Short Communication

A Microbial Sensor Based on Direct Electron Transfer at Shewanella Sp. Drop-Coated Screen-Printed Carbon Electrodes Kariate Sudhakara Prasad,a A. B. Arun,b P. D. Rekha,b Chiu-Chung Young,b Jen-Lin Chang,a Jyh-Myng Zena* a

Department of Chemistry, National Chung Hsing University, Taichung 402, Taiwan Department of Soil and Environmental Sciences, National Chung Hsing University, Taichung 402, Taiwan *e-mail: [email protected]


Received: February 5, 2009 Accepted: April 22, 2009 Abstract Aerobically grown Shewanella sp. bacterial suspension drop-coated on a disposable screen-printed carbon electrode was found to possess electroactivity without the aid of redox mediator. Cyclic voltammetric studies revealed the characteristics of a mixed diffusion adsorption-controlled electrochemical process for direct electron transfer at the bacteria-modified electrode. Both FE-SEM and ATR FT-IR experiments were carried out to investigate the surface characteristics. The electroanalytical applicability was further demonstrated for electrocatalytic reduction of arsenite, hydrogen peroxide and nitrite. Low cost and very simple manufacturing procedure allow for the proposed bacterial sensor to be applied as disposable devices. Keywords: Electrochemistry, Shewanella sp., Screen-printed carbon electrode, Electron transfer DOI: 10.1002/elan.200904605

Previous studies have reported that metal-reducing bacteria, such as Geobacter, Rhodoferax, and Shewanella, can transfer electron to electrode to provide a wide variety of applications [1 – 3]. Among these metal-reducing bacteria, Shewanella sp. is known for the role in biogeochemical cycling of iron, manganese, trace elements and phosphates. The applications in microbial fuel cell and bioremediation of water and sediment contaminated with organics, metals and radionuclides were also reported earlier [3 – 7]. The physical contact of the outer membrane or membrane appendage of the bacteria is believed to play an important role in the electron transfer process between the electrode and bacteria [5, 7 – 8]. Normally direct electron transfer can be observed by cyclic voltammetry of either the bacterial cell suspension or the bacteria-immobilized graphite electrode or by a fuel cell type electrochemical cell [3, 9]. We report here an easy method with the applicability of a disposable screen-printed carbon electrode (SPCE) to study the direct electron transfer reaction at Shewanella sp. bacteria. The SPCE was modified with Shewanella sp. CCGIMA-1 by a simple drop-coating procedure and cyclic voltammetric techniques were applied for further studies. Note that the growing of bacterial biofilm on electrode material is laborious and time consuming. Similar to graphite electrode, the SPCE presents a suitable hydrophobic surface able to adsorb many organic redox couples, enabling to study electrochemical charge transfer processes [10]. While most of the works relate to solution electrochemistry, it is of interest to study this species adsorbed or immobilized on electrode surface. The present work has been aimed to

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study the direct electron transfer of Shewanella sp. coated on a disposable SPCE for sensor application. Bacterial strain CC-GIMA-1 used in this study was isolated by incubating the gut content of a sea abalone after appropriate dilution and subsequent cultivation on marine agar 2216 plates (Difco) at 37 8C. The 16S rRNA gene of this strain CC-GIMA-1 was a continuous stretch of 1448 bp (DDBJ/EMBL/GenBank accession number EU794725), analyzed as described previously [11]. A comparison of the sequence with those of representatives of the genera classified in the family Shewanellaceae of Gammaproteobacteria showed that the organism fells within the evolutionary radiation occupied by the genus Shewanella. Sequence similarity calculations based on pair-wise alignment obtained using EzTaxon database [12] indicated highest degree of similarity to Shewanella algae OK-1T (GenBank accession no; AF005249) and Shewanella haliotis DW01T (GenBank accession no; EF178282) sharing a 16S rRNA gene sequence similarity of 99.5% and 99.3%, respectively. Strain CC-GIMA-1 was grown on marine broth 2216 plates and incubated for 48 h at 30 8C on a shaker at 150 rpm under aerobic conditions. After incubation period, cells were harvested by centrifugation, washed twice using sterile water and resuspended in sterile 0.1 M, pH 7.4 phosphate buffer solution (PBS). Bacterial numbers were determined by dilution plating method after spreading cells on marine agar 2216 plates and incubating at 30 8C. Here, 7 mL of Shewanella sp. CC-GIMA-1 bacterial suspension (prepared in 0.1 M, pH 7.4 PBS) was drop-coated on the electrode surface and allowed to settle under room temperature for 1 h, as illustrated in Figure 1A. The bacteria-coated SPCEs  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


