U.S. patent application number 12/333932 was filed with the patent office on 2009-05-21 for system for the production of methane from co2.
Invention is credited to Laurens Mets.
Application Number | 20090130734 12/333932 |
Document ID | / |
Family ID | 40642378 |
Filed Date | 2009-05-21 |
United States Patent
Application |
20090130734 |
Kind Code |
A1 |
Mets; Laurens |
May 21, 2009 |
SYSTEM FOR THE PRODUCTION OF METHANE FROM CO2
Abstract
A method of converting CO.sub.2 gas produced during industrial
processes comprising contacting methanogenic archaea with the
CO.sub.2 gas under suitable conditions to produce methane.
Inventors: |
Mets; Laurens; (Chicago,
IL) |
Correspondence
Address: |
MICHAEL BEST & FRIEDRICH LLP
ONE SOUTH PINCKNEY STREET, P O BOX 1806
MADISON
WI
53701
US
|
Family ID: |
40642378 |
Appl. No.: |
12/333932 |
Filed: |
December 12, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2007/071138 |
Jun 13, 2007 |
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12333932 |
|
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60813020 |
Jun 13, 2006 |
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61028413 |
Feb 13, 2008 |
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Current U.S.
Class: |
435/167 ;
435/300.1 |
Current CPC
Class: |
C12P 5/023 20130101;
C12M 47/18 20130101; C12M 21/04 20130101; C12M 29/24 20130101; C12M
45/06 20130101; C12M 43/04 20130101; Y02E 50/343 20130101; Y02E
50/30 20130101 |
Class at
Publication: |
435/167 ;
435/300.1 |
International
Class: |
C12P 5/02 20060101
C12P005/02; C12M 1/00 20060101 C12M001/00 |
Claims
1. A method of converting carbon dioxide produced during an
industrial process to methane comprising: a) preparing a culture of
hydrogenotrophic methanogenic archaea in a bioreactor; b) supplying
an output gas from an industrial process to the bioreactor; wherein
the output gas comprises CO.sub.2 and between 0.02% and 6.7% oxygen
(moles/volume of output gas); and c) wherein the hydrogenotrophic
methanogenic archaea converts the output gas to continuously
produce methane.
2. The method of claim 1 wherein the culture is a substantially
pure culture of one hydrogenotrophic methanogenic archaea
species.
3. The method of claim 1 wherein H.sub.2 is supplied in an amount
to maintain a redox potential in the bioreactor under -100 mV or
less, and wherein no additional constituent other than the H.sub.2
gas is added to the bioreactor to maintain the redox potential in
the bioreactor under -100 mV or less.
4. The method of claim 1 wherein the industrial process is coal
gasification, biomass gasification, or liquid fuel production by
biomass fermentation.
5. The method of claim 2 wherein the hydrogenotrophic methanogenic
archea species is selected from the group consisting of
Methanobacterium alcaliphilum, Methanobacterium bryantii,
Methanobacterium congolense, Methanobacterium defluvii,
Methanobacterium espanolae, Methanobacterium formicicum,
Methanobacterium ivanovii, Methanobacterium palustre,
Methanobacterium thermaggregans, Methanobacterium uliginosum,
Methanobrevibacter acididurans, Methanobrevibacter arboriphilicus,
Methanobrevibacter gottschalkii, Methanobrevibacter olleyae,
Methanobrevibacter ruminantium, Methanobrevibacter smithii,
Methanobrevibacter woesei, Methanobrevibacter wolinii,
Methanothermobacter marburgensis, Methanothermobacter
thermautotrophicum, Methanothermobacter thermoflexus,
Methanothermobacter thermophilus, Methanothermobacter wolfeii,
Methanothermus sociabilis, Methanocorpusculum bavaricum,
Methanocorpusculum parvum, Methanoculleus chikuoensis,
Methanoculleus submarinus, Methanogenium frigidum, Methanogenium
liminatans, Methanogenium marinum, Methanosarcina acetivorans,
Methanosarcina barkeri, Methanosarcina mazei, Methanosarcina
thermophila, Methanomicrobium mobile, Methanocaldococcus
jannaschii, Methanococcus aeolicus, Methanococcus maripaludis,
Methanococcus vannielii, Methanococcus voltaei, Methanothermococcus
thermolithotrophicus, Methanopyrus kandleri, Methanothermobacter
thermoautotroiphicus, Methanocaldococcus fervens,
Methanocaldococcus indicus, Methanocaldococcus infernus, and
Methanocaldococcus vulcanius.
6. The method of claim 5 wherein the hydrogenotrophic methanogenic
archea is selected from the group consisting of Methanosarcina
barkeri and Methanococcus maripaludis.
7. The method of claim 6 wherein the conditions include a
temperature of about 35.degree. C. to about 37.degree. C.
8. The method of claim 5 wherein the hydrogenotrophic methanogenic
archea is Methanothermobacter thermoautotroiphicus.
9. The method of claim 8 wherein the conditions include a
temperature of about 60.degree. C. to about 65.degree. C.
10. The method of claim 5 wherein the hydrogenotrophic methanogenic
archea is selected from the group consisting of Methanocaldococcus
fervens, Methanocaldococcus indicus, Methanocaldococcus infernus,
and Methanocaldococcus vulcanius.
11. The method of claim 10 wherein the conditions include a
temperature of about 80.degree. C. to about 100.degree. C.
12. The method of claim 1 wherein the industrial output gas
comprises at least 0.8% oxygen (moles/volume of output gas).
13. The method of claim 1 wherein the industrial output gas
comprises at least 1.7% oxygen (moles/volume of output gas).
14. The method of claim 1 wherein the industrial output gas
comprises at least 3.4% oxygen (moles/volume of output gas).
15. The method of claim 1 wherein the industrial output gas
comprises between 3.4%-6.7% oxygen (moles/volume of output
gas).
16. The method of claim 1 wherein the industrial output gas further
comprises carbon monoxide.
17. The method of claim 16, wherein the industrial output gas
comprises at least about 8% carbon monoxide by volume.
18. The method of claim 16, wherein the industrial output gas
comprises at least about 16% carbon monoxide by volume.
19. The method of claim 16, wherein the industrial output gas
comprises at least about 60% carbon monoxide by volume.
20. A method of converting carbon dioxide produced during an
industrial process to methane comprising: a) contacting a culture
comprising hydrogenotrophic methanogenic archaea with H.sub.2 gas
and an output gas from an industrial process comprising CO.sub.2
gas in a bioreactor; b) supplying an amount of H.sub.2 gas to
maintain a redox potential in the bioreactor under -100 mV or less,
wherein no additional constituent other than the H.sub.2 gas is
added to the bioreactor to maintain the redox potential in the
bioreactor under -100 mV or less; and c) wherein the
hydrogenotrophic methanogenic archaea converts the H.sub.2 gas and
the CO.sub.2 gas to methane.
21. A method of converting carbon dioxide produced during an
industrial process to methane comprising: a) preparing an initial
culture of hydrogenotrophic methanogenic archaea and placing the
culture in a bioreactor; b) supplying an output gas from an
industrial process to the bioreactor; wherein the output gas
comprises CO.sub.2; c) supplying fresh medium to the culture; d)
wherein the hydrogenotrophic methanogenic archaea converts the
output gas to continuously produce methane; and e) wherein no
sulfur is added in the method, other than the presence of sulfur in
the initial culture.
22. A method of converting carbon dioxide produced during an
industrial process to methane using a cascaded bioreactor process,
the process comprising: a) preparing a culture of hydrogenotrophic
methanogenic archaea, wherein the culture is present in a first and
second reactor vessel; b) supplying fresh medium to the first and
second reactor vessels through a first medium feed line attached to
the first reactor vessel, and a second medium feed line attached to
the second reactor vessel; c) supplying H.sub.2 gas to the first
reactor vessel through hydrogen gas feed line; d) supplying an
output gas from an industrial process to the first reactor vessel;
wherein the output gas comprises CO.sub.2, through an output gas
feed line; e) wherein the hydrogenotrophic methanogenic archaea
culture in the first reactor converts H.sub.2 gas and CO.sub.2 gas
to produce methane; f) transferring at least a portion of gas in
the first reactor vessel to the second reactor vessel by a first
gas feed line; g) wherein the hydrogenotrophic methanogenic archaea
culture in the second reactor converts H.sub.2 gas and CO.sub.2 gas
to produce methane.
