U.S. patent application number 10/294263 was filed with the patent office on 2003-07-24 for electrochemical methods for generation of a biological proton motive force and pyridine nucleotide cofactor regeneration.
Invention is credited to Jain, Mahendra K., Shin, Hyoun S., Zeikus, Gregory J..
Application Number | 20030138674 10/294263 |
Document ID | / |
Family ID | 25158852 |
Filed Date | 2003-07-24 |
United States Patent
Application |
20030138674 |
Kind Code |
A1 |
Zeikus, Gregory J. ; et
al. |
July 24, 2003 |
Electrochemical methods for generation of a biological proton
motive force and pyridine nucleotide cofactor regeneration
Abstract
Disclosed are methods using neutral red to mediate the
interconversion of chemical and electrical energy. Electrically
reduced neutral red has been found to promote cell growth and
formation of reduced products by reversibly increasing the ratio of
the reduced:oxidized forms of NAD(H) or NADP(H). Electrically
reduced neutral red is able to serve as the sole source of reducing
power for microbial cell growth. Neutral red is also able to
promote conversion of chemical energy to electrical energy by
facilitating the transfer of electrons from microbial reducing
power to a fuel cell cathode.
Inventors: |
Zeikus, Gregory J.; (Okemos,
MI) ; Shin, Hyoun S.; (Lansing, MI) ; Jain,
Mahendra K.; (Lexington, NY) |
Correspondence
Address: |
QUARLES & BRADY LLP
411 E. WISCONSIN AVENUE
SUITE 2040
MILWAUKEE
WI
53202-4497
US
|
Family ID: |
25158852 |
Appl. No.: |
10/294263 |
Filed: |
November 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10294263 |
Nov 14, 2002 |
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09793025 |
Feb 26, 2001 |
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6495023 |
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09793025 |
Feb 26, 2001 |
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09350072 |
Jul 8, 1999 |
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6270649 |
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60092190 |
Jul 9, 1998 |
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60092191 |
Jul 9, 1998 |
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Current U.S.
Class: |
429/2 ; 429/401;
429/422; 429/431 |
Current CPC
Class: |
Y02E 60/50 20130101;
C12N 13/00 20130101; H01M 8/16 20130101; Y02E 60/527 20130101; C12Q
1/004 20130101 |
Class at
Publication: |
429/2 ; 429/43;
429/13 |
International
Class: |
H01M 008/16 |
Goverment Interests
[0002] This invention was made with U.S. Government support in the
form of the United States Department of Energy grant
DE-FG02-93ER20108. The United States may have certain rights in
this invention.
Claims
We claim:
1. A method for generating electricity using a biological system
comprising the steps of: (a) providing an electrochemical fuel cell
system comprising an anode compartment and a cathode compartment
separated by a cation-selective membrane, wherein each compartment
is equipped with an electrode, wherein the electrodes are connected
by a wire to a multimeter; (b) placing an anolyte in the anode
compartment, the anolyte comprising a suitable concentration of
neutral red and a biological catalyst selected from the group
consisting of resting cells, growing cells, and anaerobic sludge
comprising cells, or a combination thereof; (c) placing a suitable
catholyte in the cathode compartment; and (d) allowing the neutral
red-mediated conversion of chemical reducing power to
electricity.
2. The method of claim 1, wherein the biological catalyst comprises
photosynthetic bacteria.
3. The method of claim 1, wherein the biological catalyst comprises
lithotrophic bacteria.
4. The method of claim 1, wherein the biological catalyst comprises
organotrophic cells.
5. The method of claim 1, further comprising the step of
supplementing the anolyte with an energy source that can be used by
the biological catalyst.
6. The method of claim 1, wherein the energy source comprises
light, organic compounds, or molecular hydrogen (H.sub.2).
7. A method for detecting the presence of a specific organic or
inorganic test compound in a sample comprising: (a) providing
biosensor comprising an electrochemical fuel cell system having an
anode compartment and a cathode compartment separated by a
cation-selective membrane, wherein each compartment is equipped
with an electrode, wherein the electrodes are connected by a wire
to a multimeter; (b) placing an anolyte in the anode compartment,
the anolyte comprising the sample, a suitable concentration of
neutral red, and a biological catalyst selected from the group
consisting of whole cells and an enzyme, wherein the biological
catalyst is able to oxidize the test compound; (c) placing a
suitable catholyte in the cathode compartment; (d) allowing
oxidation of at least a portion of any test compound present in the
sample and reduction of at least a portion of oxidized neutral red;
(e) allowing the transfer of electrons from reduced neutral red to
the cathode; and (f) detecting the generation of an electrical
current.
8. The method of claim 7, further comprising the step of
determining the concentration of the test compound in the
sample.
9. A method for measuring the chemical oxygen demand in waste water
comprising the steps of: (a) providing an electrochemical fuel cell
system comprising an anode compartment and a cathode compartment
separated by a cation-selective membrane, wherein each compartment
is equipped with an electrode, wherein the electrodes are connected
by a wire to a multimeter; (b) placing an anolyte in the anode
compartment, the anolyte comprising a suitable concentration of
neutral red and waste water comprising or supplemented with a
biological catalyst; (c) placing a suitable catholyte in the
cathode compartment; (d) allowing the neutral red-mediated
conversion of chemical reducing power to electricity; and (e)
measuring the electrical current generated by the fuel cell system.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. Ser. No. 09/793,025
filed Feb. 26, 2001 which is a continuation-in-part of U.S. Ser.
No. 09/350,072, filed Jul. 8, 1999, now U.S. Pat. No. 6,270,649,
which claims priority to U.S. Provisionals Ser. No. 60/092,190 and
Ser. No. 60/092,191, both filed Jul. 9, 1999. These applications
are incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0003] Microbial fermentation and biotransformation reactions are
being employed with increasing frequency in the production of a
number of commercially and industrially important products. There
is also growing interest in developing alternative energy sources
through microbial fermentation of waste materials. The economic
feasibility of these processes depends on maximizing the efficiency
of the fermentation or biotransformation reactions.
[0004] Bacterial species are able to use various energy sources,
including light and diverse organic and inorganic chemicals, for
growth and metabolism. These energy sources are used to produce an
electrochemical gradient that provides an electron donor for
metabolism and allows maintenance of a membrane potential and
proton motive force. The energetics of living systems are driven by
electron transfer processes in which electrons are transferred from
a substrate, which is thereby oxidized, to a final electron
acceptor, which is thereby reduced.
[0005] In microbial metabolism, the energy produced from the
driving force of electrons is directly proportional to the
potential energy difference (.DELTA. E.sub.0') between the initial
electron donor (the first biochemical dehydrogenating reaction) and
final electron acceptor (e.g., the final biochemical hydrogenating
reaction).
[0006] Certain microorganisms (e.g., Escherichia and
Actinobacillus) are able to grow using H.sub.2 as an electron donor
to reduce fumarate into succinate in an anaerobic respiration
process. These bacteria obtain free energy and reducing power from
the electron driving force generated by the E.sub.0' difference
between the coupled oxidoreduction half reactions of
[2H.sup.+/H.sub.2] and [fumarate/succinate].
[0007] Methanogens are strict anaerobic archea that can couple
H.sub.2 or HCOOH oxidation to CO.sub.2 reduction into methane.
Methanogenesis produces less free energy than other anaerobic
respiration processes (e.g., fumarate, nitrate, or sulfate
reduction) because the E.sub.0' difference between the half
oxidation reduction reactions of [2H.sup.+/H.sub.2] and
[CO.sub.2/CH.sub.4] is relatively small.
[0008] Hydrogen oxidation by microbial hydrogenases can be coupled
to reduction of various biological electron carriers including
NAD.sup.+, cytochromes, and quinones or to certain artificial redox
dyes, such as methyl-viologen and neutral red (NR) (Annous, et al.,
1996, Appl. Microbiol. Biotechnol. 45:804-810, Kim, et al., 1992,
J. Microbiol. Biotechnol. 2:248-254). The effect of redox dyes,
with or without electrochemical reduction systems, on metabolite
patterns and H.sub.2 production has been examined in several
microbial processes, including the glutamate (Hongo, et al., 1979,
Agric. Biol. Chem. 43:2083-2986), butanol (Girbal, et al., 1995,
Microbiol. Rev. 16:151-162 and Kim, et al., 1992, J. Microbiol.
Biotechnol, 2:268-272), and butyrate (Shen, et al., 1996, Appl.
Microbiol, Biotechnol, 45:355-362) fermentations.
[0009] The specific activities of redox enzymes involved in
bacterial catabolism, such as hydrogenase or fumarate reductase,
can be measured using their in vivo electron carriers (e.g., NAD or
menanquinone) or with artificial redox dyes (e.g., benzyl viologen)
(Cecchini, et al., 1986, Proc. Natl. Acad. Sci. USA 83:8898-8902,
Dickie, et al., 1979, Can. J. Biochem., 57:813-821, Kemner, et al.,
1994, Arch. Microbiol., 161:47-54, Petrov, et al., 1989, Arch.
Biochem. Bio-phys. 268:306-313, and Wissenbach, et al., 1990, Arch.
Microbiol. 154:60-66). Bacteria that produce succinic acid as a
major catabolic end product (e.g., E. coli, Wolinella succinogenes
and other species) have a fumarate reductase (FRD) complex that
catalyzes fumarate-dependent oxidation of menaquinone. This
reaction is coupled to the generation of a transmembrane proton
gradient that is used by the organism to support growth and
metabolic function (Kortner, et al., 1992, Mol. Microbiol.
4:855-860 and Wissenbach, et al., 1992, Arch. Microbiol.
158:68-73). The fumarate reductase of E. coli is composed of four
nonidentical subunits: FRDA, FRDB, FRDC, and FRDD. The subunits are
arranged in two domains: (i) the FRDAB catalytic domain and the
FRDCD membrane anchor domain, which is essential for electron
transfer and proton translocation reactions involving menaquinone
(Cecchini, et al., 1995, J. Bacteriol. 177:4587-4592, Dickie, et
al., 1979, Can. J. Biochem., 57:813-821, and Westenberg, et al.,
1990, J. Biol. Chem. 265:19560-19567). Subunits FRDA and FRDB
retain catalytic activity in solubilized membrane preparations.
[0010] Electrochemical techniques employing redox dyes are useful
for investigating the oxidation-reduction characteristics of
biological systems and provide information about biological energy
metabolism (Moreno, et al., 1993, Eur. J. Biochem. 212:79-86 and
Sucheta, et al., 1993, Biochemistry 32:5455-5465). Redox dyes that
are useful in bioelectrochemical systems must easily react with
both the electrode and the biological electron carriers. Many
biological electron carriers, such as NAD (Miyawaki, et al., 1992,
Enzyme Microb. Technol. 14:474-478 and Surya, et al., 1994,
Bioelectrochem. Bioenerg. 33:71-73), c-type cytochromes (Xie, et
al., 1992, Bioelectrochem. Bioenerg. 29:71-79), quinones (Sanchez,
et al., 1995, Bioelectrochem. Bioenerg. 36:67-71), and redox
enzymes, such as nitrite reductase (White, et al., 1987,
Bioelectro-chem. Bioenerg. 26:173-179), nitrate reductase (Willner,
et al., 1992, Bioelectrochem. Bioenerg. 29:29-45), fumarate
reductase (Sucheta, et al., 1993, Biochemistry. 32:5455-5465),
glucose-6-phosphate dehydrogenase (Miyawaki, et al., 1992, Enzyme
Microb. Technol. 14:474-478), ferredoxin-NADP reductase (Kim, et
al., 1992, J. Microbiol. Biotechnol. 2:2771-2776) and hydrogenase
(Schlereth, et al., 1992, Bioelectrochem. Bioenerg. 28:473-482)
react electrochemically with the redox dyes.
