WO2014144308A2

WO2014144308A2 - Sensor and performance and usage data array for electrogenic bioreactor

Info

Publication number: WO2014144308A2
Application number: PCT/US2014/028655
Authority: WO
Inventors: Jason E. Barkeloo
Original assignee: Tauriga Sciences, Inc.
Priority date: 2013-03-15
Filing date: 2014-03-14
Publication date: 2014-09-18
Also published as: WO2014144308A3

Abstract

The present invention relates to alternative clean energy resources, and in particular to systems for efficiently utilizing and generating electrical energy from renewable energy sources for the home, office and complexes of buildings. The present invention provides using a electrogenic bioreactor as a hub to collect sensor and performance data and providing it to the end user.

Description

SENSOR AND PERFORMANCE AND USAGE DATA ARRAY FOR ELECTROGENIC

BIOREACTOR

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Appl. No. 61/794,864, filed March 15, 2013. The content of the aforesaid application is relied upon and incorporated by reference in its entirety.

INCORP ORATION-BY-REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readable sequence listing submitted concurrently herewith and identified as follows: One (12, 177 Byte ASCII (Text)) file named "Sequence_listing_ST25.txt," created on March 14, 2014.

FIELD

[0001] The field relates to alternative clean energy resources, and in particular to systems for efficiently utilizing and generating electrical energy from renewable energy sources for home, office and complexes of buildings.

BACKGROUND

[0002] Traditional houses rely primarily on electrical energy supplied by a national grid.

Even in with houses that have solar panels, electrical energy supplied by the panels is typically used during the day to offset electrical energy bills, while at night the house relies on electrical power supplied by the national grid. Similar problems are also encountered with other renewable energy systems such as wind power, as well as tidal and wave derived energy generating systems, which all tend to suffer from transient bursts in energy generating capacity followed by relatively long periods of reduced electrical output.

[0003] Furthermore such systems typically require the implementation of large dedicated systems to store excess energy during peak production times for use when electrical demand is highest, which are expensive to implement, and impractical for many houses. Accordingly completely stand-alone electrical systems based on renewable energy sources are rarely implemented, or even practical in most houses and offices. [0004] Accordingly there remains a need for environmentally friendly energy sources, which can provide stable and reliable electrical power supplies for homes, offices and collections of buildings that is cost effective to implement, and can regulate energy generation with local power needs. Microbial bioreactor fuel cells offer the potential to provide such energy sources, while at the same time offering the ability to be integrated with other energy generation systems to provide a sustainable means for generating electrical power.

[0005] Microbial Fuel Cells - Some bacteria can gain energy by transferring electrons from a low-potential substrate such as for example, glucose, to a high- potential electron acceptor such as for example, molecular oxygen ((¾) in a process commonly referred to as respiration. In eukaryotic cells, mitochondria obtain energy in the form of ATP through the processes of oxidation and phosphorylation, commonly referred to as oxidative phosphorylation or cellular respiration. Gram-negative bacteria such as Pseudomonas aeruginosa function similarly to the eukaryotic mitochondria in producing energy. P. aeruginosa is a Gram-negative, rod-shaped bacterium with a single polar flagellum. An opportunistic human pathogen, P. aeruginosa is also an opportunistic pathogen of plants. P. aeruginosa is capable of growth at ranges of 4°C to ~43°C. It can live in diesel and jet fuel where it becomes a hydrocarbon utilizing microorganism. It can also metabolize high nitrogen (e.g., nitrate, amino acids)-containing organic materials. P. aeruginosa derive electrons from a myriad (at least 300) of carbon sources and can derive electrons in aerobic, anaerobic and anaerobic fermentative processes (e.g., with arginine or pyruvate). In anaerobic respiratory growth, especially, P. aeruginosa cells continue to couple oxidation and phosphorylation to gain energy at roughly 60% the ATP-generating capacity of aerobic bacteria.

[0006] In some types of microbial fuel cells, a microbe donates electrons to an anode rather than the natural recipient molecule such as oxygen, nitrate, or sulfate. Various types of microbes including bacteria and fungi have been demonstrated to generate electrical energy during metabolism, but microbial fuel cells most commonly utilize bacteria such as Geobacter or Shewanella. Geobacter cells respond to high microbial density in such a way as to interfere with large surface area biofilm formation.

[0007] In some types of microbial fuel cells, metabolic processes in the microbe generate energy in the form of electrons, especially in the anaerobic biofilm mode of growth. Rather than utilizing the energy, the microbe donates the electrons from a myriad of metabolized substrates to the anode for transfer through an electrical circuit. The electrical circuit carries electricity through a load, which represents work to be performed by the electron flow. The load may be a light emitting device, machinery, LCD, electrical appliance, battery charger, cell phone, computer and many other devices.

[0008] Generally, microbes such as bacteria utilize a coenzyme known as nicotinamide adenine dinucleotide or NAD⁺ to accept electrons from, and thus oxidize, a feedstock or substrate. The NAD⁺ cleaves two hydrogen atoms from a reactant substrate. The NAD⁺ accepts one of the hydrogen atoms to become NADH and gains an electron in the process. A hydride ion, or cation, is released. The equation is as shown below, where RH₂ is oxidized, thereby reducing NAD⁺ to NADH. RH₂ could represent an organic substrate such as glucose or other organic matter such as protein extracts, commercially available bacterial broths, or any suitable carbohydrate, alcohol, hydrocarbon, or oxidizable substance.

[0009] Eq. 1 RH₂ + NAD⁺→ NADH + H⁺ + R

[0010] NADH, the 2-electron reduction product of NAD+, is a strong reducing agent that the bacteria uses to donate electrons when reducing another substrate. NADH reduces the other substrate and is concurrently reoxidized into NAD⁺. In the natural state, the other substance may be oxygen or sulphate in a microbe-dependent fashion. In a microbial fuel cell the other substance may be a mediator or an anode. A mediator transfers electrons to the anode. The electrons, prevented from moving directly from the anode to the cathode, transfer to the cathode through an external electrical circuit and through the load perform useful work.

[0011] The present inventors have advantageously discovered that microbial bioreactor fuel cells can be integrated into energy distribution systems that provide for a reliable, cost effective and environmentally friendly means for supplying energy to houses, offices and other buildings, or complexes of buildings. The microbes of the microbial fuel cell are genetically modified to generate energy by breaking down, metabolizing, fermenting, or digesting the feedstock, thereby providing a safe and environmentally friendly source of clean energy, that can be rapidly up-regulated to meet local transient energy needs, or to compensate for periodic decreases in energy output from other renewable energy sources such as wind energy, solar power, geothermal power and tidal or wave energy.

BRIEF SUMMARY

[0012] In one aspect, the invention provides one or more sensor and performance and usage data arrays for an electrogenic bioreactor. In some embodiments, within each microbial bioreactor there may be an array of sensors. These sensors are designed to provide operating data about the bioreactor to an integrated circuit (IC) and/or processor on a network interface card (NIC). The NIC may be capable of a conducting a connection (telephone, Ethernet, etc.) to an external network operations center (NOC) where the status, usage, and other operational information may be downloaded.

[0013] In some embodiments, the downloaded information can be used by any number of parties (end-user, utility, etc.) to make adjustments to the energy distribution network. That information can be parsed into reports and provided back to the subscribed party(ies) via mobile text messaging, email, telephone, etc.

[0014] In some embodiments, the microbial bioreactor may serve as a hub. For example, the bioreactor's hub can collect data from those appliances and devices connected to the bioreactor for the primary purpose of gaining electricity. The data provided by the appliances may be wired or wireless (for example, using Zigbee). The data providing device may be embedded within the appliance, attached to the appliance, or placed between the plug and outlet.

[0015] In some embodiments, the data provided by appliances and devices gaining their energy from the bioreactor may be forwarded to the NOC. In such a case, that information may assist appliance and device vendors determine usage, power requirements, etc.

[0016] In some embodiments, other forms of energy generation (such as wind, solar, geothermal, etc.) may plug-in to the bioreactor's hub (internal/external and wired/wireless location of the connection) and that energy generating platform usage and status information can be provided to the NOC via the NIC for decision-making and monetization.

[0017] In some embodiments, the invention provides a water-energy network (WEN) powered by an on-site cleantech electrogenic bioreactor. In some aspects, the WEN may utilize a sensor array that collects information on the status and usage of the bioreactor and connected appliances. This information can then be used to optimize usage. For example: when energy usage is low, excess electricity produced by the bioreactor can be fed into the power grid. When the energy demand in the home is at peak, additional electricity can be fed into the WEN from the power grid; when hydrogen or methane gas is produced, it can be used locally or distributed externally; carbon and renewable energy credits are trackable and auditable.

[0018] In some embodiments, the appliances can have their status fed into the WEN/EBR. In such a case the EBR serves as a data collection hub. For example, gas furnace performance and usage status information is provided to the EBR. The EBR is then able to provide that information to the owner of the gas furnace.

[0019] In further embodiments, other energy generating utilities like solar, wind, geothermal, can have their status information provided to the EBR. Similar to appliances, in such a case the EBR serves as a data collection hub.

[0020] In some embodiments, the EBR can serve as a cyber-secure data collection hub within the local WEN. That data may be provided back to the end-user, owner, or other subscribers.

[0021] The inventor has surprisingly found that the bioreactor harnesses genetically- enhanced bacteria that generate direct current (DC) electricity and hydrogen gas from their metabolism of organic compounds. One of the substrates that the bacteria can use is on-site generated wastewater, making the bioreactor an on-site wastewater remediation, gas or chemical producer, and power generator. This provides the installation with values that are not dependent on the weather or time of day, like solar and wind power.

[0022] Besides the sensor array in the bioreactor the WEN can also include a user interface and an external control panel as described in the following paragraphs:

1. Sensor Array

[0023] The sensor array may be located in the bioreactor and may inter alia, provide information on: temperature, amount and purity of hydrogen produced, amount of DC electricity generated, bacterial density on anode, percentage of feedstock present, Biochemical Oxygen Demand/Chemical Oxygen Demand, pH, and the amount of DC electricity used by connected devices. There can also be a platform for adding other sensors. 2. Network Interface Card (NIC)

[0024] In some embodiments, this component of the WEN collects data from the sensors on the performance of the bioreactor and the usage of appliances. These data are then sent to the Network Operations Center (NOC) for evaluation.

3. Network Operations Center (NOC)

[0025] The NOC is an external device and is a database across a web/IP connection that receives and provides a parsing feature for data mining, status, and usage reporting. For example, the collected data can be used to balance power supply and demand in the home. This would allow vendors to determine usage status of appliances to provide early warning of maintenance issues.

[0026] In one embodiment, the present invention comprises methods of generating electrical energy for a building, or group of buildings, comprising;

1) connecting a microbial fuel cell bioreactor, with a means for transferring electrical energy from the bioreactor to an energy distribution system for the building or the group of buildings, wherein the bioreactor comprises,

a) a housing with one or more openings to facilitate contact of feedstock and fuel cell components;

b) a cathode;

c) an anode in electrical communication with the cathode; and

d) a biofilm functionally associated with the anode;

wherein the biofilm comprises genetically modified microbes that break down the feedstock and donate electrons to the anode, thereby generating electrical energy;

2) connecting the bioreactor to a first plurality of sensors, wherein the first plurality of sensors provide operating data about the bioreactor, and can transmit the data to an external network operations center,

3) connecting a second plurality of sensors to a plurality of devices gaining their energy from the microbial fuel cell, wherein the second plurality of sensors provide operating data about the plurality of devices and can transmit the data to the external network operations center, wherein the external network operations center makes adjustments to the energy distribution system, or bioreactor, based on the data received from the first plurality of sensors, and the second plurality of sensors.

[0027] In one aspect of the method, the energy distribution system is further connected to at least one additional form of local energy generation selected from the group consisting of solar energy, wind energy, thermal energy, chemical energy, wave energy, and tidal energy.

[0028] In another aspect of the method, the additional form of local energy generation is connected to a third plurality of sensors that provide operating data about the additional form of local energy generation, and can transmit the data to the external network operations center; wherein the network operations center makes adjustments to the energy distribution system, or the bioreactor, or the additional form of local energy generation, based on the data received from the first plurality of sensors, or the second plurality of sensors, or the third plurality of sensors.

[0029] In another aspect of any of these methods, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the P. aeruginosa has at least one genetic modification that results in the loss of functional expression, or the suppression in expression in at least one endogenous gene selected from the group consisting of pilT (SEQ ID NO: l), pilA (SEQ ID NO:2), nirS (SEQ ID NO:3), bdlA (SEQ ID NO:4), lasi (SEQ ID NO:5), lasR (SEQ ID NO:6), ftsZ (SEQ ID NO:7), fliC (SEQ ID NO:8) and combinations thereof, wherein said microbes exhibit decreased expression of the gene interest and exhibit an altered electrogenic efficacy.

[0030] In one aspect of this method, the genetically modified microbes comprise Pseudomonas aeruginosa in which a combination of the genes are disrupted or suppressed.