23. The method of claim 22 further comprising: a) preparing a
culture of hydrogenotrophic methanogenic archaea, wherein the
culture is present in a third reactor vessel; b) supplying fresh
medium to the third reactor vessels through a third medium feed
line attached to the third reactor vessel; c) transferring at least
a portion of gas in the second reactor vessel to the third reactor
vessel by a second gas feed line; d) wherein the hydrogenotrophic
methanogenic archaea culture in the third reactor converts H.sub.2
gas and CO.sub.2 gas to produce methane.
24. The method of claim 22 wherein the output gas further comprises
between 0.02% and 6.7% oxygen (moles/volume of output gas) and
wherein the hydrogenotrophic methanogenic archaea in the first and
second reactor vessels continuously produce methane.
25. The method of claim 22 wherein H.sub.2 is supplied in an amount
to maintain a redox potential in the first reactor vessel under
-100 mV or less, and wherein no additional constituent other than
the H.sub.2 gas is added to the first reactor vessel to maintain
the redox potential in the first reactor vessel under -100 mV or
less.
26. The method of claim 22 wherein no sulfur is in the process
other than sulfur in the culture medium.
27. The method of claim 22 wherein the hydrogenotrophic
methanogenic archea comprises one or more species selected from the
group consisting of Methanobacterium alcaliphilum, Methanobacterium
bryantii, Methanobacterium congolense, Methanobacterium defluvii,
Methanobacterium espanolae, Methanobacterium formicicum,
Methanobacterium ivanovii, Methanobacterium palustre,
Methanobacterium thermaggregans, Methanobacterium uliginosum,
Methanobrevibacter acididurans, Methanobrevibacter arboriphilicus,
Methanobrevibacter gottschalkii, Methanobrevibacter olleyae,
Methanobrevibacter ruminantium, Methanobrevibacter smithii,
Methanobrevibacter woesei, Methanobrevibacter wolinii,
Methanothermobacter marburgensis, Methanothermobacter
thermautotrophicum, Methanothermobacter thermoflexus,
Methanothermobacter thermophilus, Methanothermobacter wolfeii,
Methanothermus sociabilis, Methanocorpusculum bavaricum,
Methanocorpusculum parvum, Methanoculleus chikuoensis,
Methanoculleus submarinus, Methanogenium frigidum, Methanogenium
liminatans, Methanogenium marinum, Methanosarcina acetivorans,
Methanosarcina barkeri, Methanosarcina mazei, Methanosarcina
thermophila, Methanomicrobium mobile, Methanocaldococcus
jannaschii, Methanococcus aeolicus, Methanococcus maripaludis,
Methanococcus vannielii, Methanococcus voltaei, Methanothermococcus
thermolithotrophicus, Methanopyrus kandleri, Methanothermobacter
thermoautotroiphicus, Methanocaldococcus fervens,
Methanocaldococcus indicus, Methanocaldococcus infernus, and
Methanocaldococcus vulcanius.
28. A cascaded bioreactor comprising: a first reactor vessel a
second reactor vessel a culture of hydrogenotrophic methanogenic
archaea which is present in the first and second reactor vessels; a
source of an output gas from an industrial process comprising CO2
that feeds into the first reactor vessel; a source of hydrogen gas
that feeds into the first reactor vessel; a gas feed from the first
reactor vessel to the second reactor vessel; a feed to the first
reactor vessel for providing fresh medium; a feed to the second
reactor vessel for providing fresh medium; a feed to the first
reactor vessel for removing the hydrogenotrophic methanogenic
archaea culture; and a feed to the second reactor vessel for
removing the hydrogenotrophic methanogenic archaea culture.
29. The cascaded bioreactor of claim 28 further comprising: a third
reactor vessel; a culture of hydrogenotrophic methanogenic archaea
which is present in the third reactor vessel; a gas feed from the
second reactor vessel to the third reactor vessel; a feed to the
third reactor vessel for providing fresh medium; and a feed to the
third reactor vessel for removing the hydrogenotrophic methanogenic
archaea culture.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part under 35 U.S.C.
1.111 of International Application No. PCT/US2007/071138, filed
Jun. 13, 2007, which claims the benefit of priority to U.S.
Provisional Application No. 60/813,020, filed Jun. 13, 2006. This
application also claims the benefit of priority to U.S. Provisional
Application No. 61/028,413, filed Feb. 13, 2008, all of which are
incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Energy self-sufficiency and sustainable energy systems with
lower environmental impacts are critical national goals. Increased
use of biomass-derived ethanol as a fuel is advantageous because it
uses solar energy, rather than fossil fuel energy, as a portion of
its energy input and CO.sub.2 obtained by photosynthesis from the
environment as a portion of its material requirement for energy
carriers. At present ethanol production from corn requires
significant energy input from fossil fuels for distillation of the
final product and for drying of fermentation residues for use in
animal feed. Present domestic ethanol production methods,
therefore, are not energetically or economically competitive with
ethanol produced abroad from sugar cane. In addition, one third of
the carbon in the corn starch is released as a concentrated
CO.sub.2 stream during ethanol production. The U.S. Department of
Energy has identified that increasing the energy efficiency and
reducing the CO.sub.2 emissions of the fuel ethanol production
process is essential for increasing the role of ethanol in meeting
our energy needs. Currently, fuel ethanol production relies on
federal subsidies for its economic viability. Therefore, it will be
important to achieve greater economic efficiency in the ethanol
production process if the industry is to be viable and
self-sustaining.
[0003] The present invention provides a system that reduces the
CO.sub.2 emissions from industrial processes, including ethanol
production, by using a bioreactor system that uses the emissions to
produce methane (natural gas).
SUMMARY OF THE INVENTION
[0004] The present invention provides a system that converts the
CO.sub.2 into methane (natural gas). The present invention utilizes
CO.sub.2 produced by industrial processes. Examples of processes
that that produce CO.sub.2 are biomass fermentation to produce
liquid fuels and coal and biomass gasification processes.
Gasification is a process that converts carbonaceous materials,
such as coal, petroleum, petroleum coke or biomass (living or dead
biological material), into carbon monoxide, hydrogen and carbon
dioxide. In the system of the present invention, CO.sub.2
industrial waste-gas streams, such as those formed during the
production of ethanol or those produced by combined cycle coal
fired energy plants, is combined with hydrogen and undergoes a
microbial fermentation process catalyzed by methanogenic archaea,
producing methane and water. Hydrogen gas may be produced from a
variety of sources. In one embodiment, inexpensive electric power
can be used to produce hydrogen from water via electrolysis. The
integrated electrolysis/methane fermentation system can be viewed
as converting an intermittent energy source (e.g. inexpensive
off-peak electricity from power plants) to a stable chemical energy
store, using hydrogen as an intermediate and methane as the final
energy carrier.
[0005] The present invention uses a bioreactor containing
methanogenic archaea to catalyze the following chemical
reaction:
CO.sub.2+4H.sub.2.fwdarw.CH.sub.4+2H.sub.2O
[0006] This reaction occurs with high efficiency with >95%
conversion of CO.sub.2 to methane at moderate temperatures.
Suitably the bioreactor conditions will allow a reaction vessel
that is 1/10 or less the volume of the ethanol fermentation system
to handle all of the CO.sub.2 stream.
[0007] In one embodiment, the present invention provides a method
of converting carbon dioxide produced during an industrial process
to methane comprising contacting a culture comprising methanogenic
archaea with H.sub.2 gas and an output gas from an industrial
process comprising CO.sub.2 gas in a bioreactor under suitable
conditions to produce methane. The industrial process can be coal
gasification, biomass gasification, or liquid fuel production by
biomass fermentation, suitably ethanol production from a biomass
such as corn.
[0008] Any suitable methanogenic archaea can be used, and a
suitable temperature and pressure for the bioreactor condition can
be selected depending at least in part on the methanogenic archaea
selected. In some embodiments, suitably pressures within the
bioreactor range from about 0.5 atmospheres to about 500
atmospheres. The bioreactor can also contain a source of
intermittent agitation of the culture.
[0009] The culture conditions should suitably maintaining a redox
potential of about -100 mV or less. In one embodiment, this redox
potential is maintained by supplying a suitable amount of hydrogen
gas.
[0010] Also in one embodiment, the methane gas removed from the
bioreactor suitably comprises less than about 450 ppm hydrogen
sulfide, or alternatively less than about 400 ppm, 300 ppm, 200
ppm, 150 ppm, 100 ppm, 50 ppm or 20 ppm of hydrogen sulfide.
[0011] Further, in certain embodiments the industrial output gas at
least intermittently further comprises air and/or carbon monoxide.
