significantly affecting the

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activity and stability of activated sludge.22 EEB were found to compete with SRB for

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electron donors in estuarine sediments.29 These findings imply that EEB might also

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play some yet unknown roles in activated sludge process. This warrants further

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investigations.

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Due to lack of efficient detection tools, acquired information about the

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distribution and abundance of EEB in natural and engineered environments is scarce,

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which leads to poor understanding about the roles of EEB in bioremediation and

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bio-electrochemical processes and has limited their practical application. The

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WO3-MPN method developed in this work can be used to explore the ecology and 11



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distribution of EEB in natural and engineered environments and to facilitate a better

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play of their roles in biogeochemical cycling, environmental bioremediation and

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wastewater treatment systems.

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ASSOCIATED INFORMATION

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Supporting Information Available. Detailed descriptions about microbial cultures

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and MPN medium preparation; abiotic, positive and negative control tests for the

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WO3 method; analytical methods of environmental samples; sampling sites in Chao

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Lake and the WWTP (Figure S1); WO3 test results with model bacteria, carbon

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sources, fermentation products and different concentrations of inorganic or organic

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species (Figure S2); current densities of the MECs and MFCs with E. coli inoculation

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(Figure S3); cell counting of S. oneidensis MR-1 (Table S1); cell counting of G.

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sulfurreducens DL-1 (Table S2), and several bacterial detection methods and their

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features (Table S3). This information is available free of charge via the Internet at

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http://pubs.acs.org/.

229 230

AUTHOR INFORMATION

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*Corresponding authors:

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Dr. Feng Zhang, Fax: +86 551 63601592; E-mail: [email protected];

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Prof. Han-Qing Yu, Fax: +86 551 63601592; E-mail: [email protected]

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Notes

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The authors declare no competing ﬁnancial interest. 12


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ACKNOWLEDGEMENTS

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This work is supported by the National Natural Science Foundation of China

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(21477120, 21590812 and 51538012), the Program for Changjiang Scholars, Ministry

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of Education, China.

241 242

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Table 1 Distribution of EEB, NH3-N, Nitrite, Phosphate, OM (Organic Matter), Ferrous and Ferric in the Sediments from Different Sampling Sites. Sample

Abundance

NH3-N

Nitrate

P

site

cells/g

mg/kg

mg/kg

mg/kg

NFH

(4.12±3.47)×106

506.5

2.77

3975.3

8.16 20945.5

905.7

AB

(2.05±0.89)×106

329.6

0.18

759.9

3.31

5744.4

333.9

NFHK

(5.78±2.10)×105

98.4

2.63

1411.8

4.37

6940.1

1191.6

XHX

(9.94±4.72)×104

71.2

4.45

1542.8

4.39 12600.3

987.2

HX

(2.86±3.24)×105

47.5

3.27

402.1

4.41 12179.9 1277.2

DHX

(3.13±1.21)×105

36.6

1.79

144.7

4.18 10300.0 1905.7

0.98

-0.31

0.81

0.76

ra a

OM Ferrous Ferric %

mg/kg

0.58

mg/kg

-0.50

Pearson’s correlation of the abundance of EEB and environmental factors.

Abundance is the average of three replicate of MPN counts.

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Page 19 of 22


Table 2 Distribution of EEB, Total Bacteria (TB), Temperature (T), Dissolved Oxidation (DO), pH, Oxidation-Reduction Potential (ORP), Suspended Solids (SS) and Volatile Suspended Solids (VSS), Total Organic Carbon (TOC), NH3-N, Nitrate and Phosphate in the Activated Sludge Samples from Different Sections of the WWTPa. Sample

Abundance

TB

site

cells/mL

cells/mL

INF

(7.5±0.3)×105

AN

o

DO

pH

ORP

TOC

SS

VSS NH3-N nitrate

P

mV

mg/L

g/L

g/L

mg/L

mg/L

mg/L

C

mg/L

1.3×108

14.2

0.9

7.3

87

20.9

3.2

2.0

23.8

0.4

2.4

(4.12±0.3)×105

1.7×109

9.2

1.7

7.2

67

10.9

7.3

4.2

17.9

0.2

9.1

ANO

(8.7±0.5)×104

1.3×109

9.1

1.3

6.9

65

8.6

6.2

2.8

0.3

6.8

0.1

AER

(7.5±0.3)×104

1.4×109

14.0

1.3

6.6

64

12.5

5.2

1.3

0.3

6.9

0.1

EFF

(1.0±0.2)×103

1.6×106

14.3

3.8

6.5

63

9.4

0.2

0.2

0.1

13.5

0.0

-0.70

-0.07

-0.53

0.93

0.83

0.77

0.24 0.54

0.99

-0.90

0.66

r a

T

INF for influent of wastewater treatment plant, AN for the sludge collected from the anaerobic tank, ANO

for the sludge collected from the anoxic tank, AER for the sludge collected from the aerobic tank, EFF for the plant effluent.

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Figure Captions

Figure 1. Color change of WO3-nanocluster after 48-h incubation. S: sterilized control incubated with steriled cell suspension of S. oneiensis MR-1; P: positive control incubated with cell suspension of S. oneiensis MR-1; B: black control incubated with sterilized deioned water (a); Variations of color intensity over time for different dilutions of S. oneidensis MR-1. The error bars indicate the standard deviations of five replications (b).

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Page 20 of 22

Page 21 of 22


Figure 1

21



Table of Contents (TOC)

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Page 22 of 22

Rapid Detection and Enumeration of Exoelectrogenic Bacteria in Lake

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