[0031] In another aspect of any of these methods, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the expression at least two genes are disrupted or suppressed, wherein the at least two genes are selected from the group consisting οΐρϊΙΤ (SEQ ID NO: l) and bdlA (SEQ ID NO:4); bdlA (SEQ ID NO:4) and nirS (SEQ ID NO:3); bdlA (SEQ ID NO:4) and lasi (SEQ ID NO:5); nirS (SEQ ID NO:3) and pilT (SEQ ID NO: l); nirS (SEQ ID NO:3) and lasi (SEQ ID NO:5); lasi (SEQ ID NO:5) and pilT (SEQ ID NO: (SEQ ID NO:7) and pilT (SEQ ID NO: 1); ftsZ (SEQ ID NO:7) and bdlA (SEQ ID N0:4); ftsZ (SEQ ID N0:7) and nirS (SEQ ID N0:3); and fisZ (SEQ ID N0:7) and lasi (SEQ ID N0:5).

[0032] In another aspect of any of these methods, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the expression at least three genes are disrupted or suppressed, wherein the at least three genes are selected from the group consisting of pilT (SEQ ID NO: l), bdlA (SEQ ID NO:4) and ftsZ (SEQ ID NO:7); bdlA (SEQ ID NO:4), nirS (SEQ ID NO:3) and fisZ (SEQ ID NO:7); bdlA (SEQ ID NO:4), lasi (SEQ ID NO:5) and ftsZ (SEQ ID NO:7); nirS (SEQ ID NO:3), pilT (SEQ ID NO: 1) and ftsZ (SEQ ID NO:7); nirS (SEQ ID NO:3), lasi (SEQ ID NO:5) and ftsZ (SEQ ID NO:7); lasi (SEQ ID NO:5), pilT (SEQ ID NO: 1) and ftsZ (SEQ ID NO:7); pilT (SEQ ID NO: l), bdlA (SEQ ID NO:4) and nirS (SEQ ID NO:3); pilT (SEQ ID NO: l), bdlA (SEQ ID NO:4) and lasi (SEQ ID NO:5); and bdlA (SEQ ID NO:4), lasi (SEQ ID NO:5) and nirS (SEQ ID NO:3).

[0033] In another aspect of any of these methods, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the expression at least four genes are disrupted or suppressed, wherein the at least four genes are selected from the group consisting of^ Γ (SEQ ID NO: l), bdlA (SEQ ID NO:4), nirS (SEQ ID NO:3) and ftsZ (SEQ ID NO:7); pilT (SEQ ID NO: l), bdlA (SEQ ID NO:4), lasi (SEQ ID NO:5) and ftsZ (SEQ ID NO:7); bdlA (SEQ ID NO:4), lasi (SEQ ID NO:5), nirS (SEQ ID NO:3) and ftsZ (SEQ ID NO:7); and pilT (SEQ ID NO: l) bdlA (SEQ ID NO:4), lasi (SEQ ID NO:5) and nirS (SEQ ID NO:3).

[0034] In another aspect of any of these methods, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the expression at least five genes are disrupted or suppressed, wherein the at least five genes are selected from the group consisting oipilT (SEQ ID NO: 1), bdlA (SEQ ID NO:4), lasi (SEQ ID NO:5), nirS (SEQ ID NO:3) and ftsZ (SEQ ID NO: 7).

[0035] In another aspect of any of these methods the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the Pseudomonas aeruginosa has a reduced proliferative capability as compared to a non-genetically modified cell.

[0036] In another aspect of any of these methods the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the Pseudomonas aeruginosa has a reduced virulence as compared to a non-genetically modified cell. In one aspect of this method, wherein the reduced virulence is in mammals or plants.

[0037] In another aspect of any of these methods, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the Pseudomonas aeruginosa exhibits reduced motility as compared to a non-genetically modified cell.

[0038] In another aspect of any of these methods, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the Pseudomonas aeruginosa exhibits altered pilus sticking as compared to a non-genetically modified cell.

[0039] In another aspect of any of these methods, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the Pseudomonas aeruginosa exhibits altered twitching motility as compared to a non-genetically modified cell.

[0040] In another aspect of any of these methods, the cathode is coated with an agent to prevent a biofilm from growing on the cathode.

[0041] In another aspect of any of these methods, the agent is a 17-mer terminal fragment of a pilus PilA protein derived from the bacteria.

[0042] In another aspect of any of these methods, the biofilm further comprises Nitrobacter and Nitrosomonas microbes.

[0043] In another aspect of any of these methods, the genetic modification produces a phenotype selected from the group consisting of a reduction in cytotaxis, an increase in the number of pili on the bacteria, a reduction in the rate of cell division, and combinations thereof.

[0044] In another aspect of any of these methods, at least one of the cathode and anode are fabricated from porous material. In one aspect of this method, the porous material comprises porous stainless steel.

[0045] In another aspect of any of these methods, the microbial fuel cell comprises a plurality of anodes selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 anodes.

[0046] In another aspect of any of these methods, the genetically modified bacteria further express an uncoupling protein. [0047] In another aspect of any of these methods, the thickness of the biofilm is between 30 μιη and 100 μιη.

[0048] In another aspect of any of these methods, the microbial fuel cell lacks a membrane.

[0049] In another aspect of any of these methods, the microbial fuel cell is in electrical communication to ultracapacitor.

[0050] In another aspect of any of these methods, at least one of the first plurality of sensors, or at least one of the second plurality of sensors, or if present, at least one of the third plurality of sensors is connected via wireless communication to one or more monitors.

[0051] In another aspect of any of these methods, at least one of the first plurality of sensors, or at least one of the second plurality of sensors, or if present, at least one of the third plurality of sensors is connected via wireless communication to the external network operations center.

[0052] In another aspect of any of these methods, the external network operations center comprises a computer implemented system that i) monitors the activity from one or more of the first, second or third plurality of sensors and ii) displays the activity from the one or more sensors to one or more registered users of the external network operations center.

[0053] In another aspect of any of these methods, the external network operations center further comprises an analysis and reporting module that provides updates and reports based on data from the one or more sensors.

[0054] In another aspect of any of these methods, the external network operations center further comprises a communication function that allows the registered users to modulate the status or activity of the bioreactor, or one or more of the plurality of devices, or if present, the additional form of local energy generation.

[0055] In one aspect of any of these methods, the bioreactor feedstock is selected from the group consisting of carbohydrates, hydrocarbons, alcohols, amino acids, and mixtures thereof. In one aspect the feedstock is derived from household wastewater, or sewage. In one aspect, the feedstock comprises a high-energy feedstock such as bacterial growth medium. In one aspect, the bacterial growth medium is selected from but not limited to the high-energy group consisting of the Terrific Broth, SuperBroth, Tryptone Broth, and LB media. In another aspect, the feedstock further comprises a nitrate source, and in one aspect the nitrate source comprises between about 0.5 % and about 5 % of a nitrate salt. In one aspect, the nitrate salt is selected from sodium and potassium. In one aspect of any of these methods, the addition of the feedstock to the bioreactor is modulated by the activity of the external operations center.

[0056] In another embodiment, the current invention includes a system for generating electrical energy for a building, or group of buildings, comprising 1) a microbial bioreactor fuel cell, 2) a computer implemented external network operations center, 3) an implemented distributed network of sensors, and 4) an energy distribution system,

[0057] wherein the microbial bioreactor fuel cell comprises,

a. a housing with at least one opening to facilitate contact facilitate contact of feedstock and fuel cell components;

b. a cathode;

c. an anode in electrical communication with the cathode; and

d. a biofilm functionally associated with the anode,

wherein the biofilm comprises genetically modified microbes that break down the feedstock and donate electrons to the anode, thereby generating electrical energy;,

[0058] wherein the computer implemented external network operations center comprises an activity module that monitors the activity from one or more of the sensors from the distributed network of sensors, and a display module that displays the activity from the one or more sensors to one or more registered users of the external network operations center,

[0059] wherein the distributed network of sensors provide operating data about the bioreactor, and energy usage in the building, or the group of buildings, and can transmit the data to the external network operations center,

[0060] wherein the energy distribution system connects the bioreactor to one or more energy consuming devices associated with the building or the group of buildings, and wherein the energy distribution system is also connected to at least one alternative energy source, [0061] wherein the computer external network operations center is adapted to make adjustments to the energy distribution system, or the bioreactor based on the data received from the distributed network of sensors.

[0062] In one aspect of this system, the alternative energy source is selected from solar energy, wind energy, thermal energy, chemical energy, wave energy, tidal energy, and an electrical distribution grid.

[0063] In another aspect of any of these systems, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the P. aeruginosa has at least one genetic modification that results in the loss of functional expression, or the suppression in expression in at least one endogenous gene selected from the group consisting of pilT (SEQ ID NO: l), pilA (SEQ ID NO:2), nirS (SEQ ID NO:3), bdlA (SEQ ID NO:4), lasi (SEQ ID NO:5), lasR (SEQ ID NO:6), ftsZ (SEQ ID NO:7), fliC (SEQ ID NO:8) and combinations thereof, wherein said microbes exhibit decreased expression of the gene interest and exhibit an altered electrogenic efficacy.

[0064] In another aspect of any of these systems, the genetically modified microbes comprise Pseudomonas aeruginosa in which a combination of the genes are disrupted or suppressed.

[0065] In another aspect of any of these systems, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the expression at least two genes are disrupted or suppressed, wherein the at least two genes are selected from the group consisting οΐρϊΙΤ (SEQ ID NO: l) and bdlA (SEQ ID NO:4); bdlA (SEQ ID NO:4) and nirS (SEQ ID NO:3); bdlA (SEQ ID NO:4) and lasi (SEQ ID NO:5); nirS (SEQ ID NO:3) and pilT (SEQ ID NO: l); nirS (SEQ ID NO:3) and lasi (SEQ ID NO:5); lasi (SEQ ID NO:5) and pilT (SEQ ID NO:

(SEQ ID NO:7) and pilT (SEQ ID NO: 1); ftsZ (SEQ ID NO:7) and bdlA (SEQ ID NO:4); ftsZ (SEQ ID NO:7) and nirS (SEQ ID NO:3); and ftsZ (SEQ ID NO:7) and lasi (SEQ ID NO:5).

[0066] In another aspect of any of these systems, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the expression at least three genes are disrupted or suppressed, wherein the at least three genes are selected from the group consisting οΐρϊΙΤ (SEQ ID NO: l), bdlA (SEQ ID NO:4) and ftsZ (SEQ ID NO:7); bdlA (SEQ ID NO:4), nirS (SEQ ID NO:3) and ftsZ (SEQ ID NO:7); bdlA (SEQ ID NO:4), lasi (SEQ ID NO:5) and ftsZ (SEQ ID NO:7); nirS (SEQ ID NO:3), pilT (SEQ ID NO: 1) and ftsZ (SEQ ID NO:7); nirS (SEQ ID N0:3), lasI (SEQ ID N0:5) and ftsZ (SEQ ID N0:7); lasI (SEQ ID N0:5), pilT (SEQ ID NO: 1) and fisZ (SEQ ID NO:7); pilT (SEQ ID NO: l), bdlA (SEQ ID NO:4) and nirS (SEQ ID NO:3); pilT (SEQ ID NO: l), bdlA (SEQ ID N0:4) and lasI (SEQ ID N0:5); and bdlA (SEQ ID N0:4), lasI (SEQ ID N0:5) and nirS (SEQ ID N0:3).

[0067] In another aspect of any of these systems, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the expression at least four genes are disrupted or suppressed, wherein the at least four genes are selected from the group consisting of pilT (SEQ ID NO: l), bdlA (SEQ ID NO:4), nirS (SEQ ID NO:3) and ftsZ (SEQ ID NO:7); pilT (SEQ ID NO: l), bdlA (SEQ ID NO:4), lasI (SEQ ID NO:5) and ftsZ (SEQ ID NO:7); bdlA (SEQ ID NO:4), lasI (SEQ ID NO:5), nirS (SEQ ID NO:3) and ftsZ (SEQ ID NO:7); and pilT (SEQ ID NO: l) bdlA (SEQ ID NO:4), lasI (SEQ ID NO:5) and nirS (SEQ ID NO:3).

[0068] In another aspect of any of these systems, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the expression at least five genes are disrupted or suppressed, wherein the at least five genes are selected from the group consisting of pilT (SEQ ID NO: 1), bdlA (SEQ ID NO:4), lasI (SEQ ID NO:5), nirS (SEQ ID NO:3) and ftsZ (SEQ ID NO: 7).

[0069] In another aspect of any of these systems, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the Pseudomonas aeruginosa has a reduced proliferative capability as compared to a non-genetically modified cell.

[0070] In another aspect of any of these systems, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the Pseudomonas aeruginosa has a reduced virulence as compared to a non-genetically modified cell. In one aspect of this method, wherein the reduced virulence is in mammals or plants.

[0071] In another aspect of any of these systems, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the Pseudomonas aeruginosa exhibits reduced motility as compared to a non-genetically modified cell.

[0072] In another aspect of any of these systems, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the Pseudomonas aeruginosa exhibits altered pilus sticking as compared to a non-genetically modified cell. [0073] In another aspect of any of these systems, the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the Pseudomonas aeruginosa exhibits altered twitching motility as compared to a non-genetically modified cell.

[0074] In another aspect of any of these systems, the cathode is coated with an agent to prevent a biofilm from growing on the cathode.

[0075] In another aspect of any of these systems, the agent is a 17-mer terminal fragment of a pilus PilA protein derived from the bacteria.

[0076] In another aspect of any of these systems, the biofilm further comprises Nitrobacter and Nitrosomonas microbes.

[0077] In another aspect of any of these systems, the genetic modification produces a phenotype selected from the group consisting of a reduction in cytotaxis, an increase in the number of pili on the bacteria, a reduction in the rate of cell division, and combinations thereof.

