The Capability of Micro-Organism Shewanella Oneidensis MR-1 to Produce Electricity - Lab Report Example

?"Microbial Fuel Cells" Electri has become the most vital component of human survival. To meet the mounting demands for electri new possibilities are constantly being excavated. Micro-organisms are exploited to meet these rising demands. Intensive studies have been carried out in recent years to utilize micro-organisms as a resource for energy generation. The present experiment was conducted to understand the capability of micro-organism Shewanella oneidensis MR-1 to produce electricity, as it is reported to possess electron accepting capacities allocating them to combine organic decomposition with reduction of terminal electron acceptors in their atmosphere. Microbial fuel cells (MFCs) signify an innovative method of procuring renewable energy in the era of energy crisis. Shewanella oneidensis MR-1 is capable of growing in 50 days in MFCs, the organism is capable of partly oxidizing lactate to acetate with enhanced recovery of the electrons producing electricity. Electricity was generated with lactate or hydrogen on their complete oxidation through electrigens. The cells are attached to the anode, the electrode, thereby conserving energy for growth as the cell is capable of donating its electrons to the electrode. Introduction It is evident that fossil fuels are limited sources for energy on the planet and they are on the verge of exhaustion. Consumption of fossil fuel has augmented climate changes, enhancing temperatures, floods or droughts on the planet causing global warming. Burning involves consumption of oxygen, on flaming fossil fuels which are chiefly carbon, the gas evolved is mainly carbon-di-oxide and obnoxious carbon mono-oxide. These two gases are hazardous and are chief pollutants. Therefore it is imperative to exploit other means and resources for the production of energy. The new source of energy is retrieved from microbial cells, which are biodegradable and environment friendly (Noam M, 2005). Present era witnesses the generation of electricity from biodegradable compounds, for instance pure chemicals and wastewater. Recently, the production of electricity from microbes paves the way for generating microbial fuel cells capable of producing electricity from complex organic wastes and renewable biomass. Wastes and renewable biomass are attractive sources of energy because both of them encompass natural carbon being fixed in recent times, thus impact on the atmosphere could be reduced. ‘A microbial fuel cells is a mimic of a biological system in which bacteria do not directly transfer their produced electrons to their characteristic electron acceptor’. (Rabaey K, Lissens G, Steven D, Siciliano S and Verstraete W. 2003). MFCs are able to generate electricity through oxidation of organic matter by means of bacteria. Electricity could be generated from a range of biodegradable substrates such as glucose, acetate, ethanol, butyrate, lactate and organic matter. The biggest advantage of MFCs is that they are capable of functioning at room temperature and can be designed to perform numerous functions at different temperatures. In addition, MFCs have also special enzymes to generate electricity. The enzymes produced by MFCs can produce high level of power (Lovely DR, 2006). Enormous factors influence MFCs, these are kind of microbe, type of the proton, resistance and chemical substrate. MFCs also can be coupled with wastewater treatment and thereby exploiting the metabolic potentials of the MFCs, as they are able to metabolize many carbon sources. Chief bacterial population associated with MFCs encompasses Aeromonas, Clostridium, Geobacter, Enterococcus and Shewanella. There are four methods that make the electrons reach the anode they are- (1) Direct membrane associated transfer, (2) Nanowires (conductive pili), (3) Endogenous electron mediators (or electron shuttles) and (4) Exogenous chemical mediators (or electron shuttles). Nanowires plays an important role in the transport of the electrons. Nanowires might help electrons transfer from the bacteria to the metal oxides without the direct