Suitably the industrial output gas comprises about 32% or less air
by volume, or between from about 0.1% to about 32% air by volume,
or less than about 4% air by volume, or at least about 4%, 8% or
16% air by volume. Suitably the industrial output gas can also
comprise less than about 40% carbon monoxide by volume, or less
than about 8% carbon monoxide by volume, or at least about 8% or
16% carbon monoxide by volume.
[0012] An another embodiment, the bioreactor comprises a culture of
methanogenic archaea, a source of an output gas from an industrial
process comprising CO.sub.2 that feeds into the bioreactor, a
source of hydrogen gas that feeds into the bioreactor, a gas feed
from the bioreactor for removing gas from the bioreactor, a feed
for providing fresh medium, and a feed for removing the
culture.
DRAWINGS
[0013] FIG. 1 shows a design schematic of one embodiment of a
CO.sub.2 recapture and methane production plant.
[0014] FIG. 2 shows a design schematic of one embodiment of a
stratified bioreactor.
[0015] FIG. 3 shows a design schematic of a system of bioreactors
set up in cascaded serial arrangement.
[0016] FIG. 4 is a chart showing the growth curve of methane
production of Methanococcus maripaludis.
[0017] FIG. 5 is a chart demonstrating the effects of agitation of
a culture of Methanococcus maripaludis with respect to methane
production.
[0018] FIG. 6 is a chart showing the changes in hydrogen conversion
to methane catalyzed by Methanococcus maripaludis with respect to
changes in the feed rate hydrogen gas into the bioreactor.
[0019] FIG. 7 is a chart showing the recovery of methanogenesis in
a culture Methanosarcina barkeri after exposure to 10 minutes of
100% air.
[0020] FIG. 8 is a chart showing the recovery of methanogenesis in
a culture Methanosarcina barkeri after exposure to 10 minutes of
100% air.
[0021] FIG. 9 is a chart showing the recovery of methanogenesis in
a culture Methanosarcina barkeri after exposure to 90 minutes of
100% air.
[0022] FIG. 10 is a chart showing the recovery of methanogenesis in
a culture Methanosarcina barkeri after exposure to 15 hours of 100%
air.
[0023] FIG. 11 is a chart showing the recovery of methanogenesis in
a culture Methanococcus maripaludis after exposure to 10 minutes of
100% air.
[0024] FIG. 12 is a chart showing the recovery of methanogenesis in
a culture Methanococcus maripaludis during exposure of a mixture of
air and hydrogen gas.
[0025] FIG. 13 is a chart showing the recovery of methanogenesis in
a culture Methanococcus maripaludis during exposure of a mixture of
carbon monoxide and hydrogen gas.
[0026] FIG. 14 is a chart showing the recovery of methanogenesis in
a culture Methanococcus maripaludis during exposure of a mixture of
carbon monoxide and hydrogen gas.
[0027] FIG. 15 is a chart showing a projected model of the extent
of the conversion of hydrogen to methane in a two and three reactor
cascade (Xcascade) divided by the extent of the conversion of
hydrogen to methane in an equivalent total volume single reactor
(Xsingle), the ratio plotted against Xsingle. Each of the single
reactor cascade, 2 reactor cascade and 3 reactor cascade have the
same total volume and same hydrogen input rate.
[0028] FIG. 16 is a chart showing the recovery of
Methanothermobacter thermoautotrophicus after exposure to air.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The present invention comprises a bioreactor system that can
be integrated with industrial processes that produce CO.sub.2 gas
as a byproduct. In one embodiment such a process is the production
of ethanol from biomass. The invention comprises a bioreactor
containing a microbial culture capable of hydrogenotrophic
methanogenesis (i.e. the conversion of CO.sub.2 gas plus hydrogen
gas to methane gas). The bioreactor is coupled to a hydrogen source
and a CO.sub.2 gas source. Suitably the CO.sub.2 gas source is the
CO.sub.2 gas stream that is emitted by the production of ethanol.
The hydrogen source is suitably hydrogen produced by the
electrolysis of water. Suitably this hydrolysis is powered by
electricity used in off peak times. The methane produced by the
system can be fed back into the ethanol production facility to
power various processes, and/or can be stored and sold as fuel.
[0030] Microbial cultures suitable for practice of the invention
are readily obtainable from public collections of organisms or can
be isolated from a variety of environmental sources. Such
environmental sources include anaerobic soils and sands, bogs,
swamps, marshes, estuaries, dense algal mats, both terrestrial and
marine mud and sediments, deep ocean and deep well sites, sewage
and organic waste sites and treatment facilities, and animal
intestinal tracts and feces. Many pure cultures of single species
are suitable. Classified pure cultures are all members of the
Archaeal domain [Woese et al. Proc Natl Acad Sci USA 87:4576-4579
(1990) "Towards a natural system of organisms: Proposal for the
domains Archaea, Bacteria, and Eucharya.", incorporated herein by
reference] and fall within 4 different classes of the Euryarchaea
kingdom. Examples of suitable organisms have been classified into 4
different genera within the Methanobacteria class (e.g.
Methanobacterium alcaliphilum, Methanobacterium bryantii,
Methanobacterium congolense, Methanobacterium defluvii,
Methanobacterium espanolae, Methanobacterium formicicum,
Methanobacterium ivanovii, Methanobacterium palustre,
Methanobacterium thermaggregans, Methanobacterium uliginosum,
Methanobrevibacter acididurans, Methanobrevibacter arboriphilicus,
Methanobrevibacter gottschalkii, Methanobrevibacter olleyae,
Methanobrevibacter rum inantium, Methanobrevibacter smithii,
Methanobrevibacter woesei, Methanobrevibacter wolinii,
Methanothermobacter marburgensis, Methanothermobacter
thermautotrophicum (also known as Methanothermobacter
thermoautotroiphicus), Methanothermobacter thermoflexus,
Methanothermobacter thermophilus, Methanothermobacter wolfeii,
Methanothermus sociabilis), 5 different genera within the
Methanomicrobia class (e.g. Methanocorpusculum bavaricum,
Methanocorpusculum parvum, Methanoculleus chikuoensis,
Methanoculleus submarinus, Methanogenium frigidum, Methanogenium
liminatans, Methanogenium marinum, Methanosarcina acetivorans,
Methanosarcina barkeri, Methanosarcina mazei, Methanosarcina
thermophila, Methanomicrobium mobile), 7 different genera within
the Methanococci class (e.g. Methanocaldococcus jannaschii,
Methanococcus aeolicus, Methanococcus maripaludis, Methanococcus
vannielii, Methanococcus voltaei, Methanothermococcus
thermolithotrophicus, Methanocaldococcus fervens,
Methanocaldococcus indicus, Methanocaldococcus infernus,
Methanocaldococcus vulcanius), and one genus within the Methanopyri
class (e.g. Methanopyrus kandleri). Suitable cultures are available
from public culture collections (e.g. the American Type Culture
Collection, the Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH, and the Oregon Collection of Methanogens). Many
suitable hydrogenotrophic methanogens, isolated in pure culture and
available in public culture collections, have not yet been fully
classified. Preferred pure culture organisms include
Methanosarcinia barkeri, Methanococcus maripaludis, and
Methanothermobacter thermoautotrophicus.
[0031] Suitable cultures of mixtures of two or more microbes are
also readily isolated from the specified environmental sources
[Bryant et al. Archiv Microbiol 59:20-31 (1967) "Methanobacillus
omelianskii, a symbiotic association of two species of bacteria.",
incorporated herein by reference]. Suitable mixtures may be
consortia in which cells of two or more species are physically
associated or they may be syntrophic mixtures in which two or more
species cooperate metabolically without physical association. Mixed
cultures may have useful properties beyond those available from
pure cultures of known hydrogenotrophic methanogens. These
properties may include, for instance, resistance to contaminants in
the gas feed stream, such as oxygen, ethanol or other trace
components, or aggregated growth, which may increase the culture
density and volumetric gas processing capacity of the culture.
[0032] Suitable cultures of mixed organisms may also be obtained by
combining cultures isolated from two or more sources. One or more
of the species in a suitable mixed culture should be an Archaeal
methanogen. Any non-Archael species may be bacterial or
eukaryotic.
[0033] Suitable cultures may also be obtained by genetic
modification of non-methanogenic organisms in which genes essential
for supporting hydrogenotrophic methanogenesis are transferred from
a methanogenic microbe or from a combination of microbes that may
or may not be methanogenic on their own. Suitable genetic
modification may also be obtained by enzymatic or chemical
synthesis of the necessary genes.