[0078] In another aspect of any of these systems, at least one of the cathode and anode are fabricated from porous material. In one aspect of this system, the porous material comprises porous stainless steel.

[0079] In another aspect of any of these systems, the microbial fuel cell comprises a plurality of anodes selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 anodes.

[0080] In another aspect of any of these systems, the genetically modified bacteria further express an uncoupling protein.

[0081] In another aspect of any of these systems, the thickness of the biofilm is between 30 μιη and 100 μιη.

[0082] In another aspect of any of these systems, the microbial fuel cell lacks a membrane.

[0083] In another aspect of any of these systems, the microbial fuel cell is in electrical communication to ultracapacitor.

[0084] In another aspect of any of these systems, at least one of the distributed network of sensors is connected via wireless communication to one or more monitors. [0085] In another aspect of any of these systems, at least one of the distributed network of sensors is connected via wireless communication to the external network operations center.

[0086] In another aspect of any of these systems, the external network operations center further comprises an analysis and reporting module that provides updates and reports based on data from the one or more sensors.

[0087] In another aspect of any of these systems, the external network operations center further comprises a communication module that allows the registered users to modulate the status or activity of the bioreactor or an alternative energy source.

BRIEF DESCRIPTION OF THE FIGURES

[0088] FIG. 1 presents electrogenic test data from experiments utilizing a 2.5 cm diameter anode. The panels present voltages measured on four different channels corresponding to microbial fuel cells comprising different microbes. In all the panels voltage is indicated on the y axis in volts. Time progression is indicated on the x axis; the microbial fuel cells were monitored for 3.5 days. Panel A presents voltages obtained from Shewenella; the voltage increases throughout the monitoring period, reaching almost 0.2 V. Panel B (channel 1) presents voltages obtained from a genetically modified P. aeruginosa; the voltage fluctuates throughout the monitoring period with an early peak between 0.2 and 0.22 V. Panel C (channel 2) presents voltages obtained from a second genetically modified P. aeruginosa; the voltage is high (0.3 V) early in the monitoring period and rapidly drops. Panel D (channel 3) presents voltages obtained from a third genetically modified P. aeruginosa; the voltage fluctuates between 0.005 V and 0.05 V. Panel E (channel 4) presents voltages obtained from a fourth genetically modified P. aeruginosa, a pilT mutant; the voltage starts near 0.5 V then decreases as the feedstock is consumed. The results from the fourth genetically modified P. aeruginosa indicate that the voltage produced exceeds the voltage produced in the microbial fuel cell containing Shewanella.

[0089] FIG. 2 depicts an example microbial fuel cell.

[0090] FIG. 3 depicts an example microbial fuel cell system.

[0091] FIG. 4 presents voltages obtained from genetically modified P. aeruginosa, a PilT mutant; the voltage starts below 0.05 V and rises to nearly 0.45 V. These voltometric measurements are from a single 13 ml fuel cell. [0092] FIG. 5 depicts a three example microbial fuel cell connected in series. This setup is shown coupled to a load.

[0093] FIG. 6 presents voltages obtained from genetically modified P. aeruginosa, a PUT mutant; the voltage starts between 0.3 and 0.5 V and rises to nearly 1 V. These voltometric measurements are from three 13 ml fuel cells connected in series.

[0094] FIG. 7 depicts an exemplary electrogenic bioreactor (EBR) Proposed Network Architecture

[0095] FIG. 8 depicts an exemplary water-energy network.

[0096] FIG. 9 depicts an exemplary data hub with a microbial bioreactor fuel cell.

DETAILED DESCRIPTION

[0097] In one embodiment, the present invention includes a method of generating electrical energy for a building, or group of buildings, comprising;

1) connecting a microbial fuel cell bioreactor, with a means for transferring electrical energy from the bioreactor to an energy distribution system for the building or the group of buildings, wherein the bioreactor comprises, a) a housing with one or more openings to facilitate contact of feedstock and fuel cell components; b) a cathode; c) an anode in electrical communication with the cathode; and d) a biofilm functionally associated with the anode; wherein the biofilm comprises genetically modified microbes that break down the feedstock and donate electrons to the anode, thereby generating electrical energy;

3) connecting a second plurality of sensors to a plurality of devices gaining their energy from the microbial fuel cell, wherein the second plurality of sensors provide operating data about the plurality of devices and can transmit the data to the external network operations center,

wherein the external network operations center makes adjustments to the energy distribution system, or bioreactor, based on the data received from the first plurality of sensors, and the second plurality of sensors. [0098] In another embodiment, the current invention includes a system for generating electrical energy for a building, or group of buildings, comprising 1) a microbial bioreactor, 2) a computer implemented external network operations center, 3) an implemented distributed network of sensors, and 4) an energy distribution system,

[0099] wherein the microbial bioreactor comprises, a) a housing with at least one opening to facilitate contact of feedstock and fuel cell components; b) a cathode; c) an anode in electrical communication with the cathode; and d) a biofilm functionally associated with the anode, wherein the biofilm comprises genetically modified microbes that break down the feedstock and donate electrons to the anode, thereby generating electrical energy,

[00100] wherein the computer implemented external network operations center comprises an activity module that monitors the activity from one or more of sensors from the distributed network of sensors, and a display module that displays the activity from the one or more sensors to one or more registered users of the external network operations center,

[00101] wherein the distributed network of sensors provide operating data about the bioreactor, and energy usage in the building, or the group of buildings, and can transmit the data to the external network operations center,

[00102] wherein the energy distribution system connects the bioreactor to one or more energy consuming devices associated with the building or the group of buildings, and wherein the energy distribution system is also connected to at least one alternative energy source,

[00103] wherein the computer external network operations center is adapted to make adjustments to the energy distribution system, or the bioreactor based on the data received from the distributed network of sensors.

[00104] In any of these methods and systems, the bioreactor comprises a biofilm functionally associated with the anode. By "biofilm" is intended a complex surface attached growth comprising multiple cells that are typically enmeshed or embedded within a polysaccharide/protein matrix. Biofilms occur in varying thickness; such thickness may change over time and may vary in different areas of the biofilm. Preferred thickness of a biofilm is within a range between 1 μιη and 300 μιη, particularly between 10 μιη and 200 μιη and more particularly between 30 and 100 μιη. Biofilms may be comprised of multiple cell types, a single cell type, or a clonal population of cells. Multiple cell types may refer to cells of different species, cells of different strains of the same species, and cells with different genetic alterations. Several biofilm-related characteristics impact electrogenic efficacy. Biofilm-related characteristics that impact electrogenic efficacy include, but are not limited to, the number of bacteria in the biofilm, the bacterial density in the biofilm, and the number of pili attached to the anode. In an embodiment a biofilm may be attached to, growing on, adhered to, coating, touching, covering or adjacent to the surface of an anode or anode chamber. The biofilm may improve survival of cells comprising the biofilm in adverse conditions including, but not limited to, non-preferred temperatures, pH ranges, heavy metal concentration and the like. Modulating the feedstock may modulate biofilm robustness.

[00105] In some embodiments, the thickness of the biofilm is between 1-500 μιη. In some embodiments, the biofilm thickness is between 10-250 μιη, 20-200 μιη, or 30-100 μιη.

[00106] The biofilm comprises genetically modified microbial cells that can donate electrons to an anode and thereby generate electrical current from a metabolic process such as, for example, oxidative phosphorylation. The microbial cells include bacteria and fungi. Bacterial cells that can transfer electrons to an external component include, but are not limited to Synechocystis sp PCC 6803, Brevibacillus sp. PTHl, Pseudomonas sp., Psuedomonas aeruginosa (P. aeruginosa), Pseudomonas putida, Shewanella sp, Shewanella oneidensis MR-1, Shewanell putrefaciens IR-1, Shewanella oneidensis DSP10, Geobacter sp., Geobacter sulfurreducens, Geobacter metallireducens, Peletomaculum thermopropionicum, Methanothermobacter thermautotrophicus , Ochrobactrum anthropi, Clostridium butyricum EG3, Desulfuromonas acetoxidans, Rhodoferax ferrireducens , Aeromonas hydrophila A3, Desulfobulbus propionicus, Geopsychrobacter electrodiphilus , Geothrix fermentans, Escherichia coli, Rhodopseudomonas palustris, Ochrobactrum anthropi YZ-1, Desulfovibrio desulfuricans, Acidiphilium sp.3.2Sup5, Klebsiella pneumonia L17. Fungal cells that can generate electrical current from a metabolite include, but are not limited to Pichia anomala. See for example, Prasad et al. (2007) Biosens. Bioelectron. 22:2604-2610; Gorby et al. (2006) Proc. Natl. Acad. Sci. USA 103 : 11358-1 1363; Pham et al (2008) Appl. Microbiol. Biotechnol. 77: 1 119-1129; and El-Naggar et al (2008) Biophys J. 95:L10-L12; herein incorporated by reference in their entirety. Microbial cells that are capable of exocellular electron transfer are sometimes described as "exoelectrogens", "electrochemically active microbes", "electricigens", "anode respiring microbes", "electrochemically active bacteria", and "anode respiring bacteria".

[00107] In some embodiments, the biofilm comprises a largely uniform population of genetically modified microbes. In other embodiments, the population of microbes that comprise the biofilm is diverse, and can include various combinations of microbial species, and include both natural and genetically modified microbes. Other microbes can also comprise the biofilm that do not necessarily generate electrical current and donate electrons to the anode. In some embodiments, the biofilm can further comprise natural and/or genetically modified microbes selected from the group consisting of Nitrobacter and Nitrosomonas and combinations thereof.

[00108] In some embodiments, the biofilm comprises genetically modified P. aeruginosa, Nitrobacter spp. and Nitrosomonas spp.

[00109] In some embodiments, the genetically modified microbial cells of the fuel cell exhibit an altered electrogenic efficacy. By "electrogenic efficacy" is intended the capability to transfer electrons to or from an anode or a cathode. Such a transfer may be direct or indirect via a mediator. With regard to the electrogenic efficacy of a microbial cell, numerous components or characteristics of the cell can impact electrogenic efficacy. A component or characteristic that impacts electrogenic efficacy is an electrogenic component or electrogenic characteristic. Such electrogenic-related characteristics include, but are not limited to, biofilm related characteristics such as biofilm forming abilities, biofilm density, tolerance for existence in a biofilm, cell packing characteristics, quorum sensing characteristic, cell growth rate, cell division rate, cell motility, substrate attachment, substrate adhesion, enzymatic processing of a feedstock, oxidation, phosphorylation, reduction, electron transfer, twitching motility, piliation, cell to cell adhesion, nanowire formation, nanowire structure, the ability to disperse from the biofilm and mediator related characteristics. Electrogenic efficacy can be measured using volt or current measuring devices known in the art (multimeters and computer-based measuring techniques).

[00110] One means by which bacteria can donate electrons is via the synthesis of tiny, hollow and retractable surface appendages termed type 4 pili that emanate from the surface of many bacteria. Pili that conduct electrons have been termed "nano wires." In this aspect, the formation of a biofilm on the anode enables the transfer of electrons to the anode directly via the pili and without the need for a mediator component.

[0011 1] Pili normally make direct contact with surfaces and mediate a form of surface movement termed twitching motility. In some embodiments, the bacteria are genetically modified to optimize transfer of electrons by means of Type 4 bacterial pili.

[00112] In some embodiments, the genetic modification produces a phenotype selected from the group consisting of a reduction in cytotaxis, an increase in the number of pili on the bacteria, a reduction in the rate of cell division, and combinations thereof.

[00113] An electrogenic component can include any polypeptide, peptide, or compound involved in electrogenesis including but not limited to transporters, ion transporters, pilus components, membrane components, cytochromes, quorum sensors, redox active proteins, electron transfer components, pyocyanin, pyorubrin, pyomelanin, 1 -hydroxy - phenazine or homogentisate, uncoupler proteins (UCPs), and enzymes, pilin, pilT, bdlA, lasl, lasR, nirS, ftsZ, pilA and fliC. In some embodiments, the genetically modified microbes express an uncoupling protein.

[00114] In some embodiments, the microbial cell is genetically modified by transformation with a nucleic acid molecule. In some embodiments, the isolated nucleic acid molecule comprises an expression cassette comprising a promoter operably linked to a heterologous nucleotide sequence of interest. The cell may exhibit altered expression of the heterologous nucleotide sequence of interest such as expression of a new or different polypeptide in the cell. Expression of the heterologous nucleotide sequence of interest may be increased in the genetically modified (mutant) cell as compared to expression of the nucleotide sequence of interest in a non-genetically modified cell (often referred to as wild-type). In some embodiments, the promoter is selected from the group consisting of inducible promoters and constitutive promoters. In some embodiments, the genetically modified microbial cell can further comprise a second expression cassette. In some embodiments, the first expression cassette comprises a second heterologous nucleotide sequence of interest.

[00115] In some embodiments, the microbial cell is transformed with an electrogenic efficacy cassette. The electrogenic efficacy cassette is an isolated nucleic acid molecule comprising an expression cassette comprising a promoter operably linked to a first nucleotide sequence of interest wherein the first nucleotide sequence of interest encodes a polypeptide that alters electrogenic efficacy. The isolated nucleic acid molecule may further comprise a second nucleotide sequence of interest wherein the second nucleotide sequence of interest also encodes a polypeptide that alters electrogenic efficacy.

