U.S. patent application number 12/660244 was filed with the patent office on 2010-11-04 for microbial fuel cell.
Invention is credited to Jason E. Barkeloo, Daniel J. Hassett, Randall T. Irvin.
Application Number | 20100279178 12/660244 |
Document ID | / |
Family ID | 43030614 |
Filed Date | 2010-11-04 |
United States Patent
Application |
20100279178 |
Kind Code |
A1 |
Barkeloo; Jason E. ; et
al. |
November 4, 2010 |
Microbial fuel cell
Abstract
Microbial fuel cells may include anode(s), cathode(s) and a
biofilm attached to at least the anode. The biofilm may include
bacterial cells adapted to facilitate transfer of a plurality of
electrons to the anode from a feedstock. In an example embodiment,
a microbial fuel surface may include a large surface area to volume
ratio in order to increase power (electron) generation and/or
transfer.
Inventors: |
Barkeloo; Jason E.;
(Cincinnati, OH) ; Hassett; Daniel J.;
(Cincinnati, OH) ; Irvin; Randall T.; (Sherwood
Park, CA) |
Correspondence
Address: |
TAFT, STETTINIUS & HOLLISTER LLP
SUITE 1800, 425 WALNUT STREET
CINCINNATI
OH
45202-3957
US
|
Family ID: |
43030614 |
Appl. No.: |
12/660244 |
Filed: |
February 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61154464 |
Feb 23, 2009 |
|
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Current U.S.
Class: |
429/401 |
Current CPC
Class: |
Y02E 60/527 20130101;
Y02E 60/50 20130101; H01M 8/16 20130101; C12N 15/78 20130101 |
Class at
Publication: |
429/401 |
International
Class: |
H01M 8/16 20060101
H01M008/16 |
Claims
1. A microbial fuel cell, comprising: at least one cathode; at
least one anode in electrical communication with the at least one
cathode, the at least one anode comprising an electrically
conductive material; a biofilm comprising a plurality of bacterial
cells, the biofilm operably coupled to the at least one anode,
wherein the biofilm facilitates transfer of a plurality of
electrons from the biofilm to the at least one anode.
2. The microbial fuel cell of claim 1, wherein the at least one
cathode is contained within a cathodic chamber; and wherein the at
least one anode is contained within an anodic chamber.
3. The microbial fuel cell of claim 2, further comprising: a
barrier between the anodic chamber and the cathodic chamber, the
barrier adapted to restrict the direct transfer of electrons
between the at least one anode and the at least one cathode.
4. The microbial fuel cell of claim 1, wherein the biofilm is
exposed to a feedstock capable of being metabolized by the
biofilm.
5. The microbial fuel cell of claim 1, wherein the biofilm is
exposed to a feedstock comprising one or more of sewage, fertilizer
run-off, waste-water, animal waste, gaseous material, and/or a
greenhouse gas.
6. The microbial fuel cell of claim 1, wherein the at least one
anode is detachably coupled to the at least one cathode.
7. The microbial fuel cell of claim 1, wherein the electrically
conductive material comprises a porous material.
8. The microbial fuel cell of claim 7, wherein the porous material
comprises at least one of 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 and/or aluminum.
9. The microbial fuel cell of claim 1, wherein the electrically
conductive material comprises a nanomaterial.
10. The microbial fuel cell of claim 1, wherein the at least one
anode comprises a plurality of anodes operably coupled
together.
11. The microbial fuel cell of claim 1, wherein the at least one
anode comprises at least one of a planar shape, a cylindrical
shape, a layered spiral cylindrical shape, a curved shape, an
angled shape and a geometrical shape.
12. The microbial fuel cell of claim 1, wherein the at least one
anode comprises a sheet, a plurality of sheets, a wire mesh
structure, a porous tube and/or a matrix structure.
13. The microbial fuel cell of claim 1, further comprising: a first
electrode operably coupled to the at least one anode, the first
electrode further operably coupled to a load; a second electrode
operably coupled to the load, the second electrode further operably
coupled to the at least one cathode.
14. The microbial fuel cell of claim 13, wherein the load comprises
at least one of a light emitting device, a machine, a display, an
electrical appliance, and/or a battery charger.
15. The microbial fuel cell of claim 1, further comprising an
ultracapacitor operably coupled to the at least one anode, the
ultracapacitor adapted to store at least a portion of the plurality
of electrons.
16. The microbial fuel cell of claim 1, wherein the at least one
anode comprises a plurality of electrically conductive sheets
oriented substantially parallel to each of the other plurality of
electrically conductive sheets.