[0034] The bioreactor system may provide continuous or
discontinuous methane production using a continuous
hydrogenotrophic methanogenic culture operating under stable
conditions. An example of such suitable conditions is set forth
below in the examples and is also provided in Schill, N., van
Gulik, M., Voisard, D., & von Stockar, U. (1996) Biotechnol
& Bioeng 51:645-658. "Continuous cultures limited by a gaseous
substrate: development of a simple, unstructured mathematical model
and experimental verification with Methanobacterium
thermoautotrophicum", incorporated herein by reference. Culture
media may be comprised of dilute mineral salts, and should be
adapted to the particular culture in use.
[0035] The medium should be replenished at a rate suitable to
maintain a useful concentration of essential minerals and to
eliminate any metabolic products that may inhibit methanogenesis.
Dilution rates below 0.1 culture volume per hour are suitable,
since they yield high volumetric concentrations of active methane
generation capacity. Surprisingly, dilution rates of less than
0.001 volumes was found to provide active methane generating
capacity.
[0036] Total gas delivery rates (CO.sub.2 plus H.sub.2) in the
range of 0.2 to 4 volume of gas (STP) per volume of culture per
minute are suitable, since they both maintain and exploit high
volumetric concentrations of active methane generation
capacity.
[0037] In one embodiment, the redox potential is maintained below
-100 mV or lower during methanogenesis. The method of the present
invention encompasses conditions in which the redox potential is
transiently increased to above -100 MV, as for example when air is
added to the system.
[0038] In the examples below the temperature of the culture was
maintained near the optimum for growth of the organism used in the
culture (e.g. about 35.degree. C. to about 37.degree. C. for
mesophilic organisms such as Methanosarcinia barkeri and
Methanococcus maripaludis or about 60.degree.-65.degree. C. for
thermophiles such as Methanothermobacter thermoautotrophicus, and
about 85.degree. C.-90.degree. C. for organisms such
asMethanocaldococcus jannaschii, Methanocaldococcus fervens,
Methanocaldococcus indicus, Methanocaldococcus infernus, and
Methanocaldococcus vulcanius.). However, it is envisioned that
temperatures above or below the temperatures for optimal growth may
be used. In fact, higher conversion rates of methane may be
obtained at temperatures above the optimal growth rate
temperature.
[0039] In one embodiment of the invention, a reducing agent is
introduced into the fermentation process along with CO.sub.2 and
hydrogen, this reducing agent can suitably be hydrogen sulfide or
sodium sulfide. In one embodiment, a 4:1 mixture of H.sub.2 and
CO.sub.2 gases can be provided at a total gassing rate (vvm) of
from 0.1 L gas per L culture per minute [L/(L-min)] to >1.0
L/(L-min), with greater than 95% of the CO.sub.2 converted to
methane and the rest of the CO.sub.2 in the input being converted
to cellular biomass.
[0040] In another embodiment, hydrogen itself can be used as a
reductant to maintain the redox potential of the culture in the
range (<-100 mV) necessary for optimum performance of
hydrogenotrophic methanogenesis. Generally, hydrogen gas is
provided in the method in concentrations effective in allowing for
at least a portion of the carbon dioxide in the bioreactor to be
converted into methane.
[0041] In another embodiment, the redox potential of the culture
can be maintained at <-100 mV via an electrochemical cell
immersed in the medium.
[0042] In another embodiment, the system comprises various methods
and/or features that reduce the presence of oxygen in the CO.sub.2
stream that is fed into the bioreactor. When obligate anaerobic
methanogenic microorganisms are used to catalyze methane formation,
the presence of oxygen may be detrimental to the performance of the
process and contaminates the product gas. Therefore the reduction
of the presence of oxygen in the CO.sub.2 stream is helpful for
improving the process. In one embodiment, the oxygen level is
reduced prior to entry of the gas into the fermentation vessel by
passing the mixed H.sub.2/CO.sub.2 stream over a palladium
catalyst, which converts any trace oxygen to water. In this
embodiment, H.sub.2 is provided in an amount above the amount
needed in the culture by a 2:1 ratio relative to the contaminating
oxygen. In another embodiment, the oxygen is removed by
pre-treatment of the gas stream in a bioreactor. In this
embodiment, the reductant may be provided either by provision of a
source of organic material (e.g. glucose, starch, cellulose,
fermentation residue from an ethanol plant, whey residue, etc.)
that can serve as substrate for an oxidative fermentation. The
microbial biological catalyst is chosen to oxidatively ferment the
chosen organic source, yielding CO.sub.2 from the contaminant
oxygen. In this embodiment, additional H.sub.2 would be provided to
enable conversion in the anaerobic fermentor of this additional
CO.sub.2 to methane. In another embodiment, oxygen removal is
accomplished in the main fermentation vessel via a mixed culture of
microbes that includes one capable of oxidative fermentation of an
added organic source in addition to the hydrogenotrophic methanogen
necessary for methane production. An example of a suitable mixed
culture was originally isolated as "Methanobacillus omelianskii"
and is readily obtained from environmental sources [Bryant et al.
Archiv Microbiol 59:20-31 (1967) "Methanobacillus omelianskii, a
symbiotic association of two species of bacteria.", incorporated
herein by reference]. In another embodiment, an oxygen tolerant
methanogen is used in the bioreactor to improve the stability of
the methane formation process in the presence of contaminating
oxygen. Both Methanosarcinia barkeri and Methanococcus maripaludis
are sufficiently oxygen tolerant in the presence of contaminating
oxygen.
[0043] FIG. 1 depicts one embodiment of a CO.sub.2 recapture and
methane production plant using the methods set forth above. An
industrial CO.sub.2 source (A)--e.g. fuel ethanol plant--with
CO.sub.2 effluent and natural gas demand, vents CO.sub.2 to a
CO.sub.2 collection and storage tank (B). A hydrolyzer (C) produces
hydrogen, suitably from electrolysis. Hydrogen produce by the
hydrolyzer (C) is collected in a hydrogen storage tank (D). The
hydrogen and CO.sub.2 from their respective storage tanks are fed
through an oxygen scrubber (E) for removal of oxygen from the
CO.sub.2 effluent stream. After passing through the oxygen scrubber
(E), the hydrogen and CO.sub.2 are feed into a fermentor/bioreactor
system (F) for conversion of CO.sub.2+H.sub.2 to methane. A storage
tank providing medium (I) is also connected to the
fermentor/bioreactor system (F) to provide for replenishment of
nutrients in the fermentor. The methane gas vented from the
fermentor/bioreactor (F) passes through a sulfur scrubber (G) for
recovering sulfur from the product methane stream. The methane gas
can then be stored in a methane storage tank (H).
[0044] A bioreactor, also known as a fermentor vessel, as set forth
in the invention is any suitable vessel in which methanogenesis can
take place. Suitable bioreactors to be used in the present
invention should be sized relative to the volume of the CO.sub.2
source. Typical streams of 2,200,000 lb CO.sub.2/day from a
100,000,000 gal/yr ethanol plant would require a CO.sub.2
recovery/methane production fermentor of about 750,000 gal total
capacity. Fermentor vessels similar to the 750,000 gal individual
fermentor units installed in such an ethanol plant would be
suitable.
[0045] FIG. 2 depicts one embodiment of a stratified bioreactor
that can be used in the present invention. In this embodiment, the
bioreactor has the CO.sub.2 and hydrogen entering into the bottom
of the bioreactor along with the nutrients for the bioreactor. A
mechanical impeller is positioned on the top of the bioreactor and
is used to move a mixing apparatus within the bioreactor. The
bioreactor has three zones, A, B and C. Zone A at the bottom of the
reactor is a high CO.sub.2 zone. Zone B, in the middle of the
bioreactor has a decreased CO.sub.2 presence, and Zone C at the top
end of the reactor has little if any CO.sub.2. The methane
produced, and the spent medium is removed from the top of the
bioreactor.
[0046] FIG. 3 depicts one embodiment of a cascaded bioreactor that
can be used in the present invention. In this embodiment, the
hydrogen, CO.sub.2 and cell nutrients are fed into the bottom of a
first compartment (A). In this compartment (A), even after
fermentation, there is still a high level of CO.sub.2. The gas
produced by the fermentation reaction in the first compartment (A)
is then transferred from the top of the first compartment to the
bottom of a second compartment (B) along with cell nutrients. In
this second compartment (B) the CO.sub.2 level is decreased from
the levels found in the first compartment (A). The gas produced by
the fermentation reaction in the second compartment (B) is
transferred from the top of the second compartment (B) to the
bottom of a third compartment (C) along with cell nutrients. In
this third compartment (C) most if not all of the CO.sub.2 has been
removed and only the methane gas is left to be removed from the top
of the compartment. In each of the compartments, spent medium can
be removed from the compartments.
