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Electricity generation by Enterobacter cloacae SU-1 in mediator less microbial fuel cell Antony V. Samrot a, P. Senthilkumar a, K. Pavankumar a, G.C. Akilandeswari a, N. Rajalakshmi b,*, K.S. Dhathathreyan b a
Department of Biotechnology, Sathyabama University, Rajiv Gandhi Salai, Chennai, Tamilnadu, India Center for Fuel Cell Technology ARCI, IITM Research Park, Phase I, 2nd Floor, 6 Kanagam Road, Tharamani, Chennai 600 113, Tamilnadu, India b
article info
abstract
Article history:
We have investigated a Enterobacter cloacae SU-1, bacteria for mediator less microbial fuel
Received 27 February 2010
cell with different carbon sources and is found to be more effective as the microorganism is
Received in revised form
able to transfer electrons directly (exo-electrogenic organism) via the cytochromes or the
12 April 2010
ubiquinone. These carriers of electrons are in form of stable reversible redox couples, not
Accepted 8 May 2010
biologically degraded and not toxic to cell. The major advantage of mediator less microbial
Available online 11 June 2010
fuel cells emphasize that additives in the anolyte is not compatible with the purpose of water purification. The anode chamber with the bacteria is maintained under anaerobic
Keywords:
conditions so that the bacteria will undergo anaerobic biochemical pathways like Glycol-
Fuel cells
ysis, TCA cycle, Electron Transport Chain (ETC) where electrons and protons are released.
Microbial fuel cells
Here protons are released in TCA cycle and whereas electrons are released from ETC. The
Mediator less
mediator less microbial fuel cell delivered an open circuit potential (OCP) of 0.93 V and
Enterobacter cloacae
power of 3 mW/sq cm. During power generation from the microbes, there was a drop in coulombic efficiency in terms of fluctuations during drawing power, as the carbon source is being utilized for the cell growth. ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Energy has become inevitable source for this modern world and fossil fuel is the major source for our energy needs. The fossil fuel is in verge of depletion and hence we have to come up with novel ideas for sustainable development. One such ideal and appropriate form would be generating electricity from bacteria by employing the microbial fuel cell. The microbial fuel cell is one in which the bio-convertible substrate is converted to electricity without any noxious emission. The energy produced by the organism during the degradation of bio-convertible substrate can be used by the microbial fuel cell.
The microbial fuel cell is a bio-electrochemical device catalyzed by the microorganism, which is capable of converting the energy in bio-convertible substrate to electricity. Bacteria gain energy by transferring electrons from an electron donor (glucose or acetate) to an electron acceptor (oxygen). The larger the difference in potential between donor and acceptor, the higher is the growth of the organism which can be proportionately affecting the couloumbic efficiency and in turn electricity generation. Hence microbial fuel cell makes use of potential microbial energy to generate electricity. In the microbial fuel cell organism is inoculated in pure culture or mixed culture [1]. The yield of electricity is
* Corresponding author. E-mail address:
[email protected] (N. Rajalakshmi). 0360-3199/$ e see front matter ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2010.05.047
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overwhelming in the case of microbial fuel cell inoculated with the mixed culture. By virtue the by-product of one organism is been made useful by others [2]. For example Pseudomonas aeruginosa in a mixed consortium, produces pyocyanin and several more shuttling compound which are used by other electrochemically inactive organisms for electron transfer. But there are also some major setbacks with this approach which predominately includes the risk of contamination. The microbial fuel cell is broadly classified as mediator less microbial fuel cell and mediator fuel cell. The mediator less microbial fuel cell is found to be more effective as the microorganism is able to transfer electrons directly (exoelectrogenic organism) via the cytochromes or the ubiquinone. These carriers of electrons are in form of stable reversible redox couples, not biologically degraded and not toxic to cell [3,4]. Examples of such bacteria are Geobacter [5,6] and Rhodoferax ferrireducens [7]. Further the sediments from eroded beds (marine as well as lake bed) have the consortia of exo-electrogenic organism [8]. These organisms have been reported to form biofilms on the electrode surface. The non exo-electrogenic microorganism transfer electrons from its cell wall to mediators which can then be trapped by electrode. The most profound mediators are the neutral red and some