Team:Northeastern/Description
Project Description
Microbial Electrolysis Cells and Geobacter sulfurreducens
Microbial Fuel Cells harness the metabolic energy of bacteria, typically Geobacter sulfurreducens, to produce a voltage difference across a galvanic cell. In Microbial Electrolysis Cells this voltage is supplemented by an outside power source to drive an electrolysis reaction and produce hydrogen gas.
This method of hydrogen production is relatively recent and to date most of the investigations into improving the technique have revolved around the physical design of the chambers. However the technique relies fundamentally on the biology of the microbes used in the reaction, and thus we investigated avenues of improving the biologics of the system using a genetically engineered E. coli partner bacteria. We attempted to characterize two parts, BBa_K773002 and BBa_K2182000.
BBa_K773002 codes for the expression of Proteorhodopsin, a light-activated, membrane-bound proton pump. The export of H+ ions into the microbial electrolysis cell makes the electron exporting action of Geobacter sulfurreducens more efficient, thus theoretically increasing hydrogen yield. Proteorhodopsin requires retinal in order to fold correctly, since retinal is not naturally produced in the strain of E. coli that was used we generated retinal by adding beta-carotene to the culture and using Blh (BBa_K1604021), a beta-carotene 15,15'-dioxygenase expressed upstream of proteorhodopsin. We included the Blh gene but not the full retinal synthesis pathway for initial tests because beta carotene is much easier to acquire than retinal and this design decreases the size and complexity of the plasmid without sacrificing proteorhodopsin function.
BBa_K2182000 is a novel part that we are submitting to the registry. It codes for NADPH Oxidase which catalyzes the formation of water from NADPH and O2. This oxygen scavenging activity improves the activity of the microbial electrolysis cell by preventing oxygen from reaching the cathode to react with hydrogen or from stressing Geobacter, which is an obligate anaerobe. This reduces the time it takes for the MEC to return to full production after the introduction of oxygen, which would periodically be required to prevent the growth of hydrogen consuming species in a wastewater-fed system. Oxygen scavenging also decreases the ability of E. coli partner bacteria to outcompete Geobacter by metabolising oxygen.
Proteorhodopsin
The proteorhodopsin used in this project is from marine gamma proteobacterium EBAC31A08, with close homologues found in many marine bacterial species. The protein is composed of seven transmembrane alpha helix domains and bound to the chromophore retinal. The all trans form of retinal undergoes a conformational change to 13-cis in the presence of 520nm light that in turn causes a chain reaction in the structure of proteorhodopsin that shifts a proton from one side of the membrane to the other. Proton export outside the cell drives a transmembrane proton gradient used to power the generation of ATP, a major molecule for energy transfer, by ATP synthase. The energy in entering light is thus used to concentrate hydrogen ions outside the cell. In an MEC, that energy would be diverted from ATP production towards hydrogen generation by the reduction of protons at the cathode.
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NADH Oxidase
Our Nox part comes from a large family of NADH oxidases, all of which catalyze the recombination of hydrogen from the cellular energy carrier NADH with diatomic oxygen through redox reactions. This replenishes NAD+, the oxidized form of NADH needed to accept electrons in cellular respiration, and for some species may assist in survival in aerobic conditions by preventing oxidative stress. The protein from Streptococcus sanguinis SK36, identified as an H2O-forming nox, is rare in that it strictly catalyses the formation of water instead of hydrogen peroxide, which is itself a reactive oxygen species that can damage cellular processes.
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