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Harvesting energy of interaction between bacteria and bacteriophage in a membrane-less fuel cell
Bioresource Technology 147 (2013) 654–657
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Harvesting energy of interaction between bacteria and bacteriophage in a membrane-less fuel cell Ragini Gupta a,b, Wasihun Bekele a,1, Animangsu Ghatak a,b,⇑ a b
Department of Chemical Engineering and DST Unit on Soft Nanofabrication, Indian Institute of Technology Kanpur, 208016, India Center for Environmental Science and Engineering, Indian Institute of Technology Kanpur, 208016, India
h i g h l i g h t s This study is first to use bacteria–phage interaction for potential generation. Potential generation is not due to bacterial lysis by bacteriophage. Potential generation does not depend upon bacteria–phage specificity. Our microbial fuel cell does not require oxygen.
a r t i c l e
i n f o
Article history: Received 10 June 2013 Received in revised form 13 August 2013 Accepted 14 August 2013 Available online 23 August 2013 Keywords: Bacteria Bacteriophage Energy Membrane-less microbial fuel cell Potential
a b s t r a c t When a fuel and oxidant flow in laminar contact through a micro-fluidic channel, a sharp interface appears between the two liquids, which eliminate the need of a proton exchange membrane. This principle has been used to generate potential in a membrane-less fuel cell. This study use such a cell to harvest energy of interaction between a bacteria having negative charge on its surface and a bacteriophage with positive and negative charges on its tail and head, respectively. When Klebsiella pneumoniae (Kp6) and phage (P-Kp6) are pumped through a fuel cell fitted with two copper electrodes placed at its two sides, interaction between these two charged species at the interface results in a constant open circuit potential which varies with concentration of charged species but gets generated for both specific and non-specific bacteria and phage system. Oxygenation of bacteria or phage however diminishes the potential unlike in conventional microbial fuel cells. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Microorganisms like bacteria can be used for converting complex organic matter to simple organic and inorganic species such as carbon dioxide, electron and proton. This principle has been used to disinfect organic waste present in water while also generating electrical energy in a fuel cell (Rabaey and Verstraete, 2005; Wang et al., 2013). Fuel cells of this kind, known as microbial fuel cell (MFC), essentially consists of two separate chambers, anode and cathode, for the fuel and the oxidant respectively, separated by a proton exchange membrane (PEM). In a continuo us operation, the microorganisms mixed with the organic material are pumped into the anode chamber where they oxidize the substrate releasing electrons and protons. The electrons get collected at the anode and are transported to the cathode by the external circuit while the ⇑ Corresponding author at: Department of Chemical Engineering and DST Unit on Soft Nanofabrication, Indian Institute of Technology Kanpur, 208016, India. Tel.: +91 512 2597146. E-mail address: [email protected] (A. Ghatak). 1 Present address: Defence Engineering College, Debre Zeit, Ethiopia. 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.08.091
protons are transferred through the PEM to the cathode chamber where it accepts the electrons and reacts with the oxygen to produce water (Lovley, 2006). Since their invention, several new designs of MFCs have been developed to increase their efficiencies. The new generation MFCs could be single or double chambered, with or without PEM or a separator (Davis and Higson, 2007; Kim et al., 2008; Lin et al., 2013; Qian et al., 2011). While these MFCs all involve oxidation of an organic nutrient by a microbe, there is no example in which an electrode potential is generated as a result of interaction between two microbial species of different types. This article explores this possibility by bringing in contact a media containing bacteria with one containing the bacteriophage (or phage). Phages attach to the specific receptors on the surface of bacteria, such as lipopolysaccharides, teichoic acids, proteins or flagella, hence infects only certain bacteria bearing the specific receptors, which in turn determines the host range of the phage (Rakhuba et al., 2010). The specific interaction between bacteria and phage has been used in several applications: for eliminating bacterial infection in food items, e.g., that of Listeria in poultry foods (Anany et al., 2011), as a therapeutic strategy in which phage is used to kill a targeted bacteria for curing infection (Gupta and
R. Gupta et al. / Bioresource Technology 147 (2013) 654–657
Prasad, 2011), for protecting live stocks in veterinary, agriculture and aquaculture from bacterial infections (Monk et al., 2010), for enhancing antibacterial effect via conjugation of the capsid protein of phage with strong antibacterial metal particles like silver, for immobilizing phage on biocompatible surfaces like cellulose membrane for variety of purposes (Anany et al., 2011; Cademartiri et al., 2010), for developing biosensors (Singh et al., 2013) and so on. This study describes the use of facultative anaerobic pathogenic bacteria, the Klebsiella pneumoniae, known to cause disease Klebsiella pneumonia leading to lung inflammation and haemorrhage, and the bacteriophage specific to it, to show that it may be possible to harvest energy via their interaction. In essence, media containing bacteria and phage, respectively were pumped through the two inlets of a ‘‘Y’’ shaped microchannel at steady laminar flow. A sharp interface forms between the two liquids at which the two charged species interact which finally result in an open circuit potential. This potential remains a strong function of the concentration of the bacteria and the phage in the respective medium. It is assumed that a charge gradient is formed across each side which finally results in an electrode potential (Arun et al., 2013; Chang et al., 2006). 