dyes. Toxic and degrading nature of mediators is the main disadvantage and can be circumvented since the mediator is not released from the electrode material [9,10]. To render the anode more susceptible for receiving electrons from the bacteria, electrochemically active compounds can be incorporated in the electrode material [11,12]. Park and Zeikus [3] incorporated dyes such as neutral red and metals (such as Mn4þ, Fe3þ) to the graphite anodes and investigated. The generation of electricity has been made to increase many folds by using immobilization of organism on the electrodes. The MFCs were considered to be used for treating wastewater early in 1991 [13]. Municipal wastewater contains a multitude of organic compounds that can fuel MFCs. The amount of power generated by MFCs in the wastewater treatment process can potentially meet out approximately 50% of the electricity needed in a conventional treatment process that consumes a lot of electric power for aerating activated sludge. MFCs yield 50e90% less solids to be disposed of [14]. Furthermore, organic molecules such as acetate, propionate, and butyrate can be thoroughly broken down to CO2 and H2O. A hybrid incorporating both electrophiles and anodophiles are especially suitable for wastewater treatment because more organic wastes can be biodegraded by a variety of organisms. MFCs using certain microbes have a special ability to remove sulfides as required in wastewater treatment [15]. MFCs can enhance the growth of bio-electrochemically active microbes during wastewater treatment thus they have good operational stabilities. Continuous flow singlecompartment MFCs and membrane-less MFCs are favored for wastewater treatment due to concerns in scale-up. Sanitary wastes, food processing wastewater, swine wastewater and corn stover are all great biomass sources for MFCs because they are rich in organic matters [12]. Up to 80% of the COD can be removed in some cases and a Coulombic efficiency as high as 80% has been reported [16,17]. Daniel et al. [18] studied the
construction and operation of a microbial fuel cell for electricity generation from wastewater using Pseudomonas and a mediator with cells were connected in series delivering a maximum power output and current density of 979 mW/m2 and 1.15 mA/m2 respectively. Sharma and Li [19] optimized the energy harvest in wastewater treatment by combining anaerobic hydrogen producing biofermentor (HPB) and microbial fuel cell (MFC). It was demonstrated that the combination of HPB and MFC improved the ECE and COD removal with the maximum total ECE of 29% and COD removal of 71%. The kinetic analysis was conducted for the HPBeMFC hybrid system. The maximum hydrogen production was projected to be 2.85 mol H2/mole glucose. The maximum energy recovery and COD removal efficiency from MFC were projected to be 559 J/L and 97%, respectively. Harnessing the microorganism for electricity generation using the microbial fuel cell has been widely worked out only with the media containing simple sugar. There are only a few commendable works that have been successful while employing complex sugars like cellulose. It is evident that, when wastewater containing these complex sugars used as substrate the energy generation is low; but it is worthy to note that during the electricity generation the wastewater from paper industry or diary industry is also degraded in the presence of readily oxidizable substrate like glucose and starch. [8,11,12]. The effect of ionic strength, cation exchanger and inoculum age on the performance of microbial fuel cells have been studied by Mohan et al. [20] and they demonstrated that both physico-chemical as well as biological parameters need to be optimized for improving the power generation in MFCs. The effective performance of the microbial fuel cell depends on the choice of each on the membrane used, the inoculated organism and on the nature of substrate and also on other physical parameters like temperature, pH. The stability of the membrane also has an impact on the power generation [21,22]. Although lot of work has been done on many bacteria and yeast for microbial fuel cells, no work has been done in pure culture without any mediator for fuel cell application using Enterobacter cloacae. Recently Mohan et al [23] have studied the same culture in mixed form using mediators like methyl viologen and methylene blue with salt bridge and obtained an OCP of 0.4 V and power generation was also limited to 56 mW. In the present work we have investigated the bacteria E. cloacae SU-1 in the anode chamber with different carbon sources, with Nafion 117 as proton exchange membrane and an oxidizing solution (10 mM KMnO4) as catholyte for power generation in microbial fuel cell. The pure culture was inoculated in the anode chamber and studied for its power generation capacity.
2.
Experimental
2.1.
Materials and methods
2.1.1.