Sensor Based on Direct Electron Transfer

were used for further experiments. The FE-SEM images, as shown in Figure 1B, verify that the modified electrode surface is covered densely with rod-shaped Shewanella sp. CC-GIMA-1 like a layer of biofilm. To further confirm the modification, ATR FT-IR spectrum of SPCE with/without bacteria-coated were recorded for comparison. As can be seen, no characteristic functional group was observed at a bare SPCE. Whereas, a band at ca. 3247 cm1 attributed to N-H (amides) and O-H vibrations and the 2800 – 3000 cm1 range representing the characteristic of CH3, > CH2 and > CH stretching vibrations in fatty acids [14] were noticed at the bacteria-coated SPCE. The mid-IR region from 1800 to 900 cm1 indicates the absorbance of phospholipids and polysaccharides and peaks in the 1700 – 1500 cm1 region correspond to the amide I and amide II bands of proteins and peptides. The main amide I peak at ca. 1638 cm1 corresponds to the stretching C ¼ O and bending CN vibration modes and 1544 cm1 belongs to amide II bending

NH and stretching CN vibration modes [15 – 16]. The band observed at ca. 1239 cm1 is assigned to PO4 asymmetric stretching vibration in the phospholipids and nucleic acids and the 1200 – 900 cm1 belongs to the CO vibration in carbohydrates of cell wall and cell membranes [15, 17]. Most importantly, surface characterization validates that simple drop-coating can indeed construct a well adsorbed bacterial layer on the surface of SPCE. This is an easy alternative procedure compared to the growing of bacterial colonies on the electrode surface under specified conditions for long duration. Note that the survivability of the cells was confirmed by plating (marine agar 2216, Difco) the coated bacteria after suspending the electrodes in sterile PBS. The direct electrochemistry of Shewanella sp. CC-GIMA1 modified electrode was next studied. A well-defined cathodic reduction peak can only be observed at the bacteria-coated electrode (Fig. 2A). In other words, the aerobically grown Shewanella sp. CC-GIMA-1 is in fact

Fig. 1. A) Schematic representation of the modification process of the SPCE strip with a built-in three-electrode configuration. B) ATR FT-IR spectrum and FE-SEM images of SPCE before (a) and after (b) coated with the Shewanella sp. bacteria.  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


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K. S. Prasad et al.