[00116] In some embodiments, the genetically modified microbial cell exhibits decreased expression of the endogenous gene of interest. In some embodiments, the genetically modified microbial cell has a disruption in an endogenous nucleotide sequence or gene of interest. The disruption can include, for example, one or more point mutations, an insertion or a deletion. In some embodiments, a nucleotide sequence or gene of interest or fragment thereof is deleted from the genome and the cell stably maintains the deletion of the endogenous nucleotide sequence or gene of interest.

[00117] It is recognized that the genetically modified microbes can contain multiple genetic alterations or mutations that inactivate or suppress the expression of the gene of interest; these may include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more stably incorporated mutations. Such a stably incorporated mutation can introduce a heterologous nucleotide sequence of interest or disrupt an endogenous nucleotide sequence.

[00118] In some embodiments, the microbe is transformed with an isolated nucleic acid molecule comprising a regulatable expression cassette. In some embodiments, expression cassettes comprise a transcriptional initiation region comprising a promoter nucleotide sequences operably linked to a heterologous nucleotide sequence of interest whose expression is to be controlled by the promoter. In some embodiments, the expression cassette is provided with at least one restriction site for insertion of the nucleotide sequence to be under the transcriptional regulation of the regulatory regions. In some embodiments, the expression cassette additionally comprises one or more selectable marker genes.

[00119] In some embodiments, expression vectors can also include anti-sense, ribozymes or siRNA polynucleotides to reduce the expression of target sequences such as, for example, to reduce the level of expression of any of specific, or combination of genes of particular interest (See, e.g., Sioud M, & Iversen, Curr. Drug Targets (2005) 6 (6):647- 53; Sandy et al, Biotechniques (2005) 39 (2):215-24).

[00120] In some embodiments, the expression cassette includes in the 5'-to-3' direction of transcription, a transcriptional and translational initiation region, and a heterologous nucleotide sequence of interest. In addition to containing sites for transcription initiation and control, expression cassettes can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome-binding site for translation. Other regulatory control elements for expression can include initiation and termination codons. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

[00121] In some embodiments, the expression cassette comprising the promoter sequence operably linked to a heterologous nucleotide sequence may also contain at least one additional nucleotide sequence for a gene to be co-transformed into the organism. Alternatively, the additional sequence(s) can be provided on another expression cassette.

[00122] The regulatory sequences include promoters for directing mRNA transcription.

These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.

[00123] In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers.

[00124] Where appropriate, the heterologous nucleotide sequence whose expression is to be under the control of the promoter sequence and any additional nucleotide sequence(s) may be optimized for increased expression in the transformed microbe. That is, these nucleotide sequences can be synthesized using species preferred codons for improved expression. Methods are available in the art for synthesizing species-preferred nucleotide sequences. See, for example, Wada et al. (1992) Nucleic Acids Res. 20 (Suppl), 211 1- 21 18; Butkus et al. (1998) Clin Exp Pharmacol Physiol Suppl. 25:S28-33; and Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., herein incorporated by reference.

[00125] Additional sequence modifications are known to enhance gene expression in a cellular host. The G-C content of the heterologous nucleotide sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. [00126] In some embodiments, the expression cassettes additionally contain 5' leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) Proc. Nat. Acad. Set USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986)); MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20); and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353 :90-94). Other methods known to enhance translation and/or mRNA stability can also be utilized, for example, introns, and the like.

[00127] In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose; in vitro mutagenesis; primer repair; restriction; annealing; substitutions, for example, transitions and transversions; or any combination thereof may be involved.

[00128] Reporter genes or selectable marker genes may be included in the expression cassettes. Examples of suitable reporter genes known in the art can be found in, for example, Ausubel et al. (2002) Current Protocols in Molecular Biology. John Wiley & Sons, New York, New York, herein incorporated by reference.

[00129] Selectable marker genes for selection of transformed cells or tissues can include genes that confer antibiotic resistance. Examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to gemtamicin. Schweizer, H.P. 1993. Small broad-host-range gentamicin resistance gene cassettes for site-specific insertion and deletion mutagenesis Biotechniques 15:831-833., carbenicillin Parvatiyar et al, 2005. Global analysis of cellular factors and responses involved in Pseudomonas aeruginosa resistance to arsenite. J Bacteriol 187:4853-64., chloramphenicol (Herrera Estrella et al. (1983) EMBO J. 2:987-992); methotrexate (Herrera Estrella et al. (1983) Nature 303 :209-213; Meijer et al. (1991) Plant Mol. Biol. 16:807-820); hygromycin (Waldron et al. (1985) Plant Mol. Biol. 5: 103-108; Zhijian et al. (1995) Plant Science 108:219-227); streptomycin (Jones et al. (1987) Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res. 5: 131-137); bleomycin (Hille et al. (1990) Plant Mol. Biol. 7: 171-176); sulfonamide (Guerineau et al. (1990) Plant Mol. Biol. 15: 127-136); puromycin (Abbate et al (2001) Biotechniques 31 :336-40; cytosine arabinoside (Eliopoulos et al. (2002) Gene Ther. 9:452-462); 6-thioguanine (Tucker et al. (1997) Nucleic Acid Research 25:3745-46).

[00130] Other genes that could serve utility in the recovery of transgenic events but might not be required in the final product would include, but are not limited to, examples such as levansucrase (sacB), GUS (beta-glucoronidase; Jefferson (1987) Plant Mol. Biol. Rep. 5:387); GFP (green fluorescence protein; Wang et al. (2001) Anim Biotechnol 12: 101-1 10; Chalfie et al. (1994) Science 263:802), BFP (blue fluorescence protein; Yang et al. (1998) J. Biol. Chem. 273:8212-6), CAT; and luciferase (Riggs et al. (1987) Nucleic Acid Res. 15 (19):81 15; Luchrsen et al. (1992) Methods Enzymol. 216: 397-414).

[00131] A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. A variety of inducible promoter systems have been described in the literature and can be used in the present invention. These include, but are not limited to, tetracycline-regulatable systems (WO 94/29442, WO 96/40892, WO 96/01313, U.S. Application No. 10/613,728); hormone responsive systems, interferon-inducible systems, metal-inducible systems, and heat-inducible systems, (WO 93/20218); ecdysone inducible systems, and araC-Pbad- Some of these systems, including ecdysone inducible and tetracycline inducible systems are commercially available from Invitrogen (Carlsbad, Calif.) and Clontech (Palo Alto, Calif), respectively. See Qiu et al, (2008) App. & Environ Microbiology 74:7422-7426 and Guzman et al, (1995) J. Bacteriol. 177:4121-4130, herein incorporated by reference in their entirety.

[00132] By "inducible" is intended that a chemical stimulus alters expression of the operably linked nucleotide sequence of interest by at least 1%, 5%, preferably 10%, 20%, more preferably 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or more. The difference may be an increase or decrease in expression levels. Methods for assaying expression levels are described elsewhere herein. The chemical stimulus may be administered or withdrawn. Various chemical stimuli are known in the art.

[00133] One of the most widely used inducible systems is the binary, tetracycline-based system, which has been used in both cells and animals to reversibly induce expression by the addition or removal of tetracycline or its analogues. (See Bujard (1999). J. Gene Med. 1 :372-374; Furth, et al. (1994). Proc. Natl. Acad. Sci. USA 91 :9302-9306; and Mansuy & Bujard (2000). Curr. Opin. Neurobiol. 10:593-596, herein incorporated by reference in their entirety.) Another example of such a binary system is the cre/loxP recombinase system of bacteriophage P I. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992) PNAS 89:6232-6236. In the Cre/LoxP recombinase system, the activator transgene encodes recombinase. If a cre/loxP recombinase system is used to regulate expression of the transgene, microbes containing transgenes encoding both the Cre recombinase and a selected target protein are required. Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. (1991) Science 251 : 1351-1355. A single transgenic microbe may comprise multiple inducible promoters.

[00134] Methods of determining expression levels are known in the art and include, but are not limited to, qualitative Western blot analysis, immunoprecipitation, radiological assays, polypeptide purification, spectrophotometric analysis, Coomassie staining of acrylamide gels, ELISAs, RT-PCR, 2-D gel electrophoresis, microarray analysis, in situ hybridization, chemiluminescence, silver staining, enzymatic assays, ponceau S staining, multiplex RT-PCR, immunohistochemical assays, radioimmunoassay, colorimetric analysis, immunoradiometric assays, positron emission tomography, Northern blotting, fluorometric assays and SAGE. See, for example, Ausubel et al, eds. (2002) Current Protocols in Molecular Biology, Wiley-Interscience, New York, New York; Coligan et al (2002) Current Protocols in Protein Science, Wiley-Interscience, New York, New York; and Sun et al. (2001) Gene Ther. 8: 1572-1579, herein incorporated by reference. It is recognized that expression of a nucleotide sequence of interest may be assessed, analyzed, or evaluated at the RNA, polypeptide, or peptide level.

[00135] By altered expression is intended a change in expression level of the full nucleotide sequence or gene of interest as compared to an untransfomed, unmodified, non-transgenic, or wild-type microbe. Such a change may be an increase or decrease in expression. An expression level may increase approximately 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more. An expression level may decrease approximately 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. It is recognized that altered expression also includes expression of a fragment of the nucleotide sequence of interest rather than the full length nucleotide sequence or gene of interest.

[00136] A genetically modified cell can exhibit an altered cellular property including, but not limited to, an altered electrogenic efficacy. Such an alteration may be an increase or decrease in the property of interest. It is recognized that an alteration in one cellular property may alter a second cellular property; it is further recognized that an increase in one property may decrease a second property, an increase in one property may increase a second property, a decrease in one property may decrease a second property, and a decrease in one property may increase a second property. An altered cellular property may be altered by 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more as compared to that cellular property in a non-transgenic microbial cell. Methods of analyzing cellular properties are known in the art.

[00137] In some embodiments, an isolated nucleic acid molecule that disrupts an endogenous nucleotide sequence or gene of interest replaces the endogenous nucleotide sequence or gene of interest, interrupts the endogenous nucleotide sequence or gene of interest, replaces a portion of the endogenous nucleotide sequence or gene of interest, replaces a regulatory region controlling expression of the endogenous nucleotide sequence or gene of interest, interrupts the regulatory region controlling expression of the endogenous nucleotide sequence, deletes an endogenous nucleotide sequence of interest, deletes a portion of an endogenous nucleotide sequence, deletes a regulatory region, or deletes a portion of a regulatory region.

[00138] By "heterologous nucleotide sequence" is intended a sequence that is not naturally occurring with the promoter sequence. While this nucleotide sequence is heterologous to the promoter sequence, it may be homologous, or native, or heterologous, or foreign, to the host cell. Heterologous nucleotide sequences of interest include, but are not limited to, nucleotide sequences of interest encoding substances that uncouple oxidation and phosphorylation. Uncoupling, interference or disruption of the normally coupled processes, oxidation and phosphorylation alters the proton gradient from the periplasmic space to the cytoplasm. For example in bacteria treated with an exogenous uncoupler such as dinitrophenol, the rate of substrate oxidation increases and electron flow to the anode may increase. Additional uncouplers include, but are not limited to, thermogenin, UCXP-1, UCP-2, and UCP-3, that would be expressed within the microbial cell. In an embodiment, a nucleic acid molecule having a nucleotide sequence encoding an uncoupling polypeptide such as, but not limited to, thermogenin, UCXP-1, UCP-2, and UCP-3 is operably linked to an inducible promoter. The bacterial cell may then be stably transformed with an expression cassette comprising an inducible promoter operably linked to an uncoupling nucleotide sequence of interest. [00139] Various substances can be added to the biofilm. Such substances may include additional organisms compatible with the genetically modified microbe, mediator compounds, antibiotic compounds, additives for regulating or modulating an inducible promoter, and biofilm optimizers. Biofilm optimizers are compounds that modulate a metabolic property of at least one of the cells present in a biofilm, such a metabolic property may impact metabolism of available substrates or physiological cooperation between microbes within the biofilm or microbial fuel cell. Antibiotics that may be added to the biofilm are selected from the group of antibiotics to which the genetically modified microbe is resistant.

[00140] Although improved biofilm formation and maintenance is desirable, it is recognized that over-production of the bacterial cell biofilm matrix may be detrimental to a microbial fuel system. For instance, overproduction of the bacterial cells may clog the microbial fuel cell, alter the environment of the microbial fuel cell, clog a filter between the anode and cathode chambers, increase the likelihood of bacterial cell death or yield a biofilm with a non-optimal thickness. Furthermore, cell division requires energy that could be transferred to the anode. Therefore, in some embodiments exogenous regulation of cell division (or cell replication) may occur. Such regulation may involve the use of inducible promoters.

[00141] In some embodiments, the genetically modified microbial cell is selected from the group consisting of bacterial cells and fungal cells. In some embodiments, the bacterial cells are electron transferring bacteria including but not limited to Pseudomonas, Geobacter, Shewanella, Rhodoferax, and combinations thereof. In some embodiments, the bacterial cells are selected from the group consisting of Pseudomonas aeruginosa and Pseudomonas putida and combinations thereof.

[00142] P. aeruginosa metabolizes a variety of substrates to produce energy, including high nitrate organic materials, carbohydrates, alcohols, amino acids, and hydrocarbons such as diesel fuel and jet fuel; greenhouse gases, solutions or gaseous material with a high nitrate concentration. In one aspect, high energy feed stocks suitable for use in the microbial fuel cell include commercially available bacterial growth media, including for example, Terrific Broth, SuperBroth, Tryptone Broth, LB media, etc. that are also supplemented with about 0.5 % to about 5% of a nitrate salt. In one aspect the nitrate salt is sodium nitrate, or potassium nitrate. Such high energy feedstocks may be added to the bioreactor to increase its electrical output during periods when there is increased demand for electrical energy. In one aspect, such high energy feedstocks may be added to the bioreactor to enhance its output of electrical power.

