ENGINEERED IRON OXIDIZING BACTERIA

CROSS REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. Provisional Application Nos.

61/446,747, filed February 25, 2011, and 61/561,595, filed November 18, 2011, which are incorporated by reference as if disclosed herein in its entirety.

BACKGROUND

[0002] There has been interest in the development of liquid biofuels as these processes have the potential to directly fix carbon dioxide into transportation fuels, which is potentially carbon neutral and politically attractive. Cellulose based biofuels including bioethanol, algae-derived lipids, cyanobacteria, and algae derived hydrogen (H ₂ ) are among the most studied biofuels. Despite the promise of these technologies and processes, there are specific limitations that preclude their wide-spread application. For example, post-processing of algal cells and derived lipids imposes higher production costs on algal biodiesel. The production rates of H ₂ from cyanobacteria still remains low and productivity needs to be improved. Genetically engineered photosynthetic organisms have also been explored for bioethanol production. However separation of ethanol from the aqueous phase remains a challenge.

[0003] Microbial fuel cells have been under investigation and development for more than a century, as the use of cells to harvest electrical energy from waste streams is attractive for many reasons. In a biofuel cell, biological catalysts are used on an anode to oxidize biofuels, and a cathode is created that can use the generated electrons to reduce oxygen to water. These systems can either be microbial with living cells on the electrodes, or they can be enzymatic systems, with purified enzymes on the electrodes. In both designs, power can be generated from the oxidation of biofuels, and there are many advantages to these systems over conventional fuel cells and other power generation schemes. However, much research still needs to be done with microbial fuel cells to make them practical and cost-efficient. A significant limitation for both enzymatic and microbial fuel cells is the need for mediators to enable electrical contact between the biological components and inorganic electrode. In some microbial systems, these mediators are made by the organisms themselves, and in other technologies, synthetic mediators are added to the system. In some systems, cells must make physical contact with the electrodes for electron transfer. This can be a significant limitation as it reduces the cellular mass that can be used for biochemical conversion.

SUMMARY [0004] Living cells have the ability to reproduce and maintain their catalytic machinery, and their metabolic pathways can be rationally altered to meet desired process objectives. But, efficient electron transfer from the electrode to the organism can limit metabolic production, and the use of mediating species can result in a process that is not economically viable. One way to address these limitations is to explore alternative organisms that naturally utilize mediators that are more attractive. The disclosed subject matter includes the metabolic engineering of chemolitotrophic iron-oxidizing-bacteria (IOB), such as Acidithiobacillus ferrooxidans, to develop a process that can overcome these limitations. IOB have the natural ability to fix carbon dioxide while oxidizing

2_ _| _

ferrous iron (Fe ) to ferric iron (Fe ). [0005] Referring to FIGS. 1 and 2, aspects of the disclosed subject matter include the use of engineered strains of the IOB, e.g., A. ferrooxidans, for the production of biofuels. IOB fix carbon dioxide for cell-synthesis while deriving energy from the oxidation of ferrous iron to ferric iron. The ferric iron produced upon ferrous iron oxidation can be electrochemically reduced back to ferrous iron in an electrochemical reactor, and additional ferrous iron can be added from any ferrous iron-rich stream. In this way, the IOB can be grown efficiently in a bioreactor using ferrous iron as the mediator.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The drawings show embodiments of the disclosed subject matter for the purpose of illustrating the invention. However, it should be understood that the present application is not limited to the precise arrangements and instrumentalities shown in the drawings, wherein:

[0007] FIG. 1 is a schematic diagram of methods and systems according to some embodiments of the disclosed subject matter; [0008] FIG. 2 is a schematic diagram of methods and systems according to some embodiments of the disclosed subject matter;

[0009] FIGS. 3 is a schematic diagram of systems according to some embodiments of the disclosed subject matter; [0010] FIG. 4 is a chart of a method according to some embodiments of the disclosed subject matter;

[0011] FIG. 5 is a diagram showing production of isobutanol via an IOB having a modified genetic sequence according to some embodiments of the disclosed subject matter; and [0012] FIG. 6 is a chart of a method according to some embodiments of the disclosed subject matter.