electroactive in nature. Since the reduction peak is close to that of oxygen reduction reaction, the cyclic voltammogram in anaerobic condition was also studied. The fact that a similar cathodic reduction peak was observed depicts an electron transfer between the electrode and bacteria without any mediators or cross-linkers. Furthermore, the mechanically modified bacterial electrode also shows similar voltammetric behaviors obtained for Shewanella sp.colonized electrodes in anaerobic condition [18, 19]. In other words, the well adsorbed bacterial layer on the electrode exhibits an electron exchange with electrode surface under given conditions and behaves similar to a bacterial biofilm. It is well known that graphite electrodes containing carbon-oxygen functional group similar to humates provides a natural habitat for the bacteria [10]. Here, the SPCE also possesses carbon-oxygen functionalities and the oxygen to carbon atomic ratio for SPCE was found to be 0.07 in our previous XPS analysis [20]. The formation of simulated biofilm by simply drop-coating the bacteria suspension is thus an easier alternative to growing a biofilm on the electrode surface. These bacteria films can sustain themselves by using carbon-oxygen functionalities of SPCE as electron acceptors for respiration. Overall, the disposable SPCE strip is promising to study the direct microbial electron transfer. Recent studies showed that flavins secreted by Shewanella sp. mediate the extracellular electron transfer and the yield of flavins produced by Shewanella sp. was the same under both aerobic and anaerobic conditions, also a percentage of the flavins are adsorbed to the electrode whenever soluble flavins are present [18, 21]. Based on these facts, we hypothesize that the adsorbed species on Shewanella sp.coated SPCE is flavins. Nonetheless, melanin is also proposed as an endogenous electron shuttle for Shewanella algae [22] and the Shewanella sp. studied here have greatest degree of similarity to type strains of Shewanella algae OK1T. In-depth studies on the characteristics of Shewanella sp. CC-GIMA-1 can certainly resolve the uncertainty in the electroactive component. The modified electrode was electrochemically studied to find the dependence of reduction peak current (ip) to scan rate (v) by scanning between 1 mV s1 to 70 mV s1. As shown in Figure 2B, the peak potential was found to shift gradually with an increase in the scan rate and simultaneously the peak current also increased. Most importantly, a linear log ip versus log v plot with a slope value of 0.65 was observed. This would indicate the existence of an intermediate case between the diffusion-controlled redox process and the surface-bound redox reaction at the bacteriacoated SPCE [23]. Most probably the deviation from the behavior characteristic of a surface-located process is caused by relatively slow charge propagation through the layer of adsorbed riboflavin. Note that cyclic voltammogram of the washed bacteria-coated electrode after electrochemical experiments showed a reduction peak shifted much away from the original bacterial response. So the introduction of a new peak can be due to the electroactive species adsorbed on the electrode surface. Electroanalysis 2009, 21, No. 14, 1646 – 1650

Fig. 2. A) Cyclic voltammograms of SPCE before (a) and after coated with the Shewanella sp. bacteria under aerobic (b) and anaerobic (c) condition at a scan rate of 30 mV s1. B) Cyclic voltammograms of Shewanella sp. modified SPCE at different scan rates of 1, 3, 5, 10, 30, 50, and 70 mV s1, respectively, and the obtained linear log ip vs. log v plot.

To evaluate the ability of the bacteria-coated electrode for electroanalysis, the electrocatalytic responses of the Shewanella sp. modified electrode for arsenite, hydrogen peroxide and nitrite were studied. Figure 3 shows that the reduction peak current of the Shewanella sp. modified electrode is increased in the presence of respective analytes. No obvious direct reduction of samples of interest was observed at a bare SPCE. The increase in the direct reduction peak current was linear with respect to the increase in concentration of hydrogen peroxide, nitrite and arsenite. Similar voltammetric behavior was also observed for reduction of Fe(III) species (data not shown). As reported previously, the application of Shewanella sp. is important in bioremediation and microbial fuel cells as it can respire with a diverse array of electron acceptors in


 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


Sensor Based on Direct Electron Transfer

Experimental All chemicals used are of ACS-certified reagent grade. Aqueous solutions were prepared with double distilled deionized water. Electroanalytical measurements were carried out at room temperature (24  1 8C) under aerobic conditions with a BAS 50W electrochemical workstation. The integrated three-electrode strip consisting of a carbon working electrode, a silver pseudo-reference electrode and a carbon counter electrode was purchased from Zensor R&D (Taichung, Taiwan) [13]. Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) spectrums were recorded at a Perkin-Elmer Spectrum 100, FT-IR spectrometer. Morphological observation was performed at a Field Emission-Scanning Electron Microscopy (FE-SEM) (JEOL, JSM-7401F SEM, Japan). Acknowledgements The authors gratefully acknowledge financial support from the National Science Council of Taiwan. This work is supported in part by the Ministry of Education, Taiwan under the ATU plan. References

Fig. 3. Cyclic voltammetric responses of A) arsenite, B) hydrogen peroxide, and C) nitrite on Shewanella sp. modified SPCE at a scan rate of 30 mV s1.

extreme environments [6]. Since Shewanella sp. are Fe(III)reducing bacteria, so the results are correlated well to the property of the bacteria. Bacteria modified electrode has been successfully fabricated by a simple drop-coating procedure on a disposable SPCE. Electrochemical observation demonstrates the formation of an electroactive bacterial layer with direct electron transfer property. The surface morphological studies provide the evidence of the formation of bacterial layer and the adhesion of the bacteria on the SPCE surface. The Shewanella sp. CC-GIMA-1 shows good surface binding affinity towards the SPCE and can electrochemically reduce arsenite, hydrogen peroxide and nitrite. The method applied here for the preparation of bacterial electrode is simple and fast.  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

