The Use of Rhodobacter sphaeroides in Microbial Fuel Cells

Kehinde Adeyemo Howard University Wayne S Kontur, Ph.D., Rodolfo Perez, Ph.D., Dan Noguera, Ph.D. and Timothy J Donohue, Ph.D. Great Lakes Bioenergy Research Center and the Departments of Civil & Environmental Engineering & Bacteriology Colleges of Agriculture & Life Sciences & College of Engineering

Abstract
The purpose of this project is to produce a microbial fuel cell prototype that efficiently uses the bacterium Rhodobacter sphaeroides, through the activity of the nitrogenase enzyme to produce hydrogen gas and in collaboration with light energy and electrodes, electrical energy1. We employ photosynthetic bacteria because they can efficiently harness sunlight and convert it into other (chemical) forms of energy. We use Rhodobacter sphaeroides because it is relatively well characterized and understood. Also these materials were chosen because of their abundance and the fact that their use is not detrimental to the environment2. The methods being employed in this project include simple biological methods such as monitoring of cell growth, plasmid preparations and isolation, gel electrophoresis and also electrical engineering elements such as creating a circuit in order to measure electricity output of the fuel cells. Hydrogen gas has been produced by these cell cultures and when grown in the microbial fuel cells small amounts of electrical energy have been observed2.

useful alternative energy resource because it is carbon neutral and it is not a greenhouse gas. Hydrogen gas is most commonly produced by the steam reforming of fossil-derived hydrocarbons which yields carbon monoxide (a greenhouse gas) and hydrogen gas. If hydrogen gas can be renewably and carbon neutrally produced it can be used to produce energy efficiently in a fuel cell. It has been observed that certain bacteria under favorable conditions can and will produce copious amounts of hydrogen gas that can be harnessed for use in energy production. Rhodobacter sphaeroides is a bacterium that produces hydrogen gas making it a prime candidate for this research. The photosynthetic capacity of R. sphaeroides also means that there is the potential to generate hydrogen gas and thus electricity from renewable resources like sunlight and waste organic nutrients. This field is yet unexplored by many bacteriologists, as fuel cell technology is relatively new. Much of the work that has been done in this endeavor is focused on the use of microbes in waste water rather than growth of bacteria for the sole purpose of energy or current formation through hydrogen production8. It is also proposed that microbes can be genetically engineered such that more electrons can be diverted toward hydrogen gas production rather than other cell functions. Other experiments show that R. sphaeroides will reduce certain oxyanions to their elemental metals and produce hydrogen gas in the process. Another area being explored by researchers in the field is the use of bioreactors containing cyanobacteria and micro-algae to produce hydrogen gas. The purpose of this study is to explore the use of R. sphaeroides to optimally produce hydrogen gas and use it in a fuel cell to produce electricity. R. sphaeroides is a bacterium whose photosynthetic apparatus and that of plants are believed to be descended from the same recent common ancestor.

Introduction
It is proposed that electrical energy can be produced from hydrogen gas produced photosynthetically by bacteria such as Rhodobacter sphaeroides2. With rising cost of energy and energy related products, it is increasingly important to explore the use of renewable and eco-friendly resources for large scale energy production. It is important to explore the use of alternatives to fossil fuels as these materials are not carbon neutral and are contributing to the greenhouse gases in what is believed to be causing global warming. Hydrogen gas is a

Materials & Methods

Microorganisms
The microorganisms being used are R. sphaeroides and E. coli.

Cultivation
The R. sphaeroides cells being used are the wild type 2.4.1 grown in Sistroms medium without ammonium as ammonium suppresses the nitrogenase enzyme. The cells were monitored using a Klett meter and a spectrophotometer and were used for fuel cells in early log phase. The E. coli cells were grown in LB medium. The E. coli strains used were DH5
(for cloning) and S17-1 (for conjugation with 2.4.1).

Reagents
The reagents used were laboratory grade chemicals. The reagents were used according to guidelines specified by the manufacturers. The restriction enzyme digests were typically run as 30L reactions. The plasmid preparations were done using a plasmid prep kit supplied by Zymo Research Corporation.

Genetics
This aspect of the method entails the deletion of regions of certain genes in the bacterium that encode proteins predicted to drain electrons from hydrogen production. The genes being targeted for deletion were the phaC1 and phbC genes. These genes encode for PHB synthases or polymerases; and catalyze polymerization of R-(-)-3-hydroxybutyryl CoA5. These pathways take up electrons PHB sythases form PHB (a polymer) out of monomeric units; the pathway involves a reduction step that consumes electrons (that could otherwise be going toward hydrogen production). The overall aim is to see if deletion of these genes would divert more electrons to the nitogenase enzyme pathway, thereby producing more hydrogen gas and hence electricity. The deletions were made by cloning the genes in plasmid puc19 (which is good for making copies of the DNA and also for manipulating and easily growing the DNA in E. coli) and using specific restriction enzyme digests to cut the DNA. The enzymes used in this deletion were Sbf1 and Sph1 from the New England Biolabs. After the deletion has been made the genes are then transferred to a suicide vector, pk18mobsacB. To verify that the desired plasmids have been made, they are digested with specific restriction enzymes and analyzed by gel electrophoresis . The restriction enzymes are carefully selected so that the fragments of DNA are easily recognizable on a gel. The restriction enzymes used include Xba1, Sph1, Sma1, EcoRV, TthIII1 and Ssp1. The ligation enzyme being used is the T4 DNA ligase. Transformation and growth of the plasmids is done using E. coli cells.

light. The platinum density is important because this metal acts as the anode catalyst to oxidize hydrogen. The anode window should be transparent to maximize the amount of light entering the fuel cell to support hydrogen production by the bacteria. The light is provided by a light box that provides an intensity of about 10W/m2 in the part of the visible spectrum (>750nm) that is captured by R. sphaeroides. The amount of light entering the cell is critical and is affected by the platinum density of the anode as the platinum is a dark substance. The platinum density is equally critical as it plays a major role in the electricity production. This results in a need for optimization platinum density and light energy as there is a tradeoff between catalytic activity and light penetration. A group of these cubic fuel cells were tested with 3 different platinum densities to contrast hydrogen production and electrical output. These 3 different densities were duplicated so that they could be compared with one another (Figure 2 and 3).