Isolation of the bacterial strain
The bacterial strain was isolated from the rhizosphere soil of Arachis hypogea. Soil samples collected were incubated in 50 ml of LB medium for several hours. After incubation, 0.5 ml of the supernatant was inoculated into minimal media
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containing potassium dihydrogen phosphate (3 g/l), disodium hydrogen phosphate (6 g/l), ammonium chloride (2 g/l), sodium chloride (5 g/l) and magnesium sulphate (1 g/l). After 2 days of cultivation at 37 C, 0.5 ml of the culture broth was diluted with 50 ml of the same minimal medium (100 fold dilution). This strain was stored in 25% glycerol solution at 70 C for further use.
2.1.2.
16S rRNA gene analysis and DNA extraction
The isolated culture was subjected for the chromosomal DNA isolation. The chromosomal DNA was isolated using a slight modification of the method reported by pitcher et al. [24]. Small amount of biomass from the minimal media agar plates was mixed and gently homogenized in 1.5-ml tubes containing 100 ml of T.E. buffer (pH 8.0) supplemented with 50 mg/ml lysozyme (Sigma, Ltd., Poole, Dorset, United Kingdom). The resulting solutions were incubated at 37 C overnight and 500 ml of a guanidine-sarcosyl solution (Guanidine thiocyanate 60 g, 0.5 mM EDTA 20 ml, Deionized water 20 ml) was added to each preparation. The aqueous layer was separated by centrifugation and extracted with chloroform isoamyl alcohol (25:1 vol/vol). The chromosomal DNA was precipitated with 0.54 volume of isopropanol, washed in 70% (vol/vol) ethanol, and dried under a vacuum. The DNA sample was redissolved in 90 ml of T.E. buffer (pH 8.0), and 10 ml of RNase A (10 mg/ml; Sigma) was added prior to incubation at 37 C for 2 h. After the RNase treatment, the DNA sample was extracted with phenol and chloroform, precipitated by adding 3 volume of ethanol in the presence of 0.8 M lithium chloride, washed with 70% ethanol, dried and redissolved in 30 ml of water.
2.1.3. Determination of 16S rDNA sequence e phylogenetic analysis A large fragment of the 16S rRNA gene was amplified by PCR using the universal primers BAC-F-(50 -AGA GTT TGA TC(AC) TGG CTC AG-30 ) BAC-R (50 AAG GAG GTG (AT)TC CA(AG) CC-30 ) [21]. The PCR products were purified using a Wizard PCR Preps DNA Purification System (Promega, USA) according to the manufacturer’s instructions and sequenced using a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, USA) and a model 3100 automatic sequencer (Applied Biosystems, USA). The closest known relatives of the new isolates were determined by performing a sequence database search. The sequences of closely related strains were retrieved from GenBank and the Ribosomal Database Project (RDP) libraries. The nucleotide (NT) sequence similarities were calculated using the PHYLODRAW program.
2.2.
containing potassium dihydrogen phosphate (15 g/l), disodium hydrogen phosphate (64 g/l), ammonium chloride (5 g/l), sodium chloride (2.5 g/l), 1 M calcium chloride (0.1 ml/l) and 1 M magnesium sulphate (2 ml//l). Glucose or lactose was added to the medium as carbon source. The anaerobic condition was ensured by the efficient use of the PTFE tape (Poly Tetra Fluoro Ethylene) which seals the system from external environment completely. The experimental facility was shown in Fig. 1. The cathode chamber was moderately aerated with an air pump. The potential difference between electrodes and the current flow were measured using digital multimeter. The current voltage characteristic of the cell was measured using ARBIN Fuel cell test bench by constant current and constant voltage method.
3.
Results and discussion
3.1.
Culture isolation studies
E. cloacae SU-1 was isolated from rhizosphere soil of A. hypogea and identified by 16s rRNA sequence. E. cloacae SU-1 was maintained in minimal agar medium. 16S rRNA gene sequencing was carried out to identify the isolated strain and the 1037 bp sequence was determined. Based on the similarity of the 16S rRNA gene, the isolate was found to be analogous to Enterobacter sp BSRA2 (98%), E. cloacae strain CMG 3058 (98%), E. cloacae strain FR (98%) and E. cloacae strain Rs e 35 (98%). A phylogenetic tree was constructed and it is shown in Fig. 2. The isolate was determined to belong to E. cloacae based on the results of 16S rRNA gene sequencing, and was designated, E. cloacae SU-1 (GenBank accession number: 848200). The numbers at the nodes indicate the levels of bootstrap support based on a neighbor-joining analysis of 1000 resampled datasets: only the values 98% are given.