17. A microbial fuel cell, comprising: a cathode; an anode in
electrical communication with the cathode, the anode having a
bacterial biofilm at least partially coupled thereto, the bacterial
biofilm adapted to metabolize a feedstock to generate at least
electrons and hydrogen; a hydrogen capture module to capture the
hydrogen generated by the bacterial biofilm's metabolizing of the
feedstock; and an electron capture module to capture the electrons
generated by the bacterial biofilm's metabolizing of the
feedstock.
18. The microbial fuel cell of claim 17, wherein the anode
comprises at least one of 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 and/or aluminum.
19. The microbial fuel cell of claim 17, wherein the anode
comprises a sheet, a plurality of sheets, a wire mesh structure, a
porous tube and/or a matrix structure.
20. A microbial fuel cell, comprising: an anodic chamber
comprising: a plurality of anodes comprising an electrically
conductive material; a biofilm comprising a plurality of bacterial
cells adapted to facilitate transfer of a plurality of electrons to
the plurality of anodes, the biofilm covering at least a portion of
the plurality of anodes; a feedstock at least partially in contact
with the biofilm; and an anodic electrode operably coupled with at
least one of the plurality of anodes, the anodic electrode further
operably coupled to a load located outside of the anodic chamber; a
cathodic chamber comprising: at least one cathode; and a cathodic
electrode operably coupled with the at least one cathode, the
cathodic electrode further operably coupled to the load; and an
ionomer barrier separating the anodic chamber and the cathodic
chamber.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to, and the benefit of,
U.S. Provisional Patent Application No. 61/154,464, entitled
"IMPROVED MICROBIAL FUEL CELL," filed on Feb. 23, 2009, by Barkeloo
et al., the entire disclosure of which is incorporated herein by
reference in its entirety.
[0002] This application may be related to co-pending U.S. patent
application Ser. No. ______, entitled "IMPROVED MICROBIAL FUEL
CELL," filed Feb. 23, 2010, by Barkeloo et al., the entire
disclosure of which is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present disclosure relates generally to microbial fuel
cells having genetically modified organisms. More specifically, it
relates to microbial fuel cells having anode(s), cathode(s) and
biofilm adapted for improved power (electron) generation and/or
improved electron transfer.
BACKGROUND OF THE INVENTION
[0004] 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 (O2) 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. 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.degree. C. to 42.degree. C. It can live in
diesel fuel and jet fuel where it is a hydrocarbon utilizing
microorganism. It can also metabolize high nitrate-containing
organic materials. P. aeruginosa derive electrons from a myriad of
carbon sources and can derive electrons in aerobic, anaerobic and
anaerobic fermentative processes (e.g., with arginine or pyruvate).
In anaerobic growth, P. aeruginosa cells continue to couple
oxidation and phosphorylation to gain energy.
[0005] In forms 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.
[0006] In forms 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,
in a microbial fuel cell 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, and many other
devices.
[0007] 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 organic waste.
RH2+NAD+.fwdarw.NADH+H++R Eq. 1
[0008] NADH is a strong reducing agent that the bacteria use 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
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.
[0009] A biofilm on a given anode does not have an unlimited number
of bacteria. For a given anode area, the number of electrons, and
hence the current and power transferred, may be limited by several
characteristics of the bacteria and the fuel cell. These
characteristics may include the number of associated bacteria, the
number of pili attached to the anode, the bacteria's metabolic
ability to consume available substrates, and the bacteria's ability
to transfer electrons. If the opportunity to modify such
characteristics is limited, designers of a fuel cell may have only
the anode surface area available as a design variable. The designer
may increase the anode area without increasing the volume of the
anode to avoid creating a larger fuel cell than practical for the
application. Therefore, an anode with a smaller surface area may
suffer from too small power to perform the work needed, or
conversely, an anode having a large enough surface area to support
the bacteria needed to generate useful amounts power may cause the
cell to become too large to be of practical value.
[0010] Therefore, there is a need to increase the current delivered
for a given electrode volume in fuel cells. There is also a need to
increase the number of electrons passed by bacteria to an
electrode. Therefore, what is needed is a microbial fuel cell
having bacteria modified to transfer larger numbers of electrons to
a surface than were transferred before, generating more current and
power from a smaller fuel cell. What is further needed is a
microbial fuel cell having bacteria modified to transfer larger
numbers of electrons to a surface and wherein the surface is
modified to have a large surface area to volume ratio to accept
more electrons in a smaller fuel cell.