2. Methods 2.1. Bacterial strain K. pneumoniae (Kp6) was isolated from human clinical sample (urine) collected from a hospital in Uttar Pradesh, India. Sample was streaked onto the MacConkey agar medium. Lactose fermenting colony was analyzed on Klebsiella selective medium. The mucoid magenta colony was subjected to Gram staining, colony morphology and selected biochemical tests (catalase, oxidase, Voges–Proskauer, acid production from glucose, citrate utilization) following the method of Cowan and Steel (Cowan and Steel, 1975). Kp6 is a Gram-negative, rod shaped, lactose fermenting and catalase positive bacteria. Kp6 displayed positive response to Voges–Proskauer, acid production from glucose and citrate utilization tests. The bacterial count was expressed in terms of colony forming units (cfu, number of bacteria per ml of culture). All subsequent bacterial cultures K. pneumoniae (Kp6) were done in NZCYM media (Himedia). 2.2. Lytic bacteriophage To isolate lytic phage, 1 ml log phase K. pneumoniae (Kp6) culture in NZCYM broth (pH 7), 10 ml 2X NZCYM broth and 10 ml filter sterilized (0.22 lm, Millipore) sewage water were combined and incubated at 37 °C overnight in a shaking incubator at 100 revolution min1. Next day, lysate was centrifuged at 5000 revolution min1 (Remi R24) to remove cell debris and clear supernatant was filtered (0.22 lm, Millipore) to obtain bacteria-free filtrate (BFF). BFF was analyzed for the occurrence of lytic phage, if any, by soft-agar overlay method (Adams, 1959). The final titre of phage stock was 1015 pfu/ml (plaque forming unit per milliliter of stock). 2.3. Preparation of microchannel ‘‘Y’’ shaped microchannel was prepared by micro-molding of polydimethylsiloxane (PDMS) (Sylgard 184 elastomer, procured from Dow Corning) mixed with the curing agent (10:1 by weight), on a suitable template (Arun et al., 2013) (dimensions: height, H = 0.3 mm, width, w = 6 mm and length, L = 25 mm) (Fig. 1a). Followed by crosslinking of the polymer, the channel was closed by bonding the solid PDMS block onto a plasma oxidized microscope glass slide. Two thin strips of copper (thickness, 50 lm, size, 0.1 3 cm), used as cathode and anode, were connected to a data
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acquisition card (NI-6009) which was interfaced with a computer to measure the open circuit potential (OCP). 2.4. Experimental procedure Exponentially grown (109 cfu/ml) bacteria K. pneumoniae (Kp6) in NZCYM broth media (pH 7) and phage (P-Kp6, 1015 pfu/ ml) (pH 7) were injected into two separate inlets of the microchannel using a syringe pump (Harvard Apparatus, USA) at equal flow rates. The Reynolds number Re ¼ deq v q=l of flow of both the liquids was maintained low: 0.2–6, so that the flow was essentially laminar. Here, deq ¼ 2wH=ðw þ HÞ represents the hydraulic diameter of the channel, v ¼ Q =ðwH=2Þ represents the velocity of each liquid. Q is the flow rate of liquids. q and l denote the density and viscosity of the liquids, respectively. 3. Results and discussion 3.1. Potential generation is a function of flow time but not of the flow rate In contrast to conventional microbial fuel cells, here two liquids flow in parallel forming a sharp interface at which the bacteria and the phage contact. As a result of the electrostatic interaction between the negative charges on the surface of the bacteria with that of the positive charge on the tail portion of the phage, a potential develops. However, this potential does not develop instantly but over some period of time, during which the open circuit potential of the fuel cell continues to rise, finally reaching a steady state value. The data in Fig. 1b shows that for flow rate varying over an order of magnitude, i.e., Q = 20600 ll/min, the time required for reaching the final plateau value of potential, Emax = 0.29 ± 0.01 volt is found to be sE = 90 min. So, neither Emax nor sE depends on Q suggesting that the observed growth of potential does not result from pressure driven flow of the fuel and oxidant streams. 3.2. Potential generation is not due to bacterial lysis by bacteriophage The residence time of the liquid (bacteria and phage) inside the channel, s = V/Q (V and Q, respectively denotes channel volume and liquid flow rate) was between 2.25 and 0.075 min for the flow rate ranging from 20 to 600 ll. Clearly, such a small residence time was insufficient for the lysis of bacteria to take place, which in a well mixed chamber occurs over 20–60 min (Shao and Wang, 2008). 3.3. Effect of bacteria and bacteriophage concentration on potential generation In order to find the effect of bacteria and phage concentration on Emax, two sets of experiments in triplicates were carried out: in the first set of experiments, NZCYM broth media containing varying concentration of bacteria: 103, 105, 107, 109, 1011, 1013 and 1015 cfu/ml was pumped into the channel, while phage concentration was kept constant at 1015 pfu/ml. In the second set of experiments, phage was pumped at different concentrations: 103, 105, 107, 109, 1011, 1013 and 1015 pfu/ml, while the bacteria was maintained constant at 109 cfu/ml. These experiments revealed that Emax increases with bacterial concentration till an intermediate concentration of 109 cfu/ml was reached at which Emax was found to be 0.26 ± 0.01 V; for concentration of bacteria exceeding this limit, Emax decreases (Fig. 2, line graph with white diamond, }). In the other set of experiments, the potential Emax was found to increase monotonically with increase in phage concentration, reaching maximum at phage density 1015 pfu/ml, at which Emax = 0.26 ± 0.04 V was achieved (Fig. 2, line graph with black
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bacteria
bacteriophage
(b) 0.3 20 40 60 200 400 600
E max
E interface anode
cathode
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0.1 0.0 0
20
40
60
80
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(a) Fig. 1. (a) Schematic of Y-shaped microchannel used as the fuel cell. (b) Growth of potential with respect to time for different flow rates of the two streams (bacteria and phage).
interaction between bacteria and phage is essential for potential generation in our MFC.