[0011] Certain redox dyes with lower redox potentials than that of
NAD, such as methyl viologen (MV) (Kim, et al., 1988, Biotechnol.
Lett. 10:123-128, Pequin, et al., 1994, Biotechnol. Lett.
16:269-274, and White, et al., 1987, FEMS Microbiol. Lett.
43:173-176), benzyl viologen (Emde, et al., 1990, Appl. Environ.
Microbiol. 56:2771-2776), and neutral red (NR) (Girbal, et al.,
1995, FEMS Microbiol. Rev. 16:151-162 and Kim, et al., J.
Biotechnol. 59:213-220) have been correlated with alterations in
the rate of biological redox reactions in vivo. Hongo and Iwahara
(Hongo, et al., 1979, Agric. Biol. Chem. 43A:2075-2081 and Hongo,
et al., 1979, Agric. Biol. Chem. 43B:2083-2086) discovered that
including redox dyes with low .DELTA. E.sub.0' values (e.g., MV,
benzyl viologen and NR) in bacterial fermentation conducted under
cathodic reduction conditions was correlated with an increase in
L-glutamate yield (about 6%). In the method of Hongo and Iwahara, a
platinum electrode was used to deliver electricity at a level that
was sufficiently high to generate hydrogen from water. Therefore,
the source of increased reducing power in the method of Hongo and
Iwahara is not known, nor was the mechanism by which the tested
dyes affect fermentation characterized. Addition of NR to
acetone-butanol fermentations is correlated with decreased
production of acids and H.sub.2, and enhanced production of solvent
(Girbal, et al., 1995, FEMS Microbiol. Rev. 16:151-162 and Kim, et
al., 1992, J. Microbiol. Biotechnol. 2:2771-2776), an effect that
was further enhanced under electroenergized fermentation conditions
(Ghosh, et al., 1987, abstr. 79. In Abstracts of Papers, 194th ACS
National Meeting. American Chemical Society). Viologen dyes have
been used as electron mediators for many electrochemical catalytic
systems using oxidoreductases in vitro and in vivo (James, et al.,
1988, Electrochem. Bioenerg. 20:21-32, Kim, et al., 1988,
Biotechnol. Lett. 10:123-128, Moreno, et al., 1993, Eur. J.
Biochem. 212:79-86, Schlereth, et al., 1992, Bioelectrochem.
Bioenerg. 28:473-482, and White, et al., 1987, FEMS Microbiol.
Lett. 43:173-173).
[0012] An electrochemical system was used to regenerate reduced
iron for growth of Thiobacillus ferrooxidans on electrical reducing
power (Robinson, et al., 1982, Can. J. Biochem. 60:811-816).
[0013] It may be possible to control or alter metabolism by linking
biochemical processes to an external electrochemical system.
Linking biochemical and electrochemical systems may allow the use
of electricity as a source of electrons for bacterial growth and in
vivo or in vitro fermentation or biotransformation reactions.
[0014] A reversible biochemical-electrochemical link may allow
conversion of microbial metabolic or enzyme catalytic energy into
electricity. Biofuel cells in which microbial energy is directly
converted to electrical energy using conventional electrochemical
technology have been described (Roller, et al., 1984, J. Chem.
Tech. Biotechnol. 34B:3-12 and Allen, et al., 1993, Appl. Biochem.
Biotechnol. 39-40:27-40). Chemical energy can be converted to
electric energy by coupling the biocatalytic oxidation of organic
or inorganic compounds to the chemical reduction of the oxidant at
the interface between the anode and cathode (Willner, et al., 1998,
Bioelectrochem. Bioenerg. 44:209-214). However, direct electron
transfer from microbial cells to electrodes has been shown to take
place only at very low efficiency (Allen, et al., 1972, J. R.
Norris and D. W. Ribbons (eds.), Academic Press, New York,
6B:247-283).
[0015] The electron transfer efficiency can be improved by using
suitable redox mediators (Bennetto, et al., 1985, Biotechnol. Lett.
7:699-105), and most of the microbial fuel cells studied employed
electron mediators such as the redox dye thionin (Thurston, et al.,
1985, J. Gen. Microbiol. 131:1393-1401). In microbial fuel cells,
two redox couples are required for: (1) coupling the reduction of
an electron mediator to bacterial oxidative metabolism; and (2)
coupling the oxidation of the electron mediator to the reduction of
the electron acceptor on the cathode surface (where the electron
acceptor is regenerated by atmospheric oxygen) (Ardeleanu, et al.,
1983, Bioelectrochem. Bioenerg. 11:273-277 and Dealney, et al.,
1984, Chem. Tech. Biotechnol. 34B: 13-27).
[0016] The free energy produced by either normal microbial
metabolism or by microbial fuel cell systems is mainly determined
by the potential difference (.DELTA. E.sub.0') between the electron
donor and acceptor according to the equation,
-.DELTA.G=nF.DELTA.E.sub.0 in which .DELTA. G is the variation in
free energy, n is the number of electron moles, and F is the
Faraday constant (96,487 J/volt) (Dealney, et al., 1984, Chem.
Tech. Biotechnol. 34B:13-27). Coupling of the metabolic oxidation
of the primary electron donor (NADH) to the reduction of the final
electron acceptor (such as oxygen or fumarate in bacterial
respiration systems) is very similar to the coupling of
electrochemical half-reaction of the reductant (electron donor) to
the half reaction of the oxidant (electron acceptor) in a fuel cell
or battery system (Chang, et al., 1981, 2nd ed., Macmillan
Publishing, New York). Biological reducing power sources such as
NADH (E.sub.0'=-0.32 volt), FdH.sub.2 (E.sub.0'=-0.42 volt), or
FADH.sub.2 (E.sub.0'=-0.19 volt) with low redox potentials can act
as reductants for fuel cells, but they are not easily converted to
electricity because the cytoplasmic membrane must be non-conductive
to maintain the membrane potential absolutely required for free
energy (i.e., ATP) production (Thauer, et al., 1997, Bacteriol.
Rev. 41:100-180).
[0017] For electron transfer to occur from a microbial electron
carrier to an electrode, an electron mediator is required (Fultz,
et al., 1982, Anal. Chim. Acta. 140:1-18). Allen, et al. (1993,
Appl. Biochem. Biotechnol. 39-40:27-40) reported that the reducing
power metabolically produced by Proteus vulgaris or E. coli can be
converted to electricity by using electron mediators such as
thionin. Tanaka, et al. (1985, Chem. Tech. Biotechnol. 35B:191-197
and 1988, Chem. Tech. Biotechnol. 42:235-240) reported that light
energy can be converted to electricity by Anabaena variabilis using
HNQ as the electron mediator. Park, et al. (1997, Biotech. Techniq.
11:145-148) confirmed that viologen dye cross-linked with carbon
polymers and adsorbed to Desulfovibro desulfuricans cytoplasmic
membranes can mediate electron transfer from bacterial cells to
electrodes or from electrodes to bacterial cells.
[0018] There remains a need in the art for improved, more efficient
methods for converting metabolic reducing power to electrical
energy, and for converting electrical energy to metabolic reducing
power.
BRIEF SUMMARY OF THE INVENTION
[0019] One aspect of the present invention is a method of promoting
reductive processes in a bioreactor system comprising the steps of
(a) providing an electrochemical bioreactor system having a cathode
compartment equipped with a cathode and an anode compartment
equipped with an anode, the cathode and anode compartment being
separated by a cation selective membrane, wherein the cathode and
anode are connected by a conductive material to a power supply; (b)
placing a suitable amount of neutral red and a biological catalyst
in the cathode compartment.
[0020] In a particularly advantageous form of the invention, the
biological catalyst is an enzyme that uses NADH or NADPH as a
cofactor. The cathode compartment comprises NADH or NADPH and an
oxidized substrate for the enzyme. Electrically reduced neutral red
transfers electrons to NAD.sup.+ or NADP.sup.+. In a preferred form
of the invention, the enzyme is oxidoreductase, most particularly
in alcohol dehydrogenase, the oxidized substrate is an aldehyde or
ketone and the reduced product is an alcohol.
[0021] Another aspect of the invention is a method for generating
electricity using a biological system comprising the steps of (a)
providing an electrochemical fuel cell system comprising an anode
compartment and a cathode compartment separated by a
cation-selective membrane, wherein each compartment is equipped
with an electrode, wherein the electrodes are connected by a wire
to a multimeter; (b) placing an anolyte in the anode compartment,
the anolyte comprising a suitable concentration of neutral red and
a biological catalyst selected from the group consisting of
bacteria, archea, plant cells, and animal cells; (c) placing a
suitable catholyte in the cathode compartment; and (d) allowing the
neutral red-mediated conversion of chemical reducing power to
electricity.
[0022] It is an object of the invention to provide methods that
allow the interconversion of biochemical reducing power (e.g.,
NADH), biological energy (ATP), and electrical energy in an
electrochemical bioreactor or fuel cell.
[0023] It is a further object of the invention to provide an
economical method of promoting cell growth or production of desired
products using electrically reduced neutral red.
[0024] Another object of the invention is to provide a method for
converting biological reducing power into electricity.
[0025] It is an advantage of the present invention that electrical
energy may be used to promote cell growth or fermentation or
enzymatic transformation in the presence of neutral red.
[0026] Another advantage of the invention is that neutral red
promotes the generation of electrical energy from waste material
comprising mixed bacterial populations.
[0027] Other objects, features, and advantages of the present
invention will be apparent on review of the specification and
claims.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0028] FIG. 1 is a schematic diagram of a microbial fuel cell using
neutral red (NR) as an electronophore.
[0029] FIG. 2 shows the current production from NADH oxidation in a
chemical fuel cell with NR (A) or thionin (B) as the electron
mediator.
[0030] FIG. 3 is a cyclic voltammogram obtained with a glassy
carbon electrode on successive cycles following introduction of the
electrode into a 100 .mu.M NAD.sup.+ solution.
[0031] FIG. 4 shows the current and potential obtained in a glucose
fuel cell using E. coli K-12 resting cells and neutral red or
thionin.
[0032] FIG. 5 shows the electrical current and potential levels
obtained using A. svccinogenes growing or resting cells.
[0033] FIG. 6 shows the current and potential produced in a glucose
(3 g/L) fuel cell using anaerobic sewage sludge as catalyst and NR
(100 .mu.M) as the electronophore.
[0034] FIG. 7 is a proposed model of the energy flow in cells under
normal (A) or electrogenic (B) glucose metabolism.