SUMMARY OF THE INVENTION
[0011] In an example embodiment, a microbial fuel cell may include
cathode(s), anode(s) in electrical communication with the cathode,
and a biofilm comprising bacterial cells. The biofilm may be
coupled to the anode (which may be electrically conductive
material) where the biofilm facilitates transfer of a plurality of
electrons from the biofilm to the anode.
[0012] In some examples, the anode may be contained in an anodic
chamber and the cathode may be contained in a cathodic chamber. In
some examples, a barrier may be located between the anodic chamber
and the cathodic chamber. Such barrier may restrict direct transfer
of electrons between the anode and cathode.
[0013] Some examples provide that the biofilm may be exposed to a
feedstock capable of being metabolized by the biofilm. Feedstocks
may include sewage, fertilizer run-off, waste-water, animal waste,
gaseous material and/or a greenhouse gas.
[0014] In some examples, the anode may be detachably coupled to the
cathode. In some examples, the anode may be a porous material.
Examples porous materials may include 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 and/or aluminum, among others.
Further, the anode may be a nanomaterial. Some examples provide for
multiples anodes to act as the anode.
[0015] In some examples, the anode may be a planar shape, a
cylindrical shape, a layered spiral cylindrical shape, a curved
shape, an angled shape and/or a geometrical shape, among others. In
some examples, the anode may be a sheet, a plurality of sheets, a
wire mesh structure, a porous tube and/or a matrix structure, among
others. In an example embodiment, the anode may include a plurality
of sheets oriented substantially parallel to each other.
[0016] Some examples provide for the anode and cathode to be
electrically coupled to a load via one or more electrodes or wires.
In some examples, the load may be a light emitting device, a
machine, a display, an electrical appliance, and/or a battery
charger, among others. Some embodiments further include an
ultracapacitor operably coupled to the anode. Such an
ultracapacitor may store at least a portion of the electrons.
[0017] In another example embodiment, a microbial fuel cell may
include a cathode(s), an anode(s) in electrical communication with
the cathode, a bacterial biofilm coupled to the anode. The
bacterial biofilm may metabolize a feedstock to generate electrons
and/or hydrogen. Further, the microbial fuel cell may include a
hydrogen capture module and/or an electron capture module. A
hydrogen capture module may capture hydrogen generated by the
bacterial biofilm. An electron capture module may capture the
electrons generated by the bacterial biofilm.
[0018] Example anodes may be created from 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 and/or aluminum, among others. In
some examples, the anode may be a sheet, a plurality of sheets, a
wire mesh structure, a porous tube and/or a matrix structure, among
others.
[0019] In yet another example embodiment, a microbial fuel cell may
include an anodic chamber, a cathodic chamber and/or an ionomer
barrier separating the anodic and cathodic chambers. The anodic
chamber may include electrically conductive anode(s), a bacterial
biofilm for facilitating transfer of electrons to the anode(s), a
feedstock partially in contact with the biofilm and an anodic
electrode coupled to the anode(s). The biofilm may cover at least a
portion of the anode(s). The anodic electrode may couple the
anode(s) to a load located outside of the anodic chamber. The
cathodic chamber may include cathode(s) and a cathodic electrode
coupled to the cathode. The cathodic electrode may also be coupled
to the load.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] 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 transgenic 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 transgenic 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 transgenic P. aeruginosa; the voltage fluctuates between
0.005 V and 0.05 V. Panel E (channel 4) presents voltages obtained
from a fourth transgenic P. aeruginosa, a pilT mutant; the voltage
starts near 0.5 V then decreases as the feedstock is consumed. The
results from the fourth transgenic P. aeruginosa indicate that the
voltage produced exceeds the voltage produced in the microbial fuel
cell containing Shewenella.
[0021] FIG. 2 depicts an example microbial fuel cell according to
at least some embodiments.
[0022] FIG. 3 depicts an example microbial fuel cell system,
according to at least some embodiments.
[0023] FIG. 4 presents voltages obtained from transgenic 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.
[0024] FIG. 5 depicts a three example microbial fuel cells
connected in series. This setup is shown coupled to a load.\
[0025] FIG. 6 presents voltages obtained from transgenic P.
aeruginosa, a pilT mutant; the voltage starts between 0.3 and 0.4 V
and rises to nearly 1 V. These voltometric measurements are from
three 13 ml fuel cells connected in series.
DETAILED DESCRIPTION OF THE INVENTION
[0026] Microbial cells that can generate electrical current from a
metabolite include, but are not limited to bacteria and fungi.
Bacterial cells that can transfer electrical current to an external
component include, but are not limited to Synechocystis sp PCC
6803, Brevibacillus sp. PTH1, 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 metaffireducens, 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
103:11358-11363; Pham et al (2008) Appl. Microbiol. Biotechnol.