EXAMPLE 1
General Setup for Bench Scale Bioreactor
[0047] A bench-scale bioreactor was used to test a series of
variables important in the design and operation of an industrial
scale bioreactor. A 1.3 L fermenter vessel (bioreactor) (BioFlo
110, New Brunswick), fitted with an Ingold autoclavable pH
electrode for measuring pH in the medium and a Lazar Labs double
junction platinum band autoclavable ORP electrode for measuring the
oxidation-reduction potential (ORP) of the medium was used in the
following experiments. The bioreactor contained 1 L culture medium
and was stirred at 400 rpm with a Rushton impeller. With 1 L of
medium, the bioreactor has a headspace of 300 cc of gas. The
chamber was also fitted with a peristaltic pump that could control
the addition of a chemical reductant, such as Na.sub.2S. A second
peristaltic pump controlled the constant addition of fresh culture
medium to the vessel to enable continuous culture operation. A
third peristaltic pump was used to remove excess liquid from the
culture vessel, maintaining a constant volume of 1 L. The excess
liquid included the metabolic water generated during methanogenesis
as well as increased medium volume from continuous culture
operation. The temperature of the culture was controlled by a
heating blanket. Gas mixtures were introduced via a sparger at the
bottom of the vessel. The composition of the gas mixture was
controlled by three mass flow controllers, one for H.sub.2, one for
CO.sub.2, and a third that could be used for controlling addition
of air, CO, or N.sub.2. Generally, a gas composition of 1 volume
CO.sub.2 to 4 volumes of H.sub.2 was used and was passed over a
palladium catalyst (Alfa AESAR) prior to introduction to the
culture. The culture in the bioreactor was maintained at about 1
atmosphere of pressure. The gas exiting the culture vessel at
ambient atmospheric pressure was passed through a condenser at
4.degree. C. to reduce water vapor content. The composition of the
effluent gas stream was analyzed by a Cirrus quadrupole mass
spectrometer continually scanning the mass range of 1 to 50 atomic
mass units. To correct for variations in ambient pressure over
time, each scan was normalized to the sum of detected masses.
Composition of individual gasses was determined by comparison with
mixtures of various composition generated with the mass flow
control system. Measurements were made of the amount of methane
produced by a given volume of culture per unit time, as well as the
efficiency of conversion of input CO.sub.2 and H.sub.2 to
methane.
EXAMPLE 2
Bench Scale Bioreactor Using Methanococcus maripaludis
[0048] The general setup of Example 1 was used with the organism
Methanococcus maripaludis. Methanococcus maripaludis is grown at
37.degree. C. in modified McCas medium containing the following
components per L of medium: KCl 0.335 g, MgCl.sub.2.6H.sub.2O 2.75
g, MgSO.sub.4.7H2O 3.45 g, CaCl.sub.2.2H.sub.2O 0.14 g, NH.sub.4Cl
0.5 g, NaHCO.sub.3 8.4 g, NaCl 22 g, K.sub.2HPO.sub.4 0.14 g,
FeSO.sub.4.7H.sub.2O 9.5 mg, Resazurin 1 mg, Casamino acids 2 g,
cysteine.H.sub.2O.HCl 0.5 g, Na.sub.3Citrate.2H.sub.2O 21 mg,
MnSO.sub.4.2H.sub.2O 5 mg, CoCl.sub.2(0.6H.sub.2O) 1 mg,
ZnSO.sub.4(0.7H.sub.2O) 1 mg, CuSO.sub.4.5H.sub.2O 0.1 mg,
AlK(SO.sub.4).sub.2 0.1 mg, H.sub.3BO.sub.4 0.1 mg,
Na.sub.2MoO.sub.4.2H.sub.2O 1 mg, NiCl.sub.2.6H.sub.2O 0.25 mg,
Na.sub.2SeO.sub.3 2 mg, V(III)Cl 0.1 mg, Na.sub.2WO.sub.4.2H.sub.2O
1 mg, biotin 0.02 mg, folic acid 0.02 mg, pyridoxine HCl 0.10 mg,
thiamine HCl 0.05 mg, riboflavin 0.05 mg, nicotinic acid 0.05 mg,
DL-calcium pantothenate 0.05 mg, vitamin B12 0.001 mg,
p-aminobenzoic acid 0.05 mg, lipoic acid 0.05 mg. After autoclaving
and before inoculation, the medium was reduced by the addition of
0.5 g/L Na.sub.2S from a 50.times. anaerobic, sterile stock
solution, yielding an ORP of the medium below -100 mV. The medium
was equilibrated prior to inoculation with a gas phase containing
0.2 atmosphere partial pressure of CO.sub.2 to yield a pH in the
range of 7.2-7.3. The initial medium used to start the culture
contained, in addition to the above components, 1.4 g/L
NaAcetate.3H.sub.2O, but the medium reservoir used in continuous
culture conditions lacked the addition of acetate.
[0049] 1 L of the fresh medium was initially inoculated with 5 mL
of Methanococcus maripaludis in a stationary phase, and methane
production was monitored over time. As shown in FIG. 4, a gas feed
of 4:1 H.sub.2:CO.sub.2 (125 cc/min or 180 volumes of gas (STP) per
volume of culture per day (VVD), 144 vvd H.sub.2 and 36 vvd
CO.sub.2) was provided at a culture of pH 7.33 and an ORP of -140
mV. During the growth phase, the rate of methane production is
limited by the available biological catalysts for the reaction. The
methane production rate stabilized once the dissolved hydrogen in
the medium was depleted. The stabilized rate is limited by the
physical process of gas-to-liquid mass transfer of hydrogen, rather
than by biological factors. Once this transition to stable methane
production was observed, the culture was switched to continuous
culture conditions in which fresh medium was added at a constant
rate of 0.94 ml/h, or 22.5 ml/day. It was found that a slower input
of fresh culture medium led to a denser culture and hence better
volumetric performance. At the limit of no fresh medium, however,
it was found that the culture ultimately dies.
EXAMPLE 3
Effect of Agitation on Methane Production of Methanococcus
maripaludis
[0050] The turbidity of the culture obtained in Example 2 continued
to increase after the gas-to-liquid mass transfer-limited rate of
methane production was reached, providing an excess of biological
catalytic capacity. This additional catalytic capacity can be
accessed by changing physical parameters that increase the
gas-to-liquid mass transfer rate. As shown in FIG. 5, the mixing
rate in the culture was varied from the standard 400 rpm. A total
feed rate of a 4:1 H.sub.2:CO.sub.2 gas mixture (250 cc/min (288
vvd H.sub.2; 72 vvd CO.sub.2)) was used. At this gas feed rate, the
conversion efficiency of both CO.sub.2 and hydrogen reaches 55-56%
at higher mixing speeds demonstrating that higher stirring rates
increases the gas-to-liquid mass transfer and therefore higher
methane production. Other abiotic methods that may be used to
increase the gas-to-liquid mass transfer and hence the methane
production rate include 1) increased gas pressure and 2) increased
temperature. Some methanognic archaea can thrive at pressures over
500 atmospheres. With respect to different temperature conditions,
thermophilic methanogens, such as Methanothermobacter
thermoautotrophicus (at about 60.degree. C.-65.degree. C.) or
Methanocaldococcus jannaschii (at about 85.degree.-90.degree. C.),
can be used as the biological catalyst.
EXAMPLE 4
Conversion Efficiency of H.sub.2 by Methanococcus maripaludis
[0051] A culture of Methanococcus maripaludis was setup in a
bioreactor as set forth in Example 2. As shown in FIG. 6, gas was
fed to a mature culture at varying rates, maintaining a 4:1
hydrogen to carbon dioxide ratio. Once the culture was above the
gas-liquid mass transfer-limited cell density, it was found that
methane production could be increased by increasing the H.sub.2 gas
feed rate. However, at higher H.sub.2 gas feed rates, it was found
that a decreasing proportion of the H.sub.2 gas was converted to
methane. The converse was also found to be true, that at lower gas
feed rates, hydrogen was converted more efficiently to methane.