[1] D. R. Bond, D. R. Lovley, Appl. Environ. Microbiol. 2003, 69, 1548. [2] D. E. Holmes, D. R. Bond, D. R. Lovely, Appl. Environ. Microbiol. 2004, 70, 1234. [3] B. H. Kim, H. J. Kim, M. S. Hyun, D. H. Park, J. Microb. Biotech. 1999, 9, 127. [4] S. K. Chaudhuri, D. R. Lovley, Nat. Biotechnol. 2003, 21, 1229. [5] Y. A. Gorby, S. Yanina, J. S. McLean, K. M. Rosso, D. Moyles, A. Dohnalkova, T. J. Beveridge, I. S. Chang, B. H. Kim, K. S. Kim, D. E. Culley, S. B. Reed, M. F. Romine, D. A. Saffarini, E. A. Hill, L. Shi, D. A. Elias, D. W. Kennedy, G. Pinchuk, K. Watanabe, S. Ishii, B. Logan, K. H. Nealson, J. K. Fredrickson, Proc. Natl. Acad. Sci. USA 2006, 30, 11358. [6] H. H. Hau, J. A. Gralnick, Ann. Rev. Microbiol. 2007, 61, 237. [7] G. Reguera, K. McCarthy, T. Mehta, J. S. Nicoll, M. T. Tuominen, D. R. Lovley, Nature 2005, 7045, 1098. [8] Y. Qiao, C.-M. Li, S.-J. Bao, Z. Lu, Y. Hong, Chem. Commun. 2008, 11, 1290. [9] S. Srikanth, E. Marsili, M. C. Flickinger, D. R. Bond, Biotechnol. Bioeng. 2008, 99, 1065. [10] S. R. Crittenden, C. J. Sund, J. J. Sumner, Langmuir 2006, 22, 9473. [11] C.-C. Young, P. Kmpfer, F.-T. Shen, W.-A. Lai, A. B. Arun, Int. J. Syst. Evol. Microbiol. 2005, 55, 423. [12] J. Chun, J.-H. Lee, Y. Jung, M. Kim, S. Kim, B. K. Kim, Y. W. Lim, Int. J. Syst. Evol. Microbiol. 2007, 57, 2259. [13] J.-C. Chen, H.-H. Chung, C.-T. Hsu, D.-M. Tsai, A. S. Kumar, J.-M. Zen, Sens. Actuators B 2005, 110, 364. [14] D. Helm, H. Labischinski, G. Schalleh, D. Naumann, J. Gen. Microbiol. 1991, 137, 69. [15] A.-M. Melin, A. Perromet, G. Deleris, Appl. Spectrosc. 2001, 55, 23. [16] S. J. Parikh, J. Chorover, Langmuir 2006, 22, 8492. [17] S. J. Parikh, J. Chorover, Geomicrobiol. J. 2005, 22, 207.


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[18] E. Marsili, D. B. Baron, I. D. Shikhare, D. Coursolle, J. A. Gralnick, D. R. Bond, Proc. Natl. Acad. Sci. USA 2008, 105, 3968. [19] H. J. Kim, H. S. Park, M. S. Hyun, I. S. Chang, M. Kim, B. H. Kim, Enzyme Microbial Technol. 2002, 30, 145. [20] K. S. Prasad, G. Muthuraman, J.-M. Zen, Electrochem. Commun. 2008, 10, 559.

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[21] H. von Canstein, J. Ogawa, S. Shimizu, J. R. Lloyd, Appl. Environ. Microbiol. 2008, 74, 615. [22] C. E. Turick, L. S. Tisa, Jr. F. Caccavo, Appl. Environ. Microbiol. 2002, 68, 2436. [23] C. G. Granqvist, Handbook of Inorganic Electrochromic Materials, Elsevier, Amsterdam, The Netherlands 1995.


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