77:1119-1129; 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".
[0027] 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 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).
[0028] Microbes may obtain energy from a feedstock or material
through a metabolic process. In a microbial fuel cell, the
transgenic microbes have access to a feedstock. In an embodiment
the feedstock is in the anodic chamber. In an embodiment the
feedstock is circulated past the anode. In an aspect it is
recognized that the feedstock and/or the transgenic microbes are
replaced, removed, or reseeded. Greenhouse gases include, but are
not limited to, carbon dioxide (CO.sub.2), nitrous oxide
(N.sub.2O), methane, sulfur dioxide (SO.sub.2), NO.sub.2, NO.sub.3,
and SO.sub.3.
[0029] P. aeruginosa metabolizes a variety of feedstocks to produce
energy. P. aeruginosa may utilize high nitrate organic materials
including, but not limited to, sewage, fertilizer run-off, pulping
plant effluent, and animal waste; and hydrocarbons such as diesel
fuel and jet fuel; greenhouse gases, and solutions or gaseous
material with a high nitrate concentration.
[0030] 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.) growth.
[0031] An electrogenic component may include any polypeptides,
peptides, or compounds 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, bdIA, last, lasR, nirS,
ftsZ, pilA and fliC.
[0032] A transgenic microbe may exhibit an altered electrogenic
efficacy. A transgenic microbial cell is a microbial cell stably
transformed with an isolated nucleic acid molecule and that
exhibits altered expression of a nucleotide sequence of interest.
The isolated nucleic acid molecule may disrupt an endogenous
nucleotide sequence of interest resulting in altered expression of
the disrupted endogenous nucleotide sequence of interest or the
isolated nucleic acid may comprise an expression cassette
comprising a promoter operably linked to a heterologous nucleotide
sequence of interest resulting in altered expression of the
heterologous nucleotide sequence of interest. It is recognized that
the transgenic microbes may contain multiple genetic alterations or
mutations that inactivate the genes; these may include a 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 may introduce a heterogenous
nucleotide sequence of interest or disrupt an endogenous nucleotide
sequence.
[0033] By "stably transformed" is intended that the genome of the
microbe has incorporated at least one copy of the isolated nucleic
acid molecule. When a stably transformed microbe divides, both
daughter cells include a copy of the isolated nucleic acid
molecule. It is envisioned that transgenic microbes include progeny
of a stably transformed microbe. The invention encompasses isolated
or substantially purified nucleic acid compositions. An "isolated"
or substantially "purified" nucleic acid molecule, or biologically
active portion thereof, is substantially free of other cellular
material, or culture medium when produced by recombinant techniques
or substantially free of chemical precursors or other chemicals
when chemically synthesized. Isolated nucleic acid molecules may
include vectors or plasmids purified from a host cell and fragments
of a vector or plasmid purified from a host cell.
[0034] By "fragment" is intended a portion of an isolated nucleic
acid molecule. Fragments of an isolated nucleic acid molecule may
range from at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,
1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650,
1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200,
2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750,
2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300,
3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850,
3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400,
4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 4950,
4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 4950, 5000,
5050, 5100, 5150, 5200, 5250, 5300, 5350, 5400, 5450, 5500, 5550,
5600, 5650, 5700, or up to and including all the full number of
nucleotides in an isolated nucleic acid molecule.
[0035] An isolated nucleic acid molecule may comprise a regulatable
expression cassette. Expression cassettes will 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. Such
an 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. The
expression cassette may additionally contain selectable marker
genes.
[0036] The expression cassette will include 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 include initiation and
termination codons as well as polyadenylation signals. 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.).
[0037] 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.
[0038] The regulatory sequences to which the polynucleotides
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage A, 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.
[0039] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0040] Where appropriate, the heterologous nucleotide sequence
whose expression is to be under the control of the promoter
sequence of the present invention 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. (Suppl.), 2111-2118; 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.
[0041] 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.
[0042] The expression cassettes may 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. Sci. 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.
[0043] 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.
[0044] 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, N.Y., herein incorporated by reference.
[0045] 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).
[0046] 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 12:101-110; 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):8115; Luchrsen at al. (1992)
Methods Enzymol. 216: 397-414).
[0047] 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 Ser. 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.
[0048] 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.
[0049] 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 P1. For a description of the
cre/IoxP 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.
[0050] 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, N.Y.; Coligan et al (2002) Current
Protocols in Protein Science, Wiley-Interscience, New York, N.Y.;
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.