[00143] P. aeruginosa attaches directly and tightly to metal substrates by means of surface-exposed proteinaceous appendages known as pili (also referred to as nanowires). The attached pili allow electron transfer from the bacteria to the insoluble substrate, in a fashion similar to nanowires. See Yu et al. (2007) J. Bionanoscience 1 :73-83, herein incorporated by reference in its entirety. P. aeruginosa forms biofilms in a variety of conditions including both aerobic and anaerobic conditions; anaerobic conditions result in improved biofilm formation (Yoon et al, 2002. Pseudomonas aeruginosa anaerobic respiration in biofilms: relationships to cystic fibrosis pathogenesis. Dev. Cell. 3: 593- 603). During anaerobic conditions, electrons are donated to the anode surface. The protons (H+) then can react at the cathodic surface to yield hydrogen gas as a byproduct. In aerobic conditions, P. aeruginosa yields water as a byproduct at the cathode in a microbial fuel cell or during planktonic (free-swimming) growth.

[00144] In some embodiments, the genetically modified bacteria are selected from the group of electron transferring bacteria and have one or more disrupted endogenous nucleotide sequences of interest. The disrupted endogenous genes of interest are selected from the group consisting of pilT, pilA, nirS, bdlA, lasl, lasR, ftsZ, fliC, and combinations thereof.

[00145] The pilT gene encodes a polypeptide involved in regulating the number of pili on the bacterial surface; the protein, an electrically conductive polypeptide, is also known as the twitching motility protein. Twitching motility is the movement of bacteria by extending the pili, attaching the pili to an inanimate or animate surface and retracting the pili. Certain pilT mutants, such as pilT disruptions, exhibit reduced twitching motility and increased piliation or hyperpiliation. These pilT mutants exhibit improved attachment, cell to cell adhesion, and biofilm formation. Certain pilT mutants exhibit decreased virulence and decreased ability to detach from surfaces. While not limited by mechanism, reduced twitching motility appears to increase attachment and cell to cell attachment thus improving biofilm formation and increasing biofilm thickness. See Chaing & Burrows (2003) J. Bacterio. 2374-2387, herein incorporated by reference in its entirety.

[00146] bdlA or biofilm dispersion locus A is involved in bacterial dispersion from biofilms. As it is desirable to maintain biofilms on anodic surfaces, altering the bacterial cells ability to perform chemotaxis may improve biofilm formation and maintenance. Chemotaxis is the process of bacterial movement toward or away from a variety of stimuli or repellents. Disruption or deletion oi bdlA reduces the bacterial cell's ability to detach from a surface, thus improving biofilm formation and maintenance and increasing electron transfer to the anode. See Morgan et al (2006) J. Bacteriol. 7335-7343, herein incorporated by reference in its entirety.

[00147] The fliC gene encodes a polypeptide involved in swimming motility and chemotaxis. FliC disruption mutants do not have a flagellum; thus their motility is reduced. FliC disruption mutations exhibit reduced chemotaxis and improved biofilm formation.

[00148] Lasl encodes N-(3-oxododecanoyl)-L-homoserine lactone synthase, a polypeptide that, while not being limited by mechanism, and appears to be involved in the process of cell to cell signaling known as quorum sensing. Certain N-(3- oxododecanoyl)-L-homoserine lactone synthase mutants have altered biofilm characteristics. These altered biofilm formation characteristics include, but are not limited to, thinner, more compact biofilms, increased cell density, altered surface attachment properties, altered polysaccharide production, decreased polysaccharide production, and altered production of pyocyanin. Pyocyanin is redox-active, exhibits antibiotic activity, and can possibly function as a mediator of electron transfer. Deletion of lasl also alters virulence of the bacterial cell in both animal and human cells. Such an altered virulence can be a decreased virulence in a human or animal cell. See Davies et al (1998) Science, herein incorporated by reference in its entirety.

[00149] Deletion of lasR alters virulence of the bacterial cell in both animal and human cells. Such an altered virulence can be a decreased virulence in a human or animal cell. By virulence is intended the relative capacity of a pathogen to overcome a target's defenses. Microbial cells may infect any other living organism; a particular type of microbial cell may have a limited range of targets. Pseudomonas aeruginosa is capable of infecting a wide range of targets including plants, insects, mammals. Exemplary mammals include, but are not limited to humans, bovines, simians, ovines, caprines, swines, lapines, murines and camellids. Aspects of virulence include but are not limited to the scope of suitable targets, infectivity, multiplicity of infection, transfer speed from one target to another, target cell binding ability, antibiotic sensitivity, pathogenesis and antigen production. It is recognized that lowering one aspect of virulence may not impact another aspect of virulence or may increase another aspect of virulence. [00150] NirS encodes respiratory nitrate reductase (NIR) precursor. Inactivated nirS mutants exhibit an altered ability to survive anaerobic culture in biofilms (Yoon et al (2002) Dev Cell 3:593, herein incorporated by reference in its entirety.). While not being limited by mechanism, NIR may be the second enzymatic step in the overall process of nitrate reduction to nitrogen gas during anaerobic respiration. The product of respiratory NIR is nitric oxide (NO), a compound that is inherently toxic to bacteria in micromolar concentrations. NIR may catalyze both the one electron reduction of NO₂ ^" to NO and may catalyze the four-electron reduction of O₂ to 2 I¾0. While not being limited by theory, inactivation of nirS can reduce problems associated with NO in anaerobic biofilms, increase electron flow through the pili, and reduce production of nitrous oxide (N₂O). The surface-exposed Type III secretion apparatus of a nirS mutant generates lower toxin concentrations than wild-type bacteria; nirS mutants exhibit improved virulence properties. See Van Alst, N. E. et al, 2009. Nitrite reductase NirS is required for type III secretion system expression and virulence in the human monocyte cell line THP-1 by Pseudomonas aeruginosa Infect Immun 77: 4446-4454, herein incorporated by reference in its entirety.

[00151] Another genetic modification can be made through the stringent control of ftsZ, a gene encoding a protein responsible for cell division. In general, construction of an isogenic ftsZ mutant is not possible because it is an essential gene. However, in some embodiments, the precise control of the production of FtsZ can be achieved by placing the ftsZ gene under tight genetic control using promoter systems that are inducible.

[00152] In some embodiments, the genetically modified bacteria comprise at least two disrupted or suppressed endogenous genes of interest selected from the group consisting of pilT, pilA, nirS, bdlA, lasl, lasR, ftsZ, and fliC. In various aspects, the genetically modified bacteria comprises at least three, at least four, at least five, at least six, at least seven, or at least eight disrupted endogenous genes of interest selected from the group comprising pilT, pilA, nirS, bdlA, lasl, lasR, ftsZ, and fliC.

[00153] In some embodiments, the genetically modified bacteria comprise Pseudomonas aeruginosa comprising a disrupted or suppressed gene of interest selected from the group consisting of pilT (SEQ ID NO: l), pilA (SEQ ID NO:2), nirS (SEQ ID NO:3), bdlA (SEQ ID NO:4), lasl (SEQ ID NO:5), lasR (SEQ ID NO:6), ftsZ (SEQ ID NO:7), fliC (SEQ ID NO: 8), and combinations thereof.

[00154] In some embodiments, the genetically modified bacteria comprise Pseudomonas aeruginosa comprising at least two disrupted endogenous nucleotide sequences of interest selected from the group consisting oipilT (SEQ ID O: \), pilA (SEQ ID NO:2), nirS (SEQ ID NO:3), bdlA (SEQ ID NO:4), lasl (SEQ ID NO:5), lasR (SEQ ID NO:6), ftsZ (SEQ ID NO:7), and fliC (SEQ ID NO:8), and combinations thereof. In some embodiments, the genetically modified bacteria comprise Pseudomonas aeruginosa comprising at least three, at least four, at least five, at least six, at least seven, or at least eight disrupted endogenous nucleotide sequences of interest selected from the group consisting of pilT (SEQ ID NO: l), pilA (SEQ ID NO:2), nirS (SEQ ID NO:3), bdlA (SEQ ID NO:4), lasl (SEQ ID NO:5), lasR (SEQ ID NO:6), ftsZ (SEQ ID NO:7), and fliC (SEQ ID NO: 8), and combinations thereof.

[00155] In some embodiments, the genetically modified microbial cell can have an altered phenotype as compared with a non-genetically modified cell. For example, in some embodiments, the genetically modified microbial can have a reduced proliferative capability, a reduced virulence in mammals or plants, a reduced motility, altered pilus sticking, altered twitching motility, altered iron-containing cytochrome concentration, particularly the iron-containing cytochrome concentration at or near the cytoplasmic membrane of the cell, altered concentration of a channel forming protein, particularly the channel forming concentration of the outer membrane of the cell, an increased current output/bacterial cell when the bacterial cell is a component of a microbial fuel cell, exhibit increased electron transfer to an anode, directly or indirectly, and a combination thereof.

[00156] In some embodiments, the biofilm functionally associated with the anode has a thickness between 1 μιη to 300 μιη, between 10 μιη to 200 μιη, between 10 μιη to 30 μιη, or between 30 μιη to 100 μιη.

[00157] In some embodiments, the microbial fuel cell comprises a genetically modified bacterial cell selected from the group of electron transferring bacteria exhibiting an altered electrogenic efficacy, an anode chamber and a cathode chamber. In some embodiments, the genetically modified bacterial cell is Pseudomonas aeruginosa or Pseudomonas putida. In some embodiments, the anode chamber is detachable.

[00158] In some embodiments, the microbial fuel cell comprises a biofilm comprising a genetically modified bacterial cell selected from the group of electron transferring bacteria and exhibiting an altered electrogenic efficacy. In some embodiments the biofilm is attached to either the anode or the cathode. [00159] In some embodiments, anaerobic conditions are maintained around the biofilm in the anodic chamber. In some embodiments, the anodic chamber may be pretreated with a biofilm inhibitor such as a polypeptide or small peptide.

[00160] In some embodiments, the microbial fuel cell further comprises a mediator. The mediator can be an exogenous mediator or a pilus. In some embodiments, the mediator is selected from the group consisting of thionine, methyl viologen, methylene blue, humic acids, neutral red, pyocyanin, pyorubrin, pyomelanin, 1 -hydroxy -phenazine and homogentisate and combinations thereof. In some embodiments, the mediator exhibits bacteriocidal or bacteriostatic activity.

[00161] In some embodiments, the fuel cell comprises a sensor for detecting one or more predetermined compounds and an indicator. The indicator may be visual or audible or both. The indicator may indicate an abnormal level of the predetermined compound.

[00162] In some embodiments, the microbial fuel cell further comprises an ultracapacitor in electrical communication with the microbial fuel cell.

[00163] In some embodiments, the microbial bioreactor fuel cell further comprises a water transfer component. In some embodiments, the water transfer component transfers water produced by the microbial fuel cell to a water collection device or the external environment. In some embodiments, the microbial fuel cell is operated aerobically to emit water as a byproduct.

[00164] In some embodiments the microbial fuel cell further comprises a hydrogen transfer component and may further comprise a hydrogen fuel cell.

[00165] In some embodiments, microbes can obtain energy from a feedstock or material and the electrons are generated through a metabolic process, such as oxidative phosphorylation or cellular respiration. In some embodiments the feedstock is circulated past the anode.

[00166] In some embodiments the genetically modified microbes are replaced, removed, augmented with other flora or fauna, or reseeded.

[00167] Anaerobic conditions may encompass both strict anaerobic conditions with no O2 present and mild anaerobic conditions wherein the O2 concentration occurs within a range from 0 to 15%, 0.001% to 12.5%, 0.001% to 10%, 0.001% to 7.5%, 0.001% to 5%, 0.01% to 4%, 0.01% to 3%, 0.01% to 2%, 0.01% to 1%, or 0.01% to 0.05%. Thus, bacteria in an anaerobic environment metabolize feedstock differently than in aerobic conditions. In some embodiments aerobic conditions are desirable. In some embodiments anaerobic conditions are desirable. Anaerobic conditions may be established by utilizing an oxygen removing system. Oxygen removing enzyme systems include, but are not limited to, a glucose-glucose oxidase -catalase enzymatic (¾ removal system. Glucose oxidase converts glucose to uric acid and H2O2. Glucose oxidase is an oxygen-dependent enzyme. The glucose oxidase and catalase reactions collectively halve the oxygen concentrations in each cycle. By "maintaining" anaerobic conditions around a biofilm is intended the establishment of anaerobic metabolism by a given microorganism for a period of time including but not limited to, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, and 1 year. It is recognized that intermittent periods of aerobic conditions may occur particularly with regard to maintenance or introduction of feedstock to the microbial fuel cell.

[00168] Methods of inoculating an anode include, but are not limited to, immersion of the anode in a culture, addition of bacteria to the anode, and addition of a matrix comprising a genetically modified microbe.

[00169] In some embodiments of the invention, one or more components of the fuel cell are coated with an agent that prevents or inhibits a biofilm from growing on the surface. In some embodiments, the cathode is coated with the agent to prevent the biofilm from attaching and growing on the cathode. In some embodiments, the agent is a 17-mer terminal fragment of a pilus PilA protein derived from the bacteria. In some embodiments, the microbial fuel cell, particularly the anodic compartment, is incubated with a 17 amino acid polypeptide from the C-terminus of the PilA peptide. The terminal 17 amino acids of the PilA protein mediates attachment to a variety of surfaces and reduces biofilm formation. In some embodiments the anodic chamber is pretreated or coated with the 17-mer, but the anode is not.