0.3
3.5. Supply of oxygen inhibits potential generation
0.2
E 0.1
0.0
103
105
Bacteria (
107
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Fig. 2. The maximum potential, Emax, generated for varying concentration of bacteria and phage. Error bars shows the standard deviations. Experiments were conducted in triplicate.
diamond, ). The phage concentration could not be further increased, because of non-availability of phage stock denser than 1015 pfu/ml. Thus, maximum value of Emax was achieved for bacterial density 109 cfu/ml and phage density 1015 pfu/ml. It is not clear at this stage, what causes the large difference in optimum concentration of these charged species, but possibly related to large difference in the sizes of the bacteria and the phage which results in a single bacteria being interacted with large number of phage particles.
Dissolved oxygen is another parameter that affects the output of a conventional fuel cell. In order to examine its effect on the bacteria–phage fuel cell, oxygenation and de-oxygenation of both the media containing the bacteria and the phage were performed by, respectively bubbling oxygen through it for a prolonged period and by placing it in vacuum (Rollie et al., 1987). Four kinds of experiments (in triplicates) were done in which either the media, or the bacteria, or the phage, or bacteria and phage both, were oxygenated. For all these cases, the potential decreased. Combinations: bacteria–oxygenated media, oxygenated bacteria–phage, bacteria– oxygenated phage and oxygenated bacteria–oxygenated phage, produced the average Emax value of 0.093 ± 0.025, 0.006 ± 0.003, 0.077 ± 0.025 and 0.008 ± 0.003 V, respectively (Fig. 3b, bars 1–4). These results therefore signify that oxygenation inhibits generation of OCP in our MFC, unlike in the conventional MFCs where presence of oxygen at cathode is essential to complete the red-ox reaction (Franks and Nevin, 2010; Wang et al., 2013). We believe that presence of oxygen generates OH ions which neutralize the charges on the surface of the phage, thereby interfering with the
0.3 0.2 0.1
3.4. Bacteria–bacteriophage interaction in microchannel generates potential
0.0 In order to examine if OCP generation in our MFC was indeed because of interaction between the bacteria and phage, different combinations of bacteria–phage–media were injected into the two inlets of the Y-shaped channel: (1) bacteria (109 cfu/ml) and phage (1015 pfu/ml), (2) bacteria (109 cfu/ml) and media, (3) media and phage (1015 pfu/ml), (4) bacteria (109 cfu/ml) in both sides, (5) phage (1015 pfu/ml) in both sides and (6) media in both sides. Experiments were conducted in triplicates to calculate the standard deviations. While combinations 4–6 yielded negligibly small potential, combinations 2 and 3 generated small potential of 0.14 ± 0.01 V and 0.13 ± 0.03 V, respectively. The maximum potential Emax = 0.29 ± 0.02 V was obtained for combination 1 (Fig. 3a, bars 1–6). These results therefore suggest that the
1 2 3 4 5 6
1 2 3 4 5 6 7
1 2 3
1 2
(a)
(b)
(c)
(d)
Fig. 3. (a) Bar chart shows potential generated for different combinations of bacteria–phage–media. Bar 1 represents bacteria–phage, 2: bacteria–media, 3: media–phage, 4: bacteria–bacteria, 5: phage–phage, and 6: media–media, respectively. (b) Bar chart shows the effect of oxygen on potential. Bar 1 represents bacteria–oxygenated media, 2: oxygenated bacteria–phage, 3: bacteria–oxygenated phage, 4: oxygenated bacteria–oxygenated phage, 5: deoxygenated bacteria–phage, 6: bacteria–deoxygenated phage and 7: deoxygenated bacteria–deoxygenated phage. (c) Potential generated for three different bacteria–phage system: bars 1, 2 and 3 represent K. pneunoniae (Kp6)–phage (P-Kp6), E. coli–phage (P-Kp6) and and S. aureus–phage (P-Kp6), respectively. (d) Potential generated by bacteria (Kp6)–HCl (bar 1) and NaOH–phage (P-Kp6) (bar 2), respectively. Three experiments were carried out for each of the four cases (a–d).