[0051] By altered expression is intended a change in expression
level of the full nucleotide sequence of interest as compared to an
untransformed, 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 of interest.
[0052] A transgenic cell may exhibit an altered cellular property
such as, 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.
[0053] An isolated nucleic acid molecule that disrupts an
endogenous nucleotide sequence of interest may replace the
endogenous nucleotide sequence of interest, may interrupt the
endogenous nucleotide sequence, may replace a portion of the
endogenous nucleotide sequence of interest, may replace a
regulatory region controlling expression of the endogenous
nucleotide sequence of interest, may interrupt the a regulatory
region controlling expression of the endogenous nucleotide
sequence, may delete an endogenous nucleotide sequence of interest,
may delete a portion of an endogenous nucleotide sequence, may
delete a regulatory region, or may delete a portion of a regulatory
region. Endogenous nucleotide sequences of interest include, but
are not limited to, pilT, bdIA, last, IasR, nirS, ftsZ, pilA, and
fliC.
[0054] 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.
[0055] bdIA 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 of bdIA
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.
[0056] The fliC gene encodes a polypeptide involved in swimming
motility and chemotaxis. FIiC disruption mutants do not have a
flagellum; thus their motility is reduced. FIiC disruption
mutations exhibit reduced chemotaxis and improved biofilm
formation. While not being limited by theory, FliC disruption
mutants may transfer more electrons to an anode.
[0057] LasI encodes N-(3-oxododecanoyl)-L-homoserine lactone
synthase, a polypeptide that, while not being limited by mechanism,
may 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 may function as a mediator of electron
transfer. Deletion of Iasi also alters virulence of the bacterial
cell in both animal and human cells. Such an altered virulence may
be a decreased virulence in a human or animal cell. See Davies et
al (1998) Science, herein incorporated by reference in its
entirety.
[0058] Deletion of lasR alters virulence of the bacterial cell in
both animal and human cells. Such an altered virulence may 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.
[0059] 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 NO.sub.2.sup.- to NO and may catalyze the
four-electron reduction of O.sub.2 to 2H.sub.2O. Inactivation of
nirS may reduce problems associated with NO in anaerobic biofilms,
increase electron flow through the pili, and reduce production of
nitrous oxide (N.sub.2O). 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.
[0060] 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 .mu.m and 300 .mu.m, particularly between
10 .mu.m and 200 .mu.m and more particularly between 30 and 100
.mu.m. 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 transgenic 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.
[0061] Various substances may be added to the feedstock provided to
a biofilm. Such substances may include additional organisms
compatible with the transgenic 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 feedstock are selected from
the group of antibiotics to which the transgenic microbe is
resistant.
[0062] 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 an
embodiment exogenous regulation of cell division (or cell
replication) may occur. Such regulation may involve the use of
inducible promoters.
[0063] 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.
[0064] Anaerobic conditions may encompass both strict anaerobic
conditions with no O.sub.2 present and mild anaerobic conditions
wherein the O.sub.2 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 certain
embodiments aerobic conditions are desirable. In certain
embodiments anaerobic conditions are desirable. P. aeruginosa
rapidly utilizes oxygen, thus may generate anaerobic conditions.
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
O.sub.2 removal system. Glucose oxidase converts glucose to uric
acid and H.sub.2O.sub.2. 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 condition and sustaining said anaerobic conditions 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, such as but
not limited to, when sewage enters the fuel cell.
[0065] Methods of inoculating an anodic chamber include, but are
not limited to, immersion of the anode in a culture, addition of
bacteria to the anodic chamber, and addition of a matrix comprising
a transgenic bacteria of the instant application.
[0066] In an embodiment, 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 an embodiment the anodic
chamber is pretreated or coated with the 17-mer, but the anode is
not.
[0067] 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. Nanomaterials are typically less than 1 micron in
thickness.
[0068] FIG. 2 depicts an example microbial fuel cell 10 according
to some embodiments. Microbial fuel cell 10 may include a housing
(or enclosure) 12. The housing 12 may include an anodic chamber 34,
a cathodic chamber 36 and a barrier 22 between the anodic chamber
34 and cathodic chamber 36. The anodic chamber 34 may include one
or more anodes 14. The cathodic chamber 36 may include one or more
cathodes 16. In some examples, the microbial fuel cell 10 may
include a feedstock 18 in which the anode(s) 14 and/or cathode(s)
16 may be placed. In some examples, anode 14 may be covered with a
bacterial film, or biofilm 28, formed from numerous bacteria, as
discussed throughout the present disclosure.