[0035] FIG. 8 is a time course for biotransformation of
.beta.-tetralone to .beta.-tetralol by the yeast T. capitacum at 1
g/L of substrate in the presence and absence of 1.5 volt
electricity.
[0036] FIG. 9 is a time course of biotransformation of
.beta.-tetralone to .beta.-tetralol by the yeast T. capitacum at 2
g/L of substrate in the presence and absence of 1.5 volt
electricity.
[0037] FIG. 10 is the effect of pulse feeding of 2 g/L of substrate
on the biotransformation of .beta.-tetralone to
.beta.-tetralol.
[0038] FIG. 11 is the effect of pulse feeding of 1 g/L of substrate
on the biotransformation of .beta.-tetralone to
.beta.-tetralol.
[0039] FIG. 12 is the effect of ethanol concentration on the
biotransformation of .beta.-tetralone to .beta.-tetralol.
[0040] FIG. 13 is the effect of electrical potential on the
biotransformation of .beta.-tetralone to .beta.-tetralol.
[0041] FIG. 14 is the effect of electrical potential on the
biotransformation of .beta.-tetralone to .beta.-tetralol.
[0042] FIG. 15 is the biotransformation kinetics with purified
.beta.-tetralone reductase with NAD in the presence of
electricity.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The present invention provides methods for achieving the
efficient interconversion of chemical and electrical energy using
neutral red. One aspect of the invention is a method for using
electrical energy as a source of reducing power in fermentation or
enzymatic reactions. Another aspect of the invention includes a
method of using neutral red and cells or enzymes to produce
electricity.
[0044] The invention is based on the discovery that the use of
neutral red in methods directed toward regulating electron flow in
biological systems offers a number of surprising advantages, which
are disclosed in related U.S. Ser. Nos. 60/092,190 and 60/092,191;
Park and Zeikus, J. Bacteriol. 181:2403-2410, 1999; and Park, et
al. Appl. Environ. Microbiol. In press, all of which are
incorporated by reference in their entirety.
[0045] A critical factor for the control of end-product yields in
fermentation or enzymatic biotransformation reactions is regulation
of electron distribution through the NADH/NAD.sup.+ ratio. If
additional reducing power (e.g., H.sub.2 or electrochemically
produced reducing equivalents) is supplied to bacteria, an increase
in the NADH/NAD.sup.+ ratio and metabolism may be expected.
However, efficient transfer of electrons from electricity to
NAD.sup.+ requires a suitable electron mediator.
[0046] As discussed in detail in U.S. Ser. Nos. 60/092,190 and
60/092,191, neutral red was discovered to be a particularly good
electron mediator for use in the interconversion of electricity and
metabolic reducing power in electrochemical bioreactor systems.
Neutral red is able to form a reversible redox couple at the
electrode and has a highly negative E.sub.0'. The E.sub.0' value
for neutral red is very similar to that of physiological electron
carriers in the electron transport chain, including, for example,
NADH. The ability of neutral red to accept electrons from far up
the electron transport chain enhances electricity production in
biofuel cell systems. Neutral red is soluble at a neutral pH, it is
stable in both its oxidized and reduced forms, it does not
decompose during long-term redox cycling.
[0047] As disclosed in U.S. Ser. Nos. 60/092,190 and 60/092,191,
neutral red is relatively nontoxic, and can be easily adsorbed on
the cytoplasmic membrane of the cells under study, where it
functions as an electronophore, or electron shuttle, for electron
transfer across the cytoplasmic membrane. Neutral red was
demonstrated to function as an electron mediator in reversible
oxidation or reduction of compounds and to substitute for
menaquinone in the cell membrane, Surprisingly, electrically
reduced neutral red promotes growth, proton translocation and
metabolite production in cells even in the absence of other sources
of reducing power.
[0048] One aspect of the present invention provides a method for
promoting reductive processes in a bioreactor system comprising the
steps of (a) providing an electrochemical bioreactor system having
a cathode compartment equipped with a cathode and an anode
compartment equipped with an anode, the cathode and anode
compartment being separated by a cation selective membrane, wherein
the cathode and anode are connected by a conductive material to a
power supply; and (b) placing a suitable amount of neutral red and
a biological catalyst in the cathode compartment.
[0049] Preferably, the biological catalyst is selected from the
group consisting of microbial cells, plant cells, animal cells,
isolated intact cytoplasmic membranes, solubilized cytoplasmic
membranes, and an enzyme having NADH or NADPH a cofactor. To
maximize the efficiency of the interconversion of biochemical and
electrical energy, the biological catalyst is immobilized on the
cathode.
[0050] In a preferred embodiment, the method of the invention
further comprises the steps of (c) placing an anolyte solution in
the anode compartment; (d) delivering to the cathode an electric
current of suitable strength to cause reduction of at least a
portion of oxidized neutral red in the cathode compartment; and (e)
allowing the reduced neutral red to transfer electrons to an
oxidized substrate or an electron carrier.
[0051] The method of the invention is very versatile, in that it
can be adapted for use with any number of biological catalysts,
including microbial, plant, or animal cells, isolated intact cell
membranes, solubilized cytoplasmic membranes, or a preparation of
an enzyme that uses NADH or NADPH as a cofactor. Most conveniently,
the biological catalyst comprises substantially pure or mixed
cultures of cells, or an enzyme preparation. Preferably, the
biological catalyst is capable of promoting the reduction of an
oxidized substrate to a commercially or industrially important
product, such as succinate, methane, or alcohols.
[0052] When whole cells are used as the biocatalyst, electrically
reduced neutral red promotes cell growth or formation of a reduced
product by chemical reduction of an NAD.sup.+ or NADP.sup.+
cofactor, or by serving as an electronophore. Preferably, the
bioreactor system is one in which the electrically reduced neutral
red promotes cell growth, ATP synthesis, or formation of a reduced
product by chemical reduction of an NAD.sup.+ or NADP.sup.+
cofactor or by functioning as an electronophore.
[0053] In the examples below, electrically reduced neutral red is
shown to promote the reduction of fumarate to form succinic acid in
fermentation reactions using Actinobacillus succinogenes in a
bioreactor system. Because succinic acid is an important
fermentation product having many industrial uses, there is interest
in developing a more efficient fermentation process with enhanced
succinic acid yields.
[0054] It was discovered that including electrically reduced
neutral red during growth of A. succinogenes on glucose medium in a
bioreactor system promotes fumarate reduction by chemically
reducing NAD.sup.+. Furthermore, neutral red promotes succinic acid
production through its function as an electron mediator and
electronophore. The electrical reduction of neutral red
(E.sub.0'=-0.325 volt) is chemically linked to NAD.sup.+ reduction,
and it is biochemically linked to generation of a proton motive
force and succinate production. Neutral red appears to function by
replacing menaquinone (E.sub.0'=-0.073 volt) in the membrane bound
fumarate reductase complex. Preferably, the reduced neutral red is
able to increase cell growth by at least 10%, 20%, or even as much
as 40% or more, relative to a comparable bioreactor system lacking
neutral red. Electrically reduced neutral red is able to increase
glucose or fumarate consumption by at least 25%, 50%, or 100% or
more. Succinate production is increased by about 10% or even as
much as 25% or more, relative to the production levels observed in
a comparable bioreactor system lacking neutral red.
[0055] Similarly, electrically reduced neutral red is able to
substitute for H.sub.2 in promoting the growth of methanogenic
bacteria and the reduction of CO.sub.2 to methane by methanogenic
archea. Preferably, the method of the invention increases growth of
archea or methane production by at least about 25%, 50%, 100% or
even as much as 300% or more.
[0056] It is reasonable to expect that the method of the present
invention may be used with a wide range of biocatalysts to promote
cell growth or the formation of reduced products in electrochemical
bioreactor systems. It is envisioned that the method can be used
with a variety of bacteria, archea, plant cells or animal
cells.
[0057] It is expected that enzyme preparations may also be used in
the practice of the invention. A desired enzyme may be partially
purified using standard methods known to one of ordinary skill in
the art. The enzyme may be isolated from its native source or from
a transgenic expression host, or obtained through a commercial
vendor.
[0058] Useful enzymes include any enzyme that can use reducing
power from electrically reduced neutral red to form a desired
reduced product, or which can transfer reducing power to neutral
red and form a desired oxidized product. Most commonly, this
reduction is mediated by NADPH or NADH. It is reasonably expected
that any oxidoreductase may be used in the practice of the
invention. For example, isolated alcohol dehydrogenases could be
used in a bioreactor system comprising electrically reduced neutral
red, NADP.sup.+ or NAD.sup.+, and a ketone, aldehyde or carboxylic
acid that can serve as a substrate for the enzyme to form a more
reduced end product such as an alcohol. Another example of a useful
enzyme is carboxylic acid reductase, which uses NADPH and ATP to
convert a carboxylic acid to reduced products (U.S. Ser. No.
5,795,759, herein incorporated by reference). One skilled in the
art would appreciate that most enzyme-catalyzed reactions are
reversible, and that there may be applications in which one would
wish to use an oxidoreductase to obtain a desired oxidized
substrate by the method of the present invention.
[0059] In the electrochemical bioreactor used in the present
invention, the biocatalyst and neutral red are preferably
immobilized on the cathode. In the case of whole cell biocatalysts,
self-immobilization on a fine woven graphite felt electrode was
found to take place. Immobilization of the biocatalyst may be
achieved using any suitable method. Numerous techniques for
immobilizing biocatalysts are known to the art (for example, see
Woodward and Spokane, Analytical Enzymes: Biosensors in Industrial
Enzymology, 2d Edition, p. 51-59, incorporated by reference
herein). One wishing to immobilize a biocatalyst in the practice of
the present invention could do so placing the biocatalyst, neutral
red, and pyridine nucleotide cofactor between an electrode and an
outer membrane (e.g., a polymer membrane) such that the
biocatalyst, cofactor, and neutral red are sandwiched between the
electrode and membrane. Alternatively, biocatalyst, neutral red,
and pyridine nucleotide cofactor could be embedded in a matrix
polymer and coated onto the electrode.
[0060] One of ordinary skill in the art wishing to practice the
present invention could readily prepare an electrochemical
bioreactor or fuel cell using the teachings disclosed herein. It
should be appreciated that certain modifications to the disclosed
bioreactors and fuel cells are well within the ability of one
skilled in the art.
[0061] Catholytes and anolytes that may be used in electrochemical
bioreactors or in fuel cells are provided in the examples.
Catholytes that have been found to be suitable in electrochemical
bioreactors include bacterial growth media or a phosphate buffer
(50-100 mM, pH 7.0-7.2). Other suitable catholyte buffers for used
in an electrochemical bioreactor include any catholyte that is
non-denaturing to cells or enzymes.
[0062] A phosphate buffer comprising saline has been found to be
suitable for use in an electrochemical bioreactor (100 mM sodium
phosphate (pH 6.0) and 100 mM NaCl. A suitable anolyte may include
any anolyte that is nondenaturing to cells or enzymes.
[0063] For a fuel cell, neutral red (100 .mu.M) and a bacterial
cell suspension in 50 mM phosphate buffer (pH 7.0) was found to be
a suitable anolyte, with 100 mM phosphate buffer (pH 7.0) and 50 mM
ferricyanide as the catholyte.