[00170] By anode is intended an electron acceptor. The anode may be of planar, cylindrical, layered spiral cylindrical, curved, angled or other geometrical shape such as but not limited to, a sheet, multiple sheets, wire mesh, porous tube, and sponge-like matrix. It is recognized that it is desirable for the anode to provide a large surface area to volume ratio. The anode may be removable from the microbial fuel cell. Optimal operation of the microbial fuel cell may involve cleaning or replacement of the anode. An anode may be constructed of any suitable material including but not limited to, metal (stainless steel), carbon, carbon nanotubes, carbon nanofibers, carbon cloth, carbon paper, platinum, graphite, graphite rods, graphite felts, graphite foams, graphite pellets, reticulated vitreous carbon (RVC) 97% porous, synthetic diamond, gold, aluminum, or other electrically conductive material. A porous metal, such as sintered steel, may provide a large surface area to volume ratio for the anode. For example, the anode may be a planar surface, multiple thin plates in close proximity with each other or a rolled planar surface or mesh. It is recognized that anode shape and anode material may be modified or optimized for different utilities of the microbial fuel cell. It is recognized that anodes may exhibit high surface area, low resistance, high conductivity, or a combination thereof and may allow high bacterial growth density. In some embodiments, the anode comprises a graphite, or carbon nanotube core decorated with gold, iron or palladium nanomaterials. For example as described in Fan et al, (Biosens Bioelectron. 2010 May 11. [Epub ahead of print] Nanoparticle decorated anodes for enhanced current generation in microbial electrochemical cells). Nanomaterials are typically less than 1 micron in thickness.

[00171] An anode may be connected by a wire to the cathode. Suitable substances for the wire include, but are not limited to, copper or diamond.

[00172] In some embodiments, the fuel cell is embedded in a fuel cell housing with one or more openings to allow the addition of feedstock. The housing may be shaped in any convenient form, and may be configured to insert into another electrical device, or to stand alone. In one aspect, the housing may comprise plugs and receptacles to enable electrical connections to electronic devices.

[00173] The housing can be any length. In some embodiments, the housing has a length selected from the group consisting of about 0.2 to about 1.0 meters, about 1 to about 1.5 meters, about 1.5 to about 2 meters, or about 2 to about 3 meters.

[00174] In some embodiments, the housing has a width selected from the group consisting of about 0.2 to about 1.0 meters, about 1 to about 1.5 meters, about 1.5 to about 2 meters, or about 2 to about 3 meters. [00175] In some embodiments, the housing has a depth selected from the group consisting of about 0.2 to about 1.0 meters, about 1 to about 1.5 meters, about 1.5 to about 2 meters, or about 2 to about 3 meters.

[00176] In some embodiment, the housing with fuel cell has a weight selected from the group consisting of about 20 kg to about 50 kg, about 50 kg to about 100 kg, about 100 kg to about 250 kg, and about 250 kg to about 500 kg.

[00177] In some embodiments, the fuel cell comprises a series of anodes, linked together, and spaced at intervals. In some embodiments, the intervals spacing can be of from 1 to about 5 centimeters. In some embodiments, the number of anodes is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35. In some embodiments, each anode is connected by wiring that connects the anodes with the cathode and the wiring is made from a material selected from the group consisting of copper and diamond.

[00178] In some embodiments, the microbial fuel cell is configured to output less than 2000 watts of power. In some embodiment, the microbial fuel cell is configured to generate from about 1 kilowatt to about 200 kilowatts, about 200 kilo watts to about 1000 kilowatts, or about 1 megawatt to about 20 megawatts of power. In another embodiment, the microbial fuel cell can generate from about 1 mega watt to about 10 mega watts. In another embodiment, the microbial fuel cell outputs from about 40 kilowatts to about 100 kilowatts.

[00179] The cathode of a microbial fuel cell can made of an electrically conductive material including but not limited to, metal (stainless steel), carbon, carbon nanotubes, carbon nanofibers, carbon cloth, carbon paper, platinum, graphite, graphite rods, graphite felts, graphite foams, graphite pellets, reticulated vitreous carbon (RVC) 97% porous, synthetic diamond, silver, gold, aluminum, or other electrically conductive material.

[00180] In some embodiments, a barrier or membrane separates the anode and cathode.

In some embodiments a membrane such as a NAFION® membrane separates the anode and cathode. The barrier slows, decreases, or prevents electrons from moving directly from the anode to the cathode; rather, the electrons flow through the wires of the electrical circuit. The barrier may be an ionomer membrane such as but not limited to a NAFION® perfluorosulfonic acid (PFSA) membrane (DuPont Fuel Cells, Inc). Excessive deposits of the biofilm on the barrier may impair function of the microbial fuel cell. Therefore it is advisable to maintain biofilm deposits on the barrier at a moderate level. Methods of regulating biofilm deposits include, but are not limited to, regulating bacterial cell division rates and precoating the barrier with a biofilm formation inhibitor. Biofilm formation inhibitors are known in the art and include the polypeptide having the amino acid sequence of the terminal 17 amino acids of the PilA protein, also known as the PilA 17mer. Alternatively the anode and cathode may be separable components as for instance an anodic tube that may be removable from the cathode portion of the microbial fuel cell. In some embodiments, the biofilm acts as a natural membrane and the microbial fuel cell lacks a conventional membrane or barrier.

[00181] A user of a microbial fuel cell may fabricate or obtain a microbial fuel cell. The user of the microbial fuel cell could then use electrodes proceeding from the anode and cathode to attach the fuel cell to a load. Thus, the user completes an electrical circuit from the anode through the load to the cathode. The user could, for example, by engaging a switch, cause electrical current created by the genetically modified microbes to flow through the load. The genetically modified microbes transfer electrons to the anode; the electrons proceed to flow through electrodes and the load to the cathode.

[00182] The microbial fuel cell electrical system can further include an ultracapacitor connected electrically in parallel in a paired system. Ultracapacitors have the advantageous ability to store power quickly and deliver it in relatively short bursts upon demand. Pairing an ultracapacitor with a fuel cell according to an embodiment of the invention enables a user of the paired system to have a continuous flow of power when beginning to start the system.

[00183] Several microbial fuel cells can be electrically associated in series or parallel to create a battery of fuel cells. In some embodiments, one or more of the microbial fuel cells can be disassembled and cleaned. Inventors have discovered that one or more components of the microbial fuel cell may be cleaned. Such cleaning may involve chemical cleaning, mechanical cleaning, scavenging the biofilm utilizing species of Bdellovibrio, scavenging the biofilm utilizing a carnivorous organism such as but not limited to a fungi, or a combination thereof. Bdellovibrio, a bactivorous bacterium, feeds upon P. aeruginosa and temporarily reverses the polarity of the electrode to release bound pili.

[00184] Furthermore, advantageous use can be made of the chemical reactions of the microbial fuel cell. For example, the products of the chemical reaction at the cathode can include free hydrogen gas when the microbial fuel cell is operated anaerobically. The hydrogen gas can be collected and utilized to power a classical hydrogen fuel cell. Also, microbial fuel cells emit very little carbon dioxide and can utilize carbon dioxide as a feedstock or remove it to biocarbonate by carbonic anhydrase as an attachment to the anode. The microbial fuel cell can be designed to emit usable sugars that can become an energy source for other devices. The microbial fuel cell can be operated aerobically to emit water as a byproduct. The water can be transferred to a water storage device or transferred to the external environment.

[00185] In any of these methods and systems, sensors may be used to monitor the performance of the bioreactor, or any other energy source, and the electrical consumption of electrical devices within the home, office, or complexes of buildings. A sensor may be connected to a monitor, or an external network operations center via an electrical conductor, or via wireless communication (for example using Zigbee).

[00186] Sensors may be used to detect, measure, or sense a change on the level or amount of any parameter. Representative parameters for bioreactor sensors, include for example, voltage output, internal resistance, local environmental temperature, pH, ionic strength, osmolarity, viscosity, oxygen saturation level, flow rate, light level, time of year, as well as the presence, or absence, or change in concentration of any feedstock nutrient, or any other analyte. Representative sensors for electrical devices include electrical consumption, temperature, time of day, and time of year. Sensors may be attached to the electrical device, or be connected between the electrical device and the electrical outlet.

[00187] In some embodiments, the bioreactor and electrical devices are connected via an electrical distribution system. In some embodiments the electrical distribution system is also connected to at least one additional source of energy. In one aspect, the alternative energy source is selected from solar energy, wind energy, geothermal, or other source of thermal energy, chemical energy, wave energy, tidal energy, and an external electrical distribution grid.

[00188] In some embodiments, the electrical distribution system is connected to an external network operations center. In some embodiments, the external network operations center is also connected to one or more sensors, and is also in communication with the bioreactor, and optionally one or more energy sources. Connections between the operations center and the sensors, bioreactor and other energy sources may be mediated via wireless or hardwired, or any combination of wireless and hardwired connections.

[00189] In some embodiments, the external network operations center can be adapted to make adjustments to the energy distribution system, or the bioreactor based on the data received from the distributed network of sensors. Such adaptations can include systems to make, or break electrical connections within the energy distribution system to bring on line alternative energy sources, and /or to modulate (i.e. increase or decrease) the electrical output of the bioreactor, for example, by increasing rate of feedstock addition to the bioreactor, increasing the temperature of the bioreactor, or increasing the available surface area of the anode.

[00190] Thus the external network operations center enables the energy distribution system to act as a smart grid, diverting power to devices that require energy, or to storage devices, depending on the balance of electrical demand, and electrical output from one or more of the energy sources at any one time.

[00191] Additionally, the external network operations center can optimize the available energy generating capacities of different sources of energy based on transient local environmental conditions, as well as seasonal variations in weather or temperature. For example, the system can be set to rely primarily on solar power during the day, in the summer months, but may rely more extensively on the bioreactor for electrical power in the evenings and winter, when solar power is less effective. Along the same lines, the system may divert, or promote electrical generation from wave, tidal, or wind energy based on their temporal or seasonal output characteristics.

[00192] In some embodiments, the external network operations center includes a computer implemented system for automatically adjusting and optimizing the electrical distribution system. Registered users of the system can access the external network operations center through any computer means capable of communicating with the network. The computer means include personal computers, laptops, mobile phones, and personal digital assistants. In some embodiments, the interface with the computer system is through a web browser.

[00193] In some embodiments the external network operations center further comprises an analysis and reporting module that provides updates and reports based on data from the one or more sensors. Such reports can include the total amount of energy used or generated by each of the system components, as well as the cost of energy generated from each source of energy.

[00194] In some embodiments the external network operations center further comprises a communication module that allows the registered users to modulate the status or activity of the bioreactor or an alternative energy source, or to make adjustment to the energy distribution system. In some embodiments, the communication module can communicate with the registered users via text messages, to computer or mobile devices, such as cell phones, PDAs, and laptop computers.

[00195] The following examples are offered by way of illustration and not limitation.

EXAMPLES

[00196] Example 1. Development of Static Biofilms on simple glass surfaces in Feedstock

[00197] Circular glass coverslips were attached to the bottom of 35 X 10 mm polystyrene tissue culture dishes with small holes in the base (Falcon). The plates were exposed to UV irradiation overnight. (UV irradiation sterilizes the culture plates).

[00198] Bacterial cells were grown in Luria Bertani media (LB) overnight. Aerobic LB, aerobic LBN (LB + 1% KNO₃), or anaerobic LBN (3 ml) was placed in each tissue culture plate. The media was inoculated with 10⁷ cfu of bacterial cells. The plates were incubated at 37°C for 24 hours. The media was removed and the plates were washed with saline buffer. LIVE/DEAD 5acLight (Molecular Probes, Inc) bacterial viability stain (0.5 ml) was added to each plate. Images were acquired on a Zeiss LSM 510 laser scanning confocal unit attached to an Axiovert microscope with a 63 X 14 NA oil immersion objective. For two color images, samples were scanned sequentially at 488 nm and 546 nm. Syto 9 (green fluorescence) was detected through a 505-530 nm bandpass filter and propidium iodine (red fluorescence) was detected through a 560 nm longpass filter and presented in two channels of a 512 X 512 pixel, 8-bit image.

Example 2. Development of Biofilms in Circulated Feedstock

[00199] Culture Media

[00200] LB media is 10 g/liter tryptone, 5 g/liter yeast extract, and 5 g/liter NaCl.

[00201] LBN media is 10 g/liter tryptone, 5 g/liter yeast extract, 5 g/liter NaCl and 10 g/liter KNO₃. [00202] Bacteria are grown aerobically in LB at 37°C until the stationary growth phase. Bacteria are diluted 1 :50 into 1% trypticase soy broth. Flow cells are inoculated with 0.2 ml diluted bacteria. Flow cells and bacteria are incubated for 1 hour. After an hour, flow is initiated at a rate of 0.17 ml/min. The cells are incubated 3 days at room temperature. The cells are stained with a live/dead viability stain composed of SYTO 9 and propidium iodine (Molecular Probes, Inc.). Biofilm images are obtained using an LSM 510 confocal microscope (Carl Zeiss, Inc.). The excitation and emission wavelengths for green fluorescence are 488 nm and 500 nm, while those for red fluorescence are at 490 nm and 635 nm, respectively. All biofilm experiments are repeated at least 3 times. The live/dead ratios of the biofilms are calculated using the 3D for LSM (V. l .4.2) software (Carl Zeiss). Overall biofilm structure such as thickness, water channel, bacterial density (substrate coverage), roughness coefficient and total biomass in m³/m² are assessed using COMSTAT software. COMSTAT analyzes stacks of images acquired with scanning confocal laser microscopy (SCLM) to quantify the 3-dimensional nature of biofilm structures. See Heydorn et al (2000) Microbiology 146 (Pt 10):2395, herein incorporated by reference in its entirety.