cell surface to mineral contact or without dissolved electron shuttles. Nanowires encompass the same electron transfer mechanism in the microbial fuel cells. There is high respiratory flexibility in bacteria and Achaea which is represented in the diverse range of electron acceptors. This flexibility allows bacteria to thrive in diverse environments under variable conditions. The electron acceptors can be sulphur, sulphur oxy-anions, organic sulphoxides, sulphonates, nitrogen oxy-anions , nitrogen oxides, organic N-oxides, halogenated organics, metalloid oxy-anions (e.g. selenate and arsenate), transitions metals (e.g. Fe(III) and Mn (IV)) and ribonuclides (e.g. U(VI) and Tc(VII)). It is established that under aerobic conditions oxygen is the electron acceptor for some bacteria. Enzymes responsible for the respiration process are regulated under the given set of conditions where microbe is proliferating. For instance, in Escherichia coli oxygen, nitrate, nitrite, dimethylsulphoxide (DMSO), trimethylamine N-oxide (TMAO) and fumarate can act as electron acceptors under diverse conditions. Enzymes are also regulated when virulence of the microbe is influenced such as Shigella felxineri and Salmonella typhimurium. Fe(III) respiration is considered to be the foremost, it is utilized by bacteria followed by S(0) respiration. Further, it is assumed that the early electron donor in the case of Fe(III) reduction respiration was hydrogen which is extracted via an extra membranous dehydrogenase. In the case of Shewanella tetra-haem, deca-haem and c-type cytochromes form a multi-haem electron wire between the inner and outer membranes. The expression of cytochromes increase swiftly when grown on Fe(III) containing media and it was also proved that these cytochromes are encoded by multiple copies of genes within the genome. Electrons can be transferred to Fe(III) by direct contact but the presence of an outer-membrane protein with multi-haem cytochromes raised a question of the possibility that these bind a redox centre. The reduction of Fe(III) by the multi-haem cytochromes is shown to be non-specific. On the other hand, nitrates and nitrites can be used as electron donors in the process of denitrification to produce dinitrogen gas, under that influence of haem containing and in some cases copper containing enzymes. Nitrite an electron acceptor is reduced, via hydroxylamine and ammonia, to nitrate through combined action of species such as Nitrosomonas europaea (NH4+oxidizer) and Nitrobacter vulgaris (NO2?oxidizer), in the process called as nitrification. Further, nitrate can be reduced via nitrite to ammonia. In bacteria the nitrate reductase cofactor which is called bis-MGD is also used by many other respiratory enzymes. These enzymes recruit iron-sulphur proteins and membrane anchors to enable the redox reaction by the bis-MGD. The redox reactions involve the transfer of electron to or from quinines (Q)/QH2 (fig XX). There are two types on nitrate reductases in bacteria one is expressed under aerobic conditions while the other is expressed under anaerobic. Figure xx: The photo above shows the basic working of a microbial fuel cell. Bacteria is metabolizing substrates, that transfer the electrons to the anode. This is displayed through the membrane or redox shuttles. Shewanella is one of the most important bacteria because it has the ability to attach to the electrode and transfer electrons without a mediator (Surya G, Viva F, Bretschger O, Yang B, El-naggar M and Nealson K.2010). The genus Shewanella is capable of accepting electrons, from the organic matter. The metabolism of Shewanella is significant for carbon cycling, remediation of the environment and microbial fuel cells (MFCs). As an excellent model, S. oneidensis MR-1 can transfer electrons to solid metal oxides under anaerobic conditions and can use more than 10 different electron acceptors. In addition, S. oneidensis MR-1 has the ability to produce sulphide from thiosulphate and can possibly reduce elemental sulphur, but these variations are not available in case of aerobic bacteria, thereby making Shewanella spp. more versatile. In nut shell, in case of