3.2.
Fuel cell studies
The fuel cell produced maximum potential difference of 0.78 V with 2% lactose which is greater than the voltage produced by
Microbial fuel cell construction
The ‘H-shaped microbial fuel cell’ consists of two-chamber, the anaerobic anode chamber and aerated cathode chamber. The chambers were made of borosil having a capacity of about 200 ml each. These two were adjoined by the flange which is provisioned with the proton exchange membrane (Nafion e 117 DuPont) and the electrodes were connected by insulated copper wire. For both the chambers in the microbial fuel cell graphite plates were used as electrodes, ensuring the distance between the two electrodes as 6 cm, constant. The anode compartment of MFC was inoculated with 200 ml media
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Fig. 1 e Photograph of microbial fuel cell facility.
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Fig. 2 e Enterobacter cloacae SU-1, based on the 16S rDNA sequences of Enterobacteriacae showing the phylogenetic position of the isolated strain.
2% glucose. So lactose was chosen as carbon source for further studies. The pure cultures of E. cloacae SU-1 with the lactose produced a maximum voltage of about 0.78 V, 0.86 V and 0.93 V with the minimal media supplemented with 2%, 3% and 4% lactose respectively. These are shown in Figs. 3, 4 and 5. E. cloacae SU-1 inoculated in microbial fuel cells with 2% glucose showed a potential of 0.719 V after 76 h, as shown in Fig. 6. This cell showed a drop in potential after 100 h to 100 mV. Comparing these results with the same mixed culture using mediators with salt bridge [23], the open circuit potential was found to be 0.4 V, whereas in the pure culture the open circuit potential was found to be a maximum of 0.93 V. This could be attributed to the pure culture, Nafion electrolyte and there is no contamination by inorganic mediators. The yield of the electricity was acclaimed maximum only in case of the lactose with 4%, there was a steady potential difference maintained for over a long period. After attaining maximum voltage it maintained a steady potential of about 0.8 V. Whereas in case of other concentrations of lactose and 2% glucose there were high potential difference in a short period but they were not maintained for long period.
Fig. 3 e Electricity generation by Enterobacter cloacae SU-1 in lactose (2%) containing minimal media (KMnO4(10 mM) as catholyte).
Fig. 4 e Electricity generation by Enterobacter cloacae SU-1 in lactose (3%) containing minimal media (KMnO4(10 mM) as catholyte).
The fuel cell experiments conducted on an Arbin test bench at room temperature showed a maximum of 30 mW delivering a maximum current of 50 mA as shown in Fig. 7, which are three orders higher than reported for mixed Enterobacter culture with mediators [23]. From the figure one can see that after 30 mA current, there exists a lot of fluctuations in drawing current from the cell at about 0.65 V. This may be due to the competition between the power generation capacity and the cell growth, which also reduces the coulombic efficiency. The current drawing capacity with respect to time is shown in Fig. 8, where one can see that over a period of 350 s, the current was fluctuating from 35 to 50 mA, without any drop in performance, revealing that the bacteria can continue to produce power. The cycle life of the bacteria was examined by potential cycling between open circuit potential to 0.1 V and the found that the cell is able to deliver power of 30 mW for about 1 h over a span of 7 cycles, as shown in Fig. 9.
3.3.
Electron transfer mechanism
In the case of mediator based systems there exists a high electron transfer rate due to soluble additives, which are stable in two redox states, and quickly diffuse in and out of the
Fig. 5 e Electricity generation by Enterobacter cloacae SU-1 in lactose (4%) containing minimal media (KMnO4(10 mM) as catholyte).
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Fig. 6 e Electricity generation by Enterobacter cloacae SU-1 in glucose (2%) containing minimal media (KMnO4(10 mM) as catholyte).