[0064] In both the electrochemical bioreactor systems and the fuel
cell system described in the examples, the cathodic and anodic
compartments were separated by a Nafion cationic selective membrane
septum that allows the passage of protons and cations only. A
suitable membrane for separating the cathodic and anodic
compartments can be any membrane that allows transfer of only
protons or cations across the membrane.
[0065] In the electrochemical bioreactor systems described in the
examples below, the electrodes were made from fine woven graphite
felt. The woven graphite felt offers the advantage of providing a
large surface area electrode that permits immobilization of the
biocatalyst over a large area. However, other materials may be
suitable for electrodes, including conductive polymers and metallic
materials.
[0066] The electrodes were connected to a power source or to a
multimeter using a platinum wire. Other materials suitable for
connecting the electrodes to the power source or multimeter include
conducting poolymers or metallic materials.
[0067] In the electrical bioreactors described below, the current
between the anode and cathode was between about 0.4 and about 2.0
mA, with the voltage being about 1.5 V. It is envisioned the
present invention could be practiced using currents of from about
0.004 to about 200 mA.
[0068] In the fuel cell system, the resistance from the anode and
cathode was about 1,000 ohms. It is envisioned that resistances of
from about 10 to about 10,000 ohms could be used in the practice of
the invention.
[0069] Neutral red was included in the catholyte of electrochemical
bioreactors and in the anolyte of fuel cell systems at a
concentration of about 100 .mu.M. It is expected that neutral red
concentrations of between about 1 and 1000 .mu.M would be suitable
in the practice of the invention.
[0070] Neutral red can also be used as an electron mediator in the
conversion of energy derived from the metabolism of growing or
resting bacterial cells to electricity.
[0071] Using Actinobacillus succinogenes 130Z growing cells in a
fuel cell system that had neutral red as the electron mediator and
ferricyanide as the electron acceptor, the maximum current produced
using was 2.17 mA, and the potential was <100 mV in a closed
circuit configuration. After 20 hour cultivation, the fuel cell
system was converted from a closed to an open circuit system. The
potential rapidly reached the theoretical maximum value of 0.685
volt (i.e. the redox potential difference between NR).
[0072] A comparison of the efficacy of NR and thionin as electron
mediators made using A. succinogenes resting cells as the catalyst
revealed that much more electricity was produced with NR than with
thionin as the electron mediator. When NADH, NR, and ferricyanide
were used as the electron donor, electron mediator, and electron
acceptor, respectively, the current produced was proportional to
NADH concentration. In a system that employed E. coli K-12 as the
catalyst, the currents and voltages produced were similar to those
obtained using A. succinogenes as the catalyst. The current and
voltage were found to increase with increasing glucose
concentrations.
[0073] Anaerobic sewage sludge was also used as the catalyst in a
fuel cell system. The voltage and current produced in fuel cells
using sewage sludge as the catalyst were comparable to those
produced using E. coli and Actinobacillus, and they were stable for
120 hours in a closed circuit system with a 2.2 K ohms external
resistance.
[0074] It is expected that growing or resting cells of types other
than those described in the examples can be used as catalysts in a
fuel cell system to generate electricity by the method of the
present invention. Depending on the particular cell chosen as a
biocatalyst, reducing power used in the generation of electricity
may include light, inorganic compounds, or organic compounds, or
any other energy source that cells are able to use for growth or
metabolism.
[0075] It is envisioned that the neutral red-mediated
interconversion of biochemical and electrical energy may be adapted
for use in a number of different applications. For example, neutral
red oxidoreduction can be used to detect electrical levels in
biosensor systems using whole cells or enzymes.
[0076] Accordingly, the invention includes a method for detecting
the presence of a specific organic or inorganic test compound in a
sample comprising the steps of (a) providing biosensor comprising
an electrochemical fuel cell system having an anode compartment and
a cathode compartment separated by a cation-selective membrane,
wherein each compartment is equipped with an electrode, wherein the
electrodes are connected by a wire to a multimeter; (b) placing an
anolyte in the anode compartment, the anolyte comprising the
sample, a suitable concentration of neutral red, and a biological
catalyst comprising microbial cells and an enzyme, wherein the
biological catalyst is able to oxidize the test compound; (c)
placing a suitable catholyte in the cathode compartment; and (d)
allowing oxidation of at least a portion of any test compound
present in the sample and reduction of at least a portion of
oxidized neutral red; (e) allowing the transfer of electrons from
reduced neutral red to the cathode; (f) detecting the generation of
an electrical current.
[0077] In cell or enzyme biosensors known to the art, the presence
of a chemical (e.g., glucose) is detected using an enzyme (glcuose
oxidase) in a membrane-based electrode system. In the example of
glucose and glucose oxidase, the enzyme-catalyzed reaction consumes
O.sub.2 and produces peroxide. Therefore, glucose present in the
sample is correlated with a decrease in O.sub.2 concentration and
an increase in peroxide concentration, either one of which be
detected by a specific electrode. By the method of the present
invention, electrical current generated can be measured directly.
In the neutral red system, a specific compound in an unknown test
sample is tested using cells or enzymes that are capable of
oxidizing the compound to generate a detectable current upon
oxidation of the compound by the biocatalyst. Therefore, the
concentration of the compound can be determined by measuring the
electricity generated upon oxidation of the test compound. It is
well within the ability of one skilled in the art wishing to detect
a particular compound to adapt the method of the present invention
to detect the compound by selecting a suitable biocatalyst capable
of oxidizing the compound.
[0078] Another important application using neutral red provides a
method for measuring the chemical oxygen demand in waste water
comprising (a) providing an electrochemical fuel cell system
comprising an anode compartment and a cathode compartment separated
by a cation-selective membrane, wherein each compartment is
equipped with an electrode, wherein the electrodes are connected by
a wire to a multimeter; (b) placing an anolyte in the anode
compartment, the anolyte comprising a suitable concentration of
neutral red and waste water comprising or supplemented with a
biological catalyst; (c) placing a suitable catholyte in the
cathode compartment; (d) allowing the neutral red-mediated
conversion of chemical reducing power to electricity; (e) measuring
the electrical current generated by the fuel cell system.
[0079] The following nonlimiting examples are intended to be purely
illustrative.
EXAMPLES
Example 1
[0080] Electrically Reduced Neutral Red Promotes the Reduction of
Fumerate to Succinic Acid
[0081] Chemicals and Reproducibility of Results
[0082] All chemicals were reagent grade and gases were purchased
from AGA Chemicals (Cleveland, Ohio, USA). All individual
experiments were repeated two to three times with identical
results.
[0083] Electrochemical Bioreactor Systems
[0084] The ECB system I (40 ml working volume) was used for
enzymatic and chemical reduction tests and ECB system II (300 ml
working volume) was used for electrical-dependent cultivation of
cells. The ECB systems, specially designed for maintaining
anaerobic conditions and for growing bacteria, were made from Pyrex
glass by the MSU Chemistry Department, East Lansing, Mich., USA.
The ECB system was separated into anode and cathode compartments by
a cation selective membrane septum (diameter [.phi.]=22 mm for type
I and [.phi.]=64 mm for type II) (Nafion, Electrosynthesis,
Lamcosta, N.Y.); 3.5 .OMEGA.cm.sup.-2 in 0.25 N NaOH). Chemicals
and metabolites cannot be transferred across the Nafion membrane;
only protons or cations transfer. Both the anode and cathode were
made from graphite fine woven felt (6 mm thickness, 0.47
m.sup.2g.sup.-1 available surface area (Electrosynthesis, NY, USA).
A platinum wire ([.phi.] .OMEGA. 0.5 mm, <1.0 .OMEGA.cm.sup.-2;
Sigma, St. Louis, Mo., USA) was attached to the graphite felt using
graphite epoxy (<1.0 .OMEGA.cm.sup.-2, Electrosynthesis, NY,
USA). The electric resistance between anode and cathode was <1
k.OMEGA.. The weight of both electrodes was adjusted to 0.4 g
(surface area, 0.188 m.sup.2) for system I and 3.0 g (surface area,
1.41 m.sup.2) for system II. The current and voltage between anode
and cathode were measured by precision multimeter (Fluke model 45,
Everett, Wash., USA) and adjusted to 0.3-2.0 mA and 1.5 volt for
system I, and 1.0-10.0 mA and 2.0 volt for system II, respectively.
The electrochemical half oxidation of H.sub.2O was coupled to half
reduction of NR (100 .mu.M) and the oxidation of reduced NR was
coupled to bacteriological reduction of fumarate. H.sub.2 was not
produced under the electrochemical conditions used to reduce NR or
MV. For tests in ECB system I, the cathode compartment contained
the cell suspension, membrane suspension or solubilized membranes
and the anode compartment contained 50 mM phosphate buffer (pH 7.2)
and 100 mM NaCl. For growth studies in ECB system II, the cathode
compartment contained the growth medium inoculated with A.
succinogenes and the anode compartment contained 100 mM phosphate
buffer (pH 7.0) and 100 mM NaCl.
[0085] Organism and Growth Conditions
[0086] A. succinogenes type strain 130Z is maintained at MBI
International (Lansing, Mich., USA) (10, 39). Bacteria were grown
in butyl-rubber-stoppered, 158 ml serum vials containing 50 ml
medium with CO.sub.2--N2 (20%-80%, 20 psi) gas phase, unless stated
otherwise. The growth medium A contained the following (per liter
of double distilled water): yeast extract, 5.0 g; NaHCO.sub.3, 10.0
g; NaH.sub.2PO.sub.4.H.sub.2O, 8.5 g; and Na.sub.2HPO.sub.4, 12.5
g. The pH of medium was adjusted to be 7.0 after autoclaving.
Separately autoclaved solutions of glucose (final concentration 60
mM), and fumarate (final concentration 50 mM) were aseptically
added to the medium after autoclaving. Media were inoculated with
5.0% (v/v) samples of cultures grown in the same medium and
incubated at 37.degree. C.
[0087] Preparation of Cell Suspensions
[0088] Bacterial cultivation, harvest and washing were done under
strict anaerobic N.sub.2 atmosphere as described previously (39). A
16 hour A. succinogenes culture was harvested by centrifugation
(5,000.times.g, 30 minutes) at 4.degree. C. and washed three times
using a 1500 ml solution of 50 mM Na phosphate buffer (pH 7.2)
containing 1 mM dithiothreitol (DTT). The washed bacterial cells
were re-suspended in 50 mM sodium phosphate buffer with 2 mM DTT.
This suspension was used as a catalyst for H.sub.2-dependent and
electrical-dependent reduction of fumarate to succinate; and, it
was used for cyclic voltammetry and for NR absorption to cells.