Because the volume of feed gas (4 volumes of hydrogen plus 1 volume
of CO.sub.2) decreases as it is converted to methane (1 volume of
methane product), a cascade or stratified bioreactor system as
shown in FIG. 2 and FIG. 3 is advantageous. In a serial bioreactor
system as shown in FIG. 3, the residual hydrogen in the effluent
gas from a first fermenter would provide a lower feed rate to a
second fermenter and would therefore be converted at higher
efficiency in the second fermenter. This phenomenon can be used to
obtain a highly efficient conversion in a cascaded fermenter
design
EXAMPLE 5
Bench Scale Bioreactor Using Methanosarcina barkeri
[0052] The general setup of Example 1 was used with the organism
Methanosarcina barkeri. Methanosarcina barkeri (strain DSM 804
obtained from the Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH) was grown at 35.degree. C. in MS enriched medium
containing the following components per L of medium: NaHCO.sub.3
8.4 g, yeast extract 2.0 g, trypticase peptones 2.0 g,
mercaptoethanesulfonic acid 0.5 g, NH.sub.4Cl 1.0 g,
K.sub.2HPO.sub.4.7H.sub.2O 0.4 g, MgCl.sub.2.7H.sub.2O 1.0 g,
CaCl.sub.2.2H.sub.2O 0.4 g, Resazurin 1 mg, cysteine.H.sub.2O.HCl
0.25 g, Na.sub.2EDTA.2H.sub.2O 5 mg, MnCl.sub.2.4H.sub.2O 1 mg,
CoCl.sub.2(0.6H.sub.2O) 1.5 mg, FeSO.sub.4.7H.sub.2O 1 mg,
ZnCl.sub.2 1 mg, AlCl.sub.3.6H.sub.2O 0.4 mg,
Na.sub.2WO.sub.4.2H.sub.2O 0.3 mg, CuCl 0.2 mg,
NiSO.sub.4.6H.sub.2O 0.2 mg, H.sub.2SeO.sub.3 0.1 mg,
H.sub.3BO.sub.4 0.1 mg, Na.sub.2MoO.sub.4.2H.sub.2O 0.1 mg, biotin
0.02 mg, folic acid 0.02 mg, pyridoxine HCl 0.10 mg, thiamine HCl
0.05 mg, riboflavin 0.05 mg, nicotinic acid 0.05 mg, DL-calcium
pantothenate 0.05 mg, vitamin B12 0.001 mg, p-aminobenzoic acid
0.05 mg, lipoic acid 0.05 mg. After autoclaving and before
inoculation, the medium was reduced by the addition of 0.5 g/L
Na.sub.2S from a 50.times. anaerobic, sterile stock solution,
yielding an ORP of the medium below -100 mV. The medium was
equilibrated prior to inoculation with a gas phase containing 0.2
atmosphere partial pressure of CO.sub.2 to yield a pH in the range
of 6.8-7.0.
[0053] 1 L of the fresh medium was initially inoculated with 20 mL
Methanosarcina barkeri in a stationary phase, and methane
production was monitored over time. Once the transition to stable
methane production was observed, the culture was switched to
continuous culture conditions in which fresh medium was added at a
constant rate of 0.94 ml/h, or 22.5 ml/day. It was found that a
slower input of fresh culture medium led to a denser culture and
hence better volumetric performance. At the limit of no fresh
medium, however, it was found that the culture ultimately dies.
EXAMPLE 6
Recovery from Oxygen Exposure
Recovery of Methanosarcina barkeri with Exposure of 10 Minutes of
Air
[0054] Methanogenic organisms are regarded as extremely strict
anaerobes. Oxygen is known as an inhibitor of the enzyme catalysts
of both hydrogen uptake and methanogenesis. A low
oxidation-reduction potential (ORP) in the growth medium is
regarded as important to methanogenesis. Air is a possible
contaminant of carbon dioxide streams that could be used to support
energy storage in the form of methane and so the effects of air on
the capacity of the cultures to produce methane was examined.
[0055] FIG. 7 shows the recovery of the methanogenic activity of
Methanosarcina barkeri after exposure to air. A bench bioreactor
containing Methanosarcina barkeri was prepared as set forth in
Example 5. Two experiments were performed involving exposing the
culture to 100% air for 10 minutes at a flow rate of 500 cc/min.
Ambient air comprises approximately (by molar content/volume) 78%
nitrogen, 21% oxygen, 1% argon, 0.04% carbon dioxide, trace amounts
of other gases, and a variable amount (average around 1%) of water
vapor. During exposure to 100% air, methanogenesis stopped and the
ORP of the culture medium rose. The air used in the experiment also
displaces CO.sub.2 dissolved in the medium, causing the pH to rise
(not shown in this figure). Following the 10 minute exposure to
100% air, gas flows of H.sub.2 and CO.sub.2 were restored (100
cc/min H.sub.2, 25 cc/min CO.sub.2).
[0056] In the first experiment, 1.5 ml of a 2.5% solution of
sulfide (Na.sub.2S.7H.sub.2O) was added within 4 minutes of
terminating air feed and restoring the H.sub.2/CO.sub.2 gas feed.
Sulfide is widely used to control the ORP of the cultures, control
that is regarded as essential. In another experiment, so sulfide
was added. The dotted curves show a case in which no sulfide was
added and the solid line shows the recovery of the culture where
sulfide was added. In both cases, methanogenesis recovers. The
figure shows that addition of sulfide for ORP control in case 1
causes the emitted hydrogen sulfide to rise to 3000 ppm. In the
case of ORP control with hydrogen (no sulfide addition), the
hydrogen sulfide level is at or below the limit of detection of the
mass spectrometer under these operating conditions (50-100 ppm).
Methanogenesis begins to recover more quickly in the case of ORP
control with sulfide, but the experiment shows that sulfide is not
essential for recovery. The presence of the hydrogen in the gas
phase is sufficient to reduce the ORP of the culture to enable
methanogenesis, no additional control of the ORP of the culture is
required. The lack of necessity of sulfide is of note in that
methanogenic cultures are typically maintained at 10,000 ppm
hydrogen sulfide in the gas phase. Such high levels of sulfide are
not tolerated in certain industrial process, for instance, natural
gas pipeline tariffs in the United States set maximum levels of
hydrogen sulfide content of natural gas ranging from 4-16 ppm,
depending upon the pipeline system.
[0057] FIG. 8 also shows the recovery of Methanosarcina barkeri
after the air exposure above (absent the addition of sulfide) after
7 hours.
EXAMPLE 7
Recovery from Oxygen Exposure
Recovery of Methanosarcina barkeri with Exposure of 90 Minutes of
Air
[0058] FIG. 9 shows the recovery of Methanosarcina barkeri after 15
hours of exposure to air. A bench bioreactor containing
Methanosarcina barkeri was prepared as set forth in Example 5. The
culture was exposed to 100% air for 90 minutes introduced at IL/min
(1440 vvd). During exposure to 100% air, methanogenesis stopped and
the ORP of the culture medium rose. The air used in the experiment
also displaced CO.sub.2 dissolved in the medium, causing the pH to
rise. Following the 90 minute exposure to 100% air, gas flows of
H.sub.2 and CO.sub.2 were restored (100 cc/min H.sub.2 (144 vvd)
and 25 cc/min CO.sub.2 (36 vvd). Restoration of hydrogen as a
reductant was sufficient to reduce the ORP to levels that favor
methanogenesis. Methane production began within 1 hr of restoring
4:1 H.sub.2:CO.sub.2 gas phase (100 cc/min H.sub.2, 25 cc/min
CO.sub.2). Full recovery of methane production was achieved within
3-4 hrs.
EXAMPLE 8
Recovery from Oxygen Exposure
Recovery of Methanosarcina barkeri with Exposure of 15 Hours of
Air
[0059] FIG. 10 shows the recovery of Methanosarcina barkeri after
15 hours of exposure to air. A bench bioreactor containing
Methanosarcina barkeri was prepared as set forth in Example 5. The
culture was exposed to 100% air for 15.1 hours at a flow rate of 1
L/min. During exposure to 100% air, methanogenesis stopped and the
ORP of the culture medium rose to about -10 mV. The air used in the
experiment also displaced CO.sub.2 dissolved in the medium, causing
the pH to rise to 9.3. Following the 15.1 hour exposure to 100%
air, gas flows of H.sub.2 and CO.sub.2 were restored (100 cc/min
H.sub.2, 25 cc/min CO.sub.2). Restoration of hydrogen as a
reductant was sufficient to reduce the ORP to levels that favor
methanogenesis. Methane production began within 1.1 hr of restoring
4:1 H.sub.2:CO.sub.2 gas phase (100 cc/min H.sub.2, 25 cc/min
CO.sub.2). Full recovery of methane production was achieved within
3 hrs.