DETAILED DESCRIPTION

[0013] Referring again to FIGS. 1 and 2, aspects of the disclosed subject matter include methods and systems that include the application of chemolitotrophic IOB for concomitant carbon dioxide fixation, conversion of the carbon dioxide to a biofuel such as isobutanol, and oxidation of ferrous iron to ferric iron. The ferric iron produced upon ferrous iron oxidation is electrochemically reduced back to ferrous iron. Additional ferrous iron can be added from other ferrous iron-rich sources. Metabolic engineering is used to introduce a new pathway into the bacteria that starts with the precursors for amino acid synthesis to create butanols, e.g., isobutanol, etc.

[0014] Referring now to FIGS. 3-6, some embodiments include systems and methods for producing products such as biofuels and chemicals. As shown in FIG. 3, some embodiments include a system 100 for producing biofuels using genetically modified IOB 102 grown in a bioreactor 104 that are fed ferrous iron and carbon dioxide. The ferrous iron provides electrons to the IOB and the carbon dioxide is used as a base material to be fixed into a biofuel or chemical. Initially, the ferrous iron is typically provided from a first source 106 that is external to system 100, e.g., a ferrous iron-rich stream in fluid communication with bioreactor 104. Typically, but not always, the ferrous iron used in system 100 is substantially provided by a second source 108 that is generated by an electrochemical reactor 1 10. Second source 108 of ferrous iron serves as a mediator for transferring electrons to IOB 102. In some embodiments, substantially all of the ferrous iron used by bioreactor 104 is provided by a source external to system 100. [0015] Bioreactor 104 includes IOB 102 that have been genetically modified to include a particular metabolic pathway to enable them to generate a particular biofuel 112. The operating parameters of bioreactor 104 are typically optimized to maximize the production of ferric iron 114. In some embodiments, bioreactor 104 will be configured so as to be fed 70 mM ferrous iron. In some embodiments, the pH will likely be maintained in the range of about 1.5 to 4 and temperature at about 20 to 40 degrees Celsius. Methods and systems according to the disclosed subject matter have operating conditions that are optimized for optimal yield, conversion, etc. Bioreactors included in methods and systems according to the disclosed subject matter are typically operated in a continuous flow mode to maximize the conversion of the substrates to the products. [0016] Ferric iron 114, which is generated in bioreactor 104, is introduced to electrochemical reactor 1 10, which is in fluid communication with the bioreactor.

Electrochemical reactor 110 includes electrodes, i.e., an anode 116 and a cathode 118, a separator 120, and source of electrical energy 121. In some embodiments, cathode 118 is formed substantially from nickel or glassy carbon and anode 116 is formed from materials known in the art. In some embodiments, flow through or flow by porous electrodes are used. In some embodiments, separator 120 is a cation selective membrane, to allow for proton transfer across the membrane to achieve acid balance.

[0017] Electrochemical reactor 110 is typically configured to electrochemically reduce ferric iron 114 to second source 108 of ferrous iron using source of electrical energy 121. In system 100, ferric iron 114 will be continually regenerated back to ferrous iron, i.e., second source 108, and the recycle loop can be theoretically closed without the need for additional ferrous iron input from first source 106 beyond startup.

[0018] In some embodiments, a portion of the ferrous iron provided to bioreactor 104 is obtained from a ferrous iron-rich stream and a portion is obtained from electrochemical reactor 110. [0019] Some embodiments of the disclosed subject matter include systems having holding tanks for the ferrous iron rich streams and ferric iron rich streams to enable the electrochemical production of ferrous iron to operate independently of the bioreactor to take advantage of the transient pricing and availability of electricity. For example, at times during the day when electricity is least expensive, the electrochemical system would produce as much ferrous iron as possible to be stored and used slowly by the bioreactor, which will be operating continuously. This solves a major limitation encountered in photobioreactors where interruptions in light can negatively impact the process.

[0020] System 100 includes a source of water 123, a source 124 of carbon dioxide, and a source of oxygen 125, all of which are in fluid communication with bioreactor 104. In some embodiments, source 124 is carbon dioxide removed from air or energy plant emissions. In some embodiments, either in place of or in addition to carbon dioxide, carbonate, e.g., from mineral sources, is fed to bioreactor 104.

[0021] Although not included in FIG. 3, in some embodiments, system 100 includes pumps for pumping the various constituents, into, through, and out of the system. In addition, the pumps are typically programmable to allow electrochemical reactor 110 to be turned off when the price of electricity is high and turned on when the price is low. Also, the pumps typically include a separator unit to separate one or more particular constituents that are to be pumped to other components of system 100 from the other constituents. [0022] Referring now to FIGS. 4-6, some embodiments include a method 200 for producing a biofuel using genetically modified IOB. As shown in FIG. 4, at 202, IOB that have been genetically modified to include a particular metabolic pathway to enable them to generate a particular biofuel are provided.