[0089] Electrochemical Reduction of NAD.sup.+ or NADP.sup.+
[0090] ECB system I with 1 mM NAD.sup.+ or NADP.sup.+ and 100
(.OMEGA.M NR or MV was used for electrochemical reduction of
NAD.sup.+ or NADP.sup.+. The electrode potential and current were
adjusted to 2.0 volts and, 1.0-3.0 mA, respectively. Ag/AgCl and
platinum electrodes were used to measure the reactants redox
potential to check if the reaction was progressing. Generally, the
redox potential of a biochemical or electrochemical reaction is
measured using an Ag/AgCl electrode (E.sub.0' of [Ag/Ag.sup.+],
=+0.196 volt) or a Calomel electrode (E.sub.0' of [Hg/Hg.sup.+],
+0.244) as a reference electrode but it has to be expressed as the
potential vs. natural hydrogen electrode (NHE), which is used for
thermodynamical calculation of organic or inorganic compounds
(e.g., E.sub.0' of NADH/NAD.sup.+ is a -0.32 volt and
H.sub.2/2H.sup.+ is -0.42 volt). A potential measured using Ag/AgCl
electrode is converted to potential vs. NHE by adding +0.196 volt
to the measured potential (E.sub.0' vs. NHE=E.sub.0' vs.
Ag/AgCl+0.196). Oxygen was purged from the reactants and from the
redox dye solution in 50 mM Tris-HCl (pH 7.5) by bubbling with
oxygen free nitrogen for 10 minutes before supplying electricity.
The NADH concentration in the reactant was spectrophotometrically
measured at 340 mm and calculated using the millimolar extinction
coefficient 6.23 mM.sup.-1 cm.sup.-1. NADH or NADPH production was
confirmed by absorption spectra data at each sampling time.
[0091] Preparation of Purified Membranes, Solubilized Membranes and
Membrane Free Cell Extract
[0092] Cell free extracts were prepared at 4.degree. C. under an
anaerobic N.sub.2 atmosphere, as described previously (Van der
Werf, et al., 1997, Arch. Microbiol. 167:332-342). The harvested
and washed cells were resuspended in 50 mM phosphate buffer (pH
7.2) containing 1 mM DTT and 0.05 mg/ml deoxyribonuclease. Cells
were disrupted by passing twice through a French Press at 20,000
psi. The cell debris was removed by centrifugation three times at
40,000.times.g for 30 minutes. The purified membranes were obtained
from the cell free extracts by centrifugation at 100,000.times.g
for 90 minutes. The supernatant was decanted and saved as the
membrane-free cell extract. The brown and clear precipitate was
washed twice with 50 mM phosphate buffer (pH 7.2) and re-suspended
in the same buffer by homogenization. Solubilized membranes were
obtained from membrane fraction by Triton X-100 extraction (Lemire,
et al., 1983, J. Bacteriol. 155:391-397). Triton X-100 was added to
a final 1% (v/v) concentration and, the suspension was incubated
for 3 hours. Triton-solubilized protein was recovered after
removing insoluble debris by centrifugation at 100,000.times.g and
4.degree. C. for 90 minutes.
[0093] Neutral Red Binding to Cells and Membranes
[0094] The absorption of redox dyes to cells and purified membranes
was determined by measuring the residual NR and MV in solution
after mixing with cells or membrane suspensions for 30 minutes at
37.degree. C. Bacterial cell suspensions (OD.sub.660 between 0-3.0)
and the purified membrane suspension (0-10 mg/ml protein) were used
to analyze redox dye absorption (i.e., binding). NR solutions (50
.mu.M and 25 .mu.M) and MV (100 .mu.M) were used for measuring dye
binding to intact cells and membranes. MV (100 .mu.M) was used for
cell binding. The cells and membranes were removed from the
reaction mixture by centrifugation at 12,000.times.g for 10 minutes
and by ultracentrifugation at 150,000.times.g for 20 minutes,
respectively. The NR concentration was calculated using a
calibration curve spectrophotometrically pre-determined at 400 nm
and pH 7.2, and MV was determined using the millimolar extinction
coefficient (578) 9.78 mM.sup.-1 cm.sup.-1 after reduction by
addition of Elepsiden 1.5 mM dithionite at pH 7.2 (Lissolo, et al.,
1984, J. Biol. Chem. 259:11725-11729). The protein concentration of
membrane suspensions was determined by a calibration curve (protein
concentration, mg/ml=A.sub.595.times.1.3327) using Bradford Reagent
(Bio-Rad, Hercules, Calif., USA).
[0095] Measurement of Proton Translocation
[0096] Proton translocation was measured under an anoxic N.sub.2
atmosphere. H.sub.2-dependent proton translocation by cell
suspensions was measured as described by Fitz and Cypionka (Fitz,
et al., 1989, Arch. Microbiol. 152:369-376). Electrical-dependent
proton translocation was measured in an electrochemical bioreactor
system designed for measurement of proton translocation. The tube
([.phi.] 10 mm ID and 90 mm length) with a Vycor tip (ion
exchangeable hard membrane, Bas, West Lafayette, Ind., USA) was
used as an anode compartment and a graphite rod ([.phi.] 7
mm.times.70 mm) was used as an anode, and 0.05 g graphite felt
(surface area, 0.0235 m.sup.2) was used as a cathode. The pH
electrode (Orion 8103 ROSS) was placed in the cathode compartment
and was connected to a recorder (Linear) via a pH meter (Corning,
130) that converted the proton pulse into a recordable signal. Cell
suspensions were made in KKG solution (pH 7.1) which contains 100
mM KSCN, 150 mM KCl and 1.5 mM glycylglycin and placed in the
cathode. The anode contained a 50 mM phosphate buffer with 50 mM
KCl as an anolyte. The total volume and working volume of the
cathode and anode compartments were 30 ml and 5.5 ml, respectively.
The working potential and current between anode and cathode were
2.0 volt and 0.3-0.35 mA for experiments using electrical reducing
power and NR. Bacterial cells were cultivated for 16 hours in
medium A with fumarate-H.sub.2 or glucose. The cells were
anaerobically harvested by centrifugation at 5,000.times.g and
20.degree. C. for 30 minutes and washed twice with 100 mM KCl. The
cells were modified with 100 .mu.M NR to measure
electrical-dependent proton translocation and washed again with 100
mM KCl. The washed bacteria (OD.sub.660, 10) were re-suspended in
N.sub.2-saturated 150 mM KCl. Cell suspensions were allowed to
equilibrate for 30 minutes at room temperature. The incubated cells
were centrifuged at 5,000.times.g and 20.degree. C. for 30 minutes
and re-suspended in KKG solution and then the incubation was
continued for 30 minutes under H.sub.2 atmosphere before the
measurement of proton translocation. To measure
electrical-dependent proton translocation upon fumarate addition,
the cell suspension was incubated in the presence or absence of
HOQNO in the cathode compartment under N.sub.2 atmosphere and
charged with 2.0 volt electrode potential for 20 minutes.
[0097] Enzyme Assays
[0098] Enzyme activity measurements were performed under an
anaerobic N.sub.2 atmosphere, as described previously (Van der
Werf, et al., 1997, Arch. Microbiol. 167:332-342). The
membrane-free extract, purified membrane and solubilized membrane
preparations described above were used to assay hydrogenase,
diaphorase, and fumarate reductase activities. Fumarate reductase
(EC 1.3.) and hydrogenase (EC 2.12.2.2.) activities were measured
as described by van der Werf (1997, Arch. Microbiol. 167:332-342),
with a Beckman spectrophotometer (Model, DU-650). Diaphorase
activity with BV2.sup.+ and NR.sup.+ was measured under analogous
conditions with hydrogenase using NADH (0.6 mM) instead of H.sub.2
as electron donor (Schneider, et al., 1984, Eur. J. Biochem.
142:75-84). The oxidation and reduction of benzyl viologen and NR
were spectrophotometrically measured at 578 nm and 540 nm, and the
oxidation and reduction of NAD(H) were spectrophotometrically
measured at 340 nm. Reduced benzyl viologen was prepared as
described previously (Lissolo, et al., 1984, J. Biol. Chem.
259:11725-11729). The millimolar extinction coefficient of benzyl
viologen (578), NR (540) and NAD(H) (340) were 8.65 mM.sup.-1
cm.sup.-1, 7.12 mM.sup.-1 cm.sup.-1, and 6.23 mM.sup.-1 cm.sup.-1,
respectively.
[0099] Enzymatic Analysis of Fumarate Reduction Membranes and
Solubilized Membrane
[0100] Membrane suspensions (3.25 mg/ml protein) and solubilized
membranes (3.2 mg/ml protein) were used as the enzyme sources.
Serum vials (50 ml) and ECB system I was used for H.sub.2-dependent
and electrical dependent reduction of fumarate to succinate,
respectively. Anaerobically prepared 50 mM fumarate in 50 mM
phosphate buffer (pH 7.2) was used as reactant and catholyte, and
100 mM phosphate buffer with 100 mM NaCl (pH 7.0) was used as
anolyte. The reaction was started by the addition of enzyme sources
and it was maintained at 37.degree. C. Substrate and product
concentrations were analyzed by HPLC (Guerrant, et al., 1982, J.
Clin. Microbiol. 16:355-360). The influence of HOQNO on fumarate
reduction in cell suspensions and membranes were analyzed as
follows.
[0101] Cell suspensions (OD.sub.660=4.2) and membrane suspension
(2.65 mg/ml protein) were used as the enzyme sources. Serum vials
(50 ml) and ECB system I was used for H.sub.2-dependent and
electrical dependent reduction of fumarate to succinate,
respectively. Anaerobically prepared 50 mM fumarate in 50 mM
phosphate buffer (pH 7.2) was used as reactant and catholyte and
100 mM phosphate buffer with 100 mM NaCl (pH 7.0) was used as
analyte. 2 .mu.M HOQNO was used as an inhibitor for menaquinone.
The reaction was started by the addition of enzyme sources and it
was maintained at 37.degree. C. Substrate and product concentration
was analyzed by HPLC.
[0102] Cyclic Voltammetry
[0103] A 3 mm diameter glassy carbon working electrode (BAS, West
Lafayette, Ind., USA), platinum wire counter electrode (BAS), and
an Ag/AgCl reference electrode (BAS) were used in an
electrochemical cell with a working volume of 2 ml. Cyclic
voltammetry was performed using a cyclic voltametric potentiostat
(BAS, model CV50W) linked to an IBM microcomputer data acquisition
system. Prior to use, the working electrode was polished with an
alumina/water slurry on cotton wool, and the electrochemical cell
was thoroughly washed. Oxygen was purged from the cell suspension,
membrane suspension, or solubilized membrane solution by bubbling
with oxygen free N.sub.2 for 10 minutes before electrochemical
measurements. Bacterial suspensions (OD.sub.660=3.0), membrane
suspensions (2.54 mg protein/ml), and solubilized membranes (3.2 mg
protein/ml) were used as enzyme sources. The scan rate used was 25
mV/s over the range -0.3 to -0.8 volt 50 mM phosphate buffer
containing 5 mM NaCl was used as electrolyte. NR .mu.100 (M) and 50
mM fumarate was used as the electron mediator and the electron
acceptor, respectively.