EXAMPLE 9
Recovery from Oxygen Exposure
Recovery of Methanococcus maripaludis with Exposure of 10 Minutes
of Air
[0060] FIG. 11 shows the recovery of the methanogenic activity of
Methanococcus maripaludis after exposure to air. A bench bioreactor
containing Methanococcus maripaludis was prepared as set forth in
Example 2. The culture was exposed to 100% air for 10 minutes at
360 vvd. During exposure to 100% air, methanogenesis stopped and
the ORP of the culture medium rose. The air used in the experiment
also displaces CO.sub.2 dissolved in the medium, causing the pH to
rise. Following the 10 minute exposure to 100% air, gas flows of
H.sub.2 and CO.sub.2 were restored (288 vvd H.sub.2, 72 vvd
CO.sub.2). Methanogenesis in was shown to have recovered within 10
min, with full recovery of methane production rate occurring within
1.5 hours.
EXAMPLE 10
Maintained Methane Production by Methanococcus maripaludis in the
Presence of Air
[0061] FIG. 12 shows that methane production can continue even in
the presence of air provided that hydrogen is present to maintain
the ORP of the culture at productive levels. A bench bioreactor
containing Methanococcus maripaludis was prepared as set forth in
Example 2. The culture was exposed to a mixture of 4% air and 76%
hydrogen at a flow rate of 100 cc/min and CO.sub.2 at 25 cc/min for
a period of 2.3 hours. The percentage of air was then increased to
8%, providing a mixture of 8% air and 72% hydrogen at a flow rate
of 100 cc/min and CO.sub.2 at 25 cc/min for a period of 1.7 hours.
The percentage of air was then increased to 16%, providing a
mixture of 16% air and 64% hydrogen at a flow rate of 100 cc/min
and CO.sub.2 at 25 cc/min for a period of 1.1 hours. Finally, the
percentage of air was then increased to 32%, providing a mixture of
32% air and 58% hydrogen at a flow rate of 100 cc/min and CO.sub.2
at 25 cc/min for a period of 0.6 hours. Methane production
continues even in the presence of air provided that hydrogen is
present to maintain the ORP of the culture at productive levels. Up
to 4% air (0.8% oxygen) is tolerated without a persistent reduction
in methane production. The efficiency of conversion of the input
hydrogen to methane remains unaffected at 22-23% under the
conditions of the experiment until the air concentration rises from
8% to 16% of the total gas mix.
[0062] Note that the gas mixtures produced by the culture under
these conditions could be explosive, since the oxygen is not
consumed by the organisms and appears in the effluent gas stream. A
potentiostat culture system provides a method for maintaining the
ORP of the culture during air exposure without introducing hydrogen
or generating methane.
EXAMPLE 11
Recovery from Carbon Monoxide Exposure by Methanococcus
maripaludis
[0063] Carbon monoxide is another known inhibitor of enzymes
involved in both hydrogen uptake and methanogenesis. CO is a
potential contaminant of CO.sub.2 and hydrogen streams derived from
gasification of coal or biomass resources. The effect CO on methane
formation by methanogen cultures was examined. FIG. 13 shows the
recovery of the methanogenic activity of Methanococcus maripaludis
after exposure to carbon monoxide. A bench bioreactor containing
Methanococcus maripaludis was prepared as set forth in Example 2.
In this experiment, the pH of the culture was maintained constant
by keeping CO.sub.2 at 20% of the gas mix and changing only the
composition of the other 80% of the gas. The ORP of the culture
remained relatively constant between -140 and -150 mV.
[0064] The culture was exposed to a mixture of 8% CO and 72%
hydrogen at a flow rate of 100 cc/min and CO.sub.2 at 25 cc/min for
a period of 1.7 hours. Then the culture was restored to a flow of
80% hydrogen at a flow rate of 100 cc/min and CO.sub.2 at 25
cc/min. Upon removal of the CO and restoration of 50% H.sub.2,
methanogenesis recovered completely within a 5 minutes (within the
mixing time of the gas phase in the culture). This rapid recovery
suggests that the primary effect of CO under these experimental
conditions is as a reversible inhibitor of the methanogenesis
process.
[0065] The culture was then exposed to a mixture of 16% CO and 64%
hydrogen at a flow rate of 100 cc/min and CO.sub.2 at 25 cc/min for
a period of 1 hour. This higher exposure of CO showed only a 25%
inhibition of methane formation rates. This suggests that the
initial exposure caused an adaptation in the culture that reduced
its sensitivity to CO inhibition. The culture was then restored to
a flow of 80% hydrogen at a flow rate of 100 cc/min and CO.sub.2 at
25 cc/min. Recovery of methanogenesis following CO removal was
again immediate.
[0066] Finally, the culture was exposed to a mixture of 40% CO and
40% hydrogen at a flow rate of 100 cc/min and CO.sub.2 at 25 cc/min
for a period of 20 minutes. This CO exposure showed almost as much
inhibition of the adapted process as occurred in the initial low
level exposure of the un-adapted organisms. The culture was then
restored to a flow of 80% hydrogen at a flow rate of 100 cc/min and
CO.sub.2 at 25 cc/min. Recovery from this level of CO was also
immediate.
[0067] Another experiment was performed showing the effects of even
higher concentration of CO on methanogenesis. A bench bioreactor
containing Methanococcus maripaludis was prepared and maintained as
set forth above. FIG. 14 shows that when the culture was given a
doses of a mixture of 60% CO and 20% hydrogen at a flow rate of 100
cc/min and CO.sub.2 at 25 cc/min, this led to a complete loss of
methane formation in this species. Recovery from this level of CO
has an immediate phase but full recovery requires several hours.
These experiments show that methanogenesis achieved by methanogen
cultures is tolerant to levels of CO likely to be found in
industrial CO.sub.2 streams derived from coal or biomass
gasification, but that higher levels may be poorly tolerated.
EXAMPLE 12
Efficiency of Methane Production from a Cascaded Bioreactor
[0068] A mathematical projection was performed to determine the
projected efficiencies of methane production according to the
methods of the present invention in a cascaded bioreactor system
similar to what is depicted in FIG. 3. The performance of a
methanogen culture in converting hydrogen and carbon dioxide to
methane was modeled as a gas-phase continuously stirred tank
reactor (CSTR) in which the gas is homogeneously mixed with a
liquid phase in which the reaction occurs, catalyzed by the
organisms. Under mass-transfer limited conditions, the reaction is
first order in hydrogen with a rate that is governed by the
gas-liquid transfer of hydrogen. The consumption of hydrogen by the
organisms is sufficiently rapid to keep the dissolved concentration
of hydrogen well below the saturating concentration. Under this
model, the product of the first order rate constant, k, and the
residence time of gas in the gas phase, .tau., govern the extent of
the reaction of the hydrogen:
k .tau. = X 1 - X , ##EQU00001##
where x is the fraction of hydrogen converted to methane. It is
assumed that there is at least enough CO.sub.2 present to consume
all of the hydrogen in methane formation.
[0069] The extent of the reaction, X, is determined by the input
flow of hydrogen gas, F.sub.o (in units of moles/time), the mass
balance for the conversion to methane, and the first order rate
constant, assuming constant volume, pressure and temperature:
F o = k V g P g ( 1 - X ) R T X ( 5 4 - X + D ) ##EQU00002##
[0070] where V.sub.g is the volume of the gas phase that is
efficiently mixed with the liquid phase, P.sub.g is the operating
pressure, R is the universal gas constant, T is the chamber
temperature in .degree. K, and D is the total mole fraction in the
gas phase of non-reactant gases, including any excess CO.sub.2 as
well as water vapor. As input reactant gas flow is increased, more
product methane is produced, but at the expense of lower extent of
reaction because of a lower retention time.
[0071] During the reaction, 4 volumes of hydrogen and 1 volume of
carbon dioxide are consumed to produce 1 volume of methane. Hence,
as the reaction proceeds, the flow of gas out of the chamber is
less than the input flow. This strong reduction in flow during the
reaction provides a uniquely valuable advantage to a system of
cascaded reactors in which the exit gas from one reaction chamber
becomes the inlet for a successive chamber. Under steady state
conditions, the exit flow of hydrogen, F, is given by
F=F.sub.o(1-X) in a given chamber. For a cascade of two identical
chambers, the flow rate in the second chamber will be less than
that in the first, and hence the retention time and the extent of
conversion will be greater. The advantage of the cascade approach
can be appreciated by comparing the extent of conversion in a
single chamber of a fixed volume with the total extent of
conversion of the same inlet gas flow by two cascaded chambers,
each half the volume of the single chamber and with that of three
cascaded chambers, each one third the volume of the single chamber,
as shown in FIG. 15. For this illustration, the initial
hydrogen:CO.sub.2 ratio is 4:1.