[0023] As shown best in FIG. 5, in some embodiments, the IOB is substantially A. ferrooxidans, e.g., wild type A. Ferrooxidans 23270 strain or similar, and the IOB are genetically modified by including at least one of a 2-keto-acid decarboxylase gene (outlined by box) and an alcohol dehydrogenase gene or similar. The production of isobutanol in prokaryotic hosts begins with the amino acid biosynthesis pathways. These pathways produce 2-keto acids, and these are converted to aldehydes using a 2-keto-acid decarboxylase. Alcohol dehydrogenase is then used to convert the aldehydes to alcohols. In the case of isobutanol, the valine biosynthesis pathway is used, and the starting precursor is 2-keto-isovalerate.

[0024] In some embodiments, the IOB provided are genetically modified to be able to utilize hydrogen as an electron donor. The use of hydrogen as a mediator improves system efficiency because hydrogen may be cogenerated with ferrous iron during the electrochemical regeneration step. There are various hydrogenase enzymes from different organisms that can be used in microbial biohydrogen production. But other hydrogenase enzymes, found in organisms such as Metallosphaera sedula and hodopseudomonas palustris, enable hydrogen uptake and its use as a reductant. [0025] Referring again to FIG. 4, at 204, a first source of ferrous iron is fed to the IOB. At 205, water is fed to the IOB. At 206, carbon dioxide is fed to the IOB. At 207, oxygen is fed to the IOB. At 208, a biofuel, ferric iron, and an IOB biomass are produced. In some embodiments, ferric iron production is maximized during 208. In some embodiments, the biofuel is one of isobutanol, a long chain alcohol, or an alkane. At 210, the ferric iron produced is electrochemically reduced to a second source of ferrous iron.

Hydrogen is also often produced while electrochemically reducing the ferric iron. Next, at 212, the second source of ferrous iron and the hydrogen are fed to the IOB. The second source of ferrous iron serves as a mediator for transferring electrons to the IOB. Then, the process returns to 208 where additional biofuel, ferric iron, and IOB biomass are produced.

[0026] Some embodiments of the disclosed subject matter include methods and systems that do not include the electrochemical regeneration of ferrous iron. For example, where a feed rich in ferrous iron exists, the conversion of ferrous iron and C0 ₂ to a valuable product (biofuel or other chemical) can be achieved without electrochemical regeneration of ferrous iron. In some cases, e.g., when the chemical product being produced is very valuable, purchased ferrous iron will be used as a feedstock, thus eliminating the need for the electrochemical regeneration of ferrous iron.

[0027] Referring now to FIG. 6, some embodiments include a method 400 for producing a chemical using genetically modified IOB. At 402, IOB that have been genetically modified to include a particular metabolic pathway to enable them to generate a particular chemical are provided. At 404, a first source of ferrous iron is fed to the IOB. At 403, water is fed to the IOB. At 406, carbon dioxide is fed to the IOB. At 407, oxygen is fed to the IOB. At 408, a chemical, ferric iron, and an IOB biomass are produced. At 410, the ferric iron produced is electrochemically reduced to a second source of ferrous iron. Hydrogen is also often produced while electrochemically reducing the ferric iron. Next, at 412, the second source of ferrous iron and the hydrogen are fed to the IOB. Then, the process returns to 408 where additional chemical, ferric iron, and IOB biomass are produced.

[0028] Reverse microbial fuel cells according to the disclosed subject matter utilize carbon dioxide and electrical input to produce infrastructure compatible transportation fuels. The technology uses cultures of IOB, e.g., A. ferrooxidans, which are genetically modified to produce isobutanol.

[0029] Systems and methods according to the disclosed subject matter use only abundant, inexpensive redox mediators. They do not use costly rare earth elements or organic redox shuttles, and thus can be potentially deployed economically at scale. They potentially exceed an overall efficiency greater than one percent and butanol has desirable fuel properties and is compatible with transportation-fuel infrastructure.

[0030] Although the disclosed subject matter has been described and illustrated with respect to embodiments thereof, it should be understood by those skilled in the art that features of the disclosed embodiments can be combined, rearranged, etc., to produce additional embodiments within the scope of the invention, and that various other changes, omissions, and additions may be made therein and thereto, without parting from the spirit and scope of the present invention.