R. Gupta et al. / Bioresource Technology 147 (2013) 654–657
interaction between the phage and the bacteria. However, de-oxygenation of bacteria or phage or both did not result in any increase in potential. deoxygenated bacteria–phage, bacteria–deoxygenated phage and deoxygenated bacteria–deoxygenated phage produced potential, Emax, 0.23 ± 0.01, 0.28 ± 0.03 and 0.25 ± 0.03 V, respectively (Fig. 3b, bars 5–7) which are nearly equal to the Emax value obtained without de-oxygenation: 0.29 ± 0.02 V (Fig. 3a, bar 1). These results thus indicate that the oxygen present in the bacteria and/or phage stream is at miniscule level so that further removal of oxygen does not result in any change in potential. 3.6. Potential generation is not a function of bacteria–phage specificity In order to find out if specificity of bacteria–phage interaction is important for generation of the potential, following experiment was carried out: two heterogenous hosts were used: (a) Escherichia coli, Gram negative bacteria (low peptidoglycan in the cell wall, thick outer membrane with phospholipids and lipopolysaccharides imparting strong negative charge on bacterial surface), and (b) Staphylococcus aureus, Gram positive bacteria (high peptidoglycan in the cell wall with teichoic acid imparting strong negative charge on bacterial surface, no outer membrane), while using the same phage P-Kp6 as in earlier experiments. Plating of these two different bacteria with phage (P-Kp6) show that these bacteria does not get lysed by this non-specific phage (data not shown). For (a) and (b) the Emax was found to be 0.17 ± 0.02 and 0.18 ± 0.02 V (Fig. 3c, bars 2 and 3, respectively), which were somewhat smaller than that achieved when the specific host Kp-6 was used (Fig. 3c, bar 1). However, above experiments with heterogenous host signifies that potential gets generated not via lysis of bacterial cell by the phage as discussed above. 3.7. Inorganic ions can substitute bacteriophage but not the bacteria In order to examine if the supply of charged ions can substitute the bacteria and/or phage in fuel cell to produce voltage, two sets of experiments were performed, in the first set, bacteria was injected through one inlet to the channel, while through the other, solution of hydrochloric acid (pH 2) instead of phage was used as the donor for +ve ion. In the second set of experiment, solution of sodium hydroxide (pH 10) and phage were injected into the two sides of the channel. Interestingly, the potential generated in the first set of experiment was Emax = 0.21 ± 0.03 V (Fig. 3d, bar 1), almost equal to that achieved in the bacteria–phage system (Fig. 3a, bar 1). However, in the second experiment, the potential was negligibly small, 0.06 ± 0.02 V (Fig. 3d, bar 2), signifying that for the fuel cell described here, bacteria is essential while phage can be substituted by acid. 4. Conclusions Present study provides a novel mechanism of harvesting energy from interaction between two charged live microorganisms, bacteria and phage, using a membraneless microbial fuel cell which allows the two species to come in laminar contact at a sharp fluidic interface. The potential generated by this process is a strong function of both bacteria and phage concentration and both specific
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and non-specific bacteria–phage system can be used to generate the potential. Thus, this study has showed for the first time that pathogenic bacteria can be used for generating potential. 5. Conflict of interest Authors declare no conflict of interest. Acknowledgements R.G. acknowledges Department of Science and Technology, Government of India grants, Intensification of Research in High Priority Areas (IRHPA) and Fast Track Scheme For Young Scientists (SERB/ CHE/20120086), for financial support. A.G. acknowledges grant DST/CHE/20080165 from Department of Science and Technology, Government of India and Mr. and Mrs. Gian Singh Bindra Research Fellowship for this work. References Adams, M.H., 1959. Bacteriophages. Interscience, New York. Anany, H., Chen, W., Pelton, R., Griffiths, M.W., 2011. Biocontrol of Listeria monocytogenes and Escherichia coli O157:H7 in meat by using phages immobilized on modified cellulose membranes. Appl. Environ. Microbiol. 77 (18), 6379–6387. Arun, R.K., Bekele, W., Ghatak, A., 2013. Self oscillating potential generated in patterned micro-fluidic fuel cell. Electrochimica. Acta. 87, 489–496. Cademartiri, R., Anany, H., Gross, I., Bhayani, R., Griffiths, M., Brook, M.A., 2010. Immobilization of bacteriophages on modified silica particles. Biomaterials 31 (7), 1904–1910. Chang, M.H., Chen, F., Fang, N.S., 2006. Analysis of membraneless fuel cell using laminar flow in a Y-shaped microchannel. J. Power Sources 159 (2), 810–816. Cowan, S.T., Steel, K.J., 1975. Manual for Identification of Medical Bacteria. Cambridge University Press, London. Davis, F., Higson, S.P., 2007. Biofuel cells – recent advances and applications. Biosens. Bioelectron. 22 (7), 1224–1235. Franks, A.E., Nevin, K.P., 2010. Microbial fuel cells, a current review. Energies 3 (5), 899–919. Gupta, R., Prasad, Y., 2011. Efficacy of polyvalent bacteriophage P-27/HP to control multidrug resistant Staphylococcus aureus associated with human infections. Curr. Microbiol. 62 (1), 255–260. Kim, I.S., Chae, K.-J., Choi, M.-J., Verstraete, W., 2008. Microbial fuel cells : recent advances, bacterial communities and application beyond electricity generation. Environ. Eng. Res. 13 (2), 51–65. Lin, C.C., Wei, C.H., Chen, C.I., Shieh, C.J., Liu, Y.C., 2013. Characteristics of the photosynthesis microbial fuel cell with a Spirulina platensis biofilm. Bioresour. Technol. 135, 640–643. Lovley, D.R., 2006. Bug juice: harvesting electricity with microorganisms. Nat. Rev. Microbiol. 4 (7), 497–508. Monk, A.B., Rees, C.D., Barrow, P., Hagens, S., Harper, D.R., 2010. Bacteriophage applications: where are we now? Lett. Appl. Microbiol. 51 (4), 363–369. Qian, F., He, Z., Thelen, M.P., Li, Y., 2011. A microfluidic microbial fuel cell fabricated by soft lithography. Bioresour. Technol. 102 (10), 5836–5840. Rabaey, K., Verstraete, W., 2005. Microbial fuel cells: novel biotechnology for energy generation. Trends Biotechnol. 23 (6), 291–298. Rakhuba, D.V., Kolomiets, E.I., Dey, E.S., Novik, G.I., 2010. Bacteriophage receptors, mechanisms of phage adsorption and penetration into host cell. Pol. J. Microbiol. 59 (3), 145–155. Rollie, M.E., Patonay, G., Warner, I.M., 1987. Deoxygenation of solutions and its analytical applications. Ind. Eng. Chem. Res. 26 (1), 1–6. Shao, Y., Wang, I.N., 2008. Bacteriophage adsorption rate and optimal lysis time. Genetics 180 (1), 471–482. Singh, A., Poshtiban, S., Evoy, S., 2013. Recent advances in bacteriophage based biosensors for food-borne pathogen detection. Sensors (Basel) 13 (2), 1763– 1786. Wang, H., Jiang, S.C., Wang, Y., Xiao, B., 2013. Substrate removal and electricity generation in a membrane-less microbial fuel cell for biological treatment of wastewater. Bioresour. Technol. 138, 109–116.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Harvesting energy of interaction between bacteria and bacteriophage in a membrane-less fuel cell Ragini Gupta a,b, Wasihun Bekele a,1, Animangsu Ghatak a,b,⇑ a b
Department of Chemical Engineering and DST Unit on Soft Nanofabrication, Indian Institute of Technology Kanpur, 208016, India Center for Environmental Science and Engineering, Indian Institute of Technology Kanpur, 208016, India
h i g h l i g h t s This study is first to use bacteria–phage interaction for potential generation. Potential generation is not due to bacterial lysis by bacteriophage. Potential generation does not depend upon bacteria–phage specificity. Our microbial fuel cell does not require oxygen.