[0069] In some examples, anodic chamber 34 and cathodic chamber 36
may be separated by barrier 22 (e.g., an ionomer membrane). Barrier
22 may divide housing 12 into compartments and prevent the movement
of electrons from anode 14 to cathode 16 within the housing 10. An
electrical circuit may connect anode 14 and cathode 16 through a
load 24. Electrodes 26 may electrically couple anode 14 and
cathode. Suitable substances for the electrodes 26 may include, but
are not limited to, copper or diamond.
[0070] The cathode 16 of a microbial fuel cell 10 may be 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.
[0071] A barrier 22 such as a Nafion.RTM. membrane may separate the
anodic 34 and cathodic chambers 36. The barrier 22 slows,
decreases, or prevents electrons from moving directly from the
anode 14 to the cathode 16; rather, the electrons flow through the
wires 26 of the electrical circuit. The barrier 22 may be an
ionomer membrane such as but not limited to a Nafion.RTM.
perfluorosulfonic acid (PFSA) membrane (DuPont Fuel Cells, Inc).
Excessive deposits of the biofilm 28 on the barrier 22 may impair
function of the microbial fuel cell 10. Therefore it is advisable
to maintain biofilm 28 deposits on the barrier 22 at a moderate
level. Methods of regulating biofilm 28 deposits include, but are
not limited to, regulating bacterial cell division rates and
precoating the barrier 22 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 PiIA protein, also known as the PiIA 17mer.
Alternatively the anode 14 and cathode 16 may be separable
components as for instance an anodic tube that may be removable
from the cathode portion 36 of the microbial fuel cell 10.
[0072] Several microbial fuel cells could be electrically
associated in series or parallel to create a battery of fuel cells.
An example of a series fuel cell system 50 is depicted in FIG. 5,
showing three microbial fuel cells 52, 54 and 56 associated in
series. Microbial fuel cells are further coupled to load 58. Load
58 may include a light bulb or other load, for example. One or more
of the microbial fuel cells could be disassembled and cleaned. It
is recognized 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.
[0073] 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 transgenic microbes to flow through the load. The transgenic
microbes transfer electrons from the feedstock to the anode, the
electrons proceed to flow through electrodes and the load to the
cathode. A barrier blocks the electrons from flow through the
interior of the fuel cell. Microbial fuel cells may be used in
consumer electronics perhaps through a lithium/ion battery
recharger or as a replacement for lithium/ion batteries. Microbial
fuel cells may be used in electric plug-in automobiles or to
recharge electric plug-in automobiles. Microbial fuel cells may
generate power for residential and commercial buildings by tapping
into organic wastes flushed from the buildings in the outgoing
sewage pipes. Microbial fuel cells may be used for large waste
treatment, farms, and utilities.
[0074] The microbial fuel cell electrical system may 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.
[0075] FIG. 3 depicts an example microbial fuel cell system 40
including a microbial fuel cell 42, a hydrogen fuel cell 44, an
ultracapacitor 46 and a load 48, according to an embodiment of the
invention. Ultracapacitor 46 may be attached in parallel with
microbial fuel cell 42 having modified bacteria. When a need for a
power in excess of that which can be supplied by the microbial fuel
cell 42 arises, the ultracapacitor 46 may discharge for a
relatively short period of time. When the need for the increased
power subsides, ultracapacitor 46 could draw from microbial fuel
cell 42 to recharge. A need for an increased power for a relatively
short period of time could occur, for example, during a quick
acceleration of an electric vehicle, the start up of a motor
connected to the fuel cell, or during the time immediately after
beginning use of the microbial fuel cell system 40 for an emergency
backup system. Pairing microbial fuel cell 42 with ultracapacitor
46 may allow a designer of system 40 to utilize a smaller microbial
fuel cell 42 because microbial fuel cell 42 may not need to be
sized for peak power applications. Ultracapacitor 46 may also serve
to make the delivery of electrical energy to an electrical grid, a
load 48, or a device more constant. Ultracapacitor 46 may smooth
out the peaks and valleys of power delivery if the flow of energy
from fuel cell 42 becomes variable. Additionally, multiple
microbial fuel cells 42 and/or multiple ultracapacitors 46 can be
paired for increased power output. In some examples, ultracapacitor
46 may be embedded within housing 12 as one compact unit. Other
applications and/or configurations may occur to those skilled in
the art.
