CROSS REFERENCE TO RELATED APPLICATION(S)

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

61/323,595, filed April 13, 2010 and 61/426,020, filed December 22, 2010, 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 chemolithoautotrophic ammonia-oxidizing- bacteria (AOB), such as Nitrosomonas europaea, to develop a process that can overcome these limitations. AOB have the natural ability to fix carbon dioxide while oxidizing ammonia to nitrite.

[0005] Referring to FIGS. 1 and 2, aspects of the disclosed subject matter include the use of engineered strains of the AOB, e.g., N. europaea 19718, for the production of biofuels. AOB fix carbon dioxide for cell-synthesis while deriving energy from the oxidation of ammonia to nitrite. The nitrite produced upon ammonia oxidation can be electrochemically reduced back to ammonia in an electrochemical reactor, and additional ammonia can be added from any ammonia-rich stream, e.g., one derived from a wastewater treatment process. In this way, the AOB can be grown efficiently in a bioreactor using ammonia as the mediator.

[0006] Other aspects of the disclosed subject matter include a downstream two-stage bioreactor system for the fermentation of the produced bacterial biomass directly to additional useful biofuels in order to improve the viability of the process.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] 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:

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

[0009] FIG. 2 is a schematic diagram of methods and systems according to some embodiments of the disclosed subject matter;

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

[0011] FIG. 4 is a chart of a method according to some embodiments of the disclosed subject matter;

[0012] FIG. 5 is a schematic diagram of methods and systems according to some embodiments of the disclosed subject matter;

[0013] FIG. 6 is a diagram showing production of isobutanol via an AOB having a modified genetic sequence according to some embodiments of the disclosed subject matter; and

[0014] FIG. 7 is a chart of a method according to some embodiments of the disclosed subject matter.

DETAILED DESCRIPTION

[0015] Referring again to FIGS. 1 and 2, aspects of the disclosed subject matter include methods and systems that include the application of chemolithoautotrophic AOB for concomitant carbon dioxide fixation, conversion of the carbon dioxide to a biofuel such as isobutanol, and oxidation of ammonia to nitrite. The nitrite produced upon ammonia oxidation is electrochemically reduced back to ammonia. Additional ammonia can be added from other ammonia-rich sources, e.g., derived from a wastewater treatment process. Other sources of ammonia will typically be sterilized to ensure it does not foul the AOB purity and bioreactor environment. 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.

[0016] Referring now to FIG. 3-7, 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 AOB 102 grown in a bioreactor 104 that are fed ammonia and carbon dioxide. The ammonia provides electrons to the AOB and the carbon dioxide is used as a base material to be fixed into a biofuel or chemical. Initially, the ammonia is typically provided from a first source 106 that is external to system 100, e.g., an ammonia-rich stream derived from a wastewater treatment process, etc., but in fluid communication with bioreactor 104.

Typically, but not always, the ammonia used in system 100 is substantially provided by a second source 108 that is generated by an electrochemical reactor 110. Second source 108 of ammonia serves as a mediator for transferring electrons to AOB 102. In some embodiments, substantially all of the ammonia used by bioreactor 104 is provided by a source external to system 100.

[0017] Bioreactor 104 includes AOB 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 nitrite 114 and minimize the production of nitrate. In some embodiments, bioreactor 104 will be configured so as to be fed 40 mM of ammonia, 10 mM of nitrate, 300 mM of phosphate, 80 g/L of carbonate, and trace metals (at micro-g/L levels). In some embodiments, the pH will likely be maintained in the range of about 7.5 to 8.0 and temperature at about 30 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. Ammonia and nitrite toxicity, leaching of ions from the electrodes, and product (biofuel) toxicity are mitigated in the methods and systems according to the disclosed subject matter. [0018] Nitrite 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.

[0019] Electrochemical reactor 110 is typically configured to electrochemically reduce nitrite 114 to second source 108 of ammonia using source of electrical energy 121. In system 100, nitrite 114 will be continually regenerated back to ammonia, i.e., second source 108, and the recycle loop can be theoretically closed without the need for additional ammonia input from first source 106 beyond startup.

[0020] In some embodiments, a portion of the ammonia provided to bioreactor 104 is obtained from an ammonia-rich stream derived from a wastewater treatment process and a portion is obtained from electrochemical reactor 1 10.

[0021] Some embodiments of the disclosed subject matter include systems having holding tanks for the ammonia rich streams and nitrite rich streams to enable the electrochemical production of ammonia 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 ammonia 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.

[0022] System 100 includes a source 124 of carbon dioxide that is 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.

[0023] Referring now to FIGS. 4-7, some embodiments include a method 200 for producing a biofuel using genetically modified AOB. As shown in FIG. 4 (and

schematically in FIG. 5), at 202, AOB that have been genetically modified to include a particular metabolic pathway to enable them to generate a particular biofuel are provided. [0024] As shown best in FIG. 6, in some embodiments, the AOB is substantially Nitrosomonas europaea and the AOB 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.

[0025] In some embodiments, the AOB 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 ammonia 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.