a r t i c l e
i n f o
Article history: Received 10 June 2013 Received in revised form 13 August 2013 Accepted 14 August 2013 Available online 23 August 2013 Keywords: Bacteria Bacteriophage Energy Membrane-less microbial fuel cell Potential
a b s t r a c t When a fuel and oxidant flow in laminar contact through a micro-fluidic channel, a sharp interface appears between the two liquids, which eliminate the need of a proton exchange membrane. This principle has been used to generate potential in a membrane-less fuel cell. This study use such a cell to harvest energy of interaction between a bacteria having negative charge on its surface and a bacteriophage with positive and negative charges on its tail and head, respectively. When Klebsiella pneumoniae (Kp6) and phage (P-Kp6) are pumped through a fuel cell fitted with two copper electrodes placed at its two sides, interaction between these two charged species at the interface results in a constant open circuit potential which varies with concentration of charged species but gets generated for both specific and non-specific bacteria and phage system. Oxygenation of bacteria or phage however diminishes the potential unlike in conventional microbial fuel cells. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Microorganisms like bacteria can be used for converting complex organic matter to simple organic and inorganic species such as carbon dioxide, electron and proton. This principle has been used to disinfect organic waste present in water while also generating electrical energy in a fuel cell (Rabaey and Verstraete, 2005; Wang et al., 2013). Fuel cells of this kind, known as microbial fuel cell (MFC), essentially consists of two separate chambers, anode and cathode, for the fuel and the oxidant respectively, separated by a proton exchange membrane (PEM). In a continuo us operation, the microorganisms mixed with the organic material are pumped into the anode chamber where they oxidize the substrate releasing electrons and protons. The electrons get collected at the anode and are transported to the cathode by the external circuit while the ⇑ Corresponding author at: Department of Chemical Engineering and DST Unit on Soft Nanofabrication, Indian Institute of Technology Kanpur, 208016, India. Tel.: +91 512 2597146. E-mail address: [email protected] (A. Ghatak). 1 Present address: Defence Engineering College, Debre Zeit, Ethiopia. 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.08.091
protons are transferred through the PEM to the cathode chamber where it accepts the electrons and reacts with the oxygen to produce water (Lovley, 2006). Since their invention, several new designs of MFCs have been developed to increase their efficiencies. The new generation MFCs could be single or double chambered, with or without PEM or a separator (Davis and Higson, 2007; Kim et al., 2008; Lin et al., 2013; Qian et al., 2011). While these MFCs all involve oxidation of an organic nutrient by a microbe, there is no example in which an electrode potential is generated as a result of interaction between two microbial species of different types. This article explores this possibility by bringing in contact a media containing bacteria with one containing the bacteriophage (or phage). Phages attach to the specific receptors on the surface of bacteria, such as lipopolysaccharides, teichoic acids, proteins or flagella, hence infects only certain bacteria bearing the specific receptors, which in turn determines the host range of the phage (Rakhuba et al., 2010). The specific interaction between bacteria and phage has been used in several applications: for eliminating bacterial infection in food items, e.g., that of Listeria in poultry foods (Anany et al., 2011), as a therapeutic strategy in which phage is used to kill a targeted bacteria for curing infection (Gupta and
R. Gupta et al. / Bioresource Technology 147 (2013) 654–657
Prasad, 2011), for protecting live stocks in veterinary, agriculture and aquaculture from bacterial infections (Monk et al., 2010), for enhancing antibacterial effect via conjugation of the capsid protein of phage with strong antibacterial metal particles like silver, for immobilizing phage on biocompatible surfaces like cellulose membrane for variety of purposes (Anany et al., 2011; Cademartiri et al., 2010), for developing biosensors (Singh et al., 2013) and so on. This study describes the use of facultative anaerobic pathogenic bacteria, the Klebsiella pneumoniae, known to cause disease Klebsiella pneumonia leading to lung inflammation and haemorrhage, and the bacteriophage specific to it, to show that it may be possible to harvest energy via their interaction. In essence, media containing bacteria and phage, respectively were pumped through the two inlets of a ‘‘Y’’ shaped microchannel at steady laminar flow. A sharp interface forms between the two liquids at which the two charged species interact which finally result in an open circuit potential. This potential remains a strong function of the concentration of the bacteria and the phage in the respective medium. It is assumed that a charge gradient is formed across each side which finally results in an electrode potential (Arun et al., 2013; Chang et al., 2006). 