Analysis
The hydrogen gas production in the cultures was analyzed using a respirometer (AER 200 Respirometer). The electricity production in the microbial fuel cells is measured using a multimeter (NI 6225). Cell density and concentration was measured using a Klett meter and a spectrophotometer.

Other Methods
Another part of the project was to reproduce a method used in a reference where an oxyanion (Tellurite) was deposited in different concentrations in the fuel cells and measure hydrogen production in these cells 7 (Figure 3).

Results
Genetics
The desired plasmids were obtained and this was verified by doing diagnostic restriction digests specifically chosen so that fragments would be recognizable. The plasmids were then transferred into the Rhodobacter sphaeroides 2.4.1. cells via conjugation with competent E. coli cells of the S17-1 strain. The cells underwent homologous recombination that resulted in a single crossover and they are currently being tested for double crossover to obtain the desired mutants.

Oxyanion Method
The cells that were prepared using Tellurite did not produce any hydrogen gas as predicted. This experiment is being repeated with adjusted parameters.

Fuel Cell Engineering

There were 2 fuel cell prototypes that were tested; one cubic design and one test tube design. The cubic fuel cell was designed so that the anode is coated on the window through which light energy is allowed into the cell. The task is to optimize platinum density on the anode of the prototype fuel cell in which a transparent anode acts as a window for the

Cubic Fuel Cell Prototype

The initial run using these cubic fuel cells did not produce any hydrogen gas and hence no electricity .The cells were found to have lost volume either due to evaporation or leakage. We were unable to start the work on optimizing these cells

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because there was no hydrogen production in any of the cells and hence no electricity was produced.

Discussion
The genetic project was successful in that the desired plasmids were made and mobilized into R. sphaeroides. In the future, others need to test these candidates for ones lacking the test genes (i.e., those which arose by double cross over). The initial fuel cell experiments using tellurite did not produce any detectable hydrogen gas, so the experiment was inconclusive. Future work involves the adjustment of parameters to see if that affects hydrogen production. Work is ongoing with the cubic fuel cells to determine why no hydrogen gas was produced in the initial run. More prototypes are being designed for testing as potentially better fuel cells. These include a high 48-96 well plate for growing cells and measuring hydrogen production and electricity.9

restriction enzyme sites

Plasmid

pk18mobsacB S17-1 2.4.1

Figure 1. Diagram of Plasmid

Acknowledgments
I would like to thank Dr. Wayne Kontur, Dr. Timothy Donohue, Dr. Dan Noguera, Dr. Rodolfo Perez and all the members of the Donohue and Noguera labs for helping me with my work and teaching me the skills necessary to conduct this research. I would not have been able to learn all of these skills without their help and guidance. I would also like to thank Dr. Janet Branchaw the IBS-SRP Director and Brian Asen the coordinator for all of their support throughout this program. I would also like to thank my subgroup leader Dr. John Greenler and the members of the Bioenergy subgroup for their useful feedback on my work. This research was supported by the: National Science Foundation (144 PE44) University of Wisconsin-Madison Graduate School Great Lakes Bioenergy Research Center

Figure 2. Cubic Fuel Cell Prototype

Figure 3. Tellurite Ion Set up. Tellurium is being deposited in the second tube so as shown by the dark color

References
1. Tavano, Christine L and Donohue, Timothy J Development of the bacterial photosynthetic apparatus Current opinion in Microbiology 9 2006 625-631 Cho Y K, Donohue T J, Tejedor I, Anderson M A, McMahon K D, Noguera D R Development of a solarpowered microbial fuel Cell Journal of Applied Microbiology 104 (2008) 640-650 Koku H N, Eroglu L I, Gunduz U, Yucel M, Turker L Aspects of metabolism of hydrogen production by Rhodobacter sphaeroides International journal of Hydrogen Energy 27(2002) 1315-1329

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Figure 4. Cubic Fuel cells connected to multimeter

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Doe Joint genome Institute <http://genome.jgipsf.org/finished_microbes/rhosp/rhosp.home.html Accessed June 11th 2008 Kim Mi-Sun, Baek, Jin-Sook and Lee, Jeong K Comparison of H2 accumulation by Rhodobacter sphaeroides KD131 and its uptake hydrogenase and PHB synthase deficient mutant International Journal of Hydrogen Energy 31 (2006) 121-127 Pohlmann Anne, Fricke Wolfgang F, Reinecke Frank et al. Genome sequence of the bioplastic-producing Knallgas bacterium Ralstonia eutropha H16 Nature Biotechnology Moore Mark D and Kaplan Samuel Identification of Intrinsic High Level Resistance to Rare-Earth Oxides and Oxyanions in Members of the Class Proteobacteria: Characterization of Tellurite, Selenite and Rhodium Sesquioxide Reduction in Rhodobacter sphaeroides Journal of Bacteriology 174(5) 1992 p. 1505-1514 Microbial Fuel Cells http://www.microbialfuelcell.org/ Accessed June 24th 2008 Schrader Paul S, Burrows Elizabeth H and Ely Roger L High-Throughput Screening Assay for Biological Hydrogen Production Analytical Chemistry 80(11) June I,2008 P 4014-4019

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