fuel cell, devoid of oxygen, when bacteria are placed in the anode chamber, they stick to the electrode. As there is no oxygen, bacteria transfer electrons that were procured during the process of food oxidation or carbon source. In the absence of oxygen, it becomes necessary to transfer electron to some agent capable of accepting the electrons being transferred and hence anode serves the purpose. On the other side cathode is exposed to oxygen and here electrons, hydrogen and oxygen combine together to give water. Therefore the two electrodes, the cathode and the anode remain at different potentials, as long as fuel are being replenished to the bacterial population, fuel cell will work (Overview). Experimental Growth & Microbial Fuel Shewanell oneidensis strain MR-1 grows under anaerobic conditions in freshwater. It grows in the medium incorporated with lactate (20mM), Fe(III) citrate (50mM), while medium contains 0.05% (w/v) of yeast extract (Lovely et al., 1989). The cells from the culture are harvested near the end of log phase through centrifugation and then resuspended in fresh medium without yeast extract or Fe(III). The cells are then inoculated in the anode chamber (Lanthier, 2008). The electrical connection between the anode and cathode encompass resistor of 560 ? (Bond and Lovely, 2003). Current could be measured every hour by Keithley Instrument, a Digital Multimeter. The medium in the anode chamber is devoid of yeast extract but it is incorporated with l-argenine (22mg/L), L-glutamine (22mg)L and DL-serine (44mg/L) (Lanthier, 2008). The lactate concentration at initial stages is 10mM. The anode chamber constantly has Nitrogen and Carbon-di-oxide, while the cathode is effervesces with air. Protein Quantification Total protein concentration in the anode chamber could be quantified by centrifugation (Smith et al., 1985). Microscopy Cells coupled with the anode could be stained using Invitrigen-Molecular Probes Inc., Eugene, OR and then could be observed under confocal laser scanning microscope (CLSM). The live cells could be observed green and dead cells as red (Lanthier, 2008). Results and Discussion Current Production Current production increases to its peak of 0.2-0.3mA. It is observed that maximum current could be procured in the medium incorporated with yeast extract. The anode chamber shows growth and therefore it is turbid. Incorporation of yeast extract in the medium could not augment the current production but enhances the biomass. No current was observed in the absence of cells, or in the presence of 4mM acetate which serves as electron donor, but S. oneidensis is unable to utilize this under anaerobic condition. Incorporation of lactate could resume the current production (Lanthier, 2008). On analysing the stoichiometry of lactate metabolism in relation to current production, lactate consumption occur over time (Lanthier, 2008). Conclusion The ability of the metal reducer Shewanella oneidensis MR-1 to produce electricity in MFCs cascade is also dependent on the activity of type IV prepilin peptidase; PilD. Further analysis of an S. oneidensis MR-1 pilD mutant reveal that the mutant is incapable of producing pili and type II secretion (T2S). It is observed that T2S is required for metal reduction but the role of pili could not be explained so far. Mutants devoid of flagella were capable of reducing Fe (III) and were able to produce electricity in MFCs, while the T2S mutant remained deficient in both processes. Additionally, MR-1 mutants deficient in type IV pili or flagella produced additional electricity then their wild type counterparts (Bouhenni, RA, 2010). The present effort is in the direction of the conservation of natural resources and to harvest resources that are cost effective in generating the power that is necessary for life. Shewanella sp. has emerged as a boon to fulfil the ever enhancing demand of the electricity. Besides harvesting solar energy for generating power, efforts are gaining success in the direction of exploiting microbial population for the energy maintenance. The capability of Shewanella species to generate current from lactate has been noted many times (Kim et al., 1999a, b, 1999, 2002; Ringeisen et al., 2006; Biffinger et al., 2007; Cho & Ellington, 2007), the results procured in this experiment reveals that a considerable amount of the electrons derived from lactate oxidation by S. oneidensis can be procured as electrons in a MFCs. Shewanella species could utilize electrodes as a respiratory electron acceptor. MFCs conserve energy and produce growth. Microbial world has immense potential to provide great benefits to the human, plant and animal worlds. Through genomics, many genes that are encoded or present in the genome and express themselves under a particular set of conditions could be exploited for proteomics and transcriptomics to understand the microbial physiology to harness the advantage from the microbes for the survival of life on the planet. The following diagram simplifies the process. Figure from: Microbial fuel cell (Rabaey, K, 2003). References 1. Biffinger JC, Pietron J, Ray R, Little B & Ringeisen BR (2007). A biofilm enhanced miniature microbial fuel cell using Shewanella oneidensis DSP10 and oxygen reduction cathodes. Biosens Bioelec 22: 1672–1679. 2. Bouhenni, RA, Vora, GJ, Biffinger, JC, Shirodkar, S, et al. (2010). The Role of Shewanella oneidensis MR-1 outer surface structures in extracellular electron transfer. Electroanalysis; 22(7):NA 3. Cho, EJ., Ellington, AD. (2007). Optimization of the biological component of a bioelectrochemical cell. Bioelectrochem 70: 165–172. 4. Fredrickson JK, Romine MF, Beliaev AS, Auchtung JM, Driscoll ME, Gardner TS, Nealson KH, Osterman AL, Pinchuk G, Reed JL, Rodionov DA, Rodrigues JL, Saffarini DA, Serres MH, Spormann AM, Zhulin IB and Tiedje JM (2008) Towards environmental systems biology of Shewanella. Nature Reviews Microbiology; 6: 592-603. 5. Kim, BH., Ikeda, T., Park, HS., et al. (1999a). Electrochemical activity of an Fe(III)-reducing bacterium, Shewanella putrefaciens IR-1, in the presence of alternative electron acceptors. Biotechnol Tech. 13: 475–478. 6. Kim, BH., Kim, HJ., Hyun, MS., Park, DH. (1999b). Direct electrode reaction of Fe(III)-reducing bacterium, Shewanella putrefaciens. J Microbiol Biotechnol 9: 127–131. 7. Kim, HJ., Hyun, MS., Chang, IS., Kim, BH. (1999). A microbial fuel cell type lactate biosensor using a metal-reducing bacterium, Shewanella putrefaciens. J Microbiol Biotechnol 9: 365–367. 8. Kim, HJ., Park, HS., Hyun, MS., Chang, IS., Kim, M., Kim, BH. (2002). A mediator-less microbial fuel cell using a metal reducing bacterium, Shewanella putrefaciens. EnzymeMicrob Technol 30:145–152. 9. Lanthier, M., Gregory, KB., Lovely, DR., Growth with high planktonic biomass in Shewanella oneidensis fuel cells. (2008). FEMS Microbiol Lett, 28, 29-35 10. Lovely, DR. (2006). Bug juice: harvesting electricity with microorganisms. Nature Reviews; 6: 497-508. 11. Lovley, DR., Phillips, EJP., Lonergan, DJ. (1989). Hydrogen and formate oxidation coupled to dissimilatory reduction of iron or manganese by Alteromonas putrefaciens. Appl Environ Microbiol, 55: 700–706. 12. Mohr, N. (2005). A new global warming strategy how environmentalists are overlooking vegetarianism as the most effective tool against climate change in our lifetimes. (viewed 18th February). http://www.earthsave.org/news/earthsave_global_warming_report.pdf. 13. Rabaey, K., Lissens, G., Steven, D., Siciliano, S., Verstraete, W. (2003). The microbial fuel cells capable of converting glucose to electricity at high and rate efficiency. Biotechnology Letters; 25: 1531-1535. 14. Ringeisen, BR., Henderson, E., Wu, PK., et al. (2006). High power density from a miniature microbial fuel cell using Shewanella oneidensis DSP10. Environ Sci Technol 40: 2629–2634. 15. Smith, PK., Krohn, RI., Hermanson, GT., et al. (1985). Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76–85. 16. Surya, G., Viva, F., Bretschger, O., Yang, B., El-naggar, M., Nealson, K. (2010). Inoculation procedures and characterization of membrane electrode assemblies for microbial fuel cells. Journal of Power Sources; 195: 111-117. 17. Overview. Available at http://www.engr.psu.edu/ce/enve/logan/bioenergy/research_mfc.htm. [Accessed on 3rd March 2011]. Read More