2. Methods 2.1. Bacterial strain K. pneumoniae (Kp6) was isolated from human clinical sample (urine) collected from a hospital in Uttar Pradesh, India. Sample was streaked onto the MacConkey agar medium. Lactose fermenting colony was analyzed on Klebsiella selective medium. The mucoid magenta colony was subjected to Gram staining, colony morphology and selected biochemical tests (catalase, oxidase, Voges–Proskauer, acid production from glucose, citrate utilization) following the method of Cowan and Steel (Cowan and Steel, 1975). Kp6 is a Gram-negative, rod shaped, lactose fermenting and catalase positive bacteria. Kp6 displayed positive response to Voges–Proskauer, acid production from glucose and citrate utilization tests. The bacterial count was expressed in terms of colony forming units (cfu, number of bacteria per ml of culture). All subsequent bacterial cultures K. pneumoniae (Kp6) were done in NZCYM media (Himedia). 2.2. Lytic bacteriophage To isolate lytic phage, 1 ml log phase K. pneumoniae (Kp6) culture in NZCYM broth (pH 7), 10 ml 2X NZCYM broth and 10 ml filter sterilized (0.22 lm, Millipore) sewage water were combined and incubated at 37 °C overnight in a shaking incubator at 100 revolution min1. Next day, lysate was centrifuged at 5000 revolution min1 (Remi R24) to remove cell debris and clear supernatant was filtered (0.22 lm, Millipore) to obtain bacteria-free filtrate (BFF). BFF was analyzed for the occurrence of lytic phage, if any, by soft-agar overlay method (Adams, 1959). The final titre of phage stock was 1015 pfu/ml (plaque forming unit per milliliter of stock). 2.3. Preparation of microchannel ‘‘Y’’ shaped microchannel was prepared by micro-molding of polydimethylsiloxane (PDMS) (Sylgard 184 elastomer, procured from Dow Corning) mixed with the curing agent (10:1 by weight), on a suitable template (Arun et al., 2013) (dimensions: height, H = 0.3 mm, width, w = 6 mm and length, L = 25 mm) (Fig. 1a). Followed by crosslinking of the polymer, the channel was closed by bonding the solid PDMS block onto a plasma oxidized microscope glass slide. Two thin strips of copper (thickness, 50 lm, size, 0.1 3 cm), used as cathode and anode, were connected to a data
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acquisition card (NI-6009) which was interfaced with a computer to measure the open circuit potential (OCP). 2.4. Experimental procedure Exponentially grown (109 cfu/ml) bacteria K. pneumoniae (Kp6) in NZCYM broth media (pH 7) and phage (P-Kp6, 1015 pfu/ ml) (pH 7) were injected into two separate inlets of the microchannel using a syringe pump (Harvard Apparatus, USA) at equal flow rates. The Reynolds number Re ¼ deq v q=l of flow of both the liquids was maintained low: 0.2–6, so that the flow was essentially laminar. Here, deq ¼ 2wH=ðw þ HÞ represents the hydraulic diameter of the channel, v ¼ Q =ðwH=2Þ represents the velocity of each liquid. Q is the flow rate of liquids. q and l denote the density and viscosity of the liquids, respectively. 3. Results and discussion 3.1. Potential generation is a function of flow time but not of the flow rate In contrast to conventional microbial fuel cells, here two liquids flow in parallel forming a sharp interface at which the bacteria and the phage contact. As a result of the electrostatic interaction between the negative charges on the surface of the bacteria with that of the positive charge on the tail portion of the phage, a potential develops. However, this potential does not develop instantly but over some period of time, during which the open circuit potential of the fuel cell continues to rise, finally reaching a steady state value. The data in Fig. 1b shows that for flow rate varying over an order of magnitude, i.e., Q = 20600 ll/min, the time required for reaching the final plateau value of potential, Emax = 0.29 ± 0.01 volt is found to be sE = 90 min. So, neither Emax nor sE depends on Q suggesting that the observed growth of potential does not result from pressure driven flow of the fuel and oxidant streams. 3.2. Potential generation is not due to bacterial lysis by bacteriophage The residence time of the liquid (bacteria and phage) inside the channel, s = V/Q (V and Q, respectively denotes channel volume and liquid flow rate) was between 2.25 and 0.075 min for the flow rate ranging from 20 to 600 ll. Clearly, such a small residence time was insufficient for the lysis of bacteria to take place, which in a well mixed chamber occurs over 20–60 min (Shao and Wang, 2008). 3.3. Effect of bacteria and bacteriophage concentration on potential generation In order to find the effect of bacteria and phage concentration on Emax, two sets of experiments in triplicates were carried out: in the first set of experiments, NZCYM broth media containing varying concentration of bacteria: 103, 105, 107, 109, 1011, 1013 and 1015 cfu/ml was pumped into the channel, while phage concentration was kept constant at 1015 pfu/ml. In the second set of experiments, phage was pumped at different concentrations: 103, 105, 107, 109, 1011, 1013 and 1015 pfu/ml, while the bacteria was maintained constant at 109 cfu/ml. These experiments revealed that Emax increases with bacterial concentration till an intermediate concentration of 109 cfu/ml was reached at which Emax was found to be 0.26 ± 0.01 V; for concentration of bacteria exceeding this limit, Emax decreases (Fig. 2, line graph with white diamond, }). In the other set of experiments, the potential Emax was found to increase monotonically with increase in phage concentration, reaching maximum at phage density 1015 pfu/ml, at which Emax = 0.26 ± 0.04 V was achieved (Fig. 2, line graph with black
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bacteria
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(a) Fig. 1. (a) Schematic of Y-shaped microchannel used as the fuel cell. (b) Growth of potential with respect to time for different flow rates of the two streams (bacteria and phage).
interaction between bacteria and phage is essential for potential generation in our MFC.