[0104] Growth Analysis
[0105] Growth of cells suspended in the medium was determined by
measuring the suspensions (optical density at 660 nm), the growth
yield of cells absorbed onto the electrode was determined by
measuring protein concentration. The protein concentration was
converted to optical density using a predetermined calibration
curve (bacterial density=protein concentration,
mg/ml.times.1.7556). The cathode, on which the bacteria absorbed,
was washed three times, by slow agitation, in 300 ml of phosphate
buffer (50 mM, pH 7.0) for 30 minutes. The bacterial lysate was
obtained from electrodes by alkaline treatment at 100.degree. C.
for 10 minutes using 1N--NaOH. After removing cell debris from the
lysate by centrifugation at 10,000.times.g and 4.degree. C. for 30
minutes, the protein concentration of the bacterial lysate was
determined using Bradford Reagent (Bio-Rad, Hercules, Calif., USA),
and a predetermined calibration curve (protein concentration,
mg/ml=A.sub.595.times.1.3327).
[0106] Methanogenic Granules Growth and Metabolic Analysis
[0107] Methanogenic granules containing mixed cultures of fatty
acid-degrading syntropliles and methanogens were obtained from a
bench scale anaerobic sludge reactor fed on a mixture of 50 mM
acetate, butyrate, and propionate in MBI International (Lansing,
Mich.) (Wu, et al., 1993, Arch. Microbiol. 39:795-803 and Wu, et
al., 1993, Appl. Mirobiol. Biotechnol. 39:804-811). Methanogenic
granules were cultivated in PBBM prepared without organic compounds
(Kenealy, et al., 1981, J. Bacteriol. 146:133-140). The medium was
prepared without phosphate, brought to pH 7.2 with NaOH, boiled,
sparged with N.sub.2--CO.sub.2 (80:20%) or H.sub.2--CO.sub.2
(80:20%), dispensed into 158-ml Wheaton serum vials, sealed with
butyl rubber stoppers, and autoclaved. Phosphate, sulfide (0.01%),
N.sub.2--CO.sub.2 (80:20%) or H.sub.2--CO.sub.2 (80:20%), and
vitamin solution were added after autoclaving. The medium volume
was 40 ml, and the initial head space gas pressure in serum vials
was adjusted to 30 psi. Media were inoculated with 3.0% (by volume;
protein concentration, 1.995 mg/ml) methanogenic granules and
incubated at 37.degree. C. All procedures for medium preparation,
inoculation, and cultivation were the same as those used for vial
cultures except that Na.sub.2S was not added because the medium was
electrically reduced. Na.sub.2S (2%) was added to the anode
compartment as reducing agent to remove the O.sub.2 generated. NR
(100 (M) was added to the cathode compartment as electron mediator.
The current and potential between anode and cathode were 0.4 mA and
2.0 volts. CO.sub.2 and CH.sub.4 were analyzed using a gas
chromatograph equipped with a carbosphere column and flame ionized
detector. The injector and column temperatures were 50.degree. C.
and 150.degree. C., respectively, and the carrier (N.sub.2) flow
rate was 45 ml/min. Gas samples were removed with a pressure lock
syringe. CO.sub.2 consumption and CH.sub.4 production are shown as
the percentage of total gas composition in the headspace.
[0108] Bacterial Growth and Cell Preparation for Generating
Electricity
[0109] A. succinogenes 130Z and E. coli K-12 were anaerobically
grown for 16 hours and 20 hours, respectively, in medium A (10 g/L
glucose, 5 g/L yeast extract, 8.5 g/L NaH.sub.2PO.sub.4, and 10 g/L
NaHCO.sub.3) under an anaerobic N.sub.2--CO.sub.2 (80:20)
atmosphere at 37.degree. C. in 150 ml serum vials or under a
N.sub.2 (100%) atmosphere in fuel cell system with a pH controller.
The inoculum size was 3% (v/v) for both vial and fuel cell
experiments. Resting cell suspensions were prepared by harvesting
stationary phase cultures at 4.degree. C. by centrifugation at
5,000.times.g . The cells were washed twice using 50 mM phosphate
buffer (pH 7.0) under a 100% N.sub.2 atmosphere. The washed cells
were resuspended in 50 mM phosphate buffer (pH 7.0), then dissolved
O.sub.2 was removed by gassing with N.sub.2 for 30 minutes. The
cell density was adjusted to OD.sub.660 3.0.
[0110] Fuel Cell Systems for Growing or Resting Cells
[0111] A two-compartment (anode and cathode) electrochemical cell
was used as a fuel cell system for microbial electricity production
(FIG. 1). When switches one and two are off, there is an open
circuit. When switch one is on and switch two is off, a closed
circuit is formed. When switch one is off and switch two is on, a
closed circuit with external variable resistance is formed. One
hundred .mu.M NR or 300 .mu.M thionin were used as the electron
mediator. The total and working volumes of each compartment were
1,600 ml and 1,300 ml, respectively. The electrodes, each made of
12 g fine woven graphite felt (0.47 m.sup.2/g, Electrosynthesis,
NY) were connected to a precision multimeter (Fluke model 45,
Everett, Wash.) with a platinum wire ([.phi.]=0.5 mm, <1.0
.OMEGA.cm.sup.-2; Sigma, St. Louis, Mo., USA) using graphite epoxy
(<1.0 .OMEGA.cm.sup.-2, Electrosynthesis, NY). Anode and cathode
compartments were separated by a cation-selective membrane septum
([.phi.] 70 mm, Nafion, Electrosynthesis, NY). The self-electric
resistance of the fuel cell system between the anode and cathode
was approximately 1,000.OMEGA.. The resistance was adjusted using
variable resistance for controlling current production, but it was
not adjusted for measuring maximum potential or current production.
The current and voltage between the anode and cathode were measured
by a precision multimeter (Fluke model 45, Everett, Wash.). The
electrochemical half-reduction of ferric ion (as potassium
ferricyanide, E.sub.0(=+0.36 volt)--which was re-oxidized by
O.sub.2 (E.sub.0'=+0.82 volt) was coupled to neutral red or thionin
half-oxidation which was, in turn, reductively coupled to bacterial
oxidative metabolism. In the fuel cell system using resting cells,
the bacterial cell suspension (OD.sub.660, 3.0) in 50 mM phosphate
buffer (pH 7.2) containing 100 .mu.M NR or 300 .mu.M thionin, and
100 mM phosphate buffer (pH 7.0) containing 50 mM ferricyanide were
used as the anolyte and catholyte, respectively. In the fuel cell
system using growing cells, medium A containing a fresh bacterial
inoculum was the anolyte; the catholyte was the same as for resting
cells. During experiments, complete anoxygenic conditions were
maintained in the anode compartment by gassing with 100% N.sub.2
for 30 minutes before operation at N.sub.2 flow rates of 0.8
ml/min. The trace oxygen contained in the N.sub.2 gas was removed
in a furnace filled with pure copper fillings at 37.degree. C. The
cathode compartment was oxygenated by constant air bubbling and
stirring. The anode compartment was maintained at pH 7.0 using an
automatic pH controller (New Brunswick Scientific Co., model pH-40,
Edison, N.J.).
[0112] Current Production by Chemical Dye Chemical Oxidation
Coupled to NADH Oxidation
[0113] A small chemical fuel cell system (total volume 50 ml;
working volume 30 ml) consisting of an anode and cathode
compartments equipped with 0.3 g fine woven graphite felt
electrodes and a cation-selective membrane septum (.OMEGA. 20 mm,
Nafion, Electrosynthesis) was used. A 100 .mu.M NR solution in 50
mM phosphate buffer (pH 7.0) and 100 mM phosphate buffer (pH 7.0)
containing 50 mM ferricyanide were used as the anolyte and
catholyte, respectively. Oxygen was completely removed from the
anode compartment by N.sub.2 gassing for 30 minutes before adding
NADH. The concentrated NADH solution in 50 mM phosphate buffer (pH
7.0) was previously gassed with N.sub.2 to remove O.sub.2.
[0114] Cyclic Voltametry
[0115] A 3 mm-diameter glassy carbon working electrode, a platinum
wire counter electrode, and an Ag/AgCl reference electrode (all
from BAS, West Lafayette, Ind.) were used in an electrochemical
cell with a 3 ml working volume. Cyclic voltametry was performed
using a cyclic voltametric potentiostat (model CV50W, BAS) linked
to an IBM personal computer data acquisition system. Prior to use,
the working electrode was polished with an aluminum/water slurry on
cotton wool, and the electrochemical cell was thoroughly washed.
Oxygen was purged from the reactant by bubbling with oxygen-free
N.sub.2 for 10 minutes before electrochemical measurement. The
scanning rate used was 25 mV/s over the range -0.3 to -0.8 volt. A
50 mM phosphate buffer containing 5 mM NaCl was used as the
electrolyte. One hundred .mu.M NR and 100 .mu.M NAD were used as
the electron mediator and acceptor, respectively.
[0116] Generation of Electricity Using Anaerobic Sludge
[0117] The anaerobic sludge was obtained from the East Lansing
sewage treatment plant (MI, USA). The fresh anaerobic sludge was
settled under a N.sub.2 atmosphere for one day to remove solid
particles. The supernatant (1,200 ml) was used as biocatalyst and
anolyte for the fuel cell system, to which 3 g/L glucose was added
as energy source. The catholyte was 100 mM phosphate buffer (pH
7.0) containing 50 mM ferricyanide.
[0118] Results
[0119] Electricity Generation by Fuel Cells
[0120] The E.sub.0' values of the electron mediators used for
converting the reducing power generated by microbial metabolic
oxidation to electricity are important determinants of the maximum
electricity amount that can be generated in microbial fuel cells.
Chemical properties of artificial electron mediators (i.e., NR and
thionin) with those of natural electron mediators (i.e., NAD.sup.+
and menaquinone) are shown in Table 1. The electron driving force
generated from using NR is far greater than from thionin when the
redox dye is coupled to an oxidant (i.e., ferricyanide) in a
chemical or microbiological fuel cell. This difference is due to
differences between the E.sub.0' values for NR and thionin.
Consequently, the .DELTA. E.sub.0 generated from NR or thionin
oxidation coupled to ferricyanide reduction is 0.645 volt (NR) and
0.296 volt (thionin). These .DELTA. E.sub.0 values are the
theoretical maximum potentials produced in fuel cells using these
electron mediators.
[0121] Results of experiments performed demonstrate the superiority
of NR over thionin as an electron mediator and that reduced NR is
able to donate electrons to the electrode for electricity
production in a microbial fuel cell. FIG. 2 shows that the use of
NR as an electron mediator in a chemical fuel cell generates higher
current than that obtained using thionin, and that the current
produced depends on the NADH concentration used. Arrows indicate
the addition of 1 (circles) or 3.5 (squares) mM NADH. At low NADH
concentrations the current was quite low. Although thionin
reduction was faster than NR reduction when using NADH as the
reductant, the mediator oxidation rate at the electrode is
rate-limiting, because more current was produced with NR as the
electron mediator.