[0076] Moreover, advantageous use may be made of the chemical
reactions of the microbial fuel cell 42. For example, the products
of the chemical reaction at the cathode may include free hydrogen
gas when microbial fuel cell 42 is operated anaerobically. This
hydrogen gas may be captured and/or collected by a hydrogen capture
module and utilized to power a hydrogen fuel cell 44. FIG. 3 shows
a hydrogen fuel cell 44 paired with microbial fuel cell 42 to form
fuel cell system 40. Fuel cell system 40 may include many fuel
cells of each type (microbial and/or hydrogen), one of each type,
or may omit one fuel cell type, as an application may dictate. In
some examples, microbial fuel cell 42 may emit very little carbon
dioxide, thus enabling an environmentally friendly energy source.
Additionally, microbial fuel cell 42 may be designed to emit usable
sugars that may become an energy source for other devices. The
microbial fuel cell 42 may be operated aerobically to emit water as
a byproduct. The water may be transferred to a water storage device
or transferred to the external environment.
[0077] By "matrix" is intended a material in which something is
embedded or enclosed. Matrices suitable for use in the current
application include, but are not limited to, sponges, filters,
beads, powders, tissues, granules, cassettes, cartridges and
capsules.
[0078] The transgenic microbes of the instant application may be
utilized in cleaning solutions and odor reduction systems.
[0079] The following examples are offered by way of illustration
and not limitation.
EXPERIMENTAL
Example 1
Development of Static Biofilms on Simple Glass Surfaces in
Feedstock
[0080] Circular glass coverslips were attached to the bottom of
35.times.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).
[0081] Bacterial cells were grown in Luria Bertani media (LB)
overnight.
[0082] Aerobic LB, aerobic LBN (LB+1% KNO.sub.3), or anaerobic LBN
(3 ml) was placed in each tissue culture plate. The media was
inoculated with 10.sup.7 cfu of bacterial cells. The plates were
incubated at 37.degree. C. for 24 hours. The media was removed and
the plates were washed with saline buffer. LIVE/DEAD BacLight
(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.times.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.times.512 pixel, 8-bit image.
Example 2
Culture Media
[0083] LB media is 10 g/liter tryptone, 5 g/liter yeast extract,
and 5 g/liter NaCl.
[0084] LBN media is 10 g/liter tryptone, 5 g/liter yeast extract, 5
g/liter NaCl and 10 g/liter KNO.sub.3.
Example 3
Development of Biofilms in Circulated Feedstock
[0085] Bacteria are grown aerobically in LB at 37.degree. 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.1.4.2) 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 4
Construction of P. aeruginosa Deletion Mutants
[0086] The P. aeruginosa strain PAO1 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(1):77-86) An insertional mutagenesis
cassette comprising a gentamicin resistant (Gm.sup.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 5
High-throughput Microbial Fuel Cell Prototype
[0087] 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.
[0088] 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.times.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 mls
of media. However, in our experiments with the device we used 7 mls
of media. 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 6
High Through-put Microbial Fuel Cell Voltage/Current Evaluation
[0089] The above described high-throughput microbial fuel cell
prototype was used to evaluate voltage and current generation from
wildtype Pseudomonas aeruginosa (POA), Shewanella oneidensis, and a
mutant strain (pilT, bdIA, nirS, last, 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% KNO.sub.3 media (7 ml) was placed in each
well utilized in the experiment. A stationary phase grown aerobic
culture (70 .mu.l, a 1:100 dilution) for each bacterial sample
(wildtype 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.degree. C. for 24 hours under
anaerobic conditions.
[0090] 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%
KNO.sub.3 media (7 ml) was added to each well. The recording
process was repeated. Results from one such experiment are
presented in FIG. 1.
Example 7
Voltage and Current Monitoring of the High-throughput Microbial
Fuel Cell Prototype
[0091] 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 LT1101 instrumentation amplifier across the 1K
current sense resistor. By measuring the voltage drop across the
resistor and utilizing Ohm's law (I=E/R) the current following in
the cell circuit can be calculated.
[0092] 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 8
Development of Microbial Fuel Cell with Increased Anode Surface
Area
[0093] A 23-plate stainless steel 314 anode system is constructed.
The first 21 plates are of the following dimensions:
0.05.times.9.851.times.7.554 inches. This involves a total surface
area of 197.56 inches. The other two plates are
0.05.times.9.851.times.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.
[0094] A single plate of hot, isostatic pressed graphite
(GraphiteStore.com) of 0.125.times.9.6.times.4.65 inches is used
for the cathode.
[0095] After the anode is assembled, the anode is treated with 1%
bleach, then 95% ethanol, and then 70% ethanol. Small
1.times.1.times.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% KNO.sub.3 at 37.degree. C.
for 24 hours inoculated with bacteria.
[0096] Experiments are performed with wild-type bacteria or with
various mutants. The overall efficiency of the wild-type and mutant
strains is compared.