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3.5. Supply of oxygen inhibits potential generation
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Fig. 2. The maximum potential, Emax, generated for varying concentration of bacteria and phage. Error bars shows the standard deviations. Experiments were conducted in triplicate.
diamond, ). The phage concentration could not be further increased, because of non-availability of phage stock denser than 1015 pfu/ml. Thus, maximum value of Emax was achieved for bacterial density 109 cfu/ml and phage density 1015 pfu/ml. It is not clear at this stage, what causes the large difference in optimum concentration of these charged species, but possibly related to large difference in the sizes of the bacteria and the phage which results in a single bacteria being interacted with large number of phage particles.
Dissolved oxygen is another parameter that affects the output of a conventional fuel cell. In order to examine its effect on the bacteria–phage fuel cell, oxygenation and de-oxygenation of both the media containing the bacteria and the phage were performed by, respectively bubbling oxygen through it for a prolonged period and by placing it in vacuum (Rollie et al., 1987). Four kinds of experiments (in triplicates) were done in which either the media, or the bacteria, or the phage, or bacteria and phage both, were oxygenated. For all these cases, the potential decreased. Combinations: bacteria–oxygenated media, oxygenated bacteria–phage, bacteria– oxygenated phage and oxygenated bacteria–oxygenated phage, produced the average Emax value of 0.093 ± 0.025, 0.006 ± 0.003, 0.077 ± 0.025 and 0.008 ± 0.003 V, respectively (Fig. 3b, bars 1–4). These results therefore signify that oxygenation inhibits generation of OCP in our MFC, unlike in the conventional MFCs where presence of oxygen at cathode is essential to complete the red-ox reaction (Franks and Nevin, 2010; Wang et al., 2013). We believe that presence of oxygen generates OH ions which neutralize the charges on the surface of the phage, thereby interfering with the
0.3 0.2 0.1
3.4. Bacteria–bacteriophage interaction in microchannel generates potential
0.0 In order to examine if OCP generation in our MFC was indeed because of interaction between the bacteria and phage, different combinations of bacteria–phage–media were injected into the two inlets of the Y-shaped channel: (1) bacteria (109 cfu/ml) and phage (1015 pfu/ml), (2) bacteria (109 cfu/ml) and media, (3) media and phage (1015 pfu/ml), (4) bacteria (109 cfu/ml) in both sides, (5) phage (1015 pfu/ml) in both sides and (6) media in both sides. Experiments were conducted in triplicates to calculate the standard deviations. While combinations 4–6 yielded negligibly small potential, combinations 2 and 3 generated small potential of 0.14 ± 0.01 V and 0.13 ± 0.03 V, respectively. The maximum potential Emax = 0.29 ± 0.02 V was obtained for combination 1 (Fig. 3a, bars 1–6). These results therefore suggest that the
1 2 3 4 5 6
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Fig. 3. (a) Bar chart shows potential generated for different combinations of bacteria–phage–media. Bar 1 represents bacteria–phage, 2: bacteria–media, 3: media–phage, 4: bacteria–bacteria, 5: phage–phage, and 6: media–media, respectively. (b) Bar chart shows the effect of oxygen on potential. Bar 1 represents bacteria–oxygenated media, 2: oxygenated bacteria–phage, 3: bacteria–oxygenated phage, 4: oxygenated bacteria–oxygenated phage, 5: deoxygenated bacteria–phage, 6: bacteria–deoxygenated phage and 7: deoxygenated bacteria–deoxygenated phage. (c) Potential generated for three different bacteria–phage system: bars 1, 2 and 3 represent K. pneunoniae (Kp6)–phage (P-Kp6), E. coli–phage (P-Kp6) and and S. aureus–phage (P-Kp6), respectively. (d) Potential generated by bacteria (Kp6)–HCl (bar 1) and NaOH–phage (P-Kp6) (bar 2), respectively. Three experiments were carried out for each of the four cases (a–d).
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interaction between the phage and the bacteria. However, de-oxygenation of bacteria or phage or both did not result in any increase in potential. deoxygenated bacteria–phage, bacteria–deoxygenated phage and deoxygenated bacteria–deoxygenated phage produced potential, Emax, 0.23 ± 0.01, 0.28 ± 0.03 and 0.25 ± 0.03 V, respectively (Fig. 3b, bars 5–7) which are nearly equal to the Emax value obtained without de-oxygenation: 0.29 ± 0.02 V (Fig. 3a, bar 1). These results thus indicate that the oxygen present in the bacteria and/or phage stream is at miniscule level so that further removal of oxygen does not result in any change in potential. 3.6. Potential generation is not a function of bacteria–phage specificity In order to find out if specificity of bacteria–phage interaction is important for generation of the potential, following experiment was carried out: two heterogenous hosts were used: (a) Escherichia coli, Gram negative bacteria (low peptidoglycan in the cell wall, thick outer membrane with phospholipids and lipopolysaccharides imparting strong negative charge on bacterial surface), and (b) Staphylococcus aureus, Gram positive bacteria (high peptidoglycan in the cell wall with teichoic acid imparting strong negative charge on bacterial surface, no outer membrane), while using the same phage P-Kp6 as in earlier experiments. Plating of these two different bacteria with phage (P-Kp6) show that these bacteria does not get lysed by this non-specific phage (data not shown). For (a) and (b) the Emax was found to be 0.17 ± 0.02 and 0.18 ± 0.02 V (Fig. 3c, bars 2 and 3, respectively), which were somewhat smaller than that achieved when the specific host Kp-6 was used (Fig. 3c, bar 1). However, above experiments with heterogenous host signifies that potential gets generated not via lysis of bacterial cell by the phage as discussed above. 