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Fig. 8 e Fuel cell characteristics for Enterobacter cloacae SU-1 at constant voltage 0.1 V, RT. enzymatic channels, thereby effectively shuttling electrons from the enzyme active site to the electrode surface. In the present case where there are no mediators, the possible electron transfer paths between a microbe and an electrode, include direct electron transfer upon which the active centre of the membrane enzyme is directly connected to the electrode. In such a case, the electron transfer rate can be very low due to the insulation of the active site of the enzyme in the protein environment and the isolation of the enzyme from the electrode surface by its relative burial into the bacterial membrane. For some exoelectrogens species, however, the redox enzymes involved in electron transfer to electrodes may be located at the outer surface of the microorganism membrane, and oriented as to the active site at the periphery of the redox enzyme is facing towards the external medium or towards the electrode. Under these circumstances, the electron transfer rate is high. In another electron transfer path, fibrous protein structures, biological nanowires of 2e3 mm long called “pili,” presumably facilitate direct electron transfer between the microbe and the electrode. In such a case, the electron transfer rate can be very low due to the insulation of the active site of the enzyme in the protein environment and the isolation of the enzyme from the electrode surface by its
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Fig. 7 e Current voltage characteristics of microbial fuel cells containing pure culture Enterobacter cloacae SU-1 at RT.
relative burial into the bacterial membrane. However in the case of exoelectrogens species, the redox enzymes involved in electron transfer to electrodes may be located at the outer surface of the microorganism membrane, allowing a high electron transfer rate. Another direct electron may be due to biological nanowires of 2e3 mm long called pili, made of fibrous protein structures, which facilitate direct electron transfer between the microbe and the electrode. Bacteria can use a large variety of organic compounds as carbon sources and can be all processed to supply the organism with energy. They result in the production of an energy carrier molecule (ATP). Through different reactions, lipids, carbohydrates and proteins can be converted through glycolysis and related processes into the acetyl unit of acetylCoA. This molecule is then fed into the citric acid cycle, where oxidation reactions are coupled to the reduction of NADþ and FAD to their electron carrier forms, NADH and FADH2. These electron carriers then transfer electrons from the cytoplasm, where the citric acid cycle occurs, to the cell membrane. It is in the membrane where all the explanation of the need of this electron transfer resides. Indeed, before being transmitted to a terminal electron acceptor, electrons are transferred through different membrane intermediaries, some of them pumping protons out of the cell as they are reduced. The energy of the proton gradient, mediated through the ATP synthase transmembrane protein, is used by the cell to phosphorylate ADP to produce ATP, the chemical energetic currency of living organisms Bacteria are able to substitute an electrode as the terminal electron acceptor in the anodic compartment of MFCs. These electron carriers then transfer electrons from the cytoplasm, where the citric acid cycle occurs, to the cell membrane. The electrons are transmitted through different intermediate membranes and protons are pumped out of the system. They pass through the electrolyte membrane Nafion 117. The energy of the proton gradient, mediated through the ATP the chemical energetic currency of living organisms is explained well by Oliver et al. [25]. By understanding the different potentials at which electrons may be released to the
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Fig. 9 e Fuel cell Cyclic stability of the Enterobacter cloacae SU-1.
electrode [26] one can see that obtaining an open circuit potential of 0.93 V clearly reveals that the complete electron transfer occurs. Although mediated electron transfer proceeds at a much faster rate of electron transfer, the added or naturally occurring redox active species, is not compatible for microbial fuel cells, when the objective is to use them for the purpose of water purification. The present experiments reveal that the electron transfer is as high as mediator based fuel cells, as can be seen from the high open circuit potential, power generation capacity and cyclic stability.
4.
Conclusion
The microbial fuel cell consisting of pure E. cloacae SU-1 with lactose can deliver a power of 30 mW without any mediators. The carbon source of 4% lactose was found to give more open current potential than other concentrations of lactose. The OCP was found to be 0.93 V, relatively higher than the same culture in mixed form using inorganic additives with salt bridge as electrolyte. The cyclic stability of the bacteria was found to be good, delivering power for almost 1 h over a span of 7 cycles. The organism can be effectively used to treat dairy effluent and electricity generation. This also reveals that the electron transfer path is as fast as mediator based fuel cells and delivering a power of 3 mW/sq cm. Further experiments are in progress to evaluate mechanism the electron transfer path for further understanding and high power generation using mixed culture without mediators.
Acknowledgements The authors would like to acknowledge Dr. G. Sundararajan, Director ARCI, for his constant support and encouragement
and Department of Science and Technology, Govt of India for financial support.
references
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