[0097] 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% KNO.sub.3. The anodic and cathodic chambers
are treated with glucose oxidase. Glucose oxidase converts glucose
to uric acid and H.sub.2O.sub.2. H.sub.2O.sub.2 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
O.sub.2 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.
[0098] Similar experiments are performed with wild-type, single,
double, triple quadruple, and multiple 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.
[0099] All publications, patents, and patent applications mentioned
in the specification are indicative of the level of those skilled
in the art to which this invention pertains. All publications,
patents, and patent applications are herein incorporated by
reference to the same extent as if each individual publication or
patent application was specifically and individually incorporated
by reference.
[0100] The herein described subject matter sometimes illustrates
different components contained within, or coupled with, different
other components. It is to be understood that such depicted
architectures are merely examples, and that in fact many other
architectures may be implemented which achieve the same
functionality. In a conceptual sense, any arrangement of components
to achieve the same functionality is effectively "associated" such
that the desired functionality is achieved. Hence, any two
components herein combined to achieve a particular functionality
may be seen as "associated with" each other such that the desired
functionality is achieved, irrespective of architectures or
intermedial components. Likewise, any two components so associated
may also be viewed as being "operably connected", or "operably
coupled", to each other to achieve the desired functionality, and
any two components capable of being so associated may also be
viewed as being "operably couplable," to each other to achieve the
desired functionality. Specific examples of operably couplable
include but are not limited to physically mateable and/or
physically interacting components and/or wirelessly interactable
and/or wirelessly interacting components and/or logically
interacting and/or logically interactable components.
[0101] With respect to the use of substantially any plural and/or
singular terms herein, those having skill in the art may translate
from the plural to the singular and/or from the singular to the
plural as is appropriate to the context and/or application. The
various singular/plural permutations may be expressly set forth
herein for sake of clarity.
[0102] It will be understood by those within the art that, in
general, terms used herein, and especially in the appended claims
(e.g., bodies of the appended claims) are generally intended as
"open" terms (e.g., the term "including" should be interpreted as
"including but not limited to," the term "having" should be
interpreted as "having at least," the term "includes" should be
interpreted as "includes but is not limited to," etc.). It will be
further understood by those within the art that if a specific
number of an introduced claim recitation is intended, such an
intent will be explicitly recited in the claims, and in the absence
of such recitation no such intent is present. For example, as an
aid to understanding, the following appended claims may contain
usage of the introductory phrases "at least one" and "one or more"
to introduce claim recitations. However, the use of such phrases
should not be construed to imply that the introduction of a claim
recitation by the indefinite articles "a" or "an" limits any
particular claim containing such introduced claim recitation to
inventions containing only one such recitation, even when the same
claim includes the introductory phrases "one or more" or "at least
one" and indefinite articles such as "a" or "an" (e.g., "a" and/or
"an" should typically be interpreted to mean "at least one" or "one
or more"); the same holds true for the use of definite articles
used to introduce claim recitations. In addition, even if a
specific number of an introduced claim recitation is explicitly
recited, those skilled in the art will recognize that such
recitation should typically be interpreted to mean at least the
recited number (e.g., the bare recitation of "two recitations,"
without other modifiers, typically means at least two recitations,
or two or more recitations). Furthermore, in those instances where
a convention analogous to "at least one of A, B, and C, etc." is
used, in general such a construction is intended in the sense one
having skill in the art would understand the convention (e.g., "a
system having at least one of A, B, and C" would include but not be
limited to systems that have A alone, B alone, C alone, A and B
together, A and C together, B and C together, and/or A, B, and C
together, etc.). In those instances where a convention analogous to
"at least one of A, B, or C, etc." is used, in general such a
construction is intended in the sense one having skill in the art
would understand the convention (e.g., "a system having at least
one of A, B, or C" would include but not be limited to systems that
have A alone, B alone, C alone, A and B together, A and C together,
B and C together, and/or A, B, and C together, etc.). It will be
further understood by those within the art that virtually any
disjunctive word and/or phrase presenting two or more alternative
terms, whether in the description, claims, or drawings, should be
understood to contemplate the possibilities of including one of the
terms, either of the terms, or both terms. For example, the phrase
"A or B" will be understood to include the possibilities of "A" or
"B" or "A and B."
[0103] While various aspects and embodiments have been disclosed
herein, other aspects and embodiments will be apparent to those
skilled in the art. The various aspects and embodiments disclosed
herein are for purposes of illustration and are not intended to be
limiting, the true scope and spirit being indicated by the
following claims.
* * * * *