3.7. Inorganic ions can substitute bacteriophage but not the bacteria In order to examine if the supply of charged ions can substitute the bacteria and/or phage in fuel cell to produce voltage, two sets of experiments were performed, in the first set, bacteria was injected through one inlet to the channel, while through the other, solution of hydrochloric acid (pH 2) instead of phage was used as the donor for +ve ion. In the second set of experiment, solution of sodium hydroxide (pH 10) and phage were injected into the two sides of the channel. Interestingly, the potential generated in the first set of experiment was Emax = 0.21 ± 0.03 V (Fig. 3d, bar 1), almost equal to that achieved in the bacteria–phage system (Fig. 3a, bar 1). However, in the second experiment, the potential was negligibly small, 0.06 ± 0.02 V (Fig. 3d, bar 2), signifying that for the fuel cell described here, bacteria is essential while phage can be substituted by acid. 4. Conclusions Present study provides a novel mechanism of harvesting energy from interaction between two charged live microorganisms, bacteria and phage, using a membraneless microbial fuel cell which allows the two species to come in laminar contact at a sharp fluidic interface. The potential generated by this process is a strong function of both bacteria and phage concentration and both specific
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and non-specific bacteria–phage system can be used to generate the potential. Thus, this study has showed for the first time that pathogenic bacteria can be used for generating potential. 5. Conflict of interest Authors declare no conflict of interest. Acknowledgements R.G. acknowledges Department of Science and Technology, Government of India grants, Intensification of Research in High Priority Areas (IRHPA) and Fast Track Scheme For Young Scientists (SERB/ CHE/20120086), for financial support. A.G. acknowledges grant DST/CHE/20080165 from Department of Science and Technology, Government of India and Mr. and Mrs. Gian Singh Bindra Research Fellowship for this work. References Adams, M.H., 1959. Bacteriophages. Interscience, New York. Anany, H., Chen, W., Pelton, R., Griffiths, M.W., 2011. Biocontrol of Listeria monocytogenes and Escherichia coli O157:H7 in meat by using phages immobilized on modified cellulose membranes. Appl. Environ. Microbiol. 77 (18), 6379–6387. Arun, R.K., Bekele, W., Ghatak, A., 2013. Self oscillating potential generated in patterned micro-fluidic fuel cell. Electrochimica. Acta. 87, 489–496. Cademartiri, R., Anany, H., Gross, I., Bhayani, R., Griffiths, M., Brook, M.A., 2010. Immobilization of bacteriophages on modified silica particles. Biomaterials 31 (7), 1904–1910. Chang, M.H., Chen, F., Fang, N.S., 2006. Analysis of membraneless fuel cell using laminar flow in a Y-shaped microchannel. J. Power Sources 159 (2), 810–816. Cowan, S.T., Steel, K.J., 1975. Manual for Identification of Medical Bacteria. Cambridge University Press, London. Davis, F., Higson, S.P., 2007. Biofuel cells – recent advances and applications. Biosens. Bioelectron. 22 (7), 1224–1235. Franks, A.E., Nevin, K.P., 2010. Microbial fuel cells, a current review. Energies 3 (5), 899–919. Gupta, R., Prasad, Y., 2011. Efficacy of polyvalent bacteriophage P-27/HP to control multidrug resistant Staphylococcus aureus associated with human infections. Curr. Microbiol. 62 (1), 255–260. Kim, I.S., Chae, K.-J., Choi, M.-J., Verstraete, W., 2008. Microbial fuel cells : recent advances, bacterial communities and application beyond electricity generation. Environ. Eng. Res. 13 (2), 51–65. Lin, C.C., Wei, C.H., Chen, C.I., Shieh, C.J., Liu, Y.C., 2013. Characteristics of the photosynthesis microbial fuel cell with a Spirulina platensis biofilm. Bioresour. Technol. 135, 640–643. Lovley, D.R., 2006. Bug juice: harvesting electricity with microorganisms. Nat. Rev. Microbiol. 4 (7), 497–508. Monk, A.B., Rees, C.D., Barrow, P., Hagens, S., Harper, D.R., 2010. Bacteriophage applications: where are we now? Lett. Appl. Microbiol. 51 (4), 363–369. Qian, F., He, Z., Thelen, M.P., Li, Y., 2011. A microfluidic microbial fuel cell fabricated by soft lithography. Bioresour. Technol. 102 (10), 5836–5840. Rabaey, K., Verstraete, W., 2005. Microbial fuel cells: novel biotechnology for energy generation. Trends Biotechnol. 23 (6), 291–298. Rakhuba, D.V., Kolomiets, E.I., Dey, E.S., Novik, G.I., 2010. Bacteriophage receptors, mechanisms of phage adsorption and penetration into host cell. Pol. J. Microbiol. 59 (3), 145–155. Rollie, M.E., Patonay, G., Warner, I.M., 1987. Deoxygenation of solutions and its analytical applications. Ind. Eng. Chem. Res. 26 (1), 1–6. Shao, Y., Wang, I.N., 2008. Bacteriophage adsorption rate and optimal lysis time. Genetics 180 (1), 471–482. Singh, A., Poshtiban, S., Evoy, S., 2013. Recent advances in bacteriophage based biosensors for food-borne pathogen detection. Sensors (Basel) 13 (2), 1763– 1786. Wang, H., Jiang, S.C., Wang, Y., Xiao, B., 2013. Substrate removal and electricity generation in a membrane-less microbial fuel cell for biological treatment of wastewater. Bioresour. Technol. 138, 109–116.