Example 3. Construction of P. aeruginosa deletion mutants

[00203] The P. aeruginosa strain PAOl is used as the starting strain for construction of deletion mutations. Classical allelic replacement techniques are used to generate mutant strains. See Hoang et al (1998) Gene 212(l):77-86) An insertional mutagenesis cassette comprising a gentamicin resistant (Gm^R) nucleotide sequence, a green fluorescent protein (GFP) nucleotide sequence, and FLP recombinase target (FRT) sites flanking the gentamicin resistance sequence and the GFP sequence is developed for each gene of interest. After conjugal transfer or electroporation plasmid integrants are selected. The cells are grown in media containing 6% sucrose. The sucrose promotes deletion of the target sequence of interest. Mutants are confirmed via PCR or Southern blotting. Mutant cells undergo conjugal transfer with a cell containing a FLP-recombinase expressing plasmid such as pFLP2. pFLP2 contains the sacB sequence; growth on sucrose containing media cures the bacterial cells of the sacB containing plasmid. Expression of FLP recombinase allows excision of the FRT cassette. After curing of plasmid the P. aeruginosa deletion mutant strain is gentamicin sensitive. Multiple mutations such as double and triple mutants are constructed by similar methods.

Example 4. High-throughput Microbial Fuel Cell Prototype [00204] A small high-throughput microbial fuel cell (Pilus Cell) prototype was developed. A Millipore filtration apparatus of the type commonly used to collect cells on a 1 inch nitrocellulose filter was utilized to construct the Pilus Cell prototype. When used to collect cells on a filter for radioactivity measurements in a scintillation counter, a filter is placed on the sintered plastic surface of each well. The top portion of the apparatus is tightly screwed to the base portion. The top "cup" portion of the apparatus has rubber seals to prevent leakage from each well. The base portion includes a vacuum port. The Millipore filtration apparatus has 12 wells.

[00205] The filtration apparatus has been modified into a high-throughput device for screening and monitoring power generation by up to 12 different genetically engineered bacteria. Copper wires have been soldered to the base of twelve 2.54 cm X 0.2 mm circular wafers of stainless steel. The milled steel was treated with acetone and then methanol to remove residual oils. The steel wafers were brushed with a wire brush to increase the surface area of the steel available for bacterial binding. The copper wire attached to the wafer represents the anode. The copper wires from each wafer were drawn through what was formerly the vacuum port of the apparatus. The copper wires were connected to a voltage/current measuring device. Each well may hold up to 15 mis of media; in these experiments 7 mis of media were used. Two holes were drilled into each of twelve grade 6 rubber stoppers that fit snugly in the wells. An 8 inch copper wire that extends 0.25 inches into the media in the anode was placed in the main hole of the stopper. This copper wire represents the cathode. This high-throughput device allows evaluation of up to 12 samples at a time. Once assembled, each well has the capacity to be an independent microbial fuel cell.

Example 5. High Through-put Microbial Fuel Cell Voltage/Current

Evaluation

[00206] The above described high-throughput microbial fuel cell prototype was used to evaluate voltage and current generation from wild-type Pseudomonas aeruginosa (POA), Shewanella oneidensis, and a mutant strain (pilT, bdlA, nirS, lasl, or fliC pilA) . The entire high-throughput microbial fuel cell prototype was assembled and secured by a bolt on the top of the apparatus. Each well utilized in the experiment was sterilized by treatment with ethanol. The ethanol was removed and the apparatus was dried in a germ- free laminar flow hood. LB + 1% K O₃ media (7 ml) was placed in each well utilized in the experiment. A stationary phase grown aerobic culture (70 μΐ, a 1 : 100 dilution) for each bacterial sample (wild-type Pseudomonas, Shewenella, and a mutant strain) was added to the media in the well. A medium alone control well was also prepared and monitored. Rubber stoppers and copper cathode wires were treated with ethanol prior to securing the stoppers in the wells. The device was incubated at 37°C for 24 hours under anaerobic conditions.

[00207] Measurements were recorded as described elsewhere herein. The stoppers were removed and the media was aspirated away. Saline (0.9%) was gently applied to each well. The saline solution was removed by aspiration. The saline wash was performed three times. Ethanol was swabbed over the plastic regions of each well. LB + 1% KNO₃ media (7 ml) was added to each well. The recording process was repeated. Results from one such experiment are presented in Figure 6.

Example 6. Voltage and Current Monitoring of the High-throughput

Microbial Fuel Cell Prototype

[00208] Measurements were obtained using a LabJack U12, 8 channel 12 bit USB A/D for data acquisition system. Four channels were used to monitor microbial fuel cell voltages. The 3 cm copper anodes were connected to four LT1012 high input impendence buffer amplifiers. The outputs of these amplifiers were then connected to the channel AIO-AI3 inputs of the Labjack A/D. Current measurements were made by connecting a LTl lOl instrumentation amplifier across the IK current sense resistor. By measuring the voltage drop across the resistor and utilizing Ohm's law (I=E/R) the current flowing in the cell circuit can be calculated.

[00209] The measurement system utilized allows voltage and current measurements to be done remotely via the internet. The system utilizes eight different graphic monitoring systems that can be configured to monitor various combinations of voltages and currents as dictated by the experimental design.

Example 7. Development of Microbial Fuel Cell with Increased Anode Surface Area

[00210] A 23-plate stainless steel 314 anode system is constructed. The first 21 plates are of the following dimensions: 0.05 X 9.851 X 7.554 inches. This involves a total surface area of 197.56 inches. The other two plates are 0.05 X 9.851 X 7.884 inches. The two larger plates serve as "legs" facing either in or toward the Nafion membrane and adding an additional 96.2 inches of surface area. Thus the total estimated surface area is approximately 294 inches. The two larger plates provide support to the 21 plate component. The electrode from the anode to the cathode compartment is stainless steel and fitted with Swagelok fittings into similar fittings embedded within the cathode. [0021 1] A single plate of hot, isostatic pressed graphite (GraphiteStore.com) of 0.125 X 9.6 X 4.65 inches is used for the cathode.

[00212] After the anode is assembled, the anode is treated with 1% bleach, then 95% ethanol, and then 70% ethanol. Small 1 x 1 x 0.05 inch stainless steel wafers are used to monitor biofilm formation. The anode is incubated in a Coy anaerobic chamber in 1 liter of LB + 1% KNO₃ at 37°C for 24 hours inoculated with the genetically modified bacteria.

[00213] Experiments are performed with wild-type bacteria or with various mutants. The overall efficiency of the wild-type and mutant strains is compared.

[00214] The complete anode assembly with a mature biofilm attached is submerged in anaerobic 0.9% NaCl solution and removed from the solution. Submersion and removal may be repeated. (Unattached bacteria are removed by this process.) The anode with the attached mature biofilm is placed in the large microbial fuel cell assembly. Two plastic boxes, one containing the anode, the other the cathode are filled with LB+ 1% KNO₃. The anodic and cathodic chambers are treated with glucose oxidase. Glucose oxidase converts glucose to uric acid and H2O2. ¾(¾ is treated with catalase. The glucose oxidase and catalase reactions lower the oxygen concentration. The anode is poised at approximately 250-400 mV (versus Ag/AgCl). A Clark-type or World Precision Instrument O2 electrode is attached to both the anode and cathode sections. Flow of fresh anaerobic media through the anodic compartment is accomplished using peristaltic pumps at a flow rate of 0.05 ml/min.

[00215] Similar experiments are performed with wild-type, single, double, triple quadruple, quintuple, and mutiple mutant strains. Current and voltage output are monitored using a LabJack system as described above herein or an Agilent 34970-A data acquisition system that is linked to electronic databases. This system allows monitoring of current in the micro-ampere range and voltage in the micro to millivolt range.

Example 8. P. aeruginosa Mutant Strains

[00216] The PAOl strain was used to prepare mutant strains. pilT, bdlA, nirS, and lasi single disruption mutations were constructed. pilT bdlA, bdlA nirS, bdlA lasi, nirS pilT, nirS lasi, and lasi pilT double disruption mutant strains were constructed. pilT bdlA nirS, bdlA lasi pilT, nirS lasi bdlA triple disruption mutant strains were constructed. A pilT bdlA nirS lasi quadruple disruption mutant strain was constructed.

[00217] A PAOl strain stably comprising araBAD-ftsZ was constructed. The PAOl araBAD-ftsZ strain was used to prepare mutant strains. pilT, bdlA, nirS, and lasi single disruption mutations were constructed in the PAOl araBAD-ftsZ background. pilT bdlA, bdlA nirS, bdlA lasi, nirS pilT, nirS lasi, and lasi pilT double disruption mutations were constructed in the PAO 1 araBAD-ftsZ background. pilT bdlA nirS, bdlA lasi pilT, and nirS lasi bdlA triple disruption mutations were constructed in the PAOl araBAD- ftsZ background. A pilT bdlA nirS lasi quadruple disruption mutant strain was constructed in the PAOl araBAD-ftsZ background.

[00218] A PAOl strain stably comprising araBAD-ftsZ and a siRNA construct (PA0730) was constructed. The PAOl araBAD-ftsZ PA0730 strain was used to prepare mutant strains. pilT, bdlA, nirS, and lasi single disruption mutations were constructed in the PAOl araBAD-ftsZ PA0730 background. pilT bdlA, bdlA nirS, bdlA lasi, nirS pilT, nirS lasi, and lasi pilT double disruption mutations were constructed in the PAO l araBAD-ftsZ PA0730 background. pilT bdlA nirS, bdlA lasi pilT, and nirS lasi bdlA triple disruption mutations were constructed in the PAOl araBAD-ftsZ PA0730 background. A pilT bdlA nirS lasi quadruple disruption mutant strain was constructed in the PAO l araBAD-ftsZ PA0730 background.

[00219] Example 9. Cloning of a eukaryotic uncoupling protein into P. aeruginosa to increase the rate of substrate oxidation

[00220] Uncouplers are compounds that when administered to bacteria or mitochondria, separate the normally coupled processes of oxidation and phosphorylation, thereby diminishing the proton gradient from the periplasmic space to the cytoplasm. Thus, bacteria treated with the classical uncoupler, dinitrophenol, will have an equal concentration of protons in both the periplasm and cytoplasm. By dissipating the proton gradient, the rate of substrate oxidation and hence electron flow to the anode will increase substantially. The eukaryotic uncoupling proteins thermogenin, UCXP-1, UCP- 2, and UCP-3 will be cloned under a tetracycline-inducible system into another site within attB. Upon exposure to tetracycline, an increase in respiration of bacteria relative to untreated bacteria will be assayed.

[00221] Having described the invention with reference to the exemplary embodiments, it is to be understood that it is not intended that any limitations or elements describing the exemplary embodiment set forth herein are to be incorporated into the meanings of the patent claims unless such limitations or elements are explicitly listed in the claims. Likewise, it is to be understood that it is not necessary to meet any or all of the identified advantages or objects of the invention disclosed herein in order to fall within the scope of any claims, since the invention is defined by the claims and since inherent and/or unforeseen advantages of the present invention may exist even though they may not be explicitly discussed herein.

22] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

A method of generating electrical energy for a building, or group of buildings, comprising

b) a cathode;

c) an anode in electrical communication with the cathode; and

d) a biofilm functionally associated with the anode;

wherein the external network operations center makes adjustments to the energy distribution system, or bioreactor, based on the data received from the first plurality of sensors, and the second plurality of sensors.

The method of claim 1, wherein the energy distribution system is further connected to at least one additional form of local energy generation selected from the group consisting of solar energy, wind energy, thermal energy, chemical energy, wave energy, and tidal energy.

3. The method of claim 2, wherein the additional form of local energy generation is connected to a third plurality of sensors that provide operating data about the additional form of local energy generation, and can transmit the data to the external network operations center,

wherein the network operations center makes adjustments to the energy distribution system, or the bioreactor, or the additional form of local energy generation, based on the data received from the first plurality of sensors, or the second plurality of sensors, or the third plurality of sensors.

4. The method of any of claim 1-3, wherein the genetically modified microbes comprise Pseudomonas aeruginosa wherein the P. aeruginosa has at least one genetic modification that results in the loss of functional expression, or the suppression of expression in an endogenous gene selected from the group consisting of z/r (SEQ ID NO: l), pilA (SEQ ID NO:2), nirS (SEQ ID NO:3), bdlA (SEQ ID NO:4), lasl (SEQ ID NO:5), lasR (SEQ ID NO: 6), ftsZ (SEQ ID NO: 7), fliC (SEQ ID NO: 8) and combinations thereof, wherein said microbes exhibit decreased expression of the gene interest and exhibit an altered electrogenic efficacy.