1TABLE 1 Redox mediators, their structural formula, redox
potentials (E.sub.o'), and maximum absorbance wavelength
(.lambda..sub.max). Structural formula Redox mediator E.sub.o' (V)
.lambda..sub.max 1 Neutral Red -0.325 540 2 Thionine +0.064 598 3
Menaquinone -0.074 260/280 4 NAD.sup.+ -0.32 340
[0122] Cyclic voltammograms of a NR solution in the presence or
absence of NAD.sup.+ show that NR oxidation (upper) and reduction
(lower) peaks did not shift during twenty scanning cycles in the
absence of NAD.sup.+ (FIG. 3A). Both peaks increased upon NAD.sup.+
addition (FIG. 3B). NAD.sup.+ enables more electrons to pass
unidirectionally from the electrode to NR to NAD and from NADH to
the electrode via NR.
[0123] FIG. 4 compares the currents and potentials generated from
glucose by E. coli resting cells in a glucose (10 g/L) fuel cell
with either 100 .mu.M NR (circles) or 300 .mu.M thionin (squares)
in closed circuit (current) (A) and open circuit (potential) (B)
configurations. Arrows mark (1) the addition of the electron
mediator; and (2) conversion to open circuit. Under the anaerobic
conditions used, higher current and potential levels were produced
with NR than with thionin as the electronophore. In control
experiments under aerobic conditions, significant levels of current
or potential were not detectable because NR and thionin cannot
oxidize NADH through the electron transport system since O.sub.2 is
a much better electron acceptor (i.e., it has a much more positive
E.sub.0' value than the two electron mediators). Under anaerobic
conditions, E. coli normally couples NADH oxidation with reduction
of either fumarate to succinate, acetyl CoA to ethanol, or pyruvate
to lactate. These reactions are inhibited in the presence of NR in
the fuel cell, and electricity is produced in lieu of these normal
reduced metabolic end products.
[0124] Previous investigations (Allen, et al., 1993, Appl. Biochem.
Biotechnol. 39-40:27-40 and Thurston, et al., 1985, J. Gen.
Microbiol. 131:1393-1401) have shown in microbial fuel cells using
thionin as the electron mediator, that both current and potential
drop when the resting cells are depleted of glucose. We performed
experiments to determine what maximal electrical productivities and
stabilities can be generated by resting E. coli cells from
different glucose concentrations in a fuel cell with NR as the
electronophore. Table 2 shows the effect of glucose concentration
on the maximal electrical productivities and stabilities in an open
circuit versus a closed circuit, with and without a 120 ohm
external resistance. The maximal current, potential, and electrical
energy produced by the fuel cell were proportional to the glucose
(i.e., fuel) concentration. The maximum current and coulombic
yields obtained from glucose using NR as the electronophore far
exceeded those obtained with thionin in other investigations
(Dealney, et al., 1984, Chem. Tech. Biotechnol. 34B: 13-27).
[0125] Previous studies (Roller, et al., 1984, J. Chem. Tech.
Biotechnol. 34B:3-12 and Bennetto, et al., 1985, Biotechnol. Lett.
7:699-105) on microbial fuel cells with thionin as the electron
mediator were only performed with resting cell suspensions (i.e.,
cells harvested after growth had ended). Using NR (100 uM) as the
electronophore, we compared the electrical productivities (i.e.,
current and potential) of A. succinogenes growing cells (FIG. 5A)
and resting cells (FIG. 5B) in a glucose (10 g/L) microbial fuel
cell under anaerobic conditions with(FIG. 5). Control experiments
(FIG. 5A) showed that the growth yield and rate (squares) were much
higher in the absence of NR when no electricity was generated
(triangles) than in the presence of NR. The electric current (open
circles) and potential (closed circles) generated increased with
cell growth. The potentials generated by growing and resting cells
were similar, whereas the current produced by resting cells was
significantly higher (about 2-fold) than that produced by growing
cells. The specific current produced per mg cell protein per hour
was calculated at 10 hours for growing cells (1.235 mA/mg
protein/hr) and at 2 hours for resting cells (2.595 mA/mg
protein/hr) when the glucose levels were high. A total of 68
coulombs was produced by growing cells at 20 hours (after glucose
was depleted); whereas the resting cells had produced 90 coulombs
at 4 hours.
2TABLE 2 Effect of initial glucose concentration on electrical
productivity and stability of a microbial fuel cell using E. coli
resting cells and NR as the electronophore. Closed Circuit with a
120.OMEGA. Resistance Open Circuit Closed Circuit Electrical
Electrical Glucose Potential Current Potential Current Potential
Current Coulomb Energy Stability (mM) (volt) (mA) (volt) (mA)
(volt) (mA) (amp/sec) (J) (hr) 11.1 0.58 0.0 0.02 1.2 0.46 0.5 57.6
26.5 32 55.5 0.65 0.0 0.04 5.6 0.57 3.6 1049.76 598.4 81 111 0.85
0.0 0.05 17.7 0.62 4.8 2039.04 1264.2 118
[0126] Similar experiments were performed using anaerobically grown
E. coli cells in the presence or absence of electrical generation.
Electrical generation dramatically decreases growth yield, ATP
yield and metabolite production (Table 3). Table 4 compares
substrate consumption, growth and electricity production by
exponential versus stationary phase E. coli cells. These data
indicate that significantly more electricity is produced by
stationary phase cells than by exponential phase cells. This result
was expected because significant reducing power is required for
cell growth that cannot be directed to electricity generation.
3TABLE 3 Comparison of anaerobic metabolism of E. coli during
anaerobic growth in the presence or absence of electrical
generation.sup.a. Glucose Cell Ysub Theoretical ATP Electricity
Growth Consumption Mass (g cell/mol Products yield (mol/mol Energy
(J/mol Condition (mM) (g/L) substrate) (mM) sub) sub) Without 60.6
3.07 50.12 11.93 7.07 -- Electricity Generation With 66.3 1.4088
22.082 8.88 2.57 1320.0 Electricity Generation .sup.aData was
determined after 20 hours of growth in medium A with 100 .mu.M
neutral red in a standard fuel cell.
[0127]
4TABLE 4 Comparison of substrate consumption and electricity
production by anaerobic E. coli exponential phase versus stationary
phase cells in a fuel cell using neutral red as
electronophore.sup.a. Exponential Phase Cells Stationary Phase
Cells Electricity Electricity Glucose Energy Glucose Energy
Consumption Cell Mass (J/mol sub) Consumption Cell Mass (J/mol sub)
45.1 mM 1.74 g/L 100.8 15.5. mM 0.214 g/L 1207.7 (7.52 mM/hr) (0.29
g/L/hr) (2.59 mM/hr) (0.035 g/L/hr) .sup.aData for exponential
phase cells is from 0-6 hours after inoculation. Data for
stationary phase cells is 12-18 hours after inoculation.
Conditions: medium A with 100 .mu.M neutral red in the standard
fuel cell system.
[0128] Experiments were initiated using anaerobic sludge to test
its potential as a catalyst for electricity generation in a fuel
cell with NR as the electronophore. FIG. 6 shows the effect of
glucose addition on the current and potential generated by the
sewage sludge, as well as the maximum current produced in a closed
circuit configuration versus the maximum potential produced in an
open circuit configuration. The numbered arrows connote the
conversion from open to closed circuit with a 2.2 kohms resistance
(1); addition of 3 g/L glucose (2); conversion from closed to open
circuit (3); and the conversion from open to closed circuit without
external resistance (4). The electrical productivity of the glucose
fuel cell using sewage sludge as the catalyst was calculated to be
a total of 370.8 C (G of 162.82 J).
[0129] We have shown here that NR serves as a superior
electronophore or electron mediator than thionin in microbial fuel
cells using glucose as fuel. Furthermore, we have shown that
resting cells generate more electricity than growing cells, and
that mixed cultures such as sewage sludge can be robust catalysts
for electricity generation in fuel cells utilizing NR as the
electron mediator.
[0130] FIG. 7 summarizes our working model explaining E. coli (or
A. succinogenes) metabolic properties in fuel cells using NR as the
electron mediator versus during normal (A) versus electrogenic
glucose metabolism (B) of E. coli or A. succinogenes in a fuel cell
with NR as the electronophore. Cell growth, ATP synthesis, and
reduced end product formation decrease in relation to the amount of
electricity generated. In the presence of NR, cell growth is
significantly reduced and NADH is oxidized via NR-mediated
electrical generation in lieu of producing normal reduced end
products (i:e., succinate, lactate, and ethanol). Cells still
generate ATP by substrate-level phosphorylation (i.e., acetate
kinase) but grow slower because they cannot generate ATP by
electron transport-mediated phosphorylation (i.e., fumarate
reductase).
[0131] NR is superior to thionin as an electron mediator because it
enhances both the rate of electron transfer (current) and the yield
of electrons transferred (coulombic yield). The highest current
(>17 mA) produced in a microbial fuel cell using NR was
significantly higher than that achieved previously with thionin as
the electron mediator (Roller, et al., 1984, J. Chem. Tech.
Biotechnol. 34B:3-12 and Allen, et al., 1993, Appl. Biochem.
Biotechnol. 39-40:27-40); it is, however, still low in electrical
terms. There may be potential applications for low-power DC
microbial fuel cells such as to maintain telecommunications in
remote areas including outer space.
Example 2
[0132] Bioreduction of Ketones to Alcohols
[0133] Introduction
[0134] Bioreductions of ketones require either NADH or NADPH as a
co-factor and the difficulty in implementing an efficient and
economical recycling system has restricted large scale applications
to whole cell processes. Specifically, we are interested in
asymmetric bioreduction of a .beta.-tetralone to its corresponding
(S)-alcohol by the yeast Trichosporon capitatum. The alcohol,
.beta.-tetralol, is subsequently used in the synthesis of MK-0499,
a very potent potassium channel blocker which mediates polarization
of cardiac tissues.
[0135] Recently, we have developed an electrochemical co-factor
recycling technology using an electrochemical bioreactor system
(described above). The technology was examined and demonstrated in
fermentation processes producing organic acids and in reducing
CO.sub.2 to CH.sub.4 with an activated sludge, resulting in a
20-40% increase in the end product concentration.
[0136] We have evaluated the electrochemical co-factor (NAD)
recycling technology using either whole cells of Trichosporon
capitatum (strain MY 1890) or an oxidoreductase isolated from this
organism, to support the bioreduction of 6-bromo-.beta.-tetralone
(L735,707) to its corresponding alcohol 6-bromo-.beta.-tetralol. In
this Example, we present the data on increased biotransformation
rate and .beta.-tetralol concentration using this new
electrochemical co-factor technology.
[0137] Materials and Methods
[0138] Electrochemical Bioreactor System
[0139] An electrochemical bioreactor (ECB) was designed and
constructed to conduct biostransformation in the presence and
absence of an electrically reduced system. The ECB was separated
into anode and cathode compartments by a cation selective membrane
septum (.phi. 22 mm, Nafion, Electrosynthesis, NY). The anode and
cathode electrodes were made from graphite fine woven felt (6 mm
thickness, 0.47 m.sup.2g-1 of available surface area,
Electrosynthesis, NY). The weights of the electrodes were adjusted
to 0.5 g (10.times.50 mm). The voltage and current between the
anode and cathode were measured using a precision multimeter, and
were adjusted to 0.7-10.0 volt and 0.5-10 mA, respectively. The
total volume of each compartment was 70 mL with a working volume of
40 mL. The reaction mixture was placed in the cathode compartment,
whereas 100 mM phosphate buffered saline was filled in the anode
compartment.