[0026] Referring again to FIGS. 4 and 5, at 204, a first source of ammonia is fed to the AOB. At 206, carbon dioxide is fed to the AOB. At 208, a biofuel, nitrite, and an AOB biomass are produced. In some embodiments, nitrite production is maximized and nitrate production is minimized during 208. In some embodiments, the biofuel is one of isobutanol, a long chain alcohol, or an alkane. At 210, the nitrite produced is

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

[0027] Some embodiments of the disclosed subject matter include methods and systems that do not include the electrochemical regeneration of ammonia. For example, where a feed rich in ammonia exists, the conversion of ammonia and C0 ₂ to a valuable product (biofuel or other chemical) can be achieved without electrochemical regeneration of ammonia. Typical waste streams suitable for this technology would have high ammonia concentrations (in the range of 200-7000 mg-N/L or higher). In some cases, e.g., when the chemical product being produced is very valuable, purchased ammonia in the form of gas or a salt such as ammonium carbonate will be used as a feedstock, thus eliminating the need for the electrochemical regeneration of ammonia.

[0028] At 214, the AOB biomass is fermented to produce a mixture including volatile fatty acids (VFA), e.g., including acetate, propionate, and butyrate. Then at 216, the mixture of VFAs is fermented to produce a second biofuel, e.g., butanol or similar. The overall fermentation process is split into two stages, i.e., 214 and 216, to allow

independent optimization of VFA production and butanol production, which involve different microbial organisms and pathways. In some embodiments, the fermentation of cellular biomass to a mixture of VFA in stage 1 will be conducted at 37 degrees Celsius and is anticipated to be catalyzed by a broad variety of fermentative bacteria. In some embodiments, stage 2 will also be operated at 37 degrees Celsius, and will be catalyzed by an axenic culture of Clostridium beijerinckii BA101. Operation of stage 2 at 37 degrees Celsius has been shown to yield higher butanol synthesis rates compared to lower temperatures. Typically, the main operational parameter of engineered biological reactors is the solids retention time (SRT), which governs the physiological activity and metabolic rate of the resident microorganisms therein. In some embodiments, to achieve VFA synthesis from cell biomass, an SRT of about 3 to 4 days is used. However, conversion of VFA (including butyrate) to butanol requires a cell residence time in the range of about 0.4 to 1 day.

[0029] Referring now to FIG. 7, some embodiments include a method 400 for producing a chemical using genetically modified AOB. At 402, AOB 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 ammonia is fed to the AOB. At 406, carbon dioxide is fed to the AOB. At 408, a chemical, nitrite, and an AOB biomass are produced. At 410, the nitrite produced is electrochemically reduced to a second source of ammonia. Hydrogen is also often produced while electrochemically reducing the nitrite. Next, at 412, the second source of ammonia and the hydrogen are fed to the AOB. Then, the process returns to 408 where additional chemical, nitrite, and AOB biomass are produced. At 414, the AOB biomass is fermented to produce a mixture including volatile fatty acids, e.g., including acetate, propionate, and butyrate. Then at 416, the mixture of volatile fatty acids is fermented to produce a second chemical such as a valuable commodity chemical, specialty chemicals, feedstocks such as acids, amino acids, carbohydrates, and other molecules.

[0030] 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 AOB, e.g., N. europaea 19718, that are genetically modified to produce isobutanol. The AOB biomass produced from this process is fed to downstream fermentors for additional biofuel production, e.g., n-butanol. Together, the two streams, if combined, have a composition of approximately 90 % iso-buantol and 10% n-butanol.

[0031] Estimates of e-/e- efficiencies indicate that values significantly greater than one percent are possible. This approach creates a new paradigm for liquid fuel production, and impacts wastewater processes that already utilize N. europaea and other AOB to treat ammonia containing wastewater streams. [0032] Butanol has been noted as being compatible with existing infrastructure. The butanols have liquid fuel energy densities exceeding the 32 MJ/kg. They would likely be used as mixtures in, for example gasoline. Alternatively, the butanol mixtures could be further transformed to other fuels.

[0033] 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.

[0034] The use of ammonia as a mediator in a reverse microbial fuel cell is a significant advancement. Ammonia is extremely attractive for use as a mediator as it is inexpensive. Furthermore, when coupled with a wastewater treatment facility, the oxidation of ammonia to nitrite by the cells is a desirable, revenue generating process. In the absence of a wastewater stream, the nitrite produced by the cells can be reduced back to ammonia using high-throughput flow-through electrodes. The conversion of nitrite to ammonia is at least 80% efficient meaning that low cost electrical energy can be efficiently transferred to the bacterial cells for metabolic processing.

[0035] More and more wastewater facilities employ anaerobic digestion for converting the organic fraction of biomass into methane, primarily for pathogen destruction and biosolids treatment. The resulting aqueous stream from anaerobic digestion is disproportionately enriched in organic and ammonia-N (both present in the N(-III) oxidation state). The treatment of this stream is one of the biggest challenges faced by wastewater treatment plants today. Systems and methods according to the disclosed subject matters can utilize ammonia from ammonia-rich streams derived from a wastewater treatment process to not only fix C0 ₂ using chemolithoautotrophic bacteria, but also converting the fixed C0 ₂ to a desirable fuel such as isobutanol.

[0036] 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.