TABLE-US-00001 TABLE 1 Conversion efficiency Single Cascade Reactor
2 reactor 3 reactor 0.3350 0.3626 0.3729 0.4562 0.5134 0.5368
0.5502 0.6389 0.6788 0.6222 0.7376 0.7927 0.6775 0.8110 0.8750
0.7204 0.8636 0.9280 0.7542 0.9004 0.9593 0.7813 0.9262 0.9770
0.8034 0.9443 0.9868 0.8369 0.9667 0.9954 0.9042 0.9928 0.9998
[0072] In this simulation, the input flow rate and the conversion
rate constant were adjusted to give a range of extents of reaction
in a single tank reactor. The extent of reaction, X, for this
single reactor is listed in the first column of Table 1. The same
initial flow rate was then fed to 2 or three cascaded reactors of
1/2 or 1/3 the volume of the single tank reactor, respectively,
keeping the other reaction conditions constant. The extent of
reaction measured at the exit of the final reactor of the cascade
is listed in the second and third columns of the table. The same
data are also presented graphically in FIG. 15. Here, the final
extent of reaction in the cascade, Xcascade, is divided by the
extent of reaction of the equivalent total volume single reactor,
Xsingle, and the ratio is plotted against Xsingle. Conditions near
the peaks of these graphs would optimize the conversion gain of the
cascade arrangement, while conditions to the right of the peaks
would exploit the cascade for efficiently reducing the final
residual unreacted hydrogen in the product gas stream.
[0073] This model assumes a homogeneous residence time for the gas.
In practice, the bubbles suspended in the liquid act as independent
mini-reactors for some period of time, a behavior that causes a
dispersion of net conversion in different bubbles and in apparent
residence times. This dispersion of residence times can be
determined under operating conditions by injecting an inert gas
tracer into the inlet gas stream and monitoring the time that it
takes to exit. Depending on the exact shape of the residence time
distribution, this phenomenon could degrade the performance of the
single tank reactor. However, the use of cascading reactors
sharpens the residence time distribution, which favors the cascaded
reactor performance over that of a single reactor of the same total
volume.
EXAMPLE 13
Maintenance of a Methane Producing Culture of Methanothermobacter
thermoautotrophicus in Stationary Phase
[0074] Methanothermobacter thermoautotrophicus (DSMZ 3590) was
grown in a culture medium comprising: NaCl 10 mM, NH.sub.4Cl 120
mM, nitrilotriacetic acid (NTA) 1.2 mM, MgCl.sub.2.7H.sub.2O 1 mM,
KH.sub.2PO.sub.4 10 mM, CoCl.sub.2 2.5 .mu.M, Na.sub.2MoO.sub.4 2.5
.mu.M, NiCl.sub.2 5 .mu.M, FeSO.sub.4.7H.sub.2O 0.2 mM,
Na.sub.2SeO.sub.3 1 .mu.M, Na.sub.2WO.sub.4 10 .mu.M, at 60.degree.
C. in a 1.3 L BioFlo 110 fermenter vessel containing 700 ml medium
and agitated at 1000 RPM until it reached stationary phase. During
initial growth of the culture, sodium sulfide was added at a rate
that maintained hydrogen sulfide in the output gas stream at 10
ppm. The culture was sparged with a 4:1 H.sub.2:CO.sub.2 gas
mixture at a total rate of 0.25 SLPM. In the stationary phase, a
culture gassed at this rate produces 49 ml/min methane (101 vvd;
98% conversion of the input hydrogen). During methanogenesis, the
culture produced two moles of metabolic water per mole of methane,
which is a significant fraction of the medium volume. Medium,
containing cells, was removed from the fermenter to keep the liquid
volume constant. Medium components removed along with the liquid
were replaced with concentrated stock solutions. During stationary
phase, sulfide addition was necessary only for maintaining cell
replacement and was maintained at a level below 1 ppm in the output
gas stream.
EXAMPLE 14
Recovery of Methane Production by M. thermoautotrophicus Following
Exposure to Air
[0075] A stationary phase culture of M. thermoautotrophicus 3590
producing methane at 49 ml/min (101 vvd) from a 0.25 SLPM input gas
stream of 4:1 H.sub.2:CO.sub.2 was exposed to air by replacing the
hydrogen in the input gas mixture with air. The composition of the
output gas was analyzed by mass spectrometry and the output rates
of the various gases were computed in SLPM. As shown in FIG. 16,
the output gas methane production declined immediately. After 1 hr,
the air in the input gas was replaced with argon for 10 min and
then with hydrogen, restoring the original 4:1 H.sub.2:CO.sub.2 gas
mixture. After a lag of 22 min, methane production recovered
quickly, reaching 50% of the original production rate within 32 min
and full 98% conversion efficiency by 57 min.
EXAMPLE 15
Methane Production by a Stationary Phase Culture of M.
thermoautotrophicus at Different Gassing Rates
[0076] A stationary phase culture of M. thermoautotrophicus was
grown as in Example 13, except with an initial hydrogen gassing
rate in a 4:1 H.sub.2:CO.sub.2 mixture of 200 ml/min (450 vvd) and
a liquid volume of 650 ml. This arrangement provides a 1:1 ratio of
liquid culture to gaseous headspace and the agitation of 1000 RPM
is adequate to maintain thorough mixing of the headspace with the
liquid medium. The culture was then gassed at various rates with
4:1 H.sub.2:CO.sub.2 until the output gas composition stabilized. A
pulse of argon gas was introduced into the gas feed stream as a
tracer to measure the average residence time of the gas, .tau..
These data showed simple exponential decay of the gas tracer,
indicating thorough mixing of the gas and liquid phases. The
performance of the culture at each gassing rate is given in Table
2.
TABLE-US-00002 TABLE 2 H2 in CH4 out (vvd) (vvd) X (fraction
complete) .tau. (s) 450 111 0.987 299 900 215 0.955 98.8 1350 311
0.922 61.6 1800 399 0.886 47.8 2250 475 0.845 47 3150 595 0.755
35.9 4050 673 0.664 37.4 5400 758 0.562 33.3 6750 840 0.498 ND
[0077] A completely efficient catalyst (X=1.0) would yield 1012 vvd
of methane from an input flow of 4050 vvd hydrogen. In practice, X
at 4050 vvd hydrogen (1 atmosphere reactor pressure) is 0.664,
corresponding to 673 vvd methane production in a single-pass
continuously stirred tank reactor operating under the conditions
specified. As projected in Example 12, a three-stage cascade
reactor with the same total volume as the single reactor would
operate with a final conversion efficiency improved to 0.86 and a
corresponding increase in methane production to 860 vvd. A single
reactor producing the same purity of product (same value of X)
would produce only about 380 vvd methane from an input of 1700 vvd
of hydrogen. If the volume of this same reactor operating at X=0.86
and 1700 vvd hydrogen input were divided into a three-tank cascade,
it would produce 425 vvd methane at X=0.99.
[0078] While the present invention has now been described and
exemplified with some specificity, those skilled in the art will
appreciate the various modifications, including variations,
additions, and omissions that may be made in what has been
described. It is to be understood that the invention is not limited
in its application to the details of construction and the
arrangements of the components set forth in the previous
description or illustrated in the drawings. The invention is
capable of other embodiments and of being practiced or being
carried out in various ways. Accordingly, it is intended that these
modifications also be encompassed by the present invention and that
the scope of the present invention be limited solely by the
broadest interpretation that lawfully can be accorded the appended
claims.
[0079] Also, it is understood that the phraseology and terminology
used herein are for the purpose of description and should not be
regarded as limiting. The use of "including", "having" and
"comprising" and variations thereof herein is meant to encompass
the items listed thereafter and equivalents thereof as well as
additional items and equivalents thereof. It also is understood
that any numerical value recited herein includes all values from
the lower value to the upper value. For example, if a concentration
range is stated as 1% to 50%, it is intended that values such as 2%
to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in
this specification. These are only examples of what is specifically
intended, and all possible combinations of numerical values between
the lowest value and the highest value enumerated are to be
considered to be expressly stated in this application.
* * * * *