[0140] Microorganism and Medium Composition
[0141] Trichosporon capitatum MY 1890 (received from the Merck
& Co.) was grown on culture medium containing glycerol, 30 g/L;
soytone, 25 g/L and yeast extract, 10 g/L. The culture was grown in
200 mL medium in a 1 L baffled flask and was incubated on a 200 rpm
orbital shaker at 29-30.degree. C. for 48 hours.
[0142] Preparation of Biomass for Biotransformation
[0143] The cells from a 48 hour grown culture were harvested by
centrifugation at 8000 rpm for 30 minutes. The cell pellet was
resuspended in the same volume (200 ml) of 50 mM Tris buffer (pH
7.0), and washed twice with the buffer by centrifugation.
[0144] Biotransformation with Biomass
[0145] The washed cells were resuspended in 50 mM Tris buffer (pH
7.0) in an appropriate volume to achieve 1.times., 2.times. and
3.times. biomass concentration. The reaction mixture contained cell
suspension, .beta.-bromo-tetralone, ethanol, 100 nM neutral red and
50 mM Tris buffer (pH 7.0). Biotransformation was conducted in the
ECB system on a reciprocal shaking incubator (200 strokes/min) at
30.degree. C.
[0146] Analysis of Substrate and Product
[0147] .beta.-Bromo-tetralone and .beta.-bromo-tetranol were
analyzed by a Waters HPLC 640 equipped with a Zorbax RX-C8 column.
The absorbency was measured at 220 nm. The mobile phase containing
50% acetonitrile and 50% acidified water (0.1% phosphoric acid)
mixture was used at a flow rate of 1.0 ml/L.
[0148] Results and Discussions
[0149] Biotransformation of .beta.-tetralone to .beta.-tetralol
with a 48 Hour Trichosporon capitatum Culture in the Presence of
1.5 Volt Electricity
[0150] Comparison of the biotransformation in the presence of 1.5
volt to the absence of electricity was carried out with 1 g/L of
substrate in the electrochemical bioreactor system. As shown in
FIG. 9, the rate of .beta.-tetralol formation in the presence of
the electricity was significantly higher than without the
electricity. The overall reaction rate during the first 3 hour
reaction was increased by 45%. Although the reaction rate gradually
decreased, the high initial reaction rate resulted in a faster
completion of the biotransformation. In the presence of the
electricity the product formation was completed in 3 hours.
However, in the absence of the electricity, the reaction was
prolonged up to 8 hours and the final end product concentration was
lower than what was achieved with the electricity (0.53 g/L). It is
important to note the high initial reaction rate was observed
within the first hour in the presence of electricity.
[0151] Biotransformation of .beta.-tetralone at 2 g/L of
Substrate
[0152] Assuming that the biotransformation yield was limited by the
availability of the substrate, the level of substrate was increased
to 2 g/L while maintaining 1.5 volt of electricity. The product
concentration increased to 1.2 g/L in about 3-4 hours (FIG. 10).
This product concentration correlates well with 053 g/L at 1 g/L of
substrate in nearly the same reaction time. The initial reaction
rate was also dramatic in this experiment (2 g/L substrate). Based
on these results, it was decided to maintain 2 g/L substrate level
to achieve higher levels of reaction rates and the end product.
[0153] Biotransformation with Pulse Feeding of Substrate
[0154] To maintain the higher substrate concentration in the
reaction mixture, pulse feeding of 2 g/L of substrate was done at
1.5-2 hour interval for a total of 4 times. Although pulse feeding
of the substrate was followed by an increase in product formation
up to 2.2. g/L in the presence of electricity (FIG. 11), this
increase was not in proportion to the amount of total fed
substrate. Because the substrate .beta.-tetralone has a very low
solubility in the aqueous phase, a larger part of the substrate
remained in the insoluble form, and some of it was probably also
adsorbed onto the graphite felt of the electrode. Thus, only
limited substrate was converted to the end product. To reduce the
unutilized substrate, the concentration of the substrate pulse fed
to the reaction mixture was reduced to 1 g/L. The results indicate
that although product concentration decreased from 2.2 g/L (at 2
g/L substrate) to 1.8 g/L at 1 g/L substrate (pulse feeding), there
was almost no change in the reaction rate when the substrate was
pulse fed at 1 g/L (FIG. 12).
[0155] Effect of Ethanol Concentration on the Biotransformation
[0156] Since the substrate was poorly soluble in water, it was
dissolved in 5% ethanol. Assuming that a higher dissolved substrate
might result in increased end-product concentration in the ECB
system, ethanol concentration was increased from 5 to 50% to
increase the amount of soluble substrate. As the results presented
in FIG. 13, the increased ethanol concentration adversely affected
the reaction rate and resulted in a low final product formation
presumably due to ethanol toxicity. The highest reaction rate as
well as the product concentration was achieved with 5% ethanol.
Thus, another method has to be applied to increase the solubility
of the substrate to test the hypothesis that a higher dissolved
substrate will result in high biotransformation rates and product
yields.
[0157] Effect of Various Levels of Electricity on Biotransformation
Rate
[0158] Since the biochemical reactions are affected by the amount
of available reducing power, we examined the effect of various
levels of electricity on the rate of the biotransformation of
.beta.-tetralone to .beta.-tetralol. In the first experiment, 0,
0.7, 1.4 and 2.1 volt of electricity was supplied. In the second
experiment, the electricity was increased up to 10 volt. For these
two experiments, two different batches were used. As shown in FIGS.
6(1) and 6(2), electricity up to 5 volts enhanced the reaction rate
and the final product concentration. It seems that a higher
electric supply provides a higher driving force for the reductive
reaction. However, the reaction rate was drastically affected
within 20 minutes with 10 volts of electricity. It is likely that
the high electrical potential of 10 volts caused the denaturation
of protein, thus affected the cellular metabolic reactions. The
initial reaction rate with different levels of electricity are
presented in Table 5. The results demonstrate that the electricity
enhanced the reaction rate, e.g. with 5 volts electricity, the
biotransformation rate increased to two-fold in comparison to the
rate without the electricity.
5TABLE 5 Relationship between electrical potential and the
biotransformation rate Reaction rate (mg/L/min) in 20 minutes
Voltage 1.sup.st trial 2.sup.nd trial 0 (control) 17.0 14 0.7 21.0
-- 1.4 23.0 -- 2.1 26.0 21.5 3.5 -- 24.5 5.0 -- 26.9 10.0 --
12.6
[0159] Biotransformation with Variable Biomass, Voltage and
Substrate in a Matrix Design
[0160] Our results have demonstrated a high initial rate of
biotransformation of .beta.-tetralone to .beta.-tetralol in the ECB
system. We have also observed an electricity-dependent increase in
the reaction rate as well as a substrate concentration-dependent
increase in the .beta.-tetralol concentration. However, these
experiments do not explain the cumulative effect of the
electricity, substrate concentration and the amount of biomass on
the reaction rate.
[0161] Therefore, to examine the combined effect of biomass,
electricity and substrate on the biotransformation of
.beta.-tetralone, we conducted experiments according to a "BB
matrix design 6" provided by Merck & Co. Initial reaction rates
were determined within 10 minutes and these measurements were
accomplished by using 4 different batches of the culture with
internal controls. Comparison of the initial reaction rates at
different substrate concentrations shows that the specific reaction
rates were significantly affected by the electricity and biomass at
higher substrate concentration (up to 4 g/L), but not at 1 g/L
(Table 6). Similarly, a higher electricity (6 volt) resulted in a
high specific reaction rate (8.1 mg/L/min/g) at a moderate level of
biomass and high substrate concentration or vice versa. A similar
reaction rate was also achieved by a moderate level of electricity
(3.5 volt), but under high biomass (3.times.) and substrate (4 g/L)
conditions. Our results also show that low biomass concentrations
resulted in a significantly enhanced specific reaction rate (6.46
mg/L/min/g) by provide high electricity. However, an increased
biomass (2.times.) was necessary to obtain the similar reaction
rate (6.54 mg/L/min/g) if electricity was reduced from 6.0 to 3.5
volt (Table 6).
6TABLE 6 Cumulative effect of biomass, voltage and substrate on the
initial specific reaction rate in a BB Matrix Design 6. Substrate
Initial reaction Specific reaction Biomass Voltage Conc. (g/L) rate
(mg/L/min) rate (mg/L/min) Culture batch #1: Dry weight of biomass:
10.23 g/L 1.0 3.5 1.0 24.6 2.40 2.0 3.5 2.5 46.7 4.57 2.0 1.0 4.0
56.4 5.52 3.0 3.5 2.5 66.9 6.54 0.0 0.0 2.5 0.0 0 1.0 3.5 4.0 48.3
4.72 1.0 1.0 2.5 29.8 2.91 2.0 0.0 2.5 31.2 3.05 Culture batch #2:
Dry weight of biomass: 7.31 g/L 1.0 3.5 4.0 59.6 8.15 1.0 6.0 2.5
47.2 6.46 2.0 3.5 2.5 32.8 4.49 2.0 6.0 4.0 59.7 8.17 Culture batch
#3: Dry weight of biomass: 7.21 g/L 3.0 1.0 2.5 44.0 6.10 2.0 1.0
1.0 20.2 2.80 3.0 6.0 2.5 58.7 8.14 2.0 6.0 1.0 26.8 3.72 Culture
batch #4: Dry weight of biomass: 8.20 g/L 3.0 3.5 1.0 33.5 4.08 2.0
6.0 4.0 73.4 7.98 2.0 3.5 2.5 37.4 4.56 1.0 0.0 2.5 19.5 2.38
[0162] Biotransformation with Purified .beta.-tetralone
Reductase
[0163] Enzymatic biotransformation was conducted with 0.02 unit/ml
of the purified .beta.-tetralone reductase, 10 mM NAD and 10 mM
.beta.-tetralone in the presence of 0, 1.0 and 3.0 volt of
electricity. To confirm the enzyme reaction, one reaction mixture
was prepared as a control with NADH.sub.2 instead of NAD, in the
absence of electricity. The results show that 0.13 g/L of the
product was produced with 3 volt of electricity after 8 hour
incubation, whereas only a trace amount of the product was found in
the reaction mixture with 0 and 1.5 volt of electricity (FIG. 14).
In comparison, about 0.8 g/L of the product was produced with
NADH.sub.2. It is not clear why the enzymatic reaction rate with 3
volt electricity was slow.
[0164] Conclusion
[0165] In this study, it was demonstrated that the initial rate of
biotransformation of .beta.-tetralone to .beta.-tetralol and the
final product concentration were enhanced due to electricity-based
reducing power in the electrochemical bioreactor system. It was
found that the biotransformation rate was significantly affected by
the amount of biomass, substrate concentration and electrical
potential. The influence of electrical potential on the
biotransformation rates was more significantly observed at a high
substrate level of 2.5-4 g/L. The ECB system has shown a great
potential in reducing the reaction tine that is likely to result in
significant cost savings.
[0166] The present invention is not limited to the exemplified
embodiments, but is intended to encompass all such modifications
and variations as come within the scope of the following
claims.
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