5. The method of claim 4, wherein a combination of the genes are disrupted or suppressed.

6. The method of claim 5, wherein at least two genes are disrupted or suppressed and comprise a combination selected from the group consisting of pilT (SEQ ID NO: l) and bdlA (SEQ ID NO:4); bdlA (SEQ ID NO:4) and nirS (SEQ ID NO:3); bdlA (SEQ ID NO:4) and lasl (SEQ ID NO:5); nirS (SEQ ID NO:3) and pilT (SEQ ID NO: 1); nirS (SEQ ID NO:3) and lasl (SEQ ID NO:5); lasl (SEQ ID NO:5) and pilT (SEQ ID NO: l); teZ (SEQ ID NO:7) and pilT (SEQ ID NO:l); teZ (SEQ ID NO:7) and bdlA (SEQ ID NO:4); ftsZ (SEQ ID NO:7) and nirS (SEQ ID NO:3); and ftsZ (SEQ ID NO:7) and lasl (SEQ ID NO:5).

7. The method of claim 5, wherein at least three genes are disrupted or suppressed and comprise a combination selected from the group consisting oipilT (SEQ ID NO: l), bdlA (SEQ ID NO:4) and ftsZ (SEQ ID NO:7); bdlA (SEQ ID NO:4), nirS (SEQ ID NO:3) and ftsZ (SEQ ID NO:7); bdlA (SEQ ID NO:4), lasl (SEQ ID NO:5) and ftsZ (SEQ ID NO:7); nirS (SEQ ID NO:3), pilT (SEQ ID NO: 1) and teZ (SEQ ID NO:7); nirS (SEQ ID NO:3), lasl (SEQ ID NO:5) and ftsZ (SEQ ID NO:7); lasl (SEQ ID NO:5), pilT (SEQ ID NO: l) and ftsZ (SEQ ID NO:7); pilT (SEQ ID NO: l), bdlA (SEQ ID NO:4) and nirS (SEQ ID NO:3); pilT (SEQ ID NO:l), bdlA (SEQ ID N0:4) and lasl (SEQ ID N0:5); and bdlA (SEQ ID N0:4), lasl (SEQ ID N0:5) and nirS (SEQ ID N0:3).

8. The method of claim 5, wherein at least four genes are disrupted or suppressed and comprise a combination selected from the group consisting oipilT (SEQ ID NO: l), bdlA (SEQ ID NO:4), nirS (SEQ ID NO:3) and ftsZ (SEQ ID NO:7); pilT (SEQ ID NO: 1), bdlA (SEQ ID NO:4), lasl (SEQ ID NO:5) and fisZ (SEQ ID NO:7); bdlA (SEQ ID NO:4), lasl (SEQ ID NO:5), nirS (SEQ ID NO:3) and fisZ (SEQ ID NO:7); and pilT (SEQ ID NO: l) bdlA (SEQ ID NO:4), lasl (SEQ ID NO:5) and nirS (SEQ ID NO:3).

9. The method of claim 5, wherein at least five genes are disrupted or suppressed and comprise pilT (SEQ ID NO: l), bdlA (SEQ ID NO:4), lasl (SEQ ID NO:5), nirS (SEQ ID NO:3) and fisZ (SEQ ID NO:7).

10. The method of claim 4, wherein the Pseudomonas aeruginosa has a reduced proliferative capability as compared to a non-genetically modified cell.

11. The method of claim 4, wherein the Pseudomonas aeruginosa has a reduced virulence as compared to a non-genetically modified cell.

12. The method of claim 11, wherein the reduced virulence is in mammals or plants.

13. The method of claim 4, wherein the Pseudomonas aeruginosa exhibits reduced motility as compared to a non-genetically modified cell.

14. The method of claim 4, wherein the Pseudomonas aeruginosa exhibits altered pilus sticking as compared to a non-genetically modified cell.

15. The method of claim 4, wherein the Pseudomonas aeruginosa exhibits altered twitching motility as compared to a non-genetically modified cell.

16. The method of any of claims 1-3, wherein the cathode is coated with an agent to prevent a biofilm from growing on the cathode.

17. The method of claim 16, wherein the agent is a 17-mer terminal fragment of a pilus PilA protein derived from the bacteria.

18. The method of any of claims 1-3, wherein the biofilm further comprises Nitrobacter and Nitrosomonas microbes.

19. The method of any of claims 1-3, wherein the genetic modification produces a phenotype selected from the group consisting of a reduction in cytotaxis, an increase in the number of pili on the bacteria, a reduction in the rate of cell division, and combinations thereof.

20. The method of any of claims 1-3, wherein at least one of the cathode and anode are fabricated from porous material.

21. The method of claim 20, wherein the porous material comprises porous stainless steel.

22. The method of any of claims 1-3, wherein the microbial fuel cell comprises a plurality of anodes selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 anodes.

23. The method of any of claims 1-3, wherein the genetically modified bacteria further express an uncoupling protein.

24. The method of any of claims 1-3, wherein the thickness of the biofilm is between 30 μιη and 100 μιη.

25. The method of any of claims 1-3, wherein the microbial fuel cell lacks a membrane.

26. The method of any of claims 1-3, wherein the microbial fuel cell is in electrical communication to ultracapacitor.

27. The method of any of claims 1 to 3, wherein at least one of the first plurality of sensors, or at least one of the second plurality of sensors, or if present, at least one of the third plurality of sensors is connected via wireless communication to one or more monitors.

28. The method of any of claims 1 to 3, wherein at least one of the first plurality of sensors, or at least one of the second plurality of sensors, or if present, at least one of the third plurality of sensors is connected via wireless communication to the external network operations center.

29. The method of claim 28, wherein the external network operations center comprises a computer implemented system that i) monitors the activity from one more or more of the first, second or third plurality of sensors and ii) displays the activity from the one or more sensors to one or more registered users of the external network operations center.

30. The method of claim 29, wherein the external network operations center further comprises an analysis and reporting module that provides updates and reports based on data from the one or more sensors.

31. The method of claim 30, further comprising a communication function that allows the registered users to modulate the status or activity of the bioreactor, or one or more of the plurality of devices, or if present, the additional form of local energy generation.

32. A system for generating electrical energy for a building, or group of buildings, comprising i) a microbial bioreactor;

ii) a computer implemented external network operations center;

iii) an implemented distributed network of sensors; and

iv) an energy distribution system,

wherein the microbial bioreactor comprises,

1) a housing with at least one opening to facilitate contact of water from a waterway and fuel cell components;

2) a cathode;

3) an anode in electrical communication with the cathode; and

4) a biofilm functionally associated with the anode,

wherein the biofilm comprises genetically modified microbes that break down nutrients in the water and donate electrons to the anode, thereby generating electrical energy,

wherein the computer implemented external network operations center comprises an activity module that monitors the activity from one more or more of sensors from the distributed network of sensors, a display module that displays the activity from the one or more sensors to one or more registered users of the external network operations center, wherein the distributed network of sensors provide operating data about the bioreactor, and energy usage in the building, or the group of buildings, and can transmit the data to the external network operations center, wherein the energy distribution system connects the bioreactor to one or more energy consuming devices associated with the building or the group of buildings, and wherein the energy distribution system is also connected to at least one alternative energy source, wherein the computer external network operations center is adapted to make adjustments to the energy distribution system, or the bioreactor based on the data received from the distributed network of sensors.

33. The system of claim 32, wherein the alternative energy source is selected from solar energy, wind energy, thermal energy, chemical energy, wave energy, tidal energy, and an electrical distribution grid.

34. The system of claim 32, wherein the genetically modified microbes comprise Pseudomonas aeruginosa, wherein the P. aeruginosa has at least one genetic modification that results in the loss of functional expression, or the suppression of expression in an endogenous gene selected from the group consisting of pilT (SEQ ID NO: l), pilA (SEQ ID NO:2), nirS (SEQ ID NO:3), bdlA (SEQ ID NO:4), lasl (SEQ ID NO:5), lasR (SEQ ID NO:6), teZ (SEQ ID NO 7), fliC (SEQ ID NO:8) and combinations thereof, wherein said microbes exhibit decreased expression of the gene interest and exhibit an altered electrogenic efficacy.

35. The system of claim 34, wherein a combination of the genes are disrupted or suppressed.

36. The system of claim 35, wherein at least two genes are disrupted or suppressed, and comprise a combination selected from the group consisting of pilT (SEQ ID NO: l) and bdlA (SEQ ID NO:4); bdlA (SEQ ID NO:4) and nirS (SEQ ID NO:3); bdlA (SEQ ID NO:4) and lasl (SEQ ID NO:5); nirS (SEQ ID NO:3) and pilT (SEQ ID NO: 1); nirS (SEQ ID NO:3) and lasl (SEQ ID NO:5); lasl (SEQ ID NO:5) and pilT (SEQ ID NO: l); teZ (SEQ ID NO:7) and pilT (SEQ ID NO:l); teZ (SEQ ID NO:7) and bdlA (SEQ ID NO:4); ftsZ (SEQ ID NO:7) and nirS (SEQ ID NO:3); and ftsZ (SEQ ID NO:7) and lasl (SEQ ID NO:5).

37. The system of claim 36, wherein at least three genes are disrupted or suppressed and comprise a combination selected from the group consisting oipilT (SEQ ID NO: l), bdlA (SEQ ID NO:4) and ftsZ (SEQ ID NO:7); bdlA (SEQ ID NO:4), nirS (SEQ ID NO:3) and ftsZ (SEQ ID NO:7); bdlA (SEQ ID NO:4), lasl (SEQ ID NO:5) and ftsZ (SEQ ID NO:7); nirS (SEQ ID NO:3), pilT (SEQ ID NO: 1) and/teZ (SEQ ID NO:7); nirS (SEQ ID NO:3), lasl (SEQ ID NO:5) and ftsZ (SEQ ID NO:7); lasl (SEQ ID NO:5), pilT (SEQ ID NO: l) and ftsZ (SEQ ID NO:7); pilT (SEQ ID NO: l), bdlA (SEQ ID NO:4) and nirS (SEQ ID NO:3); pilT (SEQ ID NO:l), bdlA (SEQ ID NO:4) and lasl (SEQ ID NO:5); and bdlA (SEQ ID NO:4), lasl (SEQ ID NO:5) and nirS (SEQ ID NO:3).

38. The system of claim 37, wherein at least four genes are disrupted or suppressed and comprise a combination selected from the group consisting oipilT (SEQ ID NO: l), bdlA (SEQ ID NO:4), nirS (SEQ ID NO:3) and ftsZ (SEQ ID NO:7); pilT (SEQ ID NO: 1), bdlA (SEQ ID NO:4), lasl (SEQ ID NO:5) and ftsZ (SEQ ID NO:7); bdlA (SEQ ID NO:4), lasl (SEQ ID NO:5), nirS (SEQ ID NO:3) and ftsZ (SEQ ID N0:7); and pilT (SEQ ID N0: 1) bdlA (SEQ ID N0:4), lasl (SEQ ID N0:5) and nirS (SEQ ID N0:3).

39. The system of claim 38, wherein at least five genes are disrupted or suppressed and comprise pilT (SEQ ID NO: l), bdlA (SEQ ID NO:4), lasl (SEQ ID NO:5), nirS (SEQ ID NO:3) and fisZ (SEQ ID NO:7).

40. The system of claim 34, wherein the Pseudomonas aeruginosa has a reduced proliferative capability as compared to a non-genetically modified cell.

41. The system of claim 34, wherein the Pseudomonas aeruginosa has a reduced virulence as compared to a non-genetically modified cell.

42. The system of claim 41 wherein the reduced virulence is in mammals or plants.

43. The system of claim 34, wherein the Pseudomonas aeruginosa exhibits reduced motility as compared to a non-genetically modified cell.

44. The system of claim 34, wherein the Pseudomonas aeruginosa exhibits altered pilus sticking as compared to a non-genetically modified cell.

45. The system of claim 34, wherein the Pseudomonas aeruginosa exhibits altered twitching motility as compared to a non-genetically modified cell.

46. The system of claim 32, wherein the cathode is coated with an agent to prevent a biofilm from growing on the cathode.

47. The system of claim 46, wherein the agent is a 17-mer terminal fragment of a pilus PilA protein derived from the bacteria.

48. The system of claim 32, wherein the biofilm further comprises Nitrobacter and Nitrosomonas microbes.

49. The system of claim 32, wherein the genetic modification produces a phenotype selected from the group consisting of a reduction in cytotaxis, an increase in the number of pili on the bacteria, a reduction in the rate of cell division, and combinations thereof.

50. The system of claim 32, wherein at least one of the cathode and anode are fabricated from porous material.

51. The system of claim 50, wherein the porous material comprises porous stainless steel.

52. The system of claim 32, wherein the microbial fuel cell comprises a plurality of anodes selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 anodes.

53. The system of any one of claim 36, wherein the genetically modified bacteria further express an uncoupling protein.

54. The system of claim 32, wherein the thickness of the biofilm is between 30 μιη and 100 μιη.

55. The system of claim 32, wherein the microbial fuel cell lacks a membrane.

56. The system of claim 32, wherein the microbial fuel cell is in electrical communication to ultracapacitor.

57. The system of claim 32, wherein at least one of the distributed network of sensors is connected via wireless communication to one or more monitors.

58. The system of claim 32, wherein at least one of the distributed network of sensors is connected via wireless communication to the external network operations center.

59. The system of claim 32, wherein the external network operations center further comprises an analysis and reporting module that provides updates and reports based on data from the one or more sensors.

60. The system of claim 32, further comprising a communication module that allows the registered users to modulate the status or activity